Synergistic Roles of Bone Morphogenetic Protein 15 and Growth Differentiation Factor 9 in Ovarian Function

Changning Yan, Pei Wang, Janet DeMayo, Francesco J. DeMayo, Julia A. Elvin, Cecilia Carino, Sarvamangala V. Prasad, Sheri S. Skinner, Bonnie S. Dunbar, Jennifer L. Dube, Anthony J. Celeste and Martin M. Matzuk

Departments of Pathology (C.Y., M.M.M.), Molecular and Cellular Biology (P.W., J.D., F.J.D., C.C., S.V.P., S.S.S., B.S.D., M.M.M.), and Molecular and Human Genetics (J.A.E., M.M.M.) Baylor College of Medicine Houston, Texas, 77030
Department of Tissue Growth and Repair (J.L.D., A.J.C.) Genetics Institute, Inc. Cambridge, Massachusetts 02140


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-ß superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15-/-) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15-/-Gdf9-/-) display oocyte loss and cysts and resemble Gdf9-/- mutants. In contrast, Bmp15-/-Gdf9+/- female mice have more severe fertility defects than Bmp15-/- females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Although important molecular events occur during all stages of mammalian ovarian folliculogenesis, few oocyte-expressed regulatory proteins have been identified. Our group and others have used embryonic stem (ES) cell technology to produce mouse models with ovarian abnormalities (for review, see Refs. 1, 2). These knockout mouse models demonstrate defects in germ cell maintenance, proliferation, and development [e.g. Dazla knockout mice (3)], formation of primordial follicles [e.g. Fig{alpha} knockout mice (4)], formation of secondary follicles [e.g. Gdf9 knockout mice (5)], formation of antral follicles [e.g. FSHß knockout mice (6)], ovulation [e.g. cyclooxygenase 2 (7) and progesterone receptor knockout mice (8)], or postovulation [e.g. Mater knockout mice (9)]. Whereas mutations in oocyte-expressed genes (e.g. Fig{alpha}, kit receptor, and Mater) result in intrinsic defects in the oocyte or early embryo (4, 9, 10), growth differentiation factor 9 (GDF-9) is the only known oocyte-secreted growth factor that is required for somatic cell function in mice in vivo. Using Gdf9 knockout mice, we have shown that GDF-9 is directly required for granulosa cell growth and differentiation and indirectly for oocyte meiotic competence and formation of a theca (5, 11, 12).

Gdf9 mRNA and GDF-9 protein are not only expressed at the primary follicle stage but are also present in oocytes through ovulation (13, 14, 15). Since mice lacking GDF-9 have a block at the primary follicle stage, fail to form a theca, and eventually demonstrate defects in meiotic competence, it was unclear from these knockout studies whether GDF-9 also plays a role at later stages of folliculogenesis. It was therefore important to determine the function of GDF-9 in the periovulatory period since oocyte-secreted growth factors had been identified to play key regulatory functions in this period. Using recombinant mouse GDF-9, we demonstrated that GDF-9 can regulate a diverse number of genes and processes in the periovulatory stage, including cumulus expansion, induction of hyaluronan synthase 2, cyclooxygenase 2, and the EP2 PGE2 receptor, and inhibition of LH receptor and urokinase plasminogen activator (13, 15, 16). In addition, recombinant rat GDF-9 stimulates rat preantral follicle growth and also stimulates basal estradiol production in granulosa cells (17, 18). Thus, GDF-9 functions as a multipurpose oocyte-secreted growth factor during the early stages of folliculogenesis and in the periovulatory period.

Using a homology-based cloning strategy, we fortuitously cloned GDF-9 homologs from the mouse and human that we termed bone morphogenetic protein 15 (BMP-15) (19) [also called growth differentiation factor 9B (20)]. In addition to the 52% identity with GDF-9, BMP-15 had several interesting features. First, Bmp15 mRNA was exclusively expressed in oocytes in an identical pattern as Gdf9 (15, 19). Second, mouse Bmp15 and human BMP15 map to syntenic positions on the X chromosome. Third, similar to GDF-9, BMP-15 protein lacks the mature peptide cysteine that normally forms an intermolecular disulfide bond in the other TGFß superfamily members. These findings suggest that BMP-15 and GDF-9 may directly interact (i.e. form heterodimers) or functionally interact (i.e. play redundant or antagonistic roles). Recent evidence from studies in sheep suggests interacting roles of these proteins in the ovary. The BMP15 gene was cloned in sheep and shown to be mutated in Inverdale and Hanna sheep carrying the fecundity X (FecXI and FecXH) mutations (21). Both strains have mutations in the mature peptide sequence. Sheep heterozygous for these BMP15 mutations show an increased ovulation frequency resulting in more twins and triplets. Surprisingly, sheep homozygous for these mutations are infertile and have a block in folliculogenesis that phenocopies the mouse Gdf9 knockout ovarian phenotype. Thus, BMP-15 appears to be the second known oocyte-secreted growth factor that is critical for ovarian function.

