Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice

in Reproduction
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  • 1 School of Biological Sciences, AgResearch, Victoria University of Wellington, Wellington 6140, New Zealand

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Abstract

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.

Introduction

Mammalian ovulation rate, oocyte competence, and fertility are regulated by multiple paracrine and endocrine pathways, including the oocyte-derived transforming growth factor β (TGFB) superfamily signaling molecules, bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9; Eppig et al. 2002, Juengel & McNatty 2005). Mouse gene knockout models have demonstrated that GDF9 is essential for ovarian follicular development and fertility (Dong et al. 1996, Elvin et al. 1999a, 1999b), whereas BMP15 is only partially required for ovulation and fertilization and is not essential for follicular development (Yan et al. 2001). In sheep, inactivating mutations in either BMP15 or GDF9 result in sterility in homozygous mutants or increased ovulation rate in heterozygous carriers (Galloway et al. 2000, Hanrahan et al. 2004). In women, BMP15 mutations lead to premature ovarian failure (Di Pasquale et al. 2004, 2006, Dixit et al. 2006), while GDF9 mutations are also linked with sterility (Laissue et al. 2006) and dizygotic twinning (Palmer et al. 2006).

The TGFB superfamily of proteins are characteristically translated as precursor promature proteins that are enzymatically processed to produce a larger N-terminus proregion or prodomain and a smaller bioactive C-terminus mature protein. Intracellularly, the proregions of TGFB proteins are known to facilitate protein folding, dimerization, secretion, and stability while interacting noncovalently with the respective mature protein (Gray & Mason 1990, Constam & Robertson 1999, Hashimoto et al. 2005, Walton et al. 2009). Additionally, a wide range of extracellular functions for proregions of several TGFB family members interacting with mature domains has been observed (Bottinger et al. 1996, Hill et al. 2002, Haramoto et al. 2004, Jiang et al. 2004, Brown et al. 2005, Le Good et al. 2005, Anderson et al. 2008, Sengle et al. 2011). Recent evidence from the crystal structure of TGFB1 supports a number of interactions occurring between proregion and mature proteins following cleavage of the precursor protein (Shi et al. 2011). Previously, we reported that transfected human embryonic kidney cells secreted mouse BMP15 and GDF9 as mixtures of processed mature and proregion proteins, together with varying amounts of unprocessed precursor protein (McIntosh et al. 2008). Additionally, the cleaved proregions of BMP15 and GDF9 interacted noncovalently with their respective mature proteins, as well as forming heteromeric complexes between the BMP15 proregion and the GDF9 mature proteins (Liao et al. 2003, McIntosh et al. 2008). By using a proregion antibody to immunoneutralize BMP15 activity with respect to thymidine incorporation into granulosa cells, we gained further evidence that the BMP15 proregion may have important extracellular functions. Moreover, in ovine follicular fluid, the predominant forms of both GDF9 and BMP15 were the unprocessed precursor proteins (McNatty et al. 2006).

To test whether these in vitro and in vivo findings have physiological relevance, we examined the potential roles of the proregions of BMP15 and GDF9 by immunizing mice against selected proregion peptide sequences or a recombinant full-length mouse proregion GDF9 protein. The end points assessed in this study were ovarian volume, the number of antral follicles and corpora lutea (CL; experiment 1), and corresponding litter sizes (experiment 2). Mouse CL remain visible in the ovary from the four most recent ovulatory cycles, with each cycle taking 4 days (Pedersen & Peters 1968). Therefore, by allowing mice to mate (experiment 2) during the 16 days before the time of ovary collection for histological analysis (experiment 1), we were able to link the CL numbers counted with the corresponding fertility outcome.

Results

Antibody titers

During the experimental timeline (see Fig. 1), antibody titers were measured in the pre-, mid-, and post-immunization sera of animals immunized with the GDF9 full-length proregion or BMP15 proregion N-terminus peptide. Titers were observed to increase consistently throughout the immunization period; for example, titers of post-immunization sera compared with mid-immunization sera significantly increased by 11-fold for the GDF9 full-length proregion (P<0.0001) and by twofold for the BMP15 N-terminus proregion peptide (P<0.01). Post-immunization antisera from all groups were then tested for specificity to the immunization antigen and for cross-reactivity to other antigens. All immunizations with keyhole limpet hemocyanin (KLH)-conjugated BMP15 or GDF9 proregion peptides (see Fig. 2 for peptide sequences) or the full-length GDF9 proregion protein resulted in significantly higher specific antibody titers against their respective antigens than when mice were immunized with KLH alone (P<0.0001, Table 1). No cross-reactivity was observed between BMP15 antisera and GDF9 antigens, or vice versa (Table 1).

