U.S. patent application number 11/423676 was filed with the patent office on 2006-12-07 for compositions and methods for making embryonic stem cells.
This patent application is currently assigned to VANDERBILT UNIVERSITY. Invention is credited to Brigid L.M. Hogan, Guang-Quan Zhao.
Application Number | 20060275898 11/423676 |
Document ID | / |
Family ID | 26683493 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060275898 |
Kind Code |
A1 |
Hogan; Brigid L.M. ; et
al. |
December 7, 2006 |
Compositions and Methods for Making Embryonic Stem Cells
Abstract
The invention relates to cell proliferation, cell
differentiation, male infertility, male fertility and to
compositions and methods involved therein.
Inventors: |
Hogan; Brigid L.M.;
(Brentwood, TN) ; Zhao; Guang-Quan; (Columbia,
MO) |
Correspondence
Address: |
PRESTON GATES & ELLIS LLP
1900 MAIN STREET, SUITE 600
IRVINE
CA
92614-7319
US
|
Assignee: |
VANDERBILT UNIVERSITY
305 Kirland Hall
Nashville
TN
|
Family ID: |
26683493 |
Appl. No.: |
11/423676 |
Filed: |
June 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10350747 |
Jan 24, 2003 |
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11423676 |
Jun 12, 2006 |
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09685666 |
Oct 10, 2000 |
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10350747 |
Jan 24, 2003 |
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08808346 |
Feb 28, 1997 |
6251671 |
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09685666 |
Oct 10, 2000 |
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60012384 |
Feb 28, 1996 |
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60012386 |
Feb 28, 1996 |
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Current U.S.
Class: |
435/325 ;
435/366 |
Current CPC
Class: |
A61K 38/1875 20130101;
G01N 33/5073 20130101; C12N 2501/235 20130101; C12N 2501/125
20130101; A61P 17/14 20180101; G01N 33/689 20130101; C12N 2501/155
20130101; A01K 67/0276 20130101; G01N 33/5017 20130101; C12N
2501/115 20130101; C12N 15/8509 20130101; G01N 2800/367 20130101;
G01N 2510/00 20130101; A61P 43/00 20180101; A01K 2267/03 20130101;
C12N 5/061 20130101; A01K 2217/075 20130101; G01N 33/5044 20130101;
A01K 2227/105 20130101; A61P 15/08 20180101; C07K 14/51 20130101;
G01N 33/5008 20130101; A61P 15/16 20180101 |
Class at
Publication: |
435/325 ;
435/366 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C12N 5/08 20060101 C12N005/08 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was supported in part by a grant from the
U.S. Government (NIH Grant No. CA 48799) and the U.S. Government
may therefore have certain rights in the invention.
Claims
1-22. (canceled)
23. A method of providing an embryonic stem cell comprising:
incubating a population of spermatogonial stem cells in a
composition comprising a growth enhancing amount of basic
fibroblast growth factor, leukemia inhibitory factor, membrane
associated steel factor, and soluble steel factor, thereby making a
population of pluripotent embryonic stem cells.
24. A population of pluripotent embryonic stem cells produced by
the method of claim 23.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation application of
U.S. patent application Ser. No. 10/350,747 filed Jan. 23, 2003,
which is a continuation of U.S. patent application Ser. No.
09/685,666 filed Oct. 10, 2000, now abandoned, which is a
continuation of U.S. patent application Ser. No. 08/808,346 filed
Feb. 28, 1997, now U.S. Pat. No. 6,251,671, which claims the
benefit of U.S. Provisional Patent Application Ser. Nos. 60/012,386
filed Feb. 28, 1996 and 60/012,384 filed Feb. 28, 1996, all of
which are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0003] The field of the invention is human and nonhuman mammalian
spermatogenesis.
BACKGROUND OF THE INVENTION
[0004] The bone morphogenetic proteins (BMPs) are members of a
large, highly conserved, family of extracellular polypeptide
signaling molecules related to transforming growth factor-.beta.
(TGF-.beta.). There is now considerable evidence from expression
studies, and from the in vivo effects of misexpression and
mutations, that Bmp genes play key roles at many different stages
of embryonic development, in both invertebrates and vertebrates
(Kingsley et al., 1994, Dev. Biol. 166:112-122; Massague et al.,
1994, Trends Cell Biol. 4:172-178; Hogan, 1995, Sem. Dev. Biol.
6:257-265). In the mouse, both spontaneous and induced mutations in
a number of Bmp genes have shed light on their function in vivo.
The first example to be described was a series of short ear
mutations, which result from alterations in the Bmp5 gene (Green,
1968, J. Exp. Zool. 167:129-150; Kingsley et al., 1992, Cell
71:399-410; King et al., 1994, Dev. Biol. 166:112-122). Null
mutants are viable, but have defects in cartilage development in
specific parts of the skeletal system, as well as abnormalities in
the lung, kidney and ureter in some genetic backgrounds.
[0005] Mutations in other Bmp genes have been generated by
homologous recombination in embryonic stem cells. For example, Bmp7
homozygous null mutant mice die shortly after birth with major
defects in eye, kidney and limb development (Dudley et al., 1995,
Genes Dev. 9:2795-2807; Luo et al., 1995, Genes Dev. 9:2808-2820).
Most Bmp4 homozygous mutant embryos die around the time of
gastrulation and many exhibit a deficiency in extraembryonic and
posterior/ventral mesoderm (Winnier et al., 1995, Genes Dev.
9:2105-2116), a finding consistent with the effect of BMP4 on
mesoderm patterning in Xenopus embryos (Jones et al., 1992,
Development 115:639-647; Graft et al., 1994, Cell 79:169-179;
Harland, 1994, Proc. Natl. Acad. Sci. USA 91:10243-10246).
Mutations have also been described in other members of the BMP
superfamily, including mouse nodal, and Gdf5 (brachypodism) (Zhou
et al., 1993, Nature 361:543-547; Conlon et al., 1994, Development
120:1919-1928; Storm et al., 1994, Nature 368:639-643).
[0006] Spermatogenesis takes places within the confines of the
seminiferous tubules in the testis. A typical tubule is ensheathed
by an outer basal lamina. Inside the lamina and attached to it is a
layer of spermatogonial cells which continue to divide very slowly
from puberty to late adult life. The self-renewing spermatogonial
stem cell (known as an A.sub.O/A.sub.S cell) is very rare and it
gives rise to other less primitive spermatogonial cells. These give
rise to non-dividing spermatocytes which leave the basal layer and
move towards the center of the tubule. These spermatocytes undergo
meiosis and eventually give rise to mature sperm. The spermatogonia
and differentiated derivatives are in intimate contact with the
somatic Sertoli cells. Little is known about the growth
factors/cytokines which regulate the proliferation of the
spermatogonial stem cells, their differentiation into
spermatocytes, the entry of the spermatocytes into meiosis, their
differentiation into sperm and the way in which this whole complex
process is co-ordinated in vivo. It has been difficult to obtain
populations of cells having high concentrations of the most
primitive spermatogonial stem cells in part because they form a
very small proportion of the total spermatogenic cells of the
testis. Additionally, cultures of spermatogonia generally only
remain viable for short periods of time, that is, about 24-48
hours.
SUMMARY OF THE INVENTION
[0007] The invention relates to a method of proliferating mammalian
spermatogonial stem cells, comprising culturing spermatogonial stem
cells in the presence of BMP8, or a biologically active fragment or
an agonist thereof, to effect proliferation of the cells.
[0008] Also included in the invention is a method of
differentiating mammalian spermatogonial stem cells, comprising
culturing spermatogonial stem cells in the presence of BMP, or a
biologically active fragment or an agonist thereof, to effect
differentiation of the cells.
[0009] In addition, the invention relates to a method of extending
viability of a mammalian spermatogonial cell population, comprising
culturing the spermatogonial cell population in the presence of
BMP8, or a biologically active fragment or an agonist thereof,
thereby extending the viability of the cultured spermatogonial cell
population.
[0010] In another aspect of the invention, there is provided a
method of inducing apoptosis of spermatocytes in culture comprising
incubating the spermatocytes in the substantial absence of BMP8, or
a biologically active fragment or an agonist thereof, thereby
inducing apoptosis of spermatocytes.
[0011] Also included in the invention is a method of inhibiting
proliferation of spermatogonial stem cells in culture, comprising
incubating a population of spermatogenic cells in the substantial
absence of BMP8 to effect inhibition of proliferation of the
cells.
[0012] Further provided is a method of effecting the proliferation
of mammalian spermatogonial stem cells in vivo in a mammal,
comprising administering to the mammal BMP8, or a biologically
active fragment or an agonist thereof, suspended in a
pharmaceutically acceptable carrier, to effect proliferation of the
cells in the mammal.
[0013] The invention also includes a method of effecting
differentiation of mammalian spermatogonial stem cells in vivo in a
mammal, comprising administering to the mammal BMP8, or a
biologically active fragment or an agonist thereof, suspended in a
pharmaceutically acceptable carrier, to effect differentiation of
the cells in the mammal.
[0014] A method of extending viability of a mammalian
spermatogonial cell population in vivo in a mammal is also
provided. The method comprises administering to the mammal BMP8, or
a biologically active fragment or an agonist thereof, suspended in
a pharmaceutically acceptable carrier, thereby extending the
viability of the mammalian spermatogonial cell population in the
mammal.
[0015] The invention also relates to a method of inducing apoptosis
of spermatocytes in vivo in a mammal comprising administering to
the mammal BMP8, or a biologically active fragment or an agonist
thereof, suspended in a pharmaceutically acceptable carrier,
thereby inducing apoptosis of spermatocytes in the mammal.
[0016] A method of inhibiting proliferation of spermatogonial stem
cells in vivo in a mammal is further provided. The method comprises
administering to the mammal an antagonist of BMP8 suspended in a
pharmaceutically acceptable carrier, to effect inhibition of
proliferation of the cells in the mammal.
[0017] In yet another aspect, the invention includes a method of
selectively obtaining a proliferating population of spermatogonial
stem cells in culture, comprising adding BMP8 to the population of
cells, thereby selectively obtaining a proliferating population of
spermatogonial stem cells.
[0018] There is also included a method of treating infertility in a
male mammal, comprising administering to the mammal BMP8, or a
biologically active fragment or an agonist thereof, suspended in a
pharmaceutically acceptable carrier.
[0019] In a preferred embodiment, the BMP8 is administered to the
testes of the mammal.
[0020] The invention includes a mammalian male contraceptive
comprising a BMP8 antagonist.
[0021] The invention also includes a method of identifying an
antagonist of BMP8, comprising adding a test compound to a culture
of spermatogonial cells in the presence or absence of BMP8 and
measuring the level of proliferation or differentiation of the
cells, wherein a lower level of proliferation or differentiation of
the cells in the presence of the test compound, compared with the
level of proliferation or differentiation of the cells in the
absence of the test compound, is an indication that the test
compound is a BMP8 antagonist.
[0022] Further included is a method of identifying an agonist of
BMP8, comprising adding a test compound to a culture of
spermatogonial cells in the presence or absence of BMP8 and
measuring the level of proliferation or differentiation of the
cells, wherein a higher level of proliferation or differentiation
of the cells in the presence of the test compound, compared with
the level of proliferation or differentiation of the cells in the
absence of the test compound, is an indication that the test
compound is a BMP8 agonist.
[0023] In addition, the invention relates to a method of
stimulating hair growth in a mammal, comprising administering a
hair growth stimulating amount of BMP8 to the hair follicles of the
mammal.
[0024] A method of isolating a BMP8 receptor on a cell is also
provided. The method comprises binding BMP8 to a BMP8-responsive
population of cells, and isolating the protein on the cells to
which the BMP8 binds.
[0025] The invention features a purified population of
spermatogonial stem cells.
[0026] The invention also features a method of making a population
of mammalian pluripotent embryonic stem cell, comprising incubating
a population of spermatogenic cells in a composition comprising a
growth enhancing amount of basic fibroblast growth factor, leukemia
inhibitory factor, membrane associated steel factor, and soluble
steel factor, thereby making a population of pluripotential
embryonic stem cells.
[0027] A population of pluripotential embryonic stem cells produced
by the just described method is also included.
[0028] In addition a composition comprising BMP8, a fibroblast
growth factor, leukemia inhibitory factor, membrane associated
steel factor, and soluble steel factor in amounts to enhance the
growth of and allow the continued proliferation of germ cells and
the formation of pluripotent embryonic stem cells from the germ
cells is included.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a diagram depicting a comparison of the deduced
amino acid sequences of murine BMP8A/OP2 and BMP8B. Identical
residues are aligned with a dash, similar residues are aligned with
a colon.
[0030] FIG. 2 is a diagram depicting the chromosomal location of
Bmp8a and Bmp8b. These two genes were mapped using the Jackson
Laboratory interspecific backcross panel mice (C57BL/6JEixSPRET/Ei)
F1xSPRET/Ei (Rowe et al., 1994, Mamm. Genome 5:253-274). In the
case of both genes, 94 of the (C57BL/6JEixSPRET) F1xSPRET/Ei
backcross progeny were typed for inheritance of the M. domesticus
or M. spretus alleles.
[0031] FIG. 2A is a partial chromosome 4 linkage map showing, on
the right hand side, the location of the two genes in relation to
surrounding loci mapped in the backcross and, on the left hand
side, the position of surrounding genes taken from the composite
map in the Mouse Genome Database.
[0032] FIG. 2B is a haplotype map. The black boxes indicate
inheritance of an M. domesticus allele while the white boxes
indicate inheritance of an M. spretus allele. R is the
recombination distances in centimorgans, SE is the standard
error.
[0033] FIG. 3A is an image depicting detection of Bmp8a and Bmp8b
RNAs in placenta and uterus using RNase protection assays. Ten
micrograms of total RNA from 8.5-15.5 d.p.c. embryos and 9.5 and
14.5 d.p.c. placentae and uteri was used in each hybridization
mixture comprising antisense riboprobes generated from Bmp8a UTR380
and Bmp8b BN260 (as shown in FIG. 3B). Bmp8 transcripts were
detected in 9.5 d.p.c. and 14.5 d.p.c. placentae and uteri. Bmp8b
transcripts were detected only in placentae but not in the uteri.
Transcripts derived from either gene were not present at
significant levels in the embryos assayed. .beta.-actin was used as
a control for the assessment of RNA levels in the experiments.
[0034] FIG. 3B is a schematic representation of Bmp8a and Bmp8h
cDNAs. The coding regions, including the pro-region and mature
region, are boxed. Regions used in DNA constructs for the
generation of riboprobes are indicated.
[0035] FIG. 4 is a series of images depicting localization of Bmp8
transcripts in uterus and placenta by in situ hybridization. FIGS.
4A, 4B, 4D, 4E, 4G, 4H, 4J, 4K, 4M, and 4N are dark-field
photomicrographs and FIGS. 4C, 4F, 4I, 4L and 4O are brightfield
photomicrographs. Scale bar is 200 .mu.m for FIGS. 4G-4I and 800
..mu.m for FIGS. 4A-4F and 4J-4O.
[0036] FIG. 4A is a section through a 7.5 d.p.c. implantation site
depicting high levels of Bmp8a transcripts in the antimesometrial
decidual cells (DE), but not in the surrounding myometrium of the
uterus (UT).
[0037] FIG. 4B and FIG. 4C is a section through 8.5 d.p.c. uterus,
deciduum, and embryo showing high levels of Bmp8a transcripts in
the antimesometrial decidual cells (AD) and lower levels of
hybridization signals in the mesometrial decidual cells (MD). No
hybridization signals are observed in the myometrium and embryo
proper (*).
[0038] FIGS. 4D, 4E and 4F are sections through 9.5 d.p.c. uterus,
placenta, and embryonic membranes depicting high levels of Bmp8a
transcripts in the deciduum and both Bmp8a and Bmp8b transcripts in
trophoblast cells (TB), but not in the myometrium of uterus or
extraembryonic membranes (*).
[0039] FIGS. 4G, 4H and 4I depict high power magnification of the
region boxed in FIG. 4F showing Bmp8a transcripts in the deciduum,
spongiotrophoblast cells (ST), and labyrinthine region of the
placenta (LT), but not in the giant trophoblasts (GT) and
extraembryonic membranes. Bmp8b transcripts are predominantly
localized in the labyrinthine region of the placenta.
[0040] FIGS. 4J, 4K and 4L are sections through 10.5 d.p.c. uterus,
placenta, and embryo showing decreased levels of Bmp8a transcripts
in the antimesometrial deciduum and increased levels of
hybridization in the mesometrial deciduum. Both Bmp8a and Bmp8b
transcripts are detected in the labyrinthine region of the
placenta. Neither Bmp8a nor Bmp8b transcripts are detected in the
embryo proper nor in the myometrium of uterus.
[0041] FIG. 4M is a section through uterus and deciduoma depicting
Bmp8a transcripts in the decidual cells, but not in the
myometrium.
[0042] FIGS. 4N and 4O is a section through 13.5 d.p.c. placenta
showing Bmp8b transcripts in the labyrinthine trophoblasts, but not
in the spongiotrophoblasts, giant trophoblasts or the decidual
cells.
[0043] FIG. 5 is a series of images depicting localization of Bmp8
transcripts in the testes by in situ hybridization. Sections of
testes from animals at 3.5 weeks (mid puberty), 5 weeks (later
puberty), 7 weeks (young adult), and 12 weeks (adult) were used for
in situ hybridization and histology. Panels (A), (B), (D), (E),
(G), (H), and (J), (K) depict adjacent sections of testes at
different ages hybridized with riboprobes for Bmp8a and Bmp8b as
indicated on the figure. Panels (C), (F), (I), and (L) depict
sections stained with periodic acid Schiff's (PAS) reagent and
hematoxylin for accurate staging of seminiferous tubules. Bmp8a and
Bmp8b transcripts are localized to stage VI-VII seminiferous
tubules in a similar pattern in animals at the different ages
shown. Panels M-O are adjacent sections from a 3.5 week testis.
Panels M and N depict the fact that the transcripts of both genes
are predominantly localized to the round spematids (arrows) and low
levels of signals are present in some pachytene spermatocytes
(large arrow head). Panel 0 is stained with PAS and hematoxylin
showing a stage VII seminiferous tubule as judged by the morphology
of acrosomes of the round spermatids (arrow). Panels P-R are
sections from a 7 week testis hybridized with Bmp8b riboprobe.
Hybridization signals are observed in the round spermatids (arrow)
of stage VII seminiferous tubule in Panel Q, but not in the round
spermatids of stage V or stage IX seminiferous tubules. No
detectable signals are observed in the elongated spermatids (small
arrow heads). Sections, in Panels P-R were exposed for 5 days, and
all other sections were exposed for 14 days. The scale bar is 100
.mu.m for Panels A-L and 25 .mu.m for Panels M-R.
[0044] FIG. 6 is a diagram depicting targeted mutagenesis of the
mouse Bmp8a locus.
[0045] FIG. 6A is a schematic representation of the Bmp8a wild-type
allele on the top, targeting construct in the middle, and the
recombinant Bmp8a.sup.tm1blh allele at the bottom. Genomic DNA
fragments used as the short (5') and long (3') homology arms of the
targeting vector are indicated as thick solid lines. Coding exons
2, 3, 4, 5, 6, and the first half of exon 7 (Ozkaynak et al., 1992,
J. Biol. Chem. 267:25220-25227) are indicated as solid boxes
(E2-E7). The second half of exon 7 containing the 3' untranslated
region is indicated as an open box. Expression cassettes PGK-TKA+,
PGK-neo.sup.r (Rudnicki et al., 1992, Cell 71:383-390), and MC1DT-A
(Yagi et al., 1990, Proc. Natl. Acad. Sci. USA 87:9918-9922) are
also shown as boxes with arrows underneath showing direction of
transcription. Restriction enzyme abbreviations: (B) BamHI; (E)
EcoRI; (S) SalI; (X) XbaI; (Xh) XhoI.
[0046] FIG. 6B is an image of a genomic Southern blot hybridized to
a cDNA probe derived from exons 2 and 3 of Bmp8a which is almost
identical in sequence to exons 2 and 3 of Bmp8b. Therefore, EcoRI
digested DNA reveals a 9 kb fragment for the wild-type Bmp8a locus
and an 8 kb fragment for the wild-type Bmp8b locus. The 9 kb Bmp8a
allele is reduced to 5 kb in the Bmp8a.sup.tm1blh allele.
[0047] FIG. 6C is a genomic Southern blot hybridized to a cDNA
fragment containing exons 4, 5, 6, and 7 as a probe. This probe is
specific for Bmp8a and reveals two EcoRI fragments of 2.3 kb
(containing exons 4, 5, and 6) and 2.0 kb (containing part of exon
7) for the wild-type allele and only a 2.0 kb fragment for the
mutant allele.
[0048] FIG. 7 is a graph depicting a comparison of testis weight
between wild-type (+/+) and Bmp8a.sup.tm1blh mutant (+/-and -/-)
mice. Testes from each age group were dissected out, blotted, and
weighed. Several litters were used for each group. Mean.+-.S.E. is
indicated in brackets; N represents the total number of testes in
each group. Generally, one testis from each animal was weighed if
the sizes of both testes appeared comparable. If in a very few
cases, the size of the two testes appeared significantly different,
both were weighed and the average was used for statistical
analysis.
[0049] FIG. 8 is a series of images depicting a histological
comparison of testes from 2 week-old animals of wild-type (+/+),
Bmp8a-/-, Bmp8b-/-, and Bmp8 compound heterozygous genotype. Left
and right hand panels are low and high power photomicrographs,
respectively. A typical representative histology from each genotype
is shown. At this age, the testes of Bmp8b homozygous mutants are
significantly smaller than the others and, in severe cases, only
one layer of epithelium is present in the seminiferous tubule
(Panels E and F; Zhao et al., 1996, Genes and Dev. 10:1657-1669).
In contrast, in wild-type animals, Bmp8a homozygous mutant and
compound heterozygous mutant testes, multiple layers of germ cells
are present in the seminiferous epithelium. Bar is 200 .mu.m in
Panels A, C, E, and G and 50 .mu.m in Panels B, D, F, and H.
[0050] FIG. 9 is a series of images depicting a histological
comparison of wild-type and Bmp8a and Bmp8b homozygous mutant
testes depicting the most advanced germ cell degeneration observed.
Panels on the left and right are low and high power
photomicrographs, respectively. Panels A and B are sections of a
wild type testis (17 weeks of age) showing normal spermatogenesis.
in all seminiferous tubules (the various stages of each tubule are
indicated by roman numerals; Russell et al., 1990, In: Histological
and histopathological evaluation of the testis, Eds. Russell et
al., pp. 119-161, Cache River Press, Clearwater, Fla.). Panels C
and D are sections of a homozygous Bmp8a mutant testis (22 weeks of
age) with the most advanced germ cell degeneration observed. In
this testis, very few spermatids were observed in the seminiferous
tubules, while meiotic germ cells were found in most seminiferous
tubules. However, a small number of tubules did lack most germ
cells and even some of the Sertoli cells had disappeared, indicated
by the *. Apoptotic germ cells exhibiting darkly-stained condensed
nuclei were observed in most seminiferous tubules (arrows). Panels
E and F are sections of a homozygous Bmp8b mutant testis (22 weeks
of age) showing that all seminiferous tubules lack spermatogenesis
and only Sertoli cells were left in a majority of the seminiferous
tubules, indicated by the *. Some darkly stained cells (arrowhead)
were observed in the center of some seminiferous tubules. These
cells did not express Sertoli cell markers such Dhh and Cp-2
(Bitgood et al., 1996, Curr, Biol. 6:298-304; Wright et al., 1986,
Biol. Reprod. 35:761-772; Wright et al., 1993, In: Cell and
Molecular Biology of the Testis, Eds. Desjardins et al., pp.
377-399, Oxford Univ. Press NY) and were most likely the
spermatogonial germ cells which had detached from the basal lamina
(Zhao et al., 1996, Genes and Dev. 10:1657-1669). Bar is 400 .mu.m
in Panels A, C, and E and 100 .mu.m in Panels B, D, and F.
[0051] FIG. 10 is a series of images depicting examples of milder
forms of histological abnormalities observed in adult Bmp8a mutant
testes. Panels A-C are sections of a wild-type testis (22 weeks of
age) showing normal spermatogenesis. Panels A is at low power
magnification; Panel B is a stage X seminiferous tubule at high
power magnification; Panel C is a stage XII seminiferous tubule at
high power magnification; Panels D-0 are representative sections
from testes of different Bmp8a homozygous null mutants (12-30 weeks
of age). Panel D, at low power magnification, shows germ cell
degeneration in numerous seminiferous tubules, indicated by the *;
Panels E and F are high power photomicrographs of seminiferous
tubules similar to those indicated in Panel D. No spermatids and
only a few meiotic germ cells are present. Arrowhead indicates a
cluster of darkly-stained cells in the center of the degenerated
tubule which are similar to those observed in Bmp8b mutant testis
as shown in FIG. 9F. Panel G, at low power magnification, shows
germ cell degeneration in two seminiferous tubules, indicated by
the *. Panels H and I are high power photomicrographs of
seminiferous tubules similar to those indicated in Panel G. No
spematids are found in Panel H and very few in are found in Panel
I. A few apoptotic spermatocytes with condensed and darkly-stained
nuclei (small arrows) are present in Panel I. Panel J, at low power
magnification, shows germ cell degeneration in a large proportion
of the seminiferous tubules, indicated by the *. Panels K and L are
two examples depicting the fact that germ cells are blocked in
metaphase and degenerate. They have darkly stained chromatin and
eosinophilic cytoplasm (arrows) and are positively stained by TUNEL
labeling. Panel M i a section of a seminiferous tubule containing
16 apparently apoptotic germ cells with condensed and darkly
stained nuclei (arrows) in a small area of the tubule. Some of
these cells are in close proximity to the basement membrane,
suggesting that they are either dying spermatogonia or dying
preleptotene spermatocytes. Panel N is a section of a seminiferous
tubule in which no spermatids are present and most meiotic germ
cells have disappeared. However, the spermatogonia and preleptotene
spermatocyte populations seem to be relatively normal. Arrows
indicate apoptotic cells. Panel 0 is a section of a seminiferous
tubule showing most meiotic germ cells have degenerated. The
remaining primary spermatocytes have condensed darkly stained
nuclei (arrows). The presence of many elongated spermatids suggests
that the previous round of meiosis was relatively unaffected. Bar
is 200 .mu.m in Panels A, D, G and J, 50 .mu.m in Panels B, C, and
0, and 30. .mu.m in Panels E, F, H, I, K, L, M, and N.
[0052] FIG. 11 is a series of images depicting histological
abnormalities of epididymides of homozygous Bmp8a mutants and a
compound Bmp8a/Bmp8b heterozygote. Panel A depicts the histology of
wild type adult epididymis showing the efferent duct (ED), initial
segment (IS), proximal caput (PC), distal caput (DC), corpus, and
cauda regions. Panel B is a section of the cauda epididymis of a
Bmp8a homozygous mutant revealing advanced granuloma formation in
which mature sperm (indicated by the *) are surrounded by
infiltrating leukocytes (arrowhead). Panel C is a section of the
cauda epididymis of a Bmp8 compound heterozygote revealing two foci
of granuloma formation outside the epididymis tubule, with sperm
(indicated by the *) in the center and leukocyte infiltration
(arrowhead) in the periphery. Panel D is a section of the same
cauda epididymis as in Panel C depicting sperm (indicated by the *)
surrounded by infiltrating leukocytes (arrowhead) and the
degenerating epididymis tubules. Panel E is a high power
magnification of the boxed region in Panel B showing sperm
infiltrating leukocytes (arrowhead), and degenerating tubules
(arrow). Panel F is a high power magnification of the boxed region
in Panel D showing a degenerating epididymis tubule with sperm
inside (indicated by a *). The epithelial layer of the tubule
contains multiple vacuoles (arrows), suggesting cell degeneration.
Panel G is a high power magnification of a section of the same
cauda epididymis as in Panel C showing sperm (indicated by the *)
in the neighborhood of an erupted epididymal tubule (arrows). Panel
H is a section through the caput epididymis and testis of a
homozygous Bmp8a mutant. Panel I is a higher power magnification of
the boxed region in Panel H showing the vacuolated epithelium in
the tubules of the distal caput region. Panel J is a high power
magnification of the boxed region in Panel I showing the vacuolated
epithelium of the epididymis tubules (arrows). Bar is 800 .mu.m in
Panels A-C and H; 200 .mu.m in Panels D, E, and I; 50. .mu.m in
Panels F, G, and J.
[0053] FIG. 12 is a series of images depicting in situ
hybridization depicting Bmp8a and Bmp7 expression in the
epididymides of adult animals. Panel A is a dark-field
photomicrograph of a section through a wild-type testis and
epididymis hybridized with antisense RNA probe directed against the
3' untranslated region of Bmp8a mRNA. Bmp8a is expressed in the
stage 6-8 round spermatids, and therefore, shows stage-specific
hybridization in the testis. Bmp8a transcripts are also detected in
the initial segment of the caput epididymis, but at lower levels
than in the testis. Panel B is a bright-field photomicrograph of
the same section as in Panel A. Panel C is a dark-field
photomicrograph of a section through a wild-type testis and
epididymis hybridized with an antisense RNA probe against the
pro-region of Bmp7 mRNA. Bmp7 transcripts are detected in the
initial segment of the caput epididymis at high levels. Panel D is
a corresponding brightfield photomicrograph of the same section as
in Panel C. IS, initial segment; PC, proximal caput; and DC, distal
caput. Bar is 800 .mu.m.
[0054] FIG. 13 is the nucleotide sequence of cDNA encoding BMP
8A.
[0055] FIG. 14 is the amino acid sequence of Bmp8a.
[0056] FIG. 15 is the nucleotide sequence of DNA encoding
BMP8B.
[0057] FIG. 16 is the amino acid sequence of BMP8B.
DETAILED DESCRIPTION OF THE INVENTION
[0058] It has been discovered in the present invention that BMP8 is
necessary for survival of spermatocytes and for the proliferation
of spermatogonial cells. Thus, the invention provides a highly
useful means of obtaining cultures of cells enriched for early
stage, spermatogonial stem cells. Furthermore, the invention
provides a means of treating infertility in males by supplying the
BMP8 protein. Other uses and advantages of the invention are
discussed further herein.
[0059] Although it appears that only a single BMP8 gene is present
in humans, the data presented herein establishes the presence of
two highly related and closely linked genes in the mouse which are
designated herein as Bmp8a and Bmp8b. Previous studies using
Northern blot analysis of poly(A)+ RNA, had concluded that
Bmp8a/Op2 is expressed in the 8.5 and 10.5 d.p c. mouse embryo
(Zkaynak et al., supra). However, as described herein, it was not
possible to detect either Bmp8a or Bmp8b transcripts in the mouse
embryo proper between 7.5 and 10.5 d.p.c. using a variety of
different techniques such as RNAse protection, RT-PCR, cDNA library
screening and in situ hybridization. In contrast, high levels of
Bmp8 RNA were found in the placenta and decidual cells of the
uterus around the embryo. In addition, it has been discovered in
the present invention that both Bmp8a and Bmp8b are expressed in
the germ cells of the testis at specific stages of spermatogenesis.
Although three other members of the TGF-.beta. gene family, for
example, Mullerian inhibiting substance, activin, and inhibin, are
expressed in the testis, transcripts specifying these genes are
found predominantly in somatic Sertoli cells (Cate et al., 1990, In
Sporn et al. Eds., Peptide Growth Factors and Their Receptor,
Springer-Verlag, Berlin, Vol. 2, pp. 179-210); Steinberger et al.,
1976, Endocrinology 99:918-921; Bicsak et al., 1987, Mol. Cell.
Endocrinol. 49:211-217; Meunier et al., 1988, Proc. Natl. Acad.
Sci. USA 85:247-251; Bhasin et al., 1989, Endocrinology
124:987-991).
[0060] Until the present invention, it was not known that Bmp8a and
Bmp8b are expressed in the germ cells of the testis. The data
presented herein therefore establish that BMP8A and BMP8B are
required for mammalian spermatogenesis and maternal-fetal
interactions and are not required for embryonic development as was
heretofore believed.
[0061] As used in the specification and in the claims, "a" can mean
one or more, depending upon the context in which it is used.
[0062] As used throughout this application, mammals can include,
for example, rats, rabbits, guinea pigs, goats, pigs, cows, and
humans.
[0063] The term "Bmp8" refers to nucleic acid encoding BMP8
protein. Bmp8 should be construed to refer to a DNA, a cDNA or an
RNA encoding BMP8. The use of the term Bmp8 should also be
construed to refer to both Bmp8a and Bmp8b, unless each of Bmp8a
and Bmp8b is individually specified. Although the mouse Bmp8a and
Bmp8b genes are exemplified herein, the use of the term "Bmp8"
should also be construed to include homologs of mouse Bmp8, which
homologs share homology with mouse Bmp8 genes. Thus, the invention
should be construed to include Bmp8 genes from mice, and any other
mammal, including rats, rabbits, guinea pigs, goats, cows, pigs and
humans, etc.
[0064] Similarly, the use of the term "BMP8" should be construed to
include any homolog of BMP8, e.g., mouse BMP8A and/or mouse BMP8B,
human BMP8 (Genetics Institute, Cambridge, Mass.), or active
portions either alone or in a larger polypeptide. These proteins
are also referred to as OP-2, OP-3 (Creative Biomolecules,
Hopkinton, Mass.). The human OP-2 protein is homologous to mouse
BMP8A and the human OP-3 protein is homologous (about 78% identical
in amino acid sequence in the mature region of the protein) to the
mouse BMP8B protein. Homologs of BMP8 include proteins which share
homology with either of mouse BMP8A or BMP8B are described herein
and which function in a manner similar to the mouse BMP8 proteins
described herein. It is also contemplated that BMP8A and BMP8B can
be used together. BMP8 can mean either BMP8A or BMP8B, particularly
as it refers to human BMP8 proteins, unless each individual protein
is so specified.
[0065] Thus, while nucleic acid encoding mouse BMP8A and BMP8B have
been initially discovered, according to the present invention, to
be useful in the methods described herein, the use of a gene
encoding a BMP8 protein obtained from any other mammalian tissue,
preferably, a human tissue, is also included in the invention.
Further, the invention should be construed to include nucleic acid
encoding BMP8 from mammals other than humans, which BMP8 functions
in a substantially similar manner to the mouse BMP8 described
herein. Preferably, the nucleic acid encoding BMP8 is about 50%
homologous, more preferably about 70% homologous, even more
preferably about 80% homologous and most preferably about 90%
homologous to the nucleic acid encoding mouse BMP8 or BMP8B.
[0066] "Homologous" as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous at that position.
The homology between two sequences is a direct function of the
number of matching or homologous positions, e.g., if half (e.g.,
five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then the two
sequences are 50% homologous, if 90% of the positions, e.g., 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 3'ATTGCC 5' and
3'TATGCG 5' share 50% homology.
[0067] An "isolated nucleic acid", as used herein, refers to a
nucleic acid sequence, segment, or fragment which has been
separated from the sequences which flank it in a naturally
occurring state, e.g., a DNA fragment which has been removed from
the sequences which are normally adjacent to the fragment, e.g.,
the sequences adjacent to the fragment in a genome in which it
naturally occurs. The term also applies to nucleic acids which have
been substantially purified from other components which naturally
accompany the nucleic acid, e.g. RNA or DNA or proteins, which
naturally accompany it in the cell.
[0068] The invention also includes an isolated nucleic acid having
a sequence which is in the antisense orientation (i.e., is
complementary) to a portion or all of the nucleic acid encoding
BMP8. By "complementary to a portion or all of a Bmp8 gene" is
meant a sequence of nucleic acid which does not encode BMP8.
Rather, the sequence which is being expressed in the cells is
identical to the non-coding strand of the BMP8 gene and thus, does
not encode BMP8.
[0069] The terms "complementary" and "antisense" as used herein,
are not entirely synonymous. "Antisense" refers particularly to the
nucleic acid sequence of the non-coding strand of a double stranded
DNA molecule encoding a protein, or to a sequence which is
substantially homologous to the non-coding strand. "Complementary"
as used herein refers to the broad concept of subunit sequence
complementarity between two nucleic acids, e.g., two DNA molecules.
When a nucleotide position in both of the molecules is occupied by
nucleotides normally capable of base pairing with each other, then
the nucleic acids are considered to be complementary to each other
at this position. Thus, two nucleic acids are complementary to each
other when a substantial number (at least 50%) of corresponding
positions in each of the molecules. are occupied by nucleotides
which normally base pair with each other (e.g., A:T and G:C
nucleotide pairs). As defined herein, an antisense sequence is
complementary to the sequence of a double stranded DNA molecule
encoding a protein. It is not necessary that the antisense sequence
be complementary solely to the coding portion of the coding strand
of the DNA molecule. The antisense sequence may be complementary to
regulatory sequences specified on the coding strand of a DNA
molecule encoding a protein, which regulatory sequences control
expression of the coding sequences.
[0070] The invention thus should be construed to include nucleic
acid encoding BMP8 and fragments of nucleic acid encoding BMP8;
and, nucleic acid and fragments of nucleic acid which is in the
antisense orientation to nucleic acid encoding BMP8.
[0071] Fragments of nucleic acid encoding BMP8 encode portions of
BMP8 which have the biological activity of BMP8 as defined herein,
or which encode a polypeptide comprising a portion of BMP8, which
polypeptide is useful, as discussed in detail herein, in the
methods of the invention.
[0072] The invention also includes an isolated protein encoded by
Bmp8 as described herein, and other BMP8 molecules encoded by other
Bmp8 genes which may be isolated by the skilled artisan once armed
with the present invention. Preferably, the amino acid sequence of
a BMP8 protein so discovered is about 70% homologous, more
preferably about 80% homologous, even more preferably about 90%
homologous, more preferably, about 95% homologous, and most
preferably, at least about 99% homologous to the amino acid
sequence of mouse BMP8A or mouse BMP8B.
[0073] Substantially pure BMP8 protein obtained as described herein
may be purified by following known procedures for protein
purification, wherein an immunological, enzymatic or other assay is
used to monitor purification at each stage in the procedure.
[0074] As used herein, the term "substantially pure" describes a
compound, e.g., a protein or polypeptide which has been separated
from components which naturally accompany it. Typically, a compound
is substantially pure when at least 10%, more preferably at least
20%, more preferably at least 50%, more preferably at least 60%,
more preferably at least 75%, more preferably at least 90%, and
most preferably at least 99% of the total material (by volume, by
wet or dry weight, or by mole percent or mole fraction) in a sample
is the compound of interest. Purity can be measured by any
appropriate method, e.g., in the case of polypeptides by column
chromatography, gel electrophoresis or HPLC analysis. A compound,
e.g., a protein, is also substantially purified when it is
essentially free of naturally associated components or when it is
separated from the native contaminants which accompany it in its
natural state.
[0075] The present invention also provides for the use of analogs
of proteins or peptides encoded by a Bmp8 gene. Analogs can differ
from naturally occurring proteins or peptides by conservative amino
acid sequence differences or by modifications which do not affect
sequence, or by both.
[0076] For example, conservative amino acid changes may be made,
which although they alter the primary sequence of the protein or
peptide, do not normally alter its function. Conservative amino
acid substitutions typically include substitutions within the
following groups:
[0077] glycine, alanine;
[0078] valine, isoleucine, leucine;
[0079] aspartic acid, glutamic acid;
[0080] asparagine, glutamine;
[0081] serine, threonine;
[0082] lysine, arginine;
[0083] phenylalanine, tyrosine.
[0084] Modifications (which do not normally alter primary sequence)
include in vivo, or in vitro chemical derivatization of
polypeptides, e.g., acetylation, or carboxylation. Also included
are modifications of glycosylation, e.g., those made by modifying
the glycosylation patterns of a polypeptide during its synthesis
and processing or in further processing steps; e.g. by exposing the
polypeptide to enzymes which affect glycosylation, e.g., mammalian
glycosylating or deglycosylating enzymes. Also embraced are
sequences which have phosphorylated amino acid residues, e.g.,
phosphotyrosine, phosphoserine, or phosphothreonine.
[0085] Also included are polypeptides which have been modified
using ordinary molecular biological techniques so as to improve
their resistance to proteolytic degradation or to optimize
solubility properties. Analogs of such polypeptides include those
containing residues other than naturally occurring L-amino acids,
e.g., D-amino acids or non-naturally occurring synthetic amino
acids. The peptides of the invention are not limited to products of
any of the specific exemplary processes listed herein.
[0086] In addition to substantially full length polypeptides, the
present invention provides for biologically active fragments of the
polypeptides.
[0087] A BMP8 polypeptide is "biologically active" if it adequately
supports the survival of spermatocytes in the assays which are
described in the experimental details section herein.
[0088] The invention also contemplates the use of BMP8 polypeptides
that are either full length or are less than full length. Such
fragments may be biologically active, as defined herein, or may be
biologically inactive in that they do not adequately support
survival of spermatocytes. In the latter instance, such fragments
may be useful for inhibition of the biological activity of BMP8,
when added to cells, either in vitro or in vivo. The invention also
contemplates the use of mutants of BMP8, which mutants comprise one
or more mutations which render the BMP8 protein inactive.
[0089] As used herein, the term "fragment," as applied to a
polypeptide, will ordinarily be at least about fifteen contiguous
amino acids, typically at least about twenty five contiguous amino
acids, more typically at least about forty contiguous amino acids,
usually at least about forty five contiguous amino acids and
preferably at least about fifty contiguous amino acids in
length.
[0090] DNA encoding BMP8 proteins from a mammal may be obtained
following the procedures described herein using as probes in
hybridization assays, PCR reactions and the like, portions of Bmp8a
or Bmp8b. Essentially, DNA is extracted from cells obtained from
the desired mammal. The procedures for the isolation, cloning and
sequencing and other characterization of a DNA molecule are well
known in the art and are described, for example, in Sambrook et al.
(1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y.).
[0091] As the data presented herein establish, BMP8 plays at least
three distinct roles in spermatogenesis. It is required for the
survival of differentiating spermatocytes, for the proliferation of
spermatogonial cells and for their differentiation.
[0092] Thus, it has been discovered in the present invention that
administration of BMP8 to certain mammalian cells facilitates
proliferation of those cells. In particular, mammalian
spermatogonial stem cells are caused to proliferate in the presence
of this protein. Thus, there is provided in the invention a method
of proliferating mammalian spermatogonial cells. The method
comprises culturing spermatogonial stem cells in the presence of
BMP9 protein. The culture medium for culturing spermatogonial cells
and the other conditions under which such cells are maintained are
described in the experimental details section herein. Regarding the
amount of BMP8 to be added to spermatogonial stem cells to effect
their proliferation, it will be appreciated that the amount of BMP8
required in the method of the invention will depend upon the type
of mammal from which the cells and the BMP8 are derived. Generally,
BMP8 is added to cells in a concentration of between about 1
.mu.g/ml to about 10 mg/ml of culture medium. Preferably, BMP8 is
added to cells in a concentration of about 1 .mu.g/ml to about 500
.mu.g/ml of culture medium.
[0093] It has also been discovered in the present invention that
administration of BMP8 to certain mammalian cells facilitates
differentiation of those cells. In particular, mammalian
spermatogonial stem cells are caused to differentiate in the
presence of this protein. Whether a culture of spermatogonial cells
is caused to proliferate or differentiate depends on the
developmental stage of the cells at the time BMP8 is added to the
culture. Thus, spermatogonial stem cells which are in one
developmental stage may be caused to proliferate in response to
BMP8, whereas, cells which are in another developmental stage may
be caused to differentiate in the presence of BMP8. It will be
apparent to the artisan skilled in spermatogonial cell development
whether any particular culture of spermatogonial stem cells will
undergo proliferation or differentiation in the presence of
BMP8.
[0094] There is therefore also provided in the invention a method
of differentiating mammalian spermatogonial cells. The method
comprises culturing spermatogonial stem cells in the presence of
BMP8 protein. The culture medium for culturing spermatogonial cells
and the other conditions under which such cells are maintained are
described in the experimental details section herein. Regarding the
amount of BMP8 to be added to spermatogonial stem cells to effect
their differentiation, it will be appreciated that the amount of
BMP8 required in the method of the invention will depend upon the
type of mammal from which the cells and the BMP8 are derived.
Generally, BMP8 is added to cells in a concentration of between
about 1 ng/ml to about 10 mg/ml of culture medium. Preferably, BMP8
is added to cells in a concentration of about 1 .mu.g/ml to about
500 .mu.g/ml of culture medium.
[0095] Regarding differentiation of spermatogonial cells using
BMP8, cells which are caused to differentiate, any actually
terminally differentiate and therefore undergo apoptosis. The
invention thus also provides a method of inducing apoptosis of
spermatocytes
[0096] In addition to proliferation and differentiation, the
addition of BMP8 to spermatogonial stem cells effects their
maintenance in culture. Similar to the situation regarding
proliferation and differentiation, maintenance of spermatogonial
cells in culture is effected by added to the cells an amount of
BMP8 protein sufficient for the maintenance of the cells. Such
maintenance is referred to herein as "extending the viability" of
spermatogonial cells.
[0097] In general, preparations of BMP8 which are added to cells in
culture include the desired concentration of BMP8 suspended in cell
culture medium or other isotonic solution which is compatible with
the viability of the cells. Such solutions will be apparent to the
artisan skilled in protein administration to cells for the purposes
described herein.
[0098] It will be appreciated from the discussion provided herein
that incubation of cells in the absence of BMP8, or in the presence
of a substance which effects the substantial absence of BMP8 in the
cell culture, serves to cause the cells to cease to proliferate,
and/or differentiate, and/or be maintained in the culture. Such a
situation provides a scenario for the study of male infertility. In
addition, when practiced in vivo, as described herein, such a
situation provides a method of inducing infertility in a male
mammal.
[0099] Thus, according to the invention, there is provided ea
method of arresting the proliferation of spermatogonial stem cells
in culture comprising incubating the cells in the substantial
absence of BMP8.
[0100] In addition, there is provided a method of arresting the
differentiation of spermatocytes in culture comprising incubating
the cells in the substantial absence of BMP8.
[0101] Further, there is provided a method of arresting the
maintenance of spermatocytes in culture comprising incubating the
cells in the substantial absence of BMP8.
[0102] It will be appreciated that whether the property of the cell
to be affected by incubation in the substantial absence of BMP8 is
proliferation, differentiation or maintenance, will depend on the
developmental stage of the cells. The appropriate developmentaL
stage of the cells will be apparent to the artisan skilled in cell
development.
[0103] When the method of the invention calls for the incubation of
cells in the substantial absence of BMP8, this should be construed
to include incubating cells in the absence of BMP8, and to include
incubation of cells in the presence of BMP8 wherein BMP8 activity
is inhibited by any desired means. Such inhibition means include
the addition of an antibody to BMP8 to the culture; the addition of
other inhibitors of BMP8 function such as small molecules and
peptidometics; the inhibition of production of BMP8 protein, such
as by adding antisense nucleic acids to the BMP8 mRNA using
methodology well known in the art. "Substantial absence of BMP8"
means the amount of BMP8 present does not adequately support the
survival of spermatocytes.
[0104] Antibodies to BMP8 may be generated using any technology
well known in the art and described, for example, in Harlow et al.
(1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor,
N.Y.). Antibodies to BMP8 should be construed to include
polyclonal, monoclonal and synthetic antibodies.
[0105] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage. The term
should also be construed to mean an antibody which has been
generated by the synthesis of a DNA molecule encoding the antibody
and which DNA molecule expresses an antibody protein, or an amino
acid sequence specifying the antibody, wherein the DNA or amino
acid sequence has been obtained using synthetic DNA or amino acid
sequence technology which is available and well known in the
art.
[0106] To obtain a substantially pure preparation of a synthetic
antibody, the antibody may be extracted from the surface of the
phage on which it is expressed. The procedures for such extraction
are well known to those in the art of protein purification.
Alternatively, a substantially pure preparation of an antibody may
be obtained by cloning an isolated DNA encoding the antibody into
an expression vector and expressing the protein therefrom. Antibody
so expressed may be obtained using ordinary protein purification
procedures well known in the art. Procedures for the generation of
synthetic antibodies are described in Barbas (1995, Nature Medicine
1:837-839) and in de Kruif et al. (1995, J. Mol. Biol. 248:97-105)
and in the refererences cited therein.
[0107] Also provided are peptidometics having BMP8 biological
activity. Peptidometics having BMP8 biological activity include
compounds which have a sufficient BMP8 activity such that their
effects on the cells to which they are administered are beneficial
to the sell, which beneficial effects are similar to that of BMP8.
Peptidometics may also have additional advantages over BMP8 in that
they may be designed such that they are capable of accessing
targets in an animal which are normally relatively inaccessible to
BMP8.
[0108] Information describing the generation, use and
administration of peptidometics is provided in PCT/US93/01201 and
U.S. Pat. No. 5,334,702, which are hereby incorporated herein by
reference. Any of the techniques described in either of these two
references may be employed in the present invention for the
administration of peptidometics.
[0109] Given the findings provided herein, it will be appreciated
that the invention further includes a method of identifying small
molecules, nucleic acids, peptides, antibodies, hormones and other
compounds which affect BMP8 activity, i.e., which compounds are
agonists or antagonists of BMP8 activity.
[0110] To identify an agonist of BMP8 activity, spermatogonial
cells at defined stages of differentiation are incubated in the
absence of any compound as a control, in the presence of BMP8, in
the presence of a test compound, or in the presence of BMP8 and the
test compound. The extent of proliferation and/or differentiation
of the cells is measured following the incubation period. The
compound is considered to be a BMP8 agonist when the extent of
proliferation and/or differentiation of the cells in the presence
of the test compound is equal to or greater than the extent of
proliferation and/or differentiation of the cells in the presence
of BMP8. If the extent of proliferation and/or differentiation of
the cells in the presence of both BMP8 and the test compound is
greater than the extent in BMP8 alone, then the compound is an
agonist of BMP8 which acts in synergy with BMP8.
[0111] To identify an antagonist of BMP8 activity, spermatogonial
cells at defined stages of differentiation are incubated in the
absence of any compound as a control, in the presence of BMP8, in
the presence of a test compound, or in the presence of BMP8 and the
test compound. The extent of proliferation and/or differentiation
of the cells is measured following the incubation period. The
compound is considered to be a BMP8 antagonist when the extent of
proliferation and/or differentiation of the cells in the presence
of the test compound and BMP8 is less than the extent of
proliferation and/or differentiation of the cells in the presence
of BMP8 alone.
[0112] Compounds which act as agonists or antagonists of BMP8 are
useful for promoting fertility in a male mammal or for inducing
infertility in a male mammal.
[0113] The methods of the invention are applicable to the
manipulation of spermatogonial cells cultured in vitro. These
methods are also applicable for the manipulation of spermatogonial
cells in vivo in a mammal.
[0114] BMP8, fragments of BMP8, or agonists or antagonists thereof,
may be administered to cells in vivo in an animal to effect
manipulation of the cells regarding proliferation, differentiation,
maintenance, or apoptosis. Protocols for treatment of mammals with
BMP8, or an agonist or antagonists thereof, will be apparent to
those skilled in the art and will vary depending upon the situation
in the mammal to be treated. Treatment regimes which are
contemplated include a single dose or dosage which is administered
hourly, daily, weekly or monthly, or yearly. Dosages may vary from
1 .mu.g to 1000 mg/kg of body weight of the agonist or antagonist,
or of BMP8 and will be in a form suitable for delivery of the
compound. The route of administration may also vary depending upon
the disorder to be treated.
[0115] The agonist, antagonist or BMP8 is prepared for
administration by being suspended or dissolved in a
pharmaceutically acceptable carrier such as saline, salts solution
or other formulations apparent to those skilled in such
administration. The compositions of the invention may be
administered to a mammal in one of the traditional modes (e.g.,
orally, parenterally, transdermally or transmucosally), in a
sustained release formulation using a biodegradable biocompatible
polymer, or by on-site delivery using micelles, gels and liposomes,
or rectally (e.g., by suppository or enema) or nasally (e.g., by
nasal spray). The appropriate pharmaceutically acceptable carrier
will be evident to those skilled in the art and will depend in
large part upon the route of administration.
[0116] For administration to the testes of an animal, the compound
to be administered directly into the testes by injection or other
suitable means which will be apparent to those skilled in the
art.
[0117] Provided herein is a method of treating infertility in a
male mammal, comprising administering to the mammal BMP8 protein,
thus facilitating spermatogenesis and treating infertility. Also
provided is a method of treating infertility in a male mammal,
comprising administering to testis cells of the mammal BMP8
protein, thus facilitating proliferation and differentiation of the
cells and treating infertility. In particular the male mammal may
have a defect in spermatogenesis resulting from reduced levels of
functional BMP8 protein. The BMP8 protein can be administered by
administering a nucleic acid functionally encoding BMP8, thus
providing BMP8 protein. Additionally, the protein can be
administered by methods elaborated herein and other methods known
in the art. The BMP8 protein can, for example, be injected into the
rete region of the testis, thus allowing the protein to diffuse
into the seminiferous tubules.
[0118] Also provided is a method of inducing infertility in a male
mammal. Administration to a mammal of a substance which effects the
substantial absence of BMP8 in cells in the testes, or
administration to a male mammal of an antagonist of BMP8 activity
will serve to reduce or eliminate BMP8 function in the cells
thereby reducing or eliminating spermatogenesis in the mammal.
Methods, routes and formulations for administration of BMP8
antagonists or other compounds which reduce or eliminate BMP8
activity are generally the same as the methods just described for
treatment of male infertility and will be apparent to the skilled
artisan once armed with the present invention.
[0119] The invention also includes a method of isolating a BMP8
receptor on a cell comprising binding BMP8 to a BMP8-responsive
population of cells, and isolating the protein on the cells to
which the BMP8 binds.
[0120] By "BMP-responsive population of cells" as used herein, is
meant a population of cells which proliferate or differentiate in
response to BMP8 in the assays described herein.
[0121] A receptor protein may be isolated using any one of a number
of protein isolation techniques available in the art, including,
for example, immunoaffinity techniques, other biochemical affinity
techniques, and the like as described herein and as can be found in
any ordinary biochemistry manual for protein purification.