In this report, we used the previously isolated mouse Bmp15 gene sequences (19) to create male and female mice with a null mutation in the X-linked Bmp15 gene. These Bmp15 null female mice are viable but display reproductive defects. In addition, we have intercrossed these Bmp15 null mice with mice carrying a mutation in the autosomal Gdf9 gene to uncover genetic interactions. These knockout mouse models have helped us define the important roles of BMP-15 and GDF-9 in oocyte-somatic cell interactions during folliculogenesis and in the periovulatory period.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeted Mutation of the Bmp15 Gene in ES Cells and Generation of Bmp15 Null Mutant Mice
We had previously isolated the mouse Bmp15 gene from a 129SvEv genomic library and shown that it was composed of two exons with a 3.5-kb intron. Bmp15 exon 1 encodes the 17-amino acid signal peptide and 91 amino acids of the propeptide, whereas exon 2 encodes the remaining 159 amino acids of the propeptide and the 125-amino acid mature domain. To generate a mutant allele in the Bmp15 gene in ES cells, we used the 129SvEv genomic sequences to construct a targeting vector to delete exon 2 (Fig. 1AGo). Recombination of this targeting vector and the endogenous Bmp15 locus was anticipated to yield a null allele because no mature (active) BMP-15 could be synthesized if the gene sequences encoding it were deleted. A similar strategy had been employed to generate a null allele in the Gdf9 locus (5).



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Figure 1. Construct to Generate a Bmp15 Mutant Allele in ES Cells and Generation of Bmp15 Mutant Mice

A, The Bmp15 replacement targeting vector to delete exon 2 is shown. The targeting vector was electroporated into hprt-deficient AB2.1 ES cells to produce the Bmp15 recombinant mutant allele. The mutant allele can be distinguished from the wild-type allele using 5'- or 3'-probes as shown. B, Southern blot analysis of tail DNA derived from seven offspring from a litter from a mating of XBmp15tm1Y and XBmp15tm1X parents. Genomic DNA was digested with PstI and analyzed as described previously (23 ) using the 3'-probe (top panel). The probe detects a 13.0-kb wild-type band in lanes derived from wild-type (+/Y) XY males and heterozygous (+/-) XBmp15tm1X females and a 9.2-kb recombinant mutant band in lanes derived from null XBmp15tm1Y (-/Y) males, heterozygous females, and null XBmp15tm1XBmp15tm1 (-/-) females. Southern blot analysis of the same DNA as in the top panel using an exon 2 probe (bottom panel) detects the 13.0-kb wild-type allele in DNA derived from the XY or XBmp15tm1X offspring but not in the null males or females confirming the null status of the Bmp15tm1Zuk allele.

 
The Bmp15 targeting vector was electroporated into AB2.1 ES cells (XY), and 13 of 92 (14%) of the ES cell clones analyzed were targeted at the Bmp15 locus and thereby null. Two of these ES cell lines were used to produce chimeric male mice that were fertile and transmitted the X-linked Bmp15 null allele (Bmp15tm1Zuk or Bmp15- or XBmp15tm1) to females. XBmp15tm1X (heterozygous or Bmp15+/-) females were used to generate XBmp15tm1Y (null) males that were intercrossed with heterozygous females to produce XBmp15tm1XBmp15tm1 (null or Bmp15-/-) females (Fig. 1BGo, top). Using Southern blot analysis of DNA, a Bmp15 exon 2 probe demonstrated a lack of a hybridizable signal in the DNA derived from XBmp15tm1Y and XBmp15tm1XBmp15tm1 mice but not XBmp15tm1X or XY mice (Fig. 1BGo, bottom). This confirmed that the Bmp15tm1Zuk allele is a null allele.

Fertility Analysis of Bmp15 Mutant Mice
Chimeric males, null males, heterozygous females, and null females were all viable and failed to demonstrate any gross developmental defects. In addition, male chimeric and Bmp15 null males were fertile, and Bmp15 null males had normal testis size [87.58 ± 2.42 ng/testis (n = 18)] compared with wild-type controls [87.27 ± 2.53 ng/testis (n = 11)]. The viability of Bmp15 mutant mice and the fertility of the chimeric and null males are consistent with the limited adult ovary-specific expression of Bmp15 mRNA (19).

To determine whether Bmp15 plays a key ovarian function in females, heterozygous and homozygous mutant females were mated to males. In contrast to GDF-9, which is absolutely required for fertility in females, Bmp15 homozygous mutants (C57/129 hybrid background) were subfertile when bred over a 1-yr period (Table 1Go). When the Bmp15 mutation was maintained on a 129SvEv inbred background strain (in which females are normally less fertile), the Bmp15 homozygous null females displayed a consistent (although more severe) subfertility compared with the Bmp15 heterozygotes (Table 1Go). Both the number of pups per litter and the number of litters per month were reduced for the Bmp15-/- females from either hybrid or inbred genetic backgrounds. Thus, BMP-15 plays an important role in female reproduction in mice but is not as essential as GDF-9 or its sheep ortholog.