Figure 1
Figure 1

Experimental timeline for the immunization experiments. Mice were immunized four times with KLH-conjugated peptides or protein, or KLH as a control (n=30 per treatment group). Each treatment group was divided into two experiments: 1) where ovaries from ten mice per treatment group were collected a week after the final immunization for follicle and CL counts, and 2) where 20 mice per treatment group were allowed to breed and the number of pups born per litter were counted. In experiment 1, visible CL were linked to the last four ovulation cycles before ovary collection. Mating in experiment 2 was therefore allowed only during the same time period of these four ovulation cycles, in order to link the number of CL counted to fertility from the same cycles. Sera were collected after immunization for experiment 1, and pre-, mid-, and post-immunization for experiment 2.

Citation: REPRODUCTION 143, 2; 10.1530/REP-11-0336

Figure 2
Figure 2

Mouse BMP15 and GDF9 proregion peptides for immunization. N- and C-terminus proregion peptides of mouse BMP15 and GDF9 were selected so that a) C-terminus peptides encompassed a conserved tyrosine residue (underlined) that results in human infertility when mutated in human BMP15 (Di Pasquale et al. 2004); b) N-terminus peptides encompassed a hydrophobic motif (underlined) thought to bind to a mature protein sequence that is also the bone morphogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) binding site of the mature protein (Walton et al. 2009).

Citation: REPRODUCTION 143, 2; 10.1530/REP-11-0336

Table 1

Optical density (mean±s.e.m.) of post-immunization mouse sera following ELISA using specified coating antigens.

Coating antigen
Mouse antiseraGDF9 proregionGDF9 C-terminusGDF9 N-terminusBMP15 C-terminusBMP15 N-terminus
KLH0.04±0.000.01±0.000.01±0.000.01±0.000.01±0.00
GDF9 proregion1.22±0.190.06±0.030.02±0.000.01±0.000.01±0.00
GDF9 C-terminus0.81±0.111.86±0.220.01±0.000.01±0.000.01±0.00
GDF9 N-terminus0.31±0.050.01±0.001.63±0.190.01±0.000.01±0.00
BMP15 C-terminus0.04±0.000.01±0.000.02±0.001.18±0.180.01±0.00
BMP15 N-terminus0.04±0.010.01±0.000.01±0.000.01±0.000.94±0.16

Values in bold represent P<0.0001 compared with KLH control within column, n=28–30 mice per group.

Ovarian volume and number of CL and antral follicles

Ovarian volume, number of CL visible over the last four ovulation cycles and number of antral follicles were analyzed for each mouse and mean values were calculated for each immunization group (Table 2). The mean volume of ovaries from animals immunized against the C-terminus BMP15 proregion peptide was significantly larger than that for the KLH group (P<0.05). No differences were observed in ovarian volumes of animals immunized with other antigens. An increased total number of CL was observed in the GDF9 full-length proregion-immunized group compared with the KLH control group (P<0.01, Table 2). In contrast, total CL number was decreased in the groups immunized with BMP15 proregion C-terminus (P<0.01) and N-terminus (P<0.05) peptides. Since mouse CL are visible in the ovary from the four most recent ovulatory cycles (Pedersen & Peters 1968), the number of CL was compared on the basis of ovarian cycle of origin, where CL1 were from the most recent cycle and CL4 were the oldest CL. CL2 and CL3 were combined for ease of identification because it was not always possible to distinguish between these two groups using morphological criteria. Interestingly, significant differences were observed in the number of CL from ovarian cycles 2+3, but not from cycles 1 or 4 (Fig. 3), compared with those of the KLH-immunized group. No differences were observed in the mean total number of antral follicles (Table 2), number of follicles at any individual antral follicular stage, or the number of atretic follicles in any immunized group compared with the KLH-immunized control group (data not shown).

Table 2

Effect of immunization treatments on ovarian volume, number of corpora lutea (CL) and antral follicles, and litter size per mouse. Values are mean±s.e.m.

Immunization groupOvarian volume (mm3)Number of CLaNumber of antral folliclesaLitter size
KLH2.17±0.12 (10)55.1±3.3 (10)116±10 (5)4.30±0.40 (20)
GDF9 proregion2.48±0.12 (10)79.5±3.5** (10)140±12 (5)1.75±0.25** (19)
GDF9 C-terminus2.25±0.16 (10)57.3±3.3 (10)171±15 (5)3.00±0.65 (20)
GDF9 N-terminus1.80±0.09 (10)49.2±3.0 (10)176±23 (5)4.11±0.72 (19)
BMP15 C-terminus2.72±0.14* (8)41.8±1.8** (10)149±21 (5)2.88±0.55 (20)
BMP15 N-terminus1.65±0.37 (7)35.8±4.3* (9)90±22 (5)2.55±0.39* (19)

Values in bold represent *P<0.05, **P<0.01 compared with the KLH control group. The number of mice analyzed in each treatment group is shown in brackets.