[0122] To facilitate the isolation of a BMP8 receptor protein, BMP8
to be bound to the cells may be labeled with a detectable marker,
it may be tagged with an identifiable marker, or it may be
covalently bound (i.e., as a fusion protein) to a tag to facilitate
isolation of a protein bound to it. Generation of fusion proteins
for the isolation of receptor proteins is well known in the art of
molecular biology.
[0123] Also provided in the invention is a method or treating a
mammal for defects in embryo implantation in the uterus, comprising
administering to the mammal BMP8 protein, thereby facilitating
implantation. The time period for administration can preferably be
when decidual cell proliferation and hypertrophy peak (Finn, 1971,
Adv. Reprod. Physiol. 5:1-26). Compounds may be administered
directly to the uterine wall or administration may be effected
systemically. BMP8 protein may be administered or, nucleic acid
encoding BMP8 can be administered. One can modify the nucleic acid
such that it is expressed at appropriate times during development
of the cells to be manipulated, e.g., by using a promoter derived
from a gene which is normally expressed in the desired cells at the
desired time.
[0124] Also provided is a method of facilitating maintenance of the
placenta of a pregnant mammal, comprising administering to the
mammal a maintaining amount of BMP8 protein, thereby facilitating
maintenance of the placenta. Administration of the BMP8 protein can
be, e.g., by injection or direct application as described
herein.
[0125] It is apparent from the data provided herein that BMP8 may
have effects on cells other than spermatogonial cells, during
various stages in their development. In particular, hair follicle
cells are likely to be affected by BMP8, in that they are likely to
be stimulated to produce hair. Thus, the invention also provides a
method of stimulating hair growth in a mammal, comprising
administering a stimulating amount of BMP8 to the hair follicles,
thereby stimulating hair growth. Compositions can be formulated for
application of the BMP8 protein to the hair follicles, which
compositions comprise suitable carriers for topical delivery of the
BMP8 that are known the art and are described, for example, in
Martin (ed. Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, Pa.). For example, formulations such as used for the
administration of minoxidol can be utilized in the method of the
present invention.
[0126] Bmp8a homozygous null mice have the same phenotype as Bmp8b
homozygous mutants and the expression pattern of the two genes is
the same in the testis. This is evidence that the two proteins have
identical functions in mammalian spermatogenesis and will be
interchangeable experimentally and in therapeutically. Therefore,
BMP8 protein can be used therapeutically to treat infertility and
as an essential growth factor for obtaining and/or maintaining
lines of spermatogonial stem cells in culture.
[0127] Based on the data provided herein concerning the effect of
depriving mice of BMP8, BMP8 is useful as a therapeutic agent in
the treatment of human males with infertility due to specific
classes of defects in spermatogenesis in the seminiferous tubules.
For example, these abnormalities could be due to intrinsic genetic
defects in the Bmp8 gene (coding or non-coding sequences) or in
other genes encoding proteins which regulate the expression of the
Bmp8 gene. Alternatively, extrinsic hormonal or environmental
factors may inactivate the Bmp8 gene or interfere with its normal
regulation. The infertility of these patients may be treated by
administration of BMP8 either systemically or in slow-release beads
coated with or impregnated with protein. Such beads could be
implanted into one or both testes. The beads coated or impregnated
with BMP8 protein could also be administered from a high velocity
particle "gun".
[0128] BMP8 useful in the methods of the invention may be a
recombinant protein comprising dimers of the C-terminal
TGF-.beta.-like portion of the protein. This recombinant protein
may be easily generated in an insect cell/bacculovirus expression
system. Alternatively, COS cells may be used for generation of
BMP8, which cells generally modify and process proteins for
secretion in a biologically active form. Such expression systems
are widely used for the production of other cytokines, including
proteins related to BMP8 (e.g. BMP2 and BMP7/Osteogenic protein 1
are in preclinical trials).
[0129] In addition, a gene therapy approach using a retrovirus
vector, which contains for example, but not limited to, a
EF1-.alpha. promoter-driven Bmp8 gene, may be used to deliver BMP8
proteins to spermatogonia. EF1-.alpha. promoter is known to drive
specific gene expression in spermatogonia in transgenic mice
(Furuchi et al., 1996, Development 122:1703-1709). Furthermore, in
addition to the use of viral vectors, non-viral vectors, such as,
but not limited to, cationic liposomes may be used to deliver the
nucleic acid to cells.
[0130] When Bmp8 nucleic acid is to be added to cells, the nucleic
acid may be modified, for example, by adding nucleic acid sequences
encoding signal sequences to promote secretion of the protein from
cells so that surrounding cells are also affected by the
protein.
[0131] BMP8 treatment may also be useful for increasing the
fertility of endangered mammalian species in captivity, which
animals have inefficient spermatogenesis due to hormonal or
environmental factors or old age.
[0132] Transgenic animals comprising mutations in the Bmp8 gene are
useful for the study of the role of BMP8 in cell development, for
the identification and testing of compounds which affect BMP8
activity in vivo. The generation of such transgenic animals is
described herein.
[0133] The invention is further described in detail by reference to
the following experimental examples. These examples are provided
for purposes of illustration only, and are not intended to be
limiting unless otherwise specified. Thus, the invention should in
no way be construed as being limited to the following examples, but
rather, should be construed to encompass any and all variations
which become evident as a result of the teaching provided
herein.
[0134] Methods for the Isolation, Propagation and Differentiation
of Spermatogonial Stem (SS) Cell Lines from Mammalian Testis
[0135] Methods have already been well established for purifying
(into roughly homogenous populations) and culturing, for short
periods of time spermatogenic cells from rats, mice. These methods
are reviewed in "Purification, culture and fractionation of
spermatogenic cells" by Anthony R. Bellve in Methods in Enzymology
225:84-113 (1993).
[0136] BMP8 homozygous mutant mice at 1-2 weeks of age can be used
to obtain populations of cells highly enriched in the most
primitive spermatogonial stem (SS) cells which are normally very
hard to obtain because they form a very small proportion of the
total spermatogenic cells of the testis.
[0137] The isolated cell population enriched in stem cells is then
cultured in a culture dish (collagen coated) either alone or in the
presence of inactivated feeder cells (to mimic the supportive
function of Sertoli cells) in the presence of culture medium
containing low levels of serum (1-10%) and different levels of BMP8
protein (these levels could range from 0.5 to 500 ng/ml). Addition
of other growth factors in the same concentration range may also be
used, for example fibroblast growth factor (FGF), Steel factor,
LIF, Interleukins, nerve growth factor, activin. Fibroblast growth
factor is known to be expressed in spermatogonial cells in the rat
and mouse. Combinations of different classes of growth factors
(LIF, Steel Factor and FGF) may be beneficial as found for the
culture of cell lines from primordial germ cells in the fetal
gonad.
[0138] Until the present invention, spermatogonia only remained
viable in culture for 24-48 hours. According to the present
invention, the addition of BMP8 alone, or in combination with other
factors, overcomes this block and allow the cells to continue
proliferating indefinitely.
[0139] The methods described herein apply to human testes either
from newborn or very young males (donated after death) or from
adult males. Alternatively, cells may be isolated from the subject
utilizing techniques currently used to obtain cells from testes for
microinjection into eggs for fertilization. These procedures are in
the art of fertility testing and include, for example, needle
biopsy of the testis.
[0140] Testes cells so obtained, are dissociated and cultured in
the presence of a cocktail of growth factors to stimulate the
proliferation of SS cells.
[0141] Additional methods for obtaining testes cells are as
follows: Testes are dissected and the tunica removed. The testes
are then incubated at 32.degree. C. with mild shaking in buffered
saline containing bovine serum albumin and collagenase (final
concentration approximately 0.5 mg/ml). When the tissue has
dissociated the tubules are allowed to settle out and then washed
in saline several times. The collagenase treatment is repeated to
remove all the cells surrounding the tubules (Leydig cells and
connective tissue). The tubules are then washed and treated with
hyaluronidase in buffered saline (final concentration approximately
0.5 mg/ml) at 32.degree. C. until the tubules are free of adherent
material. The tubules are washed and placed onto tissue culture
dishes coated with Poly-L-lysine. The Sertoli cells attach strongly
to the dish and spread out, while the germ cells remain in
suspension. The germ cells are collected and plated onto a layer of
irradiated feeder cells comprising membrane bound and soluble stem
cell factor, LIF and basic FGF as described in U.S. Pat. No.
5,453,357, which is incorporated by reference.
[0142] A "pluripotential embryonic stem cell" as used herein means
a cell which can give rise to many differentiated cell types in an
embryo or adult, including the germ cells (sperm and eggs).
Pluripotent embryonic stem cells are also capable of self-renewal.
Thus, these cells not only populate the germ line and give rise to
a plurality of terminally differentiated cells which comprise the
adult specialized organs, but also are able to regenerate
themselves. This cell type is also referred to as an "ES cell"
herein.
[0143] A "fibroblast growth factor" (FGF) as used herein means any
suitable FGF. There are presently seven known FGFs. These FGFs
include FGF-1 (acidic fibroblast growth factor), FGF-2 (basic
fibroblast growth factor), FGF-3 (int-2), FGF-4 (hst/K-FGF), FGF-5,
FGF-6, FGF-7 and FGF-8.
[0144] Each of the suitable factors may be used directly in the
methods taught herein to produce or maintain ES cells. Each FGF can
be screened in the methods described herein to determine if the FGF
is suitable to enhance the growth of or allow continued
proliferation of ES cells or their progenitors. Various examples of
FGF and methods of producing an FGF are well known; see, for
example, U.S. Pat. Nos. 4,994,559; 4,956,455; 4,785,079; 4,444,760;
5,026,839; 5,136,025; 5,126,323; and 5,155,214.
[0145] "Steel factor" (SF) is used herein. SF is also called stem
cell factor, mast cell growth factor and c-kit ligand in the art.
SF is a transmembrane protein with a cytoplasmic domain and an
extracellular domain. Soluble SF refers to a fragment cleaved from
the extracellular domain at a specific proteolytic cleavage site.
Membrane associated SF refers to both normal SF before it has been
cleaved or the SF which has been altered so that proteolytic
cleavage cannot take place. SF is well known in the art; see
European Patent Publication No. 0 423 980 A1, corresponding to
European Application No. 90310889.1.
[0146] "Leukemia Inhibitory Factor" (LIF) is also used herein. LIF
is also known as DIA or differentiation inhibiting activity. LIF
and uses of LIF are also well known; see for example U.S. Pat. Nos.
5,187,077 and 5,166,065.
[0147] It should be recognized that BMP8, FGF, SF and LIF are all
proteins and as such certain modifications can be made to the
proteins which are silent and do not remove the activity of the
proteins as described herein. Such modifications include additions,
substitutions and deletions. Methods modifying proteins are well
established in the art (Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., 1989).
[0148] Once the ES cells are established, they can be genetically
manipulated to produce a desired characteristic. For example, the
ES cells can be mutated to render a gene non-functional, e.g.
oncogene. Alternatively, functional genes can be inserted to allow
for the production of that gene product in an animal, e.g. growth
hormones or valuable proteins.
[0149] Proliferating-enhancing amounts of FGF, LIF and SF can vary
depending on the species or strain of the cells, and type or purity
of the factors. Generally, 0.5 to 500 ng/ml of each factor within
the culture solution is adequate. In a more narrow range, the
amount is between 10 to 20 ng/ml for .beta.FGF and LIF and between
10 to 100 ng/ml for SF. Regardless of whether the actual amounts
are known, the optimal concentration of each factor can be
routinely determined by one skilled in the art. Such determination
is performed by titrating the factors individually and in
combination until optimal growth is obtained. Additionally, other
factors can also be tested to determine their ability to enhance
the effect of FGF, LIF and SF on ES cell proliferation. As
described below, such other factors, or combinations of factors
when used to enhance ES cell proliferation can be included within
the above compositions. Also, compounds and fragments of FGF, LIF
and SF which mimic the function of these factors can be used to
enhance the growth and proliferation of the cells to become ES
cells and are included within the scope of the invention.
[0150] The invention thus provides a method of making a mammalian
pluripotential embryonic stem cell comprising incubating a
population of cells from postnatal mammalian testis comprising
spermatogonial stem cells in the substantial absence of BMP8, then
incubating the resulting population of cells in a composition
comprising a growth enhancing amount of basic fibroblast growth
factor, leukemia inhibitory factor, membrane associated steel
factor, and soluble steel factor, thereby making a pluripotential
embryonic stem cell from a spermatogonial stem cell. In the testis,
these spermatogonial stem cells represent a small population of
cells capable of both self-renewal and differentiation into mature
spermatogonia.
[0151] These methods may be practiced utilizing any animal cell,
especially mammal cells including mice, rats, rabbits, guinea pigs,
goats, cows, pigs, humans, etc. The ES cell produced by this method
is also contemplated.
[0152] Additional growth factors may be found useful in enhancing
the production and proliferation of germ cells from various
animals.
[0153] The Methods used in the next set of experiments presented
herein are now described.
[0154] Isolation of Bmp8b Genomic DNA and cDNA
[0155] Approximately 5.times.10.sup.5 phage plaques of a 129/SvJ
mouse genomic DNA library in lambda Fix II (Stratagene) were spread
on NZY-agar plates and transferred to Hybond positively charged
nylon membranes (Amersham Life Science). Hybridization was done
overnight at 65.degree. C. in 0.5 M NaH.sub.2PO.sub.4 (pH 7.2), 7%
sodium dodecyl sulfate (SDS), 3 mM EDTA with a mouse Bmp8a cDNA
probe (containing exons 2-7; zkaynak et al., 1992, J. Biol. Chem.
267:25220-25227). Final washing was carried out at 65.degree. C. in
0.2.times.SSC, 0.2% SDS for 30 minutes. Two overlapping clones of
Bmp8a and seven overlapping Bmp8b clones were isolated. Bmp8b cDNAs
were isolated by 5' and 3' RACE-PCR (Frohman et al., 1988, Proc.
Natl. Acad. Sci. USA 85:8998-9002; Loh et al., 1989, Science
243:217-220; Bertchtold, 1989, Nucleic Acids Res. 17, 453) with
total RNA obtained from 14.5 d.p.c. placenta. Gene specific primers
were derived from exon 4 encoding part of the mature region of
Bmp8b (FIG. 3B, Bmp8b BN260). DNA sequencing was performed by the
dideoxynucleotide termination method (Sanger et al., 1977, Proc.
Natl. Acad. Sci. USA 74:5463-5467).
[0156] Chromosome Mapping
[0157] A 2.0 kb SalI-EcoRI Bmp8a genomic DNA fragment downstream of
exon 7 was used as a probe for Southern blot at high stringency.
Restriction fragment length variants (RFLVs) were found with MspI
digested C57BL/6J (2.7 kb) and Mus spretus (4.0 kb) genomic DNA. A
1.4 kb XbaI Bmp8b genomic DNA fragment from intron 6 was used as a
probe for Southern blot at high stringency. RFLVs were also found
with MspI digested C57BL/6J (4.1 kb) and Mus spretus (1.5 kb and
0.5 kb) genomic DNA. Both probes were hybridized with The Jackson
Laboratory interspecific backcross DNA panel (C57BL/6JEixSPRET/E1)
F1xSPRET/E1 (Rowe et al., 1994, Mamm. Genome 5,253-274) digested
with MspI. Both genes were mapped to mouse chromosome 4 and no
recombination event occurred between these two genes in this panel
of 94 DNA samples. The mapping data have been submitted to the
Jackson Laboratory Backcross Data on the World Wide Web at
htt://www.jax.org/resources/documents/cmdata.
[0158] DNA Constructs
[0159] Bmp8a STR380 (FIG. 3B) was constructed by inserting a 380 bp
Bmp8a cDNA fragment (from base pair 1273 to 1653, Zkaynak et al.,
1992, J. Biol. Chem. 267:25220-25227) into pBluescript KSII between
SmaI and EcoRI. Bmp8b BN260 was made by inserting a 260 bp genomic
DNA fragment containing exon 4 into pBluescript KS II (Stratagene).
Bmp8a 3' UTR and Bmp8b 3' UTR (FIG. 3B) were constructed by
inserting the 600 bp and 900 bp 3' UTRs of each gene (generated by
RT-PCR) into pBluescript SKII (Stratagene).
[0160] Preparation of RNA and RNase Protection Assays
[0161] Total RNA from mouse tissues and embryos was isolated by
guanidine thiocyanate-cesium chloride ultracentrifugation and
phenol/chloroform extraction. RNase protection assays were
performed with 10 .mu.g total RNA from each tissue. Antisense RNA
probes labeled with .sup.32P were synthesized using T3 or T7 RNA
polymerase and template DNAs Bmp8a STR380, Bmp8b BN260, and
pTRI-B-actin-mouse plasmid (Ambion). Full length probes were
purified on 5% polyacrylamide-urea gels. RNase protection assays
were carried out with RPAII kit (Ambion).
[0162] In Situ Hybridization
[0163] In situ hybridization was performed essentially as described
by Zhao et al., (1993, Proc. Natl. Acad. Sci. USA 90:8633-8637)
with slight modifications as follows. Freshly dissected mouse
embryos and tissues were rinsed in phosphate-buffered saline (PBS),
then fixed in freshly prepared 4% paraformaldehyde-PBS for 2-12
hours depending on the mass of the tissues. After fixation, the
tissues were rinsed in PBS and dehydrated in a series of increasing
concentrations of ethanol for a period of 3-5 hours. After being
embedded in paraplast, tissues were sectioned at 7 .mu.m. RNA
probes were labeled with [a-.sup.35S] UTP to a specific activity of
1.2.times.10.sup.9 cpm/.mu.g. Hybridization was carried out at
50-55.degree. C. with 2.times.10.sup.4 cpm/.mu.l riboprobe for
12-16 hours in 50% formamide, 300 mM NaCl, 10 mM Tris (pH 7.4), 10
mM NaH.sub.2PO.sub.4 (pH 6.8), 5 mM EDTA (pH 8.0), 0.2% Ficoll 400,
0.2% polyvinyl pyrrolidone, 10% dextran sulfate, 200 .mu.g/ml yeast
tRNA, and 50 mM dithiothreitol (DTT). Two 30 minute high-stringency
washes were carried out in 2.times.SSC, 50% formamide at
60-65.degree. C. Slides were dipped in NTB-2 Kodak emulsion, then
exposed for 5-14 days at 4.degree. C. After being developed and
fixed, slides were stained in Mayer's hematoxylin (Sigma) for 3-5
minutes and then mounted with permount for photography.
[0164] Generation of Deciduoma
[0165] Pseudopregnant ICR females were generated by mating females
in oestrus with vasectomized males. Four days later, 50 .mu.l
mineral oil was injected into one uterus horn. Five days after
injection, animals were sacrificed. Uterus and/or deciduoma were
dissected out for fixation or RNA preparation.
[0166] Sequence Comparison and GenBank Accession Number
[0167] Sequence comparison between Bmp8a and Bmp8b cDNAs and
proteins was performed with the BLAST program of the National
Center for Biotechnology Information. The Bmp8b cDNA sequence has
been submitted to GenBank under accession number U39545.
[0168] Generation of Mutant Mice
[0169] Mice with mutations in Bmp8a and Bmp8b genes have been made
using the technique of homologous recombination in embryonic stem
(ES) cells. In these mice, DNA sequences encoding specific protein
sequences have been deleted and the genes have been rendered
nonfunctional.
[0170] Bmp8a and Bmp8b genomic DNA clones from a 129/SvJ mouse
genomic library were isolated. Replacement targeting constructs for
Bmp8a and Bmp8b were generated. Targeting DNA constructs were
transfected into 129/WvJ embryonic stem (ES) cells and targeted ES
cell clones were selected by positive and negative drug selections.
Targeted ES cells were injected into mouse blastocysts to generate
chimeric animals (chimeras). Chimeras were mated with wildtype
(+/+) Blackswiss females for germ line transmission, i.e. to obtain
heterozygous (+/-) animals. Heterozygous (+/-) animals were mated
to obtain homozygous (-/-) mutant animals.
[0171] Phenotype of the Mutant Mice
[0172] Male mice homozygous for a null mutation in Bmp8b have small
testes and are infertile by 3 months of age. Analysis of the
seminiferous tubules of adult homozygous mutants shows a very high
incidence of apoptosis among the spermatocytes, establishing that
the gene product (BMP8B protein) is required for the survival of
these cells in vivo. Homozygous mutant mice also have delayed onset
of spermatogenesis in the seminiferous tubules after birth. At one
and two weeks of age the seminiferous tubules consist only of a
simple layer of spermatogonial cells in contact with the basal
lamina and the Sertoli cells. Studies on the rate of cell
proliferation by BrdU labeling of whole animals at 1 and 2 weeks of
age has established that the proliferation of spermatogonial stem
cells adjacent to the basal lamina of the seminiferous tubules is
greatly reduced in mutant mice. This establishes that the Bmp8b
gene product also plays a crucial role in
enhancing/promoting/stimulating the proliferation of spermatogonial
cells after birth.
[0173] The Results of these experiments are now described.
[0174] Analysis of the Temporal and Spatial Expression of the Genes
Encoding BMP8.
[0175] The experiments which are now described feature an analysis
of the temporal and spatial expression of the gene encoding BMP8
(Kingsley, 1994, Genes Dev. 8:133-146), also known as osteogenic
protein 2 (OP2) (zkaynak et al., 1992, J. Biol. Chem.
267:25220-25227).
[0176] Molecular Cloning of Bmp8b
[0177] A mouse Bmp8a cDNA fragment containing exons 2-7 was used to
screen a mouse 129/SvJ genomic DNA library in lambda FixII at high
stringency. Nine positive clones were purified from
5.times.10.sup.5 plaques. Two overlapping clones corresponded to
Bmp8a, starting from the middle of intron 1 and ending downstream
of exon 7. The seven remaining clones were derived from a different
genomic locus. Sequence analysis revealed that the new locus,
Bmp8b, contained DNA sequences with a high degree of identity to
those of Bmp8a. Based on the sequence information in the mature
region of the presumed exon 4, specific oligonucleotide primers
were designed 5' RACE- and 3' RACE-PCR was conducted using RNA
isolated from 14.5 d.p.c. placenta to isolate Bmp8b cDNAs. A
comparison of the deduced amino acid sequences of BMP8A and BMP8B
is shown in FIG. 1.
[0178] Bmp8 Genes Share High Levels of Sequence Identity
[0179] Sequence identity between Bmp8a and Bmp8b at the nucleotide
level is limited to the coding region plus about 20 flanking base
pairs in both the 5' untranslated region (UTR) and the 3' UTR. DNA
sequences encoding the pro-region share 91% identity while
sequences encoding the mature region share 83% identity. As shown
in FIG. 1, both BMP8A and BMP8B proteins contain 399 amino acids.
The amino acid sequences in the pro-region of these proteins share
87% identity, while those of the mature region share 76% identity.
This is exceptional among all the known closely related TGF-.beta.
superfamily members, wherein the pro-regions have higher sequence
identity than the mature regions. The sequence homology of mouse
BMP8 proteins with other TGF-.beta. superfamily members is
significantly lower than the homology shared between BMP8A and
BMP8B.
[0180] Bmp8a and Bmp8b are Closely Linked on Chromosome 4
[0181] The Jackson Laboratory interspecific backcross panel (BSS)
was used to map the chromosomal localizations of Bmp8a and Bmp8b.
RFLVs between C57BL/6 and Mus spretus were identified for each gene
using probes derived from a Bmp8a DNA fragment downstream of the 3'
UTR (exon 7) or from a Bmp8b DNA fragment in intron 6. In the case
of both genes, RFLVs were detected using the restriction enzyme
MspI. In both cases, 94 of the (C57BL/6JEixSPRET/E1) F1xSPRET/E1
backcross progeny were typed for inheritance of the Mus domesticus
or Mus spretus alleles and the distribution pattern of each allele
was used to place the locus onto the interspecific map (Rowe et
al., 1994, Mamm. Genome 5:253-274). As shown in FIG. 2, both genes
were assigned to chromosome 4, there being no recombination in 94
animals with the marker D4Mit11. These data establish that Bmp8a
and Bmp8b are closely linked and suggest that these genes arose by
duplication of a single gene.
[0182] The location of the two Bmp8 genes on chromosome 4 according
to The Jackson Laboratory backcross panel data is compared with the
composite chromosome 4 map in the Mouse Genome Database (MGD) in
FIG. 2A. The only potential candidate for a mutation in Bmp8a/b is
sks (skeletal fusions with sterility) (Handel et al., 1988, Gamete
Res.21:409-423). In males homozygous for this recessive mutation,
the majority of germ cells are arrested in late stages of
meiosis.
[0183] Expression of Bmp8 Genes During Development
[0184] To investigate the function of the Bmp8 genes, the
expression pattern of the genes was examined during mouse
embryogenesis using a battery of techniques including Northern
blotting, RNase protection assays (FIG. 3), whole mount in situ
hybridization, in situ hybridization of sections (7.5-10.5 d.p.c.
in FIG. 4), and screening of a 8.5 d.p.c. mouse embryo cDNA
library. No detectable levels of Bmp8 expression in embryos at any
of these stages were observed. These data therefore contradict a
previous report wherein high levels of Bmp8a (Op 2) transcripts
were detected in 8.5 and 10.5 d.p.c. mouse embryos by Northern
hybridization (Zkaynak et al, supra). However, when reverse
transcriptase PCR (RT-PCR) was performed using RNAs isolated from
uterus and placenta of different stages, high levels of Bmp8a
transcripts were detected in both tissues, and Bmp8b was detected
in placenta only. RNase protection assays using RNAs from these
tissues and from embryos (FIG. 3) confirmed the results obtained
using RT-PCR.
[0185] To localize Bmp8 transcripts in placenta and uterus, in situ
hybridization was performed using antisense RNA probes
corresponding to the unique 3' untranslated region of Bmp8 mRNAs.
As shown in FIG. 4, high levels of Bmp8a transcripts were detected
in decidual cells between 7.5 and 10.5 d.p.c. Lower levels of
expression were also detected in trophoblast cells (FIGS. 4D, 4G,
and 4J). Although very low levels of Bmp8a transcripts were found
by RT-PCR in the nonpregnant uterus, no strong in situ
hybridization signal was observed in 5.5 d.p.c. deciduum and
uterus. Therefore, expression of Bmp8a increases significantly
between 5.5 and 7.5 d.p.c. Bmp8a transcripts decrease to levels
undetectable by in situ hybridization after 11.5 d.p.c.
[0186] The highest levels of Bmp8a expression were observed in the
antimesometrial region of the deciduum, and a decreasing gradient
was noted mesometrially before 9.5 d.p.c. At 10.5 d.p.c., although
the overall level of expression decreased, the highest expression
was found in the mesometrial region. Bmp8a expression in decidual
cells is independent of embryo implantation. This was determined by
injecting mineral oil into the uteri of pseudopregnant mice to
generate deciduomas in the absence of an embryo. Bmp8a expression
in the deciduoma faithfully reflected the normal expression pattern
after embryo implantation.
[0187] Bmp8b transcripts were detected predominantly in trophoblast
cells in the labyrinthine region of the placenta starting at about
9.5 d.p.c., and transcripts were still detectable at 16.5 d.p.c.
Although a very low level of Bmp8b transcripts seemed to be present
in 13.5 and 15.5 d.p.c. embryos by RNase protection assays,
transcripts could not be detected in the embryo proper by in situ
hybridization.
[0188] Expression of Bmp8 Genes in Male Germ Cells
[0189] Among the adult tissues assayed by RNase protection
including heart, brain, lung, kidney, liver, spleen, thymus, skin,
skeletal muscle, testis, and ovary, only testis contained
detectable Bmp8 signals. Therefore, in situ hybridization using
.sup.35S-labeled antisense RNA probes was performed on testis
sections. During mammalian spermatogenesis, the early spermatogonia
(stem cells) divide mitotically to give rise to primary
spermatocytes. These cells in turn give rise to secondary
spermatocytes and spematids through meiosis. As reviewed in Russell
et al. (1990, In:Histological and histopathological evaluation of
the testis, Cache River Press, Clearwater, Fla., pp. 119-161),
during mouse spermatogenesis the seminiferous tubules, which
contain germ cells and somatic Sertoli cells, are arbitrarily
divided into 12 stages indicated by Roman numerals. In this staging
system, the morphology of acrosomes (stained by periodic acid and
Schiff's reagents) and nuclei (stained with hematoxylin) of the
spermatids serve as critical markers. The development of spermatids
is divided into 16 stages indicated by Arabic numerals. Strictly
regulated cellular associations among germ cells of different
stages can be observed in different regions of the seminiferous
tubules. As shown in FIG. 5, transcripts specifying both Bmp8 genes
were detected in stage 6-8 round spermatids in a similar temporal
and spatial pattern. Strong hybridization signals first appeared in
testes of 3 week-old animals, a time when round spermatids first
differentiated to stage 6. Weak signals were also detected in some
pachytene spermatocytes in the testes of younger animals before 6
weeks of age (as shown in FIG. 5, Panels M and N). The strongest
hybridization signals were observed in late stage 7 and early stage
8 round spermatids and signals decreased dramatically when
spermatids started to elongate (stage 9).
[0190] Among all of the known closely related members of the
TGF-.beta. superfamily of proteins, BMP8A and BMP8B exhibit the
highest levels of sequence identity, especially in the pro-region.
The two genes encoding these proteins are closely linked on mouse
chromosome 4 and share a similar genomic organization (intron/exon
size and boundaries), suggesting that they arose by a recent gene
duplication. However, although their expression patterns are
similar, they are not identical. In male germ cells, both genes are
expressed at the highest levels in stage 6-8 round spermatids and
at lower levels in pachytene spermatocytes. In the developing
uterus and placenta, Bmp8a is expressed at high levels in decidual
cells and at low levels in trophoblast cells, and its expression
decreases to a level undetectable by in situ hybridization after
11.5 d.p.c. In contrast, Bmp8b is not expressed in the maternal
component of the placenta but in the labyrinthine region of
trophoblasts, where its expression persists up to at least 16.5
d.p.c. Another difference in Bmp8a and Bmp8b expression patterns is
that Bmp8a is expressed at high levels in the developing hair
follicles of early postnatal animals while Bmp8b is only expressed
at a level slightly above background. Such differences in
expression patterns suggest that following duplication of Bmp8a and
Bmp8b genes, regulatory elements outside the coding exons have
diverged, allowing some elements to be lost and/or others to be
gained.
[0191] BMPs and Placental Development
[0192] The roles of BMPs during implantation and placental
development have not been well established, although transcripts
for Bmp2, Bmp4, Bmp6 (Lyons et al., 1990, Development 109:833-844)
and Bmp8a and Bmp8b have all been detected in extra-embryonic sites
by in situ hybridization. The expression of Bmp8a described here
overlaps with that of Bmp2. High levels of both transcripts are
present in the deciduum, not at the time of implantation, but
rather when decidual cell proliferation and hypertrophy peak (for
review Finn, 1971, Adv. Reprod. Physiol. 5:1-26). Bmp8a expression
decreases when the growth of the deciduum slows down. This temporal
and spatial pattern suggests that these ligands are involved in
promoting the proliferation, survival and/or differentiation of the
uterine stromal cells.
[0193] In the placenta, the trophoblast cells in the labyrinthine
region are a relatively undifferentiated and rapidly proliferating
population which gives rise to the cells in the spongiotrophoblast
layer. The outer layer of spongiotrophoblast cells further
differentiate into non-dividing trophoblast giant cells
(Billington, 1971, Adv. Reprod. Physiol. 5:27-66; Cross et al.,
1994, Science 266:1508-1518). The expression of Bmp8b is highest in
the labyrinthine trophoblast region while that of Bmp4 is highest
in the spongiotrophoblasts. This finding suggests that Bmp8b and
Bmp4 may promote the proliferation/survival of trophoblast cells
and/or regulate their differentiation into spongiotrophoblasts and
ultimately into giant trophoblasts.
[0194] In the past few years, a number of genes encoding
transcription factors have been detected in the trophoblast cells
of the developing placenta (for review see Cross et al. 1994,
Science 266:1508-1518). Among these gene products, the evidence
suggests that helix-loop-helix (HLH) transcription factors, Mash-2
(Johnson et al., 1990, Nature 346:858-861), Id-1, Id-2 (Cross et
al., 1994, Science 266, 1508-1518; Evans et al., 1993, Dev. Biol.
159, 485-499; Janaypour et al., 1994, Mol. Biol. Cell. 5:(suppl.),
453a), and Hxt (Cross et al., 1995, Development 121:2513-2523)
affect the proliferation and differentiation trophoblasts. The
newly identified eHand transcripts were also detected at high
levels in the trophoblast lineage (Cserjesi et al., 1995, Dev.
Biol. 170:664-678). The expression of BMPs and HLH proteins in the
placenta may reflect complex interactions among these two groups of
proteins in regulating the growth and differentiation of the
placenta. The fact that BMP purified from bone matrix inhibits
differentiation of myoblastic cells by suppressing the expression
of MyoD family of HLH proteins (Murray et al., 1993, J. Cell.
Biochem. 53:51-60) supports such a hypothesis.
[0195] The Role of BMP8 Proteins in Spermatogenesis
[0196] Bmp8 genes are the first of the TGF-.beta. superfamily
members shown to be expressed in male germ cells. In the testes of
young animals before any round spermatids appear, Bmp8 transcripts
were detected in the primary spermatocytes at low levels (FIG. 5,
Panels M and N for testis of 3.5 weeks). When round spermatids
develop to stages 6-8, much higher levels of expression of Bmp8a
and Bmp8b were detected in these haploid germ cells (FIG. 5 M and
N). Such an expression pattern is distinct from those of three
other known TGF-.beta. superfamily members, Muillerian inhibiting
substance (MIS), inhibin, and activin, which are all expressed
predominantly in the Sertoli cells of the testis. Furthermore,
inactivation of these genes by targeted mutagenesis does not lead
to a primary defect in male germ cells (Behringer et al., 1994,
Cell 79, 415-425; Matzuk et al., 1992, Nature 360:313-319; Vassalli
et al., 1994, Genes Dev. 8: 414-427; Matzuk et al., 1995, Nature
374:354-356). Rather, tumorigenesis of interstitial cells in the
homozygous inhibin null mutants supports an indirect role of
inhibin in testis function by inhibition of follicle stimulating
hormone (FSH) production in the pituitary gland. In the absence of
inhibin, the over production of FSH causes the over proliferation
and tumorigenesis of the interstitial cells (Matzuk et al., 1992,
supra). In the absence of MIS, the development of the female
reproduction system is not fully inhibited in male mice. Although
the presence of both male and female reproduction systems in the
male imposes anatomical incompatibility and infertility,
spermatogenesis seems relatively unaffected (Behringer et al.,
1994, Cell 79, 415-425). In the absence of activin BB subunit,
spermatogenesis is normal (Vassalli, et al., supra), while the
absence of activin BA subunit leads to perinatal lethality, so that
its role in spermatogenesis cannot be evaluated (Matzuk et al.,
1995, Nature 374:354-356). However, the-absence of the activin
receptor ActRcII only causes a reduction in the volume of
seminiferous tubules, but no primary defects in the germ cell
population, suggesting that this receptor may affect the
proliferation and differentiation of somatic Sertoli cells (Matzuk
et al., 1995, Nature 374, 356-359).
[0197] In contrast to other TGF-.beta. superfamily members, the
data presented herein establish that the expression of the Bmp8
genes in male germ cells tightly correlates with the progression of
spermatogenesis. During postnatal development of the testis, germ
cells may have one of two fates, differentiation to give rise to
mature sperm or degeneration either through necrosis or apoptosis.
About 50% of early germ cells die during their life time,
predominantly by apoptosis (Allan et al., 1987, In Potten, C. S.
(Ed.), Perspectives on mammalian cell death, Oxford University
Press, London. pp. 229-258; Allan et al., 1992, Cell Prolif.
25:241-250). The data presented below confirm that before puberty a
significant proportion of mouse gonocytes degenerate through
apoptosis. However, with the appearance of stages 6-8 round
spermtids, the levels of Bmp8 expression increase (3.5 weeks of age
or older) and the number of apoptotic germ cells decreases. Such an
inverse relationship between the level of Bmp8a and Bmp8b
expression and germ cell apoptosis favors a role of BMP8 in
inhibiting germ cell degeneration. Furthermore, in the homozygous
null Bmp8B mutant males, increased apoptosis of germ cells leads to
testis degeneration and infertility, and eventually only Sertoli
cells are left in the seminiferous tubules. These observations
suggest a non-redundant role of BMP8A and BMP8B in the survival of
germ cells by inhibiting apoptosis.
[0198] Expanding upon the experiments just described, in the next
set of experiments the in vivo functions of Bmp8 genes was
investigated using a molecular genetic approach by introducing
targeted mutations into each gene separately. As discussed herein,
in the absence of a functional Bmp8b gene, male germ cell
proliferation is significantly reduced during early puberty, and
there is also a marked increase in male germ cell apoptosis in the
adult. Eventually, the majority of the Bmp8b homozygous mutant
males show severe seminiferous tubule degeneration and become
sterile. Therefore, Bmp8b is required both for the initiation and
maintenance of spermatogenesis in the mouse. To similarly
investigate the in vivo function of Bmp8a during spermatogenesis
and pregnancy, the mouse gene was inactivated by homologous
recombination in embryonic stem (ES) cells. The data which are now
described relate to a detailed phenotypic analysis of Bmp8a mutant
mice and Bmp8a/Bmp8b compound heterozygous mice. To summarize the
results to be presented, the mouse Bmp8a gene is not essential for
deciduum formation and placenta development and is not required for
the initiation of spermatogenesis. Rather, it appears to play a
role in maintaining normal spermatogenesis and the integrity of the
epididymis during male reproduction.
[0199] The Methods used in the next set of experiments are now
described.
[0200] Construction of the Targeting Vector
[0201] Bmp8a genomic DNA clones were isolated as described herein.
Two overlapping Bmp8a genomic clones covering exons 2-7 were mapped
by restriction enzyme digestion as shown in FIG. 6A. A replacement
targeting vector was constructed using 1.2 kb 5' and 4.5 kb (5'
portion of the second phage clone) 3' homology arms as indicated.
As described herein, PGK-TKA+(Rudnicki, et al., 1992, Cell
71:383-390), and MCLDT-A (Yagi et al., 1990, Proc. Natl. Acad. Sci.
USA 87:9918-9922) cassettes were attached to the 5' and 3' ends of
the targeting vector for negative selection. In the targeted
allele, exons 4-6 are deleted and replaced with PGK-neo.sup.r
cassette (Rudnicki, et al., supra). This Bmp8a mutant allele is
designated as Bmp8.sup.atm1blh according to standard nomenclature
(Davisson 1995, In: Trends in Genetics Nomenclature Guide, Ed.
Stewart, pp. 35-3 S. Elsevier Trends Journals, Kidlington, Oxford,
UK).
[0202] Generation of Recombinant ES Cell Clones and Mouse
Chimeras
[0203] TL1 ES cells of passage 11 and 12 were transfected with
20-50 .mu.g of the linearized targeting vector by electroporation
as described herein. ES cell culture and drug selection were
performed essentially as described (Winnier et al., 1995, Genes
Dev. 9:2105-2116). Three out of one hundred drug-resistant ES
clones designated as A5, H4, and H9, exhibited a correctly
recombined Bmp8.sup.atm1blh allele. All three lines were injected
into C57BL/6 blastocysts to generate chimeras (Hogan et al., 1994,
Manipulating the mouse embryo: A Laboratory Manual. Second edition.
Cold Spring Harbor Laboratory Press, NY). Bmp8.sup.atm1blh was
transmitted from A5 and H4 cells by mating male chimeras with Black
Swiss females (Taconic). Agouti animals were genotyped by Southern
blotting.
[0204] Southern Blot Analysis
[0205] Genomic DNA was digested with EcoRI and size fractionated on
a 0.8% agarose gel. Denatured DNA was then transferred to
positively charged Nylon membrane and hybridized with two different
probes. A 5' external Bmp8a cDNA probe, containing exons 2 and 3,
hybridized to 9.0 kb and 8.0 kb bands from the wild-type Bmp8a and
Bmp8b alleles, respectively, and to a 5.0 kb band from the
Bmp8.sup.atm1blh allele (FIG. 6B). A 3' internal cDNA probe,
containing exons 4, 5, 6, and 7, hybridized with 2.0 kb and 2.3 kb
bands for the wild-type Bmp8a allele and only a 2.0 kb band for the
Bmp8.sup.atm1blh allele (FIG. 6C).
[0206] Histology and in Situ Hybridization
[0207] For histology, freshly dissected testes were weighed and
rinsed in phosphate buffered saline (PBS), then fixed either in
Bouin's fixative or in 4% paraformaldehyde-PBS for 2-24 hours
depending on the size. Sections of 7 .mu.m were mounted onto
superfrost plus slides and stained either by hematoxylin/eosin or
periodic acid-Schiff's reagent/hematoxylin. Histological analysis
of Bmp8b mutants and the initial survey of Bmp8a mutants in this
study established that the testes from a single animal, when
comparable in weight, usually have a similar histology. Therefore,
only one testis from each animal was embedded and sectioned for
most of the histological analysis. Testes of different genotypes
were embedded and sectioned in the same block and stained on the
same slides as internal controls. A third of the sections through
the whole testes were mounted and stained by taking every third
pair of sections for microscopic examination. If any abnormalities
were observed in any given section, then the adjacent sections were
mounted and stained for further examination. Seminiferous tubules
that lack germ cells, or with no spermiogenesis or obviously
compromised spermiogenesis, were considered abnormal. A
seminiferous tubule was also scored as a degenerating tubule if it
contained more than 50 germ cells with condensed nuclei and
eosinophilic cytoplasm (this was considered to be characteristic of
apoptosis) in 10 consecutive sections. Of the testes examined, 47%
(15/32) of the Bmp8a homozygous and 17% (3/18) of the Bmp8a
heterozygous mutants, and none (0/11) of the wild-type animals
showed different degrees of germ cell degeneration.
[0208] In situ hybridization was performed as described herein
being mindful of the following points. In brief, proteinase K
treatment was extended from 7 minutes to 8 minutes for better
penetrance of the probes. Hybridization temperature was raised to
60-65.degree. C. to increase the ratio of signal/background. High
stringency washes were performed at 63.degree. C. Slides were
exposed for 10 days after being dipped in NBT2 emulsion.
[0209] The results of this set of experiments are now
described.
[0210] Targeted Mutagenesis of the Mouse Bmp8a Gene
[0211] The mouse Bmp8a gene contains seven coding exons and extends
over 27 kilobases of DNA. Exons 1, 2, 3, and the first half of exon
4 encode the signal peptide and the pro-region of the precursor
protein, while the second half of exon 4, and exons 5, 6, and 7
encode the mature region (Ozkaynak et al., supra). As shown in FIG.
6, in the Bmp8.sup.atm1blh allele, an 8-kb genomic DNA fragment
encompassing exons 4, 5, and 6 is deleted and replaced by a
PGK-neo.sup.r cassette (Rudnicki et al., supra) in the opposite
transcriptional orientation. Therefore, DNA encoding part of the
pro-region, the dibasic cleavage site RXXR, and the majority of the
mature region is removed from the mouse genome. Furthermore, the
remaining exons 3 and 7 are not in frame if alternative splicing of
the PGK-neo.sup.r cassette should occur. It is therefore predicted
that no functional or dominant negative forms of BMP8A protein will
be made from the Bmp8.sup.atm1blh allele. In situ hybridization
with .sup.35S-labeled riboprobes against the specific 3'
untranslated region of Bmp8a facilitates detection of a transcript
in stage 6-8 round spermatids of homozygous mutant testis.
[0212] After drug selection, three recombinant Bmp8a mutant ES cell
lines A5, H4, and H9 were obtained and injected into C57BL/6
blastocysts to generate chimeras. The Bmp8.sup.atm1blh allele was
transmitted to offspring in lines A5 and H4. Mutant mice from both
ES cell lines exhibit similar phenotypes when maintained on a mixed
genetic background of [129 X Black Swiss] for this study. All data
reported here were generated from a combination of these two
lines.
[0213] Reproductive Performance of Bmp8a Mutants
[0214] Bmp8a is expressed in the deciduum during pregnancy, in
spermatogonia and primary spermatocytes during the initiation of
spermatogenesis, and in stage 6-8 round spermatids during the
maintenance of spermatogenesis. This expression pattern raises the
possibility that the absence of a functional Bmp8a gene would
compromise the reproductive performance of both males and females.
During initial mating tests of heterozygous Bmp8.sup.atm1blh
mutants, the expected ratio of wild-type (n=45), heterozygous
(n=104), and homozygous mutant (n=52) offspring was obtained. The
homozygous mutants grow normally to adulthood and appear healthy.
Therefore, Bmp8a is not required for embryonic and postnatal
development. The fertility of the homozygous Bmp8.sup.atm1blh
animals was further tested by mating with wild-type and
heterozygous animals. As summarized in Table 1, all homozygous
mutant females exhibited normal reproductive performance. All
mutant males exhibited normal fertility initially. However, as they
aged, some animals (2 out of 16) eventually became sterile.
Therefore, Bmp8a plays a role in the fertility of certain males,
but not of females. TABLE-US-00001 TABLE 1 Bmp8.sup.atm1blh mice
Genotype Litter size Male (n) Female (n) Litter Number (mean .+-.
S.E.) +/+ (3) -/- (18) 30 8.5 .+-. 2.2 +/- (3) +/- (11) +/- (23) 77
8.3 .+-. 2.6 +/- (8)* -/- (22) 85 8.1 .+-. 2.6 -/- (8).sup.# +/-
(20) 66 8.3 .+-. 2.8 +/+ (5) Comp (6).sup.@ +/+ (10) 27 7.4 .+-.
3.2 Comp (10)
[0215] In Table 1, 7-10 week old mice of different genotypes were
caged together for 9-20 weeks. Each male was housed with 2-4
females, and pregnant females were separated before giving birth.
The number of pups was recorded within 24 hours after birth. Most
matings resulted in litters of normal frequency and size (wild type
mating gave an average litter size of 8-8.5 pups). *One male in
this group had two litters of 7-8 pups during the initial mating
period, but was infertile thereafter, presumably due to epididymis
degeneration and granuloma formation (FIG. 11B). #One male in this
group never reproduced and histology revealed severely compromised
spermiogenesis. @ Comp for Bmp8 compound heterozygotes. In this
group, one male never reproduced and a cyst was found in the left
testis. Another male had normal reproductive performance for 8-9
weeks, but did not reproduce for the last 6 weeks. Histological
examination revealed epididymis degeneration and granuloma
formation (FIG. 11C).
[0216] Bmp8a is not Required for the Initiation of
Spermatogenesis
[0217] As shown in FIG. 7, from 1 week after birth to young
adulthood, there was no significant difference in the average
weight of testes of wild-type males compared to heterozygous or
homozygous Bmp8.sup.atm1blh mutants. This is in contrast to the
findings in Bmp8b mutants, where testes of all homozygotes from 1
to 3 weeks of age are significantly smaller than those of
wild-type. Only 1 out of 30 Bmp8a homozygous mutants examined at 2
weeks of age had a small testis weighing 7 mg. This testis and 14
more testes were from Bmp8a/Bmp8b compound heterozygous matings and
not used for the statistical analysis depicted FIG. 7. The average
weight of testis from 2 week-old normal mice is 12-15 mg. The 7 mg
testis had a histology similar to that of Bmp8b homozygous mutants
(FIGS. 8C and 8D). The testes of the remaining Bmp8a homozygous
mutants at 2 weeks of age exhibited a histology similar to that of
wild type mice, and no other mice with small testes were found in
any other age group before puberty (6 weeks of age). In summary,
histological examination of testes of more than 60 homozygous Bmp8a
mutant animals (at least 10 for each age group) revealed no obvious
abnormalities from 1 to 6 weeks of age (except for the one cited
above).
[0218] As shown in FIG. 8, histological comparison of wild-type,
Bmp8a and Bmp8b homozygous mutant, and Bmp8a/Bmp8b compound
heterozygous testes at two weeks of age, revealed an obvious germ
cell deficiency only in the Bmp8b mutant testis. Therefore, it
appears that Bmp8a is not required for the initiation of
spermatogenesis in the mouse.
[0219] Bmp8a Plays a Role in the Maintenance of Spermatogenesis
[0220] Based on testis weight and histology, homozygous Bmp8a
mutants do not show any obvious abnormalities during the initiation
of spermatogenesis. However, the testis weight of some Bmp8a
mutants older than 17 weeks of age appears to be smaller than that
of wild-type and heterozygous mice (FIG. 7). Although the
difference of the average testis weights of wild-type and
homozygous mutant testes are not statistically significant by
Student's t test (P is between 0.05 and 0.1), a larger proportion
of testes in the Bmp8a homozygous mutant group (13 out of 57) are
below 90 mg compared to either wild-type (1 out 37) or heterozygous
testes (5/86).
[0221] Histologically, about 47% (15 out of 32 testes examined of
Bmp8a homozygous mutant testes from 12-30 week-old males show
varied degrees of germ cell degeneration (FIGS. 9 and 10). As shown
in FIGS. 9C and 9D, the most severe germ cell deficiency so far
observed among the homozygous Bmp8a mutants (2 out of the 32
examined) is much milder than that seen in homozygous Bmp8b mutants
(FIGS. 9E and 9F). In a majority of seminiferous tubules of such
Bmp8a mutant testes, spermiogenesis (the maturation of spermatids)
is severely compromised, or even absent. However, spermatogenesis
(although abnormal) is still present. Increased meiotic germ cell
apoptosis can be identified in most tubules, as revealed by
condensed and darkly stained nuclei (arrows in FIG. 9D).
[0222] In the cases of milder forms of germ cell deficiency, only a
certain percentage of the seminiferous tubules (from one tubule to
50% of the tubules) of given sections show seminiferous tubule
degeneration, predominantly caused by meiotic germ cell apoptosis,
resulting in the absence of spermiogenesis (FIGS. 10D-100). These
defects are similar to many of the abnormalities associated with
the Bmp8b homozygous mutant phenotype. The characteristics of the
apoptotic germ cells. are condensed and darkly stained nuclei or
chromatin and strongly eosinophilic cytoplasm. These cells can be
labeled positively by TUNEL (Zhao et al., 1996, Genes and Dev.
10:1657-1669; Furuchi et al., 1996, Development 122:1703-1709; Dix
et al., 1996, Proc. Natl. Acad. Sci. USA 93:3264-3268; Gavrieli et
al., 1992, J. Cell. Biol. 119:493-501. Similar abnormalities are
also observed in 17% of the Bmp8a heterozygous mutant, testes (3
out of 18 examined). Of the Bmp8a/Bmp8b compound heterozygous
mutant adult males examined, about half of them (5 out of 11)
exhibit a similar germ cell degeneration phenotype.
[0223] Bmp8a Plays a Role in the Maintenance of Epididymis
Integrity
[0224] As shown in Table 1, most of the Bmp8a homozygous mutant
males show normal reproductive performance. However, one out of the
16 males tested was fertile within the first month, but produced no
offspring afterwards. This animal was sacrificed at 4 months of
age, revealing two adhesive clumps attached to the right
epididymis, one attached to the caput, the other to the cauda. No
obvious abnormality was found in the left testis and epididymis.
Histological examination revealed gross abnormalities in the
morphology of the right epididymis. As shown in FIGS. 11B and 11E,
a large granuloma-like mass, with sperm and some necrosis in the
center and massive leukocyte infiltration on the periphery, almost
replaced the cauda epididymis. Only sparsely dispersed abnormal
epididymis tubules were observed outside of the granuloma mass.
Subsequently, similar but milder pathological abnormalities were
observed in another mating homozygous Bmp8a mutant and a
Bmp8a/Bmp8b compound heterozygote (FIGS. 11C, 11D, 11F, and
11G).
[0225] One interpretation of the phenotype is that the epithelium
lining the epididymal tubules degenerated and sperm was forced out
from the collapsed tubule. Due to the antigenicity of sperm,
massive leukocyte infiltration would then accompany the eruption of
the epididymal tubule, resulting in a granuloma-like mass. Similar
degeneration of the epithelium and leukocyte infiltration was also
found in the distal caput region of the same epididymides shown in
FIGS. 11B and 11C. However, in contrast to the mating group (2 out
of 16 for Bmp8a homozygous mutants, 1 out of 6 for Bmp8a/Bmp8b
compound heterozygous mutants), no granuloma-like pathology was
found in the epididymis of more than 60 non-mating Bmp8a homozygous
and Bmp8 compound heterozygous males were examined. Only epithelial
degeneration was found in the distal caput region in 2 out of the
40 epididymides sectioned (FIG. 11H-11J), and no obvious epididymal
epithelium degeneration was found in the cauda region of the same
epididymides. Therefore, it appeared that mating exacerbated the
epididymal degeneration phenotype of Bmp8a homozygous and Bmp8
compound heterozygous mutants.
[0226] Bmp8a and Bmp7 are Expressed in the Initial Segment of the
Caput Epididymis
[0227] In light of the degeneration of the epididymal epithelium
and granuloma formation in the Bmp8a homozygous and Bmp8a/Bmp8b
compound heterozygous mutants, the expression of Bmp family members
was examined in the adult epididymis of mating and non-mating
animals (14-17 weeks of age). Among the genes examined (Bmp2, 4, 5,
6, 7, 8a, 8b, and Vgr2), only Bmp8a and Bmp7 exhibited significant
expression. As shown in FIG. 12, both Bmp8a and Bmp7 are expressed
in the same region of the epididymis, namely the initial segment of
the caput. However, the expression levels of Bmp8a in this region
are significantly lower than in the stage 68 round spermatids of
the testis (FIG. 12A). Also, the levels of Bmp7 expression in the
initial segment are much higher than those of Bmp8a.
[0228] The data just described establish that Bmp8a does not play a
major role in the initiation of spermatogenesis. Further, the data
establish that Bmp8a plays a role in the maintenance of
spermatogenesis.
[0229] The disclosures of each and every patent, patent application
and publication cited herein are hereby incorporated herein by
reference in their entirety.
[0230] While this invention has been disclosed with reference to
specific embodiments, it is apparent that other embodiments and
variations of this invention may be devised by others skilled in
the art without departing from the true spirit and scope of the
invention. The appended claims are intended to be construed to
include all such embodiments and equivalent variations.
Sequence CWU 1
1
* * * * *