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Endocrinology Vol. 148, No. 1 393-400
Copyright © 2007 by The Endocrine Society

A Novel Mutation in the Bone Morphogenetic Protein 15 Gene Causing Defective Protein Secretion Is Associated with Both Increased Ovulation Rate and Sterility in Lacaune Sheep

Loys Bodin1, Elisa Di Pasquale1, Stéphane Fabre, Martine Bontoux, Philippe Monget, Luca Persani and Philippe Mulsant

Institut National de la Recherche Agronomique, UR631, Station d’Amélioration Génétique des Animaux (L.B.), and UR444, Laboratoire de Génétique Cellulaire (P.Mu.), INRA, F-31326 Castanet-Tolosan, France; Department of Medical Sciences (E.D.P., L.P.), Laboratory of Experimental Endocrinology, Istituto Auxologico Italiano, Istituto di Ricovero e Cura a Carattere Scientifico, University of Milan, 20095 Cusano Milanino, Italy; and Institut National de la Recherche Agronomique, UMR6175, Physiologie de la Reproduction et des Comportements (S.F., M.B., P.Mo.), INRA, F-37380 Nouzilly, France

Address all correspondence and requests for reprints to: Loys Bodin, Institut National de la Recherche Agronomique, Station d’Amélioration Génétique des Animaux, BP 52627, 31326 Castanet-Tolosan, France. E-mail: loys.bodin{at}toulouse.inra.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Genetic mutations with major effects on ovulation rate and litter size in sheep were recently identified in three genes belonging to the TGFß superfamily pathway: the bone morphogenetic protein 15 (BMP15, also known as GDF9b), growth differentiation factor 9 (GDF9), and BMP receptor type IB (also known as activin-like kinase 6). Homozygous BMP15 or GDF9 mutations raise female sterility due to a failure of normal ovarian follicle development, whereas heterozygous animals for BMP15 or GDF9 as well as heterozygous and homozygous animals for BMP receptor type IB show increased ovulation rates. In the present work, a new naturally occurring mutation in the BMP15 gene in the high prolific Lacaune sheep breed is described. The identified variant is a C53Y missense nonconservative substitution leading to the aminoacidic change of a cysteine with a tyrosine in the mature peptide of the protein. As for other mutations found in the same gene, this is associated with an increased ovulation rate and sterility in heterozygous and homozygous animals, respectively. Further in vitro studies showed that the C53Y mutation was responsible for the impairment of the maturation process of the BMP15 protein, resulting in a defective secretion of both the precursor and mature peptide. Overall, our findings confirm the essential role of the BMP15 factor in the ovarian folliculogenesis and control of ovulation rate in sheep.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN SHEEP, A large range in litter size has been observed among different breeds and within breeds. In some cases, genetic studies have indicated that the litter size and ovulation rate can be genetically determined by the action of single genes with a major effect, named fecundity (Fec) genes (reviewed in Ref. 1). Three of these Fec genes were recently identified in sheep, namely bone morphogenetic protein receptor type IB (BMPRIB; or activin-like kinase 6), known as FecB on chromosome 6 (2, 3, 4); growth differentiation factor 9 (GDF9), known as FecG on chromosome 5 (5); and bone morphogenetic protein 15 (BMP15) known as FecX on the X chromosome (5, 6). Interestingly, all three Fec genes belong to the TGFß superfamily (reviewed in Ref. 7).

The mutations found on these genes have been shown to be associated with different phenotypic effects: the FecBB (Booroola) mutated allele was identified to be associated with the additive effect on ovulation rate (2, 3, 4), whereas the FecG (one mutated allele) and FecX (four different mutated alleles) mutations led to increased ovulation rates in heterozygous animals and sterility in homozygous animals. In particular, heterozygous ewe carriers of the FecGH (high fertility), FecXI (Inverdale), FecXH (Hanna), FecXB (Belclare), or FecXG (Galway) alleles exhibit one to two additional ovulations, compared with noncarriers, whereas homozygous ewes are sterile due to a blockage of the folliculogenesis process (5, 6, 8). In particular, the FecGH mutation leads to the substitution of a serine with a phenylalanine at position 77 of the mature GDF9 peptide [S77F (5)]; likewise, FecXI and FecXB mutations cause the nonconservative amino acid substitutions at positions 31 and 99, within the BMP15 mature protein, respectively. Interestingly, FecXI and FecXB mutant ewes exhibit the same phenotype as the carriers of FecXG and FecXH mutations that introduce a premature stop codon at positions 239 and 291 of the BMP15 proprotein, obviously impairing the production of the biologically active mature form. Then it was admitted that these mutations induce loss of function in the BMP15 activity, leading to increased ovulation rate or sterility, in a dosage sensitive manner (6). Additionally, in humans, several heterozygous nonconservative substitutions in the proregion of BMP15 are associated with idiopathic hypergonadotropic ovarian failure (9, 10, 11). It is noteworthy that in the mouse, the targeted deletion of the BMP15 gene causes only slight reduction in ovulation rate and litter size (12). Altogether these observations highlight the crucial role of BMP15 in the ovarian function in mammals but with variable incidence among species.

In the Lacaune population, the presence of a major gene was suspected in a flock of ewes presenting repeatedly very high litters. To validate this hypothesis, we searched for mutations in the BMP15, GDF9, and BMPR1B genes already known to be implicated in the control of ovulation rate in sheep. In this work, we describe the phenotypic and molecular characterization of a new C53Y mutation identified in the BMP15 gene in the Lacaune sheep, named FecXL. This mutation was responsible for a dramatic alteration of the intracellular processing of the peptide.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
A small nucleus of extreme hyperprolific ewes was established after extensive screening on a population of about 10,000 Lacaune adult ewes. This screening was based on natural litter size (without hormonal treatment) recorded in the database of the French National Sheep Recording Scheme. From this file, 31 ewes, which had repeatedly high litter size (twice three or four; once five), were bought from a farm and gathered in an Institut National de la Recherche Agronomique experimental farm. Ovulation rate was observed by laparoscopy on several occasions (five to 10). Known mutations in the three candidate genes BMPR1B, GDF9, and BMP15 were checked. A new mutation was discovered in the BMP15 gene on some of these prolific ewes. After mutation discovering, all rams of the Lacaune artificial insemination OVI-TEST center were genotyped, and 12 were found as hemizygous carriers.

To check whether this new mutant allele (FecXL) would be able to induce the same phenotype as the four other BMP15 mutations previously described, ovulation rates were observed on different ewe samples. First, a small group (n = 8) of specific heterozygous ewes was procreated in 2002 by insemination of noncarrier dams with semen of hemizygous carrier sires, and their ovulations were recorded on three consecutive cycles when they were 1 and 2 yr old. Second, the national recording program allowed the identification of daughters of the 12 hemizygous mutated rams, which were inseminated with semen of mutated rams, and their induced ovulations were controlled 6 d after artificial insemination. Sixteen female lambs issued from these inseminations were genotyped after birth and reared on an Institut National de la Recherche Agronomique experimental farm. Ovulation rate was assessed during the breeding season on 1- and 2-yr-old animals by laparoscopy on three successive cycles. And third, four of these ewes that did not ovulate the first year at any of the three successive cycles were killed and the ovaries were collected. All procedures were approved by the Agricultural and Scientific Research Government Committees in accordance with the guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Approval A37801).

Ovine BMP15 gene sequence analysis
A fragment of 312 bp of the BMP15 exon 2 sequence was amplified using primers obtained from the published ovine sequence (accession no. AF236079). Primer sequences were 5'-CATGATGGGCCTGAAAGTAAC (upper) and 5'-GGCAATCATACCCTCATACTCC (lower). Polymorphisms of the amplified fragments were detected by single-strand conformation analysis (SSCA). Amplification and SSCA conditions have been previously described (5). For large-scale genotype analysis on an ABI prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA), the SSCA assay was modified as follows: the amplification was carried out with fluorescent primers (6-Fam for upper and Hex for the lower) and PCR products were separated in 36-cm capillaries using a 5% GSpop polymer and 1% glycerol at 18 C. Polymorphisms were detected in 6-Fam-labeled fragments. Sequence analysis was performed on PCR products with an ABI sequencing kit (Applied Biosystem) and the amplification primers. Sequence reaction products were analyzed on an ABI prism 3700 genetic analyzer.

Histological analysis
Ovaries from noncarrier or homozygous FecXL ewes were fixed in Bouin solution and embedded in paraffin. The paraffin-embedded tissues were sectioned serially at 10 µm, and these sections were stained with Feulgen coloration for light microscopy studies.

Construction of BMP15 expression plasmids
The aminoacidic sequences of sheep and human BMP15 (genifo-identifier: 13123984 and 4885097, respectively) were aligned (BLAST; National Center for Biotechnology Information, Bethesda, MD) to verify the conservation of the cysteine at position 53 in the mature peptide in both species. The C53Y mutation was later introduced into the pCDNAhumanBMP15wt-MycHis vector by site directed mutagenesis, using the high-fidelity Taq DNA polymerase Pfu Turbo (Stratagene, La Jolla, CA) and the following specific oligonucleotides, as previously described (9): upper, 5'-TCTACACCCCAAACTACTATAAAGGAACTTGTCTCCG-3'; lower, 5'-CGGAGACAAGTTCCTTTATAGTAGTTTGGGGTGTAGA-3'.

Positive clones were checked by sequencing and enzymatic digestion with specific restriction endonucleases. Plasmidic DNA was purified using the commercial Hispeed plasmid maxikit (QIAGEN, Valencia, CA) for the following experiments.

Production and analysis of recombinant BMP15 proteins
Human epithelial kidney (HEK) 293T cells were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Euroclone, Pero, Italy), glutamine (Invitrogen), and penicillin/streptomycin (Sigma, St. Louis, MO). The purified plasmidic DNA pCDNABMP15-C53Y-MycHis was stably transfected in the HEK293T cells. A total amount of 15 µg plasmidic DNA and 25 µl Lipofectamine reagent (Invitrogen) were used. Stable clones expressing mutant BMP15C53Y were selected using 600 µg/ml Zeocin. Stable clones expressing the wild-type BMP15 were previously described (9).

Positive clones were checked by cytofluorometric analysis after cell fixation and permeabilization. The detection was performed using the monoclonal anti-Myc antibody (Invitrogen) and the fluorescein isothiocyanate-probed antimouse IgG secondary antibody. Selected clones were maintained in medium containing 10% FBS and Zeocin (400 µg/ml). At 70% confluence, the cells were switched to a medium containing 1% FBS. Total RNA and purified recombinant proteins were then obtained from cells or conditioned media and harvested after 48 h incubation.

The BMP15 recombinant proteins were extracted from either the cell extract (guanidium lysis solution) or the dialyzed conditioned media (Centricon 10,000 Da; Becton, Oxford, UK) using the Probond nickel resin system (Invitrogen). The amount of recovered protein was evaluated by colorimetric assay (BCA kit; Pierce, Rockford, IL). Five micrograms and 20 µg of purified proteins were analyzed by SDS-PAGE or denaturant nonreducing condition Western blot, respectively. In both cases the detection was performed using the ECL Plus detection kit after incubation with the monoclonal anti-Myc antibody (1:5000; Invitrogen) and the horseradish peroxidase-conjugated secondary antibody (antimouse IgG-horseradish peroxidase; Chemicon, Temecula, CA). Purified extracts from the medium of nontransfected HEK293T cells were used as the control.

Real-time PCR
Total RNA was extracted from stable transfected HEK293T cells using the Trizol reagent (Invitrogen). One microgram of total RNA was treated with DNase I amplification grade (Invitrogen), and single-strand cDNA was synthesized using the SuperScript II first-strand cDNA synthesis kit (Invitrogen). BMP15 expression was screened in 11 independent clones. Real-time PCRs were performed using Syber Green (Applied Biosystems), 40 ng of template (cDNAss), and 2 pmol each of the following specific oligonucleotides: upper, 5'-CATGGTGAGGCTGGTGAAG-3', and lower, 5'-TCCTCGGTTTGGTCTGAGAG-3'. Amplification was carried out on an ABI Prism 7900HT apparatus (Applied Biosystems) with the standard program, and a dissociation curve was added at the end of the polymerization. The raw data were analyzed with the SDS 2.1 software (Applied Biosystems), provided with a quantitative relative study ({Delta}{Delta}Ct) using the HEK293T cell sample as the calibrator and comparing the BMP15 transcript levels in mutant and wild-type cell clones. hypoxanthine-guanine phosphoribosyl transferase gene amplification was used for normalization.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mutation identification
The extreme values of litter size and mean ovulation rate (between six and 12.4) observed in ewes of the prolific nucleus of Lacaune sheep could not be explained by the sole variability of polygenic effects that controls ovulation rate. It was thus hypothesized that these animals could carry a mutation increasing their prolificacy. We failed to find the FecBB allele in the BMPR1B gene that was first found in hyperprolific Booroola sheep and that has also been encountered in Garole and Javanese sheep (13). We also searched for the FecGH allele in the GDF9 gene that has been found to be associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (5). However, the FecGH mutation was absent in all of these hyperprolific females. Then we considered the BMP15 gene previously shown to be involved in both a hyperprolific and sterile phenotype in four other prolific breeds (5, 6). SSCA effectively revealed the presence of two genotypes at the BMP15 locus (exon 2) in the Lacaune prolific nucleus, with seven ewes (of 30) heterozygous for a new allele. Sequence analysis showed that the mutation corresponds to a G to A transition, leading to a substitution of the Cys residue (TGT) at position 53 of the mature BMP15 protein with a Tyr (TAT). This new mutated allele of the BMP15/FecX gene in the Lacaune population was named FecXL and its product BMP15C53Y (Fig. 1Go). As shown in Fig. 1BGo, the substitution of this Cys residue was expected to alter the BMP15 mature peptide conformation by disrupting a potential intrachain disulfide bridge in the cystine-knot motif characteristic of TGFß family members (14).


Figure 1
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  		FIG. 1. BMP15 sequence and mutations. A, Comparison of predicted sequence of sheep BMP15 with humans. Numbering is relative to the sheep sequence. Numbers in brackets indicate amino acid positions of the mature peptide (underlined). The FecXL mutation at position 53 and the four already known mutations in sheep, FecXG, FecXH, FecXI, and FecXB, are marked with a framed residue. B, Alignment of cystine-knot motifs of BMP15 and other members of the BMP family using Multalin software (http://prodes.toulouse.inra.fr/multalin/multalin.html). All are human sequences and numbering is relative to BMP15 mature peptides. Letters in the consensus sequence indicate amino acids strictly conserved among the nine factors. Conserved Cys residues forming the cystine-knot motif are shaded in black, and intrachain disulfide bridges identified by the crystal structures of BMP7 and BMP2 (22 23 ) are indicated. It is noteworthy that the fourth Cys residue that forms the interchain disulfide bridge in the BMP7 (Cys103) or BMP2 (Cys78) homodimeric complex is absent in BMP15 and GDF9 sequences (position 91).

 
The FecXL allele was later found at the hemizygous state in 12 Lacaune sires of the OVI-TEST selection center among 300 tested animals. When available, the prolificacy breeding values of these mutant males, estimated through a progeny test, were very high (Table 1Go), which is to be expected for a mutation in an X-linked gene that affects prolificacy.


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  		TABLE 1. Breeding values (in SD) of eight FecXL carrier sires estimated on litter size of their daughters (national recording scheme)

 
In vivo effects of the mutation
Table 2Go displays the successive ovulations of the seven heterozygous FecXL ewes found in the nucleus. The individual mean is very high with a tremendous between-ewe variability (from 4.43 to 12.4) that leads to a very high repeatability (r = 0.65) estimated through an intraclass correlation coefficient. Although these sheep had very high ovulation rates, these observations did not prove the direct implication of the C53Y substitution in the BMP15 gene in the onset of their increased ovulation rate.


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  		TABLE 2. Ovulation of the seven ewes heterozygous for the FecXL mutation found in the small hyperprolific Lacaune nucleus

 
To prove that the FecXL allele was implicated in this increase in ovulation rate, heterozygous and homozygous carrier ewes were procreated. The mean ovulation rate recorded for the 12 heterozygous carrier ewes was 3.29 and 3.93 for the first and the second year, respectively (Fig. 2Go). In total, 8.4% of the ovulations were twins and 89% were triple or more. Because all daughters of carrier sires were heterozygous, it was not possible to compare carrier and noncarrier ewes in the same genetic background. However, the mean ovulation rate (152 records) of FecXL noncarrier adult Lacaune ewes recorded on the same location in similar conditions were 1.95, with 68% of twin ovulations and only 16% of triple or more ovulations. Then when present at the heterozygous state, the FecXL allele was able to increase ovulation rate in sheep.