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Table 1. Mating Data for Bmp15 and GDf9 Mutant Mice

 
Ovarian and Oocyte Physiology
To determine the cause of the subfertility in the Bmp15-/- female mice, ovaries were analyzed both morphologically and histologically at various time points. In contrast to Gdf9-/- ovaries, which were extremely small (5), Bmp15-/- ovaries were often grossly indistinguishable from either Bmp15+/- or wild-type ovaries. Histological analysis of these mutant ovaries confirmed our gross impressions. In general, Bmp15-/- ovaries at all ages up through 1 yr demonstrated all stages of follicle development and multiple corpora lutea and appeared indistinguishable from control ovaries (Fig. 2Go). These findings would be consistent with the ability of Bmp15-/- mice to become pregnant. However, occasional Bmp15-/- ovaries at different ages demonstrated very few follicles and had an increased number of zona pellucida (ZP) remnants (data not shown; see below). Single rare Bmp15-/- and Bmp15+/- mice had unilateral or bilateral cysts at 1 yr of age. These findings have never been seen in wild-type mice in our laboratory.



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Figure 2. Histological Analysis of Bmp15 Mutant Ovaries

A, Ovary from a 4-month-old Bmp15-/- 129 mouse showing normal follicular development and corpora lutea (CL). B, Ovary from a 6-month-old Bmp15+/- 129 mouse showing normal follicular development and corpora lutea except there is a single follicle with two oocytes (black arrows) and accumulation of ZP remnants (yellow arrows). C, High-power magnification of a follicle with two oocytes from a 4-month-old Bmp15-/- 129 ovary. Additional examples of multiple oocytes in a single follicle were seen in double mutant mice (see below). D, Ovary from a 1-yr-old Bmp15-/- 129 mouse showing fairly normal follicular development and corpora lutea except for the accumulation of PAS-positive material (arrows) and ZP remnants in the interstitium. There is also a reduction in the number of oocytes and follicles compared with younger mice. E, Follicle with a small, trapped denuded oocyte (arrow) from a superovulated Bmp15+/- C57/129 mouse. F, Follicle with a large, trapped denuded oocyte (arrow) from a superovulated Bmp15-/- C57/129 mouse. All sections were stained with PAS/hematoxylin; for the scale bars, white lines represent 100 µm; black lines represent 200 µm.

 
Since Bmp15-/- ovarian histology was fairly normal, we subjected the Bmp15+/- and Bmp15-/- mice (C57/129 hybrid background) to a pharmacological superovulation protocol and determined the number of eggs that were ovulated. "Normal" numbers of eggs were released by the Bmp15+/- females (36.4 ± 3.7 eggs; n = 26 females), but Bmp15-/- females ovulated about two-thirds the number of oocytes as the controls (24.1 ± 3.0 eggs; n = 21 females). To uncover the cause of the reduced ovulations, we collected and analyzed ovaries from some of these superovulated mice. Whereas it was extremely difficult to find any oocytes inside follicles after the PMSG and hCG treatment in the case of the wild-type or Bmp15+/- females (Fig. 2EGo), Bmp15-/- ovaries occasionally had denuded oocytes that were larger than normal and had few cumulus cells surrounding the oocytes (Fig. 2FGo). These findings suggested that follicular recruitment and the number of preovulatory follicles were likely normal in the Bmp15-/- mice but that a percentage of the oocytes were trapped within follicles and were not released. A similar phenotype is also seen for the EP2 PGE2 receptor knockout mice (22) whereas an absolute failure to ovulate is seen with cyclooxygenase 2 and progesterone receptor knockout mice (7, 8). In addition to these ovulation defects, Bmp15-/- mice also demonstrated a reduction of oocytes that could develop to embryos although Bmp15+/- oocytes also appeared to have some reduction compared with wild-type mice (Table 2Go; see below).


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Table 2. In Vivo "Fertilization" Data from Wild-Type and Bmp15 and Gdf9 Mutant Mice

 
Analysis of Gdf9 and Bmp15 mRNA in Mutant Mice
One possibility for the defects in the Gdf9 knockout mice is that there is altered regulation of Bmp15 mRNA whereas the less severe phenotype of the Bmp15 knockout mice could be due to increased compensation of GDF-9. To determine whether there were changes in the transcriptional regulation of Bmp15 mRNA in Gdf9 knockout ovaries or vice versa, we performed Northern blot analysis of RNA derived from control or mutant ovaries. Gdf9 mRNA expression was identical in control and Bmp15-/- ovaries (Fig. 3Go). In contrast, Bmp15 mRNA levels were higher in the Gdf9-/- ovary samples compared with control ovaries (Fig. 3Go). This relative increase in Bmp15 mRNA was due to a dramatic increase in oocytes/unit volume in the Gdf9-/- ovaries (5). We have confirmed this by showing that four other oocyte-specific genes also show a relative increase in mRNA levels in the Gdf9-/- ovaries compared with wild-type ovaries (C. Yang, P. Wang, and M. M. Matzuk, unpublished data). Thus, the abnormalities in the Gdf9 knockout ovaries are not due to transcriptional inactivation of the Bmp15 gene nor is there a compensatory increase of Gdf9 mRNA in the Bmp15 knockout ovaries.



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Figure 3. Northern Blot Analysis of Gdf9 and Bmp15 in Control and Mutant Ovaries

Analysis of the expression of Bmp15 mRNA in Gdf9-/- ovaries and controls (top left). Analysis of the expression of Gdf9 mRNA in Bmp15-/- ovaries and controls (top right). Each blot was subsequently analyzed for expression of GAPDH mRNA as a control for RNA loading and integrity (bottom panels).

 
Characterization of Bmp15-/-Gdf9+/- Double Mutant Mice
To study possible genetic, physical, and functional interactions between Bmp15 and Gdf9, we intercrossed mice carrying the Gdf9 null mutation (5) with the Bmp15 null mutants generated herein. Initial studies focused on comparing the breeding of female mice homozygous for the Bmp15 mutation and heterozygous for the Gdf9 mutation (Bmp15-/-Gdf9+/-) to either Gdf9+/- or Bmp15-/- or Bmp15+/- mutant mice. Whereas Gdf9+/- mice appear to produce more offspring per litter than Bmp15+/- mice, similar litters per month were seen (Table 1Go). In contrast, the Bmp15-/-Gdf9+/- females on a C57/129 background that gave birth produced even fewer pups per litter (3.67 ± 0.22) compared with the Bmp15-/- females (4.93 ± 0.25). Furthermore, the number of litters per month was further reduced to approximately half of the controls, and four females were completely infertile. We also saw an increased incidence of death in these breeding Bmp15-/-Gdf9+/- females (4 of 21 breeding females) but not in Bmp15-/-Gdf9+/- females that were caged without males; we attribute these deaths to a failure to deliver singlets and subsequent intrauterine infection. Recent breeding of 129 inbred Bmp15-/-Gdf9+/- females over a 6-month period failed to produce any offspring in contrast to the breeding of 129 inbred Bmp15+/-Gdf9+/- females over a 4- to 6-month period, which yielded 64 litters (0.73 litters/month) with an average litter size of 4.39 (Table 1Go). These findings for the 129 inbred Bmp15-/-Gdf9+/- females are even more dramatic than the Bmp15-/-Gdf9+/- hybrid strain data and 129 inbred Bmp15-/- female mice that breed poorly. Thus, by reducing the dosage of these two related oocyte-expressed proteins, we have uncovered important genetic interactions.

To further understand the subfertility defects of the Bmp15-/-Gdf9+/- females, we analyzed the ovaries histologically. Similar to the Bmp15-/- ovaries, normal folliculogenesis and corpora lutea could be observed in a minority of Bmp15-/-Gdf9+/- ovaries up through 1 yr (Fig. 4Go, A and F). However, abnormalities were observed in five of nine ovaries from 6- to 7-month-old mice, three of five 9-month-old mice, and 12 of fourteen 11- to 12-month-old mice. These abnormalities included decreased numbers of late-stage follicles, increased oocyte loss, and increased ZP remnants, accumulation of periodic acid Schiff (PAS)-positive material in the interstitium, follicles with multiple oocytes, and absence of corpora lutea (Fig. 4Go, B and C). This progressed to the point where there were very few oocytes and follicles in some ovaries by 1 yr of age (Fig. 4GGo). Thus, these findings suggest that BMP-15 and GDF-9 play synergistic roles in oocyte survival and folliculogenesis.



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Figure 4. Histological Analysis of Bmp15-/-Gdf9+/- Ovaries and Gdf9+/- Control Ovary

A–F, Bmp15-/-Gdf9+/- ovaries at 6 months (A–C), 4 months (D and E), 1 yr (F), and 10 months (G). Normal folliculogenesis and corpora lutea (CL) are seen in two of these ovaries (A and F). Abnormalities in folliculogenesis, a decrease in number of corpora lutea, interstitial cell proliferation (I), increased oocyte loss, and multiple ZP remnants (arrows) are seen in the other sections from independent mice (B, C, and G). Follicles with multiple oocytes are also seen (D and E). In panel D, two small oocytes are seen (top right) as well as two other oocytes of differing sizes (bottom left) surrounded by a complete layer of granulosa cells and basement membrane. In panel E, three similar size oocytes are surrounded by a single granulosa cell layer. H and I, Treatment of Gdf9+/- (H) and Bmp15-/-Gdf9+/- (I) immature mice with PMSG (48 h) and hCG (8 h) revealed normal cumulus expansion of the granulosa cells in the control (H, arrow) but an absence of cumulus cells around a large oocyte (arrow) in the Bmp15-/-Gdf9+/- mutant (I). Sections A–G were stained with PAS/hematoxylin. Sections H and I were stained with toluidine blue. For the scale bars, the red lines represent 50 µm, the white lines represent 100 µm, and the black lines represent 200 µm. The diameter of the "fixed" oocyte in panel I is 65 µm.

 
To follow up on some of the above findings, we performed immunohistological analysis of the ovaries that contained prominent PAS-positive (magenta-colored) structures, which resembled ZP after oocyte degeneration. These structures are a prominent finding in the GDF-9-deficient mice. Ovaries from five independent mice presented in Figs. 4Go and 7Go were analyzed using antibodies specific for ZP protein 1 (ZP1), 2 (ZP2), or 3 (ZP3) or a nonspecific antibody. Antibodies to ZP1, ZP2, or ZP3 stained the ZP around intact oocytes and also detected the prominent remnants centrally (Fig. 5Go, A–C), confirming that these are remnants of ZP after the oocyte had disappeared. The nonspecific antibody failed to detect any signal in the ovaries (Fig. 5DGo) confirming the specificity of the anti- ZP antibodies. These ZP remnants appear to be quite stable after oocyte loss (see below).



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Figure 7. Morphological and Histological Analysis of Bmp15-/-Gdf9-/- Ovaries (A–H)

A, Morphological analysis of large bilateral cysts (1 cm and 1.7 cm) attached to the uterine horns of a 1-yr-old Bmp15-/-Gdf9-/- mouse. B, Ovary from a 5-month-old mouse showing primary (type 3) follicles around the periphery and follicular nests that resemble small corpora lutea (arrows) in the center. The phenotype of this ovary resembles the findings seen in Gdf9-/- ovaries (5 12 ). C, Ovary from a 4-month-old mouse showing increased magenta-colored ZP remnants (arrows) throughout the center, a sign of increased oocyte turnover. D, Ovary from a 9-month-old showing few oocytes and a further accumulation of ZP remnants (arrows). E, Ovary from a 9-month-old mouse showing a central cyst, few oocytes, and increased ZP remnants. F, Ovary from a 1-yr-old mouse showing absence of oocytes. G and H, Low power (G) and high power (H) views of an ovary from a 4-month-old mouse showing multiple follicles (arrows) that resemble seminiferous tubules (T) with Sertoli-like cells. There is also interstitial (I) cell proliferation. All ovaries are derived from C57/129 hybrid mice. For the scale bars, the red line represents 50 µm, and the black lines represent 200 µm.

 


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Figure 5. Immunohistological Analysis of ZP Proteins

The Bmp15-/-Gdf9+/- ovary shown in Fig. 4BGo was analyzed with polyclonal antibodies to ZP1 (A), ZP2 (B), ZP3 (C), and a nonspecific (control) antibody (D). Both ZP around intact oocytes around the periphery and the ZP remnants centrally after oocyte loss are detected (dark staining) with all three antibodies but not the nonspecific antibody. The sections were counterstained with hematoxylin. The black scale bars represent 200 µm.

 
It was also a surprise to find follicles that contained multiple oocytes (Fig. 4Go, D and E). To quantitatively study these findings, we analyzed multiple adjacent sections (8–10 sections per pair of ovaries) of 129SvEv inbred ovaries from Bmp15-/-Gdf9+/- vs. Bmp15+/-Gdf9+/- mice (littermate controls). Whereas only two of eight Bmp15+/-Gdf9+/- mice contained follicles with multiple oocytes (two follicles total), four of eight Bmp15-/-Gdf9+/- mice contained multiple oocytes (eight follicles total). Interestingly, two Bmp15-/-Gdf9+/- mice and one Bmp15+/-Gdf9+/- mouse had follicles with three oocytes in the follicle (Fig. 4EGo). These findings are rare for wild-type inbred 129SvEv mice. Thus, the dosage of these ligands appears to somehow alter the development of the granulosa cell layers around individual oocytes, allowing the formation of these double and triple oocyte follicles.

Since folliculogenesis was relatively normal in the Bmp15-/-Gdf9+/- ovaries at early time points, we analyzed the ability of the Bmp15-/-Gdf9+/- oocytes to be pharmacologically released and fertilized in vivo. Whereas the Bmp15-/-Gdf9+/- mice had high numbers of oocytes released (Table 2Go), only 13.8% of these oocytes developed to embryos. Analysis of eggs from Bmp15-/-Gdf9+/- females that were subjected to PMSG/hCG treatment but were not mated with males revealed the likely cause of the Bmp15-/-Gdf9+/- defects. Normally, cumulus cell-egg complexes from wild-type and Gdf9+/- mice demonstrate a resilient adhesion of cumulus cells and eggs upon removal of the complexes from the oviduct (Fig. 6AGo). In contrast, cumulus cells fail to adhere to eggs isolated from the oviducts of Bmp15-/-Gdf9+/- mutant mice (Fig. 6BGo). A similar finding was also seen for many of the cumulus cell-egg complexes isolated from Bmp15-/- mice. Furthermore, treatment of immature Bmp15-/-Gdf9+/- mice with PMSG for 48 h and subsequent analysis of their ovaries 8 h after hCG injection revealed the presence of some follicles in which cumulus expansion had not occurred or examples of large denuded oocytes (oocytes that were completely lacking cumulus cells) (Fig. 4IGo). This finding was in contrast to Gdf9+/- mice (Fig. 4HGo). Analysis of individual sections of ovaries from wild-type or Gdf9+/- ovaries revealed 25 preovulatory follicles in which cumulus expansion appeared normal and 29 oocytes of antral follicles that were appropriate in size (11). However, analysis of Bmp15-/- or Bmp15-/-Gdf9+/- ovaries revealed 11 of 19 antral follicles where absence of cumulus expansion had occurred or where oocytes were larger than normal in size. In addition to these findings, there was one case in which the cumulus granulosa cells were seen invading the ZP. These findings were not unique to pharmacologically treated mice but were also observed in seven of ten 11- to 12-month-old Bmp15-/-Gdf9+/- mice in which denuded oocytes in antral follicles (total of seven oocytes with cumulus cell defect) and oocytes trapped in corpora lutea (total of eight trapped oocytes) were observed. Six of six Bmp15+/- mice of the same age failed to demonstrate these defects. We believe that these findings are part of the variation in the cumulus cell adhesion/oocyte-cumulus cell interaction phenotype. Thus, these studies demonstrate that BMP-15 and GDF-9 play functionally redundant roles in cumulus expansion and maintenance of a cohesive interaction between cumulus cells and oocytes or eggs that influences subsequent fertility.



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Figure 6. Eggs Isolated from Gdf9+/- (A) and Bmp15-/-Gdf9+/- (B) Mutant Mice

Eggs isolated from the oviducts of Gdf9+/- females after PMSG and hCG stimulation were embedded in a resilient three-dimensional extracellular matrix which contained cumulus cells (A). In contrast, the cumulus cells of Bmp15-/-Gdf9+/- mice were loosely attached to the oocyte and readily fell off the oocyte (B). Eggs were isolated from C57/129 hybrid strain mice.

 
Production of Mice Null for Both Bmp15 and Gdf9
One possible reason for the early block in folliculogenesis in the Gdf9 knockout mice could be due to persistent (unopposed) BMP-15 protein levels since the Bmp15 mRNA continued to be expressed in these mice. Thus, to ensure that the Gdf9 knockout phenotype was due to absence of GDF-9 and not secondary to unopposed BMP-15, mice lacking both BMP-15 and GDF-9 (i.e. Bmp15-/-Gdf9-/-) mice were generated. At early time points, the ovaries of these Bmp15-/-Gdf9-/- mice resembled the ovaries of Gdf9-/- mice and demonstrated a block at the type 3B primary (one-layer) follicle stage (Fig. 7BGo). Furthermore, similar to Gdf9-/- mice, unilateral and bilateral cysts were grossly (Fig. 7AGo) and microscopically (Fig. 7EGo) present. Only one cyst per ovary was present in these double mutant mice as in the Gdf9-/- mice. At later time points, there were a number of unique abnormalities found in the Bmp15-/-Gdf9-/- ovaries. These abnormalities included increased loss of oocytes, increased ZP remnants, complete absence of oocytes, and proliferation of cells in the interstitium (sometimes PAS-positive) (Fig. 7Go, C–F), and a rare mouse that displayed a transformation of the granulosa cells into Sertoli-like cells (Fig. 7Go, G and H) similar to mice lacking inhibin {alpha} (23) or estrogen receptors {alpha} and ß (24). Interestingly, accumulation of ZP remnants are seen after loss of oocytes in the Gdf9-/- ovaries (5) but are not seen after loss of oocytes in animal models that have blocks at later stages such as FSHß, cyclooxygenase 2, or progesterone receptor knockouts (6, 7, 8). It is not clear whether there are altered proteolytic processing defects in the ovaries of mice that have blocks at the primary follicle stage vs. blocks at later stages. Thus, unopposed BMP-15 is not the cause of the Gdf9 knockout phenotype but BMP-15 appears to play some additional roles along with GDF-9 in oocyte survival.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have used knockout mouse technology and genetic intercrosses to define the functions of BMP-15 and its interactions with GDF-9. Our studies have uncovered periovulatory functions of BMP-15 in females and confirm our in vitro studies (13, 15, 16) by showing that GDF-9 also plays a key role in this period. We have demonstrated that hybrid and inbred Bmp15 knockout mice are subfertile due to reduced ovulation and fertilization; Bmp15-/-Gdf9+/- mice demonstrate further defects in early follicle development compared with Bmp15-/- or Gdf9+/- mice. Previous studies had demonstrated an important role of oocyte-secreted proteins in cumulus expansion, hyaluronic acid synthesis signaling through the PGE2 receptor EP2, and suppression of urokinase plasminogen activator and LH receptor (25). Furthermore, cumulus-oocyte complexes are known to synthesize progesterone and PGE2. Using recombinant GDF-9, we have shown that GDF-9 can perform all of the functions of the oocyte-secreted protein(s) and also stimulate PGE2 through an induction of cyclooxygenase 2 in cumulus cells and progesterone via induction of PGE2 and the EP2 receptor. Our in vivo studies confirm these in vitro analyses; oocytes from Bmp15-/-Gdf9+/- fail to demonstrate a stable "adherence" of cumulus cells likely due to a reduction of key periovulatory factors (e.g. hyaluronic acid and PGE2). Bmp15-/-Gdf9+/- mice also have some of the features of the progesterone receptor and cyclooxygenase 2 knockout mice [i.e. failure to release oocytes and the presence of oocytes trapped in corpora lutea (7, 8)], further confirming the in vivo relationship of these factors. Since Gdf9+/- mice do not demonstrate these defects and some Bmp15-/- mice demonstrate some of these defects, these findings also indicate an important role of BMP-15 in at least some of these processes.

Gdf9-/- and Bmp15-/-Gdf9-/- mice develop unilateral and bilateral cysts with high frequency (Ref. 5 and the current study). These cysts can become very large (Fig. 7Go), and analysis of the cystic ovaries of these mice or after regression of the cysts demonstrates a dramatic reduction in the number of oocytes (i.e. the cysts lead to decreased oocyte survival). We believe that these findings in our knockout mice have important implications in humans. Polycystic ovarian syndrome (PCOS) is a major cause of reduced fertility in women, and our findings suggest that the presence of cysts in the ovaries of these women could likewise lead to increased oocyte loss through direct structural destruction or via indirect growth factor/hormonal effects.

Unlike Bmp15+/- mice, BMP15 heterozygous mutant sheep demonstrate increased fertility (i.e., increased twins and triplets), suggesting that the BMP-15 propeptide sequences (present as the sheep mutations are in the mature peptide encoding region) are acting in a dominant negative fashion. This BMP-15 propeptide may be somehow interfering with GDF-9 homodimer, BMP-15 homodimer, and/or GDF-9/BMP-15 heterodimer formation. For example, the BMP-15 propeptide may preferentially bind to a wild-type GDF-9 propeptide monomer to cause decreased BMP-15/GDF-9 heterodimers and shift the equilibrium toward increased BMP-15 homodimers.

Interestingly, sheep homozygous for null mutations in the BMP15 gene do not phenocopy Bmp15 knockout mice but instead resemble Gdf9-/- mice (i.e. homozygotes are infertile due to a block at the primary follicle stage). How might one explain these findings? Based on the available animal models and in vitro studies, two different models could be evoked to explain the functions of GDF-9 and BMP-15 in sheep vs. mice (Fig. 8Go). In model A (the mouse model), GDF-9 homodimers would be the most bioactive and play the major function. This model is based on our findings from Gdf9-/- mice (5, 11, 12), which display an early block in folliculogenesis, and also on the present study on the Bmp15-/- mice (which have a defect in late folliculogenesis and ovulation). Furthermore, mouse GDF-9 homodimers, but not mouse BMP-15 homodimers, are active in our mouse in vitro bioassays (13, 16). A similar situation has been shown recently by our group in the case of the activin ßA and ßB monomers (26); using a gene "knockin" strategy, we demonstrated that activin ßB (which shows 63% amino acid identity in the mature peptide sequence) can replace activin ßA for some, but not all, functions, demonstrating that ßB is less bioactive than ßA. However, in model B (the sheep model), BMP-15 homodimers would be postulated to be the most bioactive compared with either GDF-9 homodimers or BMP-15/GDF-9 heterodimers. This model would explain how the sheep BMP15 homozygous mutant phenotype mimics the Gdf9-/- mouse ovarian phenotype. Interestingly, mouse and sheep GDF-9 proteins are highly conserved whereas mouse and sheep BMP-15 proteins have diverged greatly (78% amino acid identity). Thus, it is possible that this protein divergence has altered the biopotency of these proteins, allowing BMP15 to become the more essential protein in sheep. Although these models assume signaling of the ligands through the same receptor, we cannot rule out evolutionary divergence of a common GDF-9 or BMP-15 receptor (or multiple receptors) to permit higher affinity interaction of the sheep receptor with BMP-15 homodimers to explain the in vivo findings. However, recent studies demonstrate that recombinant human BMP-15 stimulates in vitro rat granulosa cell proliferation and decreases FSH-induced progesterone production (27). This finding suggests that the BMP-15 receptors are conserved between species. Future structure-function and receptor binding studies should help us clarify the active forms of these ligands in the different mammalian species and determine whether BMP-15, GDF-9, or both are essential for human fertility.



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Figure 8. Models for the Differential Functions of BMP-15 and GDF-9 in Mammals

A, In mice, GDF-9 homodimers appear to be the major signaling protein, whereas BMP-15 homodimers and BMP-15/GDF-9 heterodimers appear to play synergistic roles in the ovary. B, In sheep, BMP-15 homodimers play a major role although we cannot rule out significant roles of GDF-9 homodimers or BMP-15/GDF-9 heterodimers. In sheep, a model in which BMP-15/GDF-9 heterodimers are the active form could also explain the findings. We have shown that mouse and human BMP-15/GDF-9 heterodimers can form in vitro (N. Wolfman, unpublished data; P. Wang and M. M. Matzuk, unpublished data). Other members of the TGFß superfamily can also form heterodimers that are more potent than the respective homodimers [e.g. heterodimers between the BMP-2/4 subgroup and the BMP-5/6/7 subgroup (36 37 38 )].

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ES Cell Technology and Southern Blot Analysis
More than 20 kb of genomic sequence encompassing the two-exon mouse Bmp15 gene were isolated from a 129SvEv genomic library (19). These genomic sequences were used to construct a targeting vector to mutate the Bmp15 gene in ES cells. The targeting vector contained 2.6 kb of Bmp15 intron 1 sequence, a positive selectable marker (the PGK-hprt expression cassette), 5.0 kb of sequence 3' to the coding portion of exon 2, and a negative selectable marker [the MC1-tk (thymidine kinase) expression cassette (Fig. 1AGo)]. Linearized vector was electroporated into the hprt-negative AB2.1 ES cell line, cell clones were selected in HAT (hypoxanthine, aminopterine, and thymidine) and FIAU [1-(2'- deoxy-2'fluoro-ß-D-arabinofuranosyl)-5-iodouracil], and DNA from the clones analyzed by Southern blot and targeted ES cell clones were expanded and injected into blastocysts as described previously (23, 28, 29). Fourteen percent of the ES cell clones were targeted at the Bmp15 locus (data not shown), and two of these ES cell clones (Bmp15-79-D7 and Bmp15-79-F7), which were injected into blastocysts, produced male chimeras that successfully transmitted the mutant Bmp15tm1Zuk allele to F1 female offspring. F1 and F2 female heterozygotes and F2 null male offspring were intercrossed to produce Bmp15 null homozygotes. Chimeras were either bred to C57BL6/J females to produce 129SvEv/C57BL6/J hybrid mice or to 129SvEv females to produce 129SvEv inbred mice. Southern blot analysis was used for genotype analysis of all Bmp15 mutant offspring as shown (Fig. 1BGo) and all Gdf9 mutant offspring as described (5).

Breeding Experiments
Bmp15 heterozygous and homozygous mutant females from both hybrid (129SvEv/C57BL6J) and inbred (129SvEv) genetic backgrounds were bred to males of the same genetic backgrounds at 6 weeks of age and breeding was continued for up to 1 yr. To generate the Bmp15/Gdf9 double mutant mice, mice carrying the Gdf9tm1Zuk mutation (5) on either hybrid or 129SvEv inbred genetic backgrounds were bred to Bmp15 mutant mice of similar genetic backgrounds. Breeding of the double mutants was also initiated at 6 weeks of age.

Immunohistological Analysis
Immunohistological analysis was carried out using epitope-selected antibodies purified from antisera of rabbits immunized with porcine ZP proteins as previously described (30, 31). To select ZP1-, 2-, and 3-specific antibodies that would recognize mouse ZP proteins, we used an epitope selection method to enhance for cross-species ZP epitopes. Antibodies recognizing all three porcine ZP proteins were epitope selected using human ZP proteins made from cDNAs expressed using the baculovirus expression system (32, 33). Briefly, antibodies were incubated with each of the three human ZP proteins that had been isolated from SF9 insect cell lines. The individual proteins were transferred to polyvinylidenefluoride (PVDF) membrane and incubated with antiserum. Nonspecific antibodies were washed from the membrane and ZP-specific antibodies were eluted with 200 mM glycine buffer, pH 2.7, which was neutralized to pH 7. Antibody specificity to each of the ZP proteins was demonstrated by SDS-PAGE and immunoblot analysis as previously described (32, 34, 35).

Other Methods
RNA isolation, Northern blot analysis, histological analysis, pharmacological superovulation, and statistical methods were performed as described previously (5, 6, 12, 13, 19) and have been described briefly in the body of the text or the figure legends.


    ACKNOWLEDGMENTS
 
We thank Ms. Shirley Baker for aid in manuscript preparation and Dr. Hua Chang for help with the figures.


    FOOTNOTES
 
Address requests for reprints to: Martin M. Matzuk, M.D., Ph.D., Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030. E-mail: mmatzuk{at}bcm.tmc.edu

These studies were supported in part by the NIH Specialized Cooperative Centers Program in Reproduction Research (Grant HD-07495) and NIH Grant HD-33438 (to M.M.M.).

Received for publication January 5, 2001. Revision received February 23, 2001. Accepted for publication March 16, 2001.


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