BMP‐15 regulation of ovulation quota in mammals

Abstract

Naturally occurring mutations in the oocyte‐specific factor, bone morphogenetic protein‐15 (BMP‐15), cause infertility in women and in ewes. In contrast to these monoovulatory mammals, the targeted deletion of BMP‐15 in polyovulatory mice results in subfertility with only minimal defects in the ovulation process. Given the established role of BMP‐15 in governing the progression of folliculogenesis, it is hypothesized that species‐specific differences in the BMP‐15 system are involved in species‐specific determination of ovulation quota and litter size. Recent data using in vitro cell transfection methodology indicate that, in contrast to human BMP‐15 which is successfully processed and secreted, the mouse BMP‐15 proprotein is resistant to proteolytic cleavage. Thus, no functional mature BMP‐15 is secreted in vitro. Further studies have shown that the functional mature form of BMP‐15 is barely detectable in mouse oocytes in vivo until just before ovulation, when it is markedly increased. The general hypothesis to emerge from these observations is that the species‐specific differences in the defects caused by mutations in the bmp15 gene between monoovulatory ewes and women and polyovulatory mice might be attributed to the timing of the production of BMP‐15 mature protein. (Reprod Med Biol 2006; 5: 245–248)

SPECIES‐SPECIFIC DIFFERENCES IN THE OVARIAN PHENOTYPE IN GENETIC MUTATIONS OF THE BMP15 GENE

A MAJOR BREAKTHROUGH in this field occurred when it was found that two naturally occurring strains of sheep called Inverdale and Hanna, exhibiting higher ovulation rates and litter sizes than their wild type counterparts, are heterozygous carriers of point mutations in the oocyte‐specific factor, BMP‐15. ¹ Specifically, the Inverdale mutation (FecX^I) is a T‐A transversion at nucleotide 92 of the bmp15 gene which substitutes a valine with an aspartic acid residue at position 31 of the mature protein. In Hanna ewes (FecX^H), a C‐T transition at nucleotide 67 of the bmp15 gene replaces a glutamic acid by a stop codon at amino acid residue 23 of the mature domain of BMP‐15, thus resulting in the synthesis of a very short peptide that is highly unlikely to be biologically active. Surprisingly, homozygous carriers of the Inverdale and Hanna BMP‐15 mutations are infertile with streak ovaries and a block in the primary stage of folliculogenesis.

The importance of BMP‐15 in sheep fertility was further confirmed by subsequent studies that identified three additional naturally occurring BMP‐15 point mutations, FecX^G, FecX^B and FecX^L that resulted in the same phenotype as the Inverdale and Hanna ewes. ² , ³ Furthermore, McNatty et al. were able to mimic both the infertile and superfertile phenotypes of the BMP‐15 mutant ewes by immunizing wild‐type ewes against BMP‐15 using various immunization protocols. ⁴ , ⁵ Collectively, these findings suggest that, in sheep, BMP‐15 plays an important role in promoting the transition of follicles through the early stages of folliculogenesis, while restraining the transition of follicles to the dominant preovulatory stage. Thus, BMP‐15 is a central player in the determination of ovulation quota and litter size in ewes. ⁶

Critical roles of BMP‐15 in female fertility have also been shown in humans. Specifically, Di Pasquale et al. identified a BMP‐15 mutation in women that is associated with hypergonadotropic ovarian failure as a result of ovarian dysgenesis. ⁷ The mutation is an A‐G transition at position 704 of the bmp15 gene and results in a non‐conserved substitution of a tyrosine with a cysteine at amino acid residue 235 of the proregion of the BMP‐15 proprotein. The patients with the BMP‐15 mutation were two sisters who inherited the mutation from their father. Interestingly, both patients had streak ovaries: a characteristic phenotype observed in the homozygous, but not heterozygous, BMP‐15 mutant ewes.

In contrast to sheep and humans, in which it is clear that BMP‐15 is critical for normal folliculogenesis, the BMP‐15 system and possibly the physiological importance of BMP‐15 in folliculogenesis appears to be quite different in mice. Specifically, the targeted deletion of the entire second exon of the bmp15 gene in mice results in only subfertility in homozygotes and there is no clear aberrant phenotype in heterozygotes. ⁸ Examination of the ovaries of homozygous BMP‐15 knockout mice showed that, unlike the BMP‐15 mutant sheep and women carrying a BMP‐15 mutation, the lack of bioactive BMP‐15 did not prevent follicles from progressing through the component stages of folliculogenesis in mice. The subfertility observed in the BMP‐15 knockout mice was attributed to defects in ovulation, resulting in follicles that have trapped oocytes that were not released from the follicles. In addition to the defects in ovulation, there was a reduction in the ability of oocytes from BMP‐15 knockout mice to develop into viable embryos. Collectively, these data show that there are species‐specific differences in the BMP‐15 system of monoovulatory sheep and humans as compared with polyovulatory mice.

ROLE OF BMP‐15 IN DETERMINING OVULATION QUOTA: A WORKING HYPOTHESIS

TRADITIONALLY, ANIMAL MODELS used for biomedical and biological research have been chosen and developed, in a large part, based on their similarities to the human system to be studied. The focus on genes and physiological systems that are highly conserved between humans and animal models is beneficial because inherently, physiological systems that are critical for the survival of a species tend to be highly conserved. However, because there are striking differences between humans and the common mammalian animal models (e.g. mice and rats) with respect to fundamental aspects of ovarian physiology, especially the mechanisms regulating ovulation quota, investigating the specific differences between the animal models and the corresponding human system might provide key insights into the physiological regulation of these poorly understood processes.

Recent research has introduced a novel concept that the oocyte plays an important, if not predominant, role in orchestrating the organization, development and function of the somatic cells of the follicle. ⁹ , ¹⁰ Accordingly, the concept that oocytes, through oocyte‐secreted factors, are actively involved in the promotion of follicular development has provided new opportunities to understand the species‐specific mechanisms governing the regulation of ovulation quota. BMP‐15 is a strong candidate molecule, having the potential to fulfil many of the requirements expected of such oocyte secreted factors. ⁶ , ¹¹

Among members of the TGF‐β superfamily, the nucleotide and amino acid sequences tend to be highly conserved between different species. For example, the amino acid sequences of the mature domain of mouse and human BMP‐2 are absolutely conserved, as are the sequences of mouse and human BMP‐4. ¹² GDF‐9 is consistent with this trend of the TGF‐β superfamily, with the amino acid sequence of the mature region of mouse and human GDF‐9 being 96% identical. ¹² In contrast, BMP‐15 does not exhibit a similar degree of cross‐species homology. At the amino acid level, mouse and human BMP‐15 mature domains share only 70% identity. ¹² The significance or consequences of the relatively low cross‐species conservation of the BMP‐15 amino acid sequence has not been directly resolved, however, we hypothesize that the divergence in the sequence of BMP‐15 might be associated with species‐specific variations in ovulation quota between mice and humans.

Our laboratory has accumulated an extensive body of data using recombinant human BMP‐15 that establishes important biological roles for this oocyte‐secreted factor in regulating ovarian function. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ Namely, we have shown that BMP‐15 acts to promote early follicle growth by stimulating granulosa cell mitosis ¹³ and kit ligand expression, ¹⁶ and also restrains follicle‐stimulating hormone (FSH)‐induced granulosa cell differentiation by suppressing the expression of FSH receptor mRNA. ¹⁴ Furthermore, BMP‐15 induces cumulus expansion in cumulus‐oocyte complexes. ²¹ We have also published results on the biochemical characteristics of recombinant human BMP‐15 including its production, processing and interactions with GDF‐9 as well as the biological effects of specific point mutations on granulosa cell function. ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ In contrast to human BMP‐15, there is almost nothing known about the mouse BMP‐15 protein function. Recently, we discovered that unlike recombinant human BMP‐15, the mature form of mouse BMP‐15 is not produced in an in vitro transfection system. ²² We showed that, in contrast to human BMP‐15 which is successfully processed and secreted, the mouse BMP‐15 proprotein is resistant to proteolytic cleavage. ²² Thus, no functional mature BMP‐15 is secreted in vitro. Furthemore, our recent studies indicated that the functional BMP‐15 mature protein is barely detectable in the mouse oocytes except for those in the preovulatory follicles following luteinizing hormone (LH)‐induced meiotic maturation. ²¹ This finding is supported by the phenotype of the BMP‐15 null mice, in which there are no defects in follicular development and yet decreased ovulation rates are observed as a result of defects in the ovulation process. ⁸

Collectively, based on the established capacity of BMP‐15 to regulate critical aspects of female reproductive physiology, ¹³ , ¹⁴ , ¹⁶ , ¹⁷ it is hypothesized that evolutionary divergence in the sequence of BMP‐15 of polyovulatory mice compared with BMP‐15 of monoovulatory humans might contribute to the differences in the ovulation quota and litter sizes of these mammals. Based on our data, together with the genetic data observed in the mutant sheep models, it is likely that in monoovulatory animals BMP‐15 is important for restricting the formation of dominant follicles through the suppression of FSH receptor expression, leading to the tight regulation of the number of ovulated ova. ¹ , ¹³ , ¹⁴ We propose the hypothesis that in polyovulatory species such as the mouse, BMP‐15 fails to restrict dominant follicle formation, allowing for the increased ovulation quota and litter sizes. Furthermore, we propose that the mechanism involves the availability of bioactive BMP‐15. Specifically, the production of functional mature BMP‐15 is highly controlled in the mouse oocytes in a timely and developmentally regulated manner after the stimulation of the ovulatory sequence by an LH surge. ²¹ Therefore, we hypothesize that the species‐specific differences in the defects caused by mutations in the bmp15 gene between monoovulatory ewes and women and polyovulatory mice might be attributed to the timing of processing of the BMP‐15 proprotein into the functional mature BMP‐15.

A clear difference in the ovarian activity between women and mice is evidenced by the fact that women generally ovulate one oocyte per menstrual cycle resulting in predominantly singleton pregnancies, whereas mice ovulate multiple oocytes and carry large litters. To date, the physiological systems that regulate ovulation quota remain largely unknown. The concept that oocytes play a critical role in governing folliculogenesis has provided a novel avenue for understanding the species‐specific regulation of ovulation quota. BMP‐15 has been shown to be a critical regulator of follicle development and ovulation. Our challenge is to determine whether species‐specific differences in the BMP‐15 system might be causal to the species‐specific differences in ovulation quota.