Figure 2
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  		FIG. 2. Distribution of ovulation numbers of 12 heterozygous FecXL ewes at 1 and 2 yr of age and of 52 adult FecX+ ewes.

 
Four homozygous FecXL adult females were found among the daughters of mutant males crossed with heterozygous mutant females. Laparoscopic observations displayed an infantile genital tract and the ovaries did not carry any obvious follicular structures, resembling the streak phenotype observed in Inverdale (FecXI) homozygous ewes (15). Moreover, these ewes did not ovulate after a synchronization treatment in full breeding season (fluorogestone acetate for 14 d). In contrast with noncarrier ovaries, histological studies of the FecXL/FecXL ovaries revealed only primordial and primary follicles (Fig. 3Go), as already observed for ewes homozygous for previously described BMP15 mutations (8, 15). The ovarian cortex of the FecXL/FecXL ovaries was densely packed with primordial follicles (Fig. 3Go, B and D) and numerous adjacent oocytes organized in clusters (Fig. 3EGo). Oocyte-free follicular structures and numerous abnormal or degenerating primary follicles were also observed (Fig. 3FGo). Many of these abnormal follicles consisted of large oocytes with thickened zona pellucida surrounded by disorganized granulosa cell layers. Thus, homozygous FecXL mutated ewes were sterile due to premature blockage at the primary stage of folliculogenesis.


Figure 3
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  		FIG. 3. Histological sections of homozygous FecXL ovaries. Photomicrographs of histological section of the FecXL noncarrier (A and C) or homozygous carrier ovaries (B and D–F). A, Evidence of follicular growth with the presence of secondary (s) and tertiary (t) follicles in normal ovaries. B, FecXL/FecXL ovaries densely packed with primordial follicles (p), compared with wild type (A and C) with no secondary or tertiary follicles. D, Ovarian cortex of FecXL/FecXL ovaries with primordial follicles (p) and numerous abnormal follicular structures (a). E, Numerous adjacent oocytes organized in clusters. F, Presence of oocyte-free follicular structures (of) and numerous abnormal primary follicles (a) exhibiting large oocytes with thickened zona pellucida surrounded by disorganized granulosa cell layers.

 
In vitro effect of the mutation
To evaluate the effects of this naturally occurring C53Y substitution on the production, processing, dimerization and secretion of BMP15, we stably transfected HEK293T cells with expression plasmids for wild-type human BMP15 (9) or its mutated form BMP15C53Y, both tagged on the C terminus with the Myc epitope (Fig. 4Go). The wild-type precursor and mature forms of BMP15 analyzed by SDS-PAGE in reducing conditions were readily detectable in culture media from transfected cells, with nontransfected cells being used as negative control (Fig. 4AGo). In contrast, the secretion of BMP15C53Y was dramatically impaired. Only a faint band corresponding to the precursor form could be seen in the mutant lane. These results were confirmed and extended by denaturant nonreducing Western blot experiments. Similar to the reducing condition, the BMP15C53Y conditioned media failed to show either a secreted mature dimer or the monomeric peptide (Fig. 4BGo). In addition, the analysis of the cell extracts revealed the presence of lower amounts of the precursor form in the mutant, compared with the wild-type one. These results were confirmed using four independent protein preparations run in separate experiments. Thus, it appears that the C53Y substitution in BMP15 could alter the production step. To rule out the possibility of transcription defects or a problem with mRNA stability produced by the transfected mutant constructs, BMP15 expression was evaluated in 11 selected HEK293T stable clones by real-time PCR experiments after reverse transcription. The quantitative analysis showed similar BMP15 transcript levels in mutant and wild-type clones (Fig. 4CGo).


Figure 4
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  		FIG. 4. Protein analysis. A, SDS-PAGE. Five micrograms of purified protein from conditioned media of the wild-type (wt), C53Y mutant, and not-transfected (nt) cells were analyzed. The BMP15 mature peptide is visible only in the wt lane, whereas a small amount of precursor is also detectable in the mutant one. B, Denaturant-not reducing Western blot. Twenty-five micrograms of purified proteins were used. On the left are shown the results for the purification from the cell extracts, in the right the analysis of the culture media. Materials from the not-transfected cells represent negative controls. Mature peptide, mature dimer, and precursor are not present in the mutant conditioned media. A faint band corresponding to a little amount of mutant precursor dimer is visible. In cellular extracts (left), a small amount of intracellular precursor is detectable. C, Real-time PCR analysis. Relative quantity (log10) of the 11 BMP15 analyzed clones, obtained with the algorithm of the relative quantity study of the SDS 2.1 software, using the HEK293T cell sample as the calibrator. All selected mutant clones present transcript levels comparable with the wild-type (wt) samples.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study reports a new nucleotide variant in the sheep BMP15 gene that leads to a nonconservative C53Y substitution in the mature protein product. This mutation brings to five the number of naturally occurring mutations in the BMP15 gene that have been associated with modulation of ovarian function in sheep. Indeed, FecXL, as with other FecX mutations, is associated with increased ovulation rate or sterility, depending on its presence at the heterozygous or homozygous state, respectively. Heterozygous FecXL carriers show about 1.5 extra ovulations, compared with noncarriers. This is higher than the about one extra ovulation seen in FecXI, FecXH, or FecXG heterozygous carriers (1, 5, 6) and could be due to an additive effect of the phenotypic background of the Lacaune breed. Homozygous carriers of FecXL show the same streak ovarian phenotype as observed for homozygous carriers of the Inverdale (FecXI) and Hanna (FecXH) mutations (15). FecXL/FecXL ovaries contain no normal developing follicles beyond the primary stage of development, justifying the sterility of these animals. In ovine ovaries, like in other mammals, the FecX/BMP15 gene is exclusively expressed in the developing oocyte from primary follicles to preovulatory follicles (6, 16, 17, 18, 19). This observation indicates that the FecXL mutation may impair the normal physiological function of the BMP15 protein in the primary to secondary follicle transition. The presence of abnormal follicles with enlarged oocytes and/or a disorganized granulosa cell layer may indicate an aberrant communication between the oocyte and the surrounding granulosa cells. Indeed, oocyte-derived BMP15 has been described to be involved in the regulation of the mitogenic activity of granulosa cells, stimulating, in these cells, the expression of the Kit ligand factor and suppressing the FSH-induced steroidogenesis by down-regulating the FSH receptor expression (20, 21).

Actually, the FecXL ovarian phenotype is difficult to distinguish from those induced by the four already known FecX mutations, indicating their similar functional consequences on the BMP15 activity. In particular, FecXG and FecXH mutations introduce a premature stop codon in the BMP15 sequence, obviously impairing the production of its biologically active form. They can then be considered as total loss of function mutations. Similarly, FecXL can be hypothesized as a total loss-of-function mutation, too. Our in vitro results on the production of the recombinant BMP15 carrying the FecXL mutation (C53Y) validate this hypothesis. Indeed, the presence of the C53Y substitution in BMP15 has been shown to drastically reduce BMP15 production, suggesting that the FecXL allele would be likely unable to produce a biologically active BMP15 product in vivo. The real-time PCR experiments have also shown similar levels of mutant and wild-type BMP15 transcripts, indicating that mRNA stability was not affected by the nucleotide variation. The identified mutation affects a cysteine residue highly conserved in the BMP15 protein across all vertebrate species where BMP15 has been evidenced. Additionally, this residue is one of the six conserved cysteines involved in the characteristic three-dimensional folding of the TGFß factors, called the cystine-knot motif (22, 23, 24). The mechanism by which this cysteine substitution causes the inhibition of the BMP15 production has not been determined yet. However, one possibility is that the mutation, breaking an intrachain disulfide bridge, would lead to the formation of unstable misfolded proteins that are subjected to targeted degradation before processing (25). On the contrary, it is also possible that the C53Y mutation would be able to damage the BMP15 processing and secretion. Due to the major action on protein production, it was not possible to conclude on a potential role of the mutation on the processing or the secretion of BMP15. However, homodimerization of the mutated precursor appeared to be possible because it represents the major form in the culture media.

Our results are in contrast with those obtained for FecXI and FecXB mutations, leading to the nonconservative aminoacidic substitutions at positions 31 and 99, respectively, within the BMP15 mature peptide. Functional analyses of these two mutations indicated that neither V31D nor S99I substitutions are able to alter the production, processing, homodimerization, or biological activity of the BMP15 protein alone (26). Despite this, FecXI and FecXB carrier ewes exhibit the same phenotype as the carriers of the other FecX mutations. This discrepancy could be explained by the possible heterodimerization of BMP15 and GDF9 (27). GDF9 is another oocyte-derived growth factor of the TGFß family, closely related to BMP15 and crucial for ovarian folliculogenesis in sheep (5, 28) and mice (29). When the mutants BMP15V31D or BMP15S99I are coexpressed with normal GDF9, the secretion of both BMP15 and GDF9 is significantly reduced (26). Then it was hypothesized that the consequences of FecXI and FecXB mutations on ovarian function in sheep went through a GDF9-dependent mechanism. Even if the action of BMP15C53Y remains to be tested on GDF9 secretion, our results suggest a mechanism involving the direct impairment of BMP15 biological action within the ovary.

Mutations on the BMP15 gene have also been described in humans, but so far they were found at the heterozygous state. The human BMP15 mutations also affect ovarian function, but instead of increasing ovulation rate as in sheep, they lead to ovarian dysgenesis or premature ovarian failure (9). In contrast to what was observed in sheep, human mutations affect the part of the gene encoding the proregion. This is known to drive the dimerization and following secretion of the active mature dimers. Moreover, Hashimoto et al. (30) recently proposed that species-specific differences in BMP15 processing may be due to its proregion and may be associated with the differences in ovulation rate between different species. Nevertheless, the functional characterization of the first BMP15 mutation identified in humans (Y235C) has been shown to affect processing leading to the production of aberrant dimers. The mutant precursor exerts a dominant-negative effect on the wild-type protein, determining a loss-of-function phenotype. However, although the different phenotypes likely depend on the degree of functional alteration generated by each mutation, there are large differences between species as quoted by Fabre et al. (7). Indeed, to obtain normal ovulation, it appears that in the monoovulating human species, two functional copies of BMP15 are required because the presence of a heterozygous mutation is sufficient for ovarian failure. In ovine species, with a large-ranging ovulation rate, only one copy is sufficient for ovarian development and ovulation; whereas the ovarian function is stopped with the lack of BMP15. Finally, in a polyovulating species such as the mouse, with an ovulation rate about 10, BMP15 is dispensable. In conclusion, our findings confirm the essential role of the BMP15 factor in ovarian folliculogenesis and the control of ovulation rate in sheep and generally in mammals.


   Acknowledgments
 
We thank the staff of the Langlade experimental farm who takes care of the animals. We also thank J. Rallières for her technical assistance and Katia Feve for SSCA.


   Footnotes
 
This work was supported by the Conseil Régional de Midi Pyrénées, the OVI-TEST Cooperative. This work was also partially supported by the Italian Ministry of Education, University and Research (PRIN 2004052155_005) and Research Funds of Instituto di Ricovero e Cura a Carattere Scientifico Istituto Auxologico Italiano (Project GENIPOF, 05C501).

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 12, 2006

1 L.B. and E.D.P. contributed equally to this work. Back

Abbreviations: BMP15, Bone morphogenetic protein 15; BMPRIB, bone morphogenetic protein receptor type IB; FBS, fetal bovine serum; Fec, fecundity; GDF9, growth differentiation factor 9; HEK, human epithelial kidney; SSCA, single-strand conformation analysis.

Received June 8, 2006.

Accepted for publication October 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Davis GH 2005 Major genes affecting ovulation rate in sheep. Genet Sel Evol 37:S11–S23
  2. Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P, Lanneluc I, Pisselet C, Riquet J, Monniaux D, Callebaut I, Cribiu E, Thimonier J, Teyssier J, Bodin L, Cognie Y, Chitour N, Elsen JM 2001 Mutation in bone morphogenetic protein receptor-IB is associated with increased ovulation rate in Booroola Merino ewes. Proc Natl Acad Sci USA 98:5104–5109[Abstract/Free Full Text]
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