For calculation of mean values per immunization group, all visible CL and antral follicles of one ovary were counted for each mouse.

Figure 3
Figure 3

Number of corpora lutea (CL) according to ovulation cycle of CL origin. (A) Morphological classification of CL into groups to identify the ovulation cycle from which the CL developed. CL1, CL from the most recent ovulation cycle 1; CL2+3, combined CL from cycles 2 and 3 before the most recent ovulation; and CL4, the oldest visible CL from cycle 4. Scale=50 μm. (B) Mean number of CL of mice within each immunization group, according to CL group, ±s.e.m. **P<0.01 and ***P<0.001 compared with KLH control groups.

Citation: REPRODUCTION 143, 2; 10.1530/REP-11-0336

Litter size

Mice immunized with either the N-terminus BMP15 proregion peptide or the full-length GDF9 proregion produced significantly smaller litters compared with the KLH-immunized control group (Table 2, P<0.05 and P<0.01 respectively). To check that the immunizations did not affect mating rate, pregnancy rate, or the number of litters produced in each group, data for these parameters were collected for χ2 analysis (Table 3). Males were housed with females for mating from the time of cycle 4 (i.e. the first mating cycle). Pregnancy occurred in 35–58% of the mice, similar to Balb/c mice pregnancy rates of 31–44% documented by The Jackson Laboratory, (Bar Harbor, Maine, USA) (http://jaxmice.jax.org/jaxnotes/archive/501d.html). No significant difference was observed between immunization groups in the proportions of animals that became pregnant or in the number of litters produced by each group per ovulatory cycle (P>0.05, Table 3). A difference between groups in the number of copulatory plugs after mating was identified (P<0.05, Table 3), which was likely due to loss of some plugs in the KLH group, since significance was lost when data from the KLH group were omitted. As the pregnancy rate for the KLH group was above the mean pregnancy rate for all groups, this difference did not translate into loss of fertility.

Table 3

Effect of immunization treatments on proportion of animals with copulatory plugs, pregnancy rate, and number of litters per ovulation cycle.

Number of litters per group, per cycle
Immunization group (n)Proportion of group with copulatory plugs (%)Pregnancy rate of group (%)Cycle 4Cycles 2+3Cycle 1
KLH (20)60*50442
GDF9 proregion (19)7540341
GDF9 C-terminus (20)9535241
GDF9 N-terminus (19)7947341
BMP15 C-terminus (20)8540432
BMP15 N-terminus (19)9558362

*P< 0.05 χ2 analysis. n, number of animals in each treatment group.

Discussion

This study provides physiological evidence to support the hypothesis that the proregions and/or precursor proteins of GDF9 and BMP15 have key regulatory functions within the mouse ovary after secretion from the oocyte, including regulation of ovulation rate as assessed by the number of CL that formed in each ovarian cycle and regulation of litter size. Immunization with the GDF9 proregion was shown to increase ovulation rate, in contrast to a reduced ovulation rate in mice immunized with the BMP15 proregion peptides, indicative of different roles for mouse GDF9 and BMP15 proregions before or during ovulation. These divergent effects are possibly linked to differing abilities of generated antibodies to modulate the concentrations of GDF9 or BMP15. For example, regulation of ovulation rate in sheep by BMP15 and GDF9 is dose dependent (Juengel et al. 2002, McNatty et al. 2007), with decreasing concentrations of biologically active BMP15 or GDF9 initially linked to increased ovulation rates until a critical threshold is reached, and with decreased ovulation rate predicted when bioactivity is below this threshold (McNatty et al. 2004).

Whether our immunizations affect ovulation rate through altering the pattern of follicular growth or through regulating the efficiency of ovulation and luteal formation is difficult to decipher. While no differences were observed in the numbers of antral follicles for any treatment at the time of autopsy, differences might have been present leading up to the cycles forming a different number of CL. The significant difference in the number of CL from cycles 2 and 3, but not from the most recent cycle 1, might have indicated that the antibody titers had decreased by the time of ovary collection. However, the steady increase in antibody titers measured throughout the study indicates that the immunoneutralization effects were likely to be increasingly compensated for over time. Underlying mechanisms for this compensation might involve intra-follicular paracrine effects since both the oocyte and the granulosa cells have membrane receptors for GDF9 and BMP15 (reviewed in Juengel & McNatty (2005) and Gilchrist et al. (2008)). Further evidence that the decreased number of CL observed in the BMP15 proregion-immunized groups could be related to interference with ovulation/luteinization was shown when normal ovulation and/or CL formation was impaired in ewes passively immunized with anti-plasma to BMP15 or GDF9 just before ovulation (Juengel et al. 2002).

Under normal physiological conditions in mice, levels of secreted BMP15 proteins are thought to be low (Hashimoto et al. 2005) until the preovulatory gonadotropin surge (Yoshino et al. 2006). In this case, it would be unlikely that BMP15 immunization would affect antral follicle numbers. The decreased ovulation rate we observed in mice immunized with BMP15 proregion peptides parallels the reported subfertility of Bmp15 knockout mice (Yan et al. 2001). Our data that GDF9 proregion-immunized mice developed increased CL numbers suggest that the GDF9 proregion might inhibit ovulation in some way, which is consistent with reports that proregions of some other TGFB family proteins inhibit or antagonize their respective mature proteins (Bottinger et al. 1996, Hill et al. 2002, Jiang et al. 2004, Makanji et al. 2011). Alternatively, removal of the GDF9 proregion may negatively affect the concentration/dose of bioactive GDF9 mature protein available for signaling. In support of this, mice injected with an Fc fusion protein of the type II BMP receptor bone morphogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) had increased the number of both antral follicles and CL (Myllymaa et al. 2010).

A key result from our studies was the decreased litter sizes of mice immunized with either the full-length GDF9 proregion or the BMP15 proregion N-terminus peptide. Since no differences between treatment groups were observed in the proportions of mice that became pregnant, or in the cycles in which fertilization occurred, it seems most likely that the proregion immunizations affected either the number of oocytes available for fertilization and/or the ability of oocytes to be fertilized and undergo normal development. Given the reduced number of CL observed in mice immunized against the BMP15 proregion N-terminus peptide, the subsequent reduced litter size is likely to be related to a decreased number of oocytes available for fertilization. A smaller litter size despite an increased CL number in mice immunized with the GDF9 proregion is consistent with the notion that some of the follicles and/or oocytes, and therefore the subsequent CL (Juengel et al. 2002), were abnormal at ovulation and compromised the pregnancy outcome. Moreover, this is also consistent with the finding that the addition of exogenous GDF9 to mouse oocytes during in vitro maturation resulted in an increased number of viable fetuses (Yeo et al. 2008).

Proregion immunoneutralization of bioactivity is likely to occur through interactions between the proregion and mature proteins. It is anticipated that the antibodies generated by immunization would bind to proregion (free or interacting with mature proteins and other regulatory proteins) and precursor proteins. However, the physiological significance of secreted BMP15 and GDF9 precursor proteins is yet unknown. The N-terminus proregion peptides used for immunization includes a hydrophobic motif identified as binding to the mature protein at the same site that also interacts with BMPR2 (Walton et al. 2009). Antibodies generated against this region in BMP15 altered CL number, but there was no effect on CL number when mice were immunized with the corresponding GDF9 N-terminus peptide, suggestive of the in vivo interactions between the proregion and the mature proteins of mouse GDF9 and BMP15 being functionally different.

In summary, we have shown that active immunization against either the GDF9 or BMP15 proregion protein or peptides altered the number of CL and decreased the number of offspring, providing evidence that the proregions of both factors are important regulators of ovarian function in vivo in mice. Discovery of the functional mechanisms of these proregions will be a vital step to understand the full effects of BMP15 and GDF9 on oocyte maturation and gonadotropin responsiveness of both cumulus and granulosa cells.

Materials and Methods

Generation of proregions and proregion peptides

Mouse proregion GDF9 was amplified from the appropriate full-length mouse gene prepared as described previously (McIntosh et al. 2008). The Gdf9 PCR product (using Gdf9 primers 5′-CACCATGTCTACTGAAGAATCC-3′, 5′-TCGACGGCGCCGGGG-3′) was blunt-end cloned into the plasmid pET101 Direction TOPO (Invitrogen). The resulting GDF9 proregion protein was purified by immobilised metal ion chromotography (IMAC) and conjugated to equal quantities of KLH (Sigma-Aldrich) using glutaraldehyde, as described previously (Juengel et al. 2002). Preparation of a stable, full-length BMP15 proregion was unsuccessful. Proregion peptides (Fig. 1) were synthesized and conjugated to KLH through a C-terminus cysteine residue by Global Peptide, Fort Collins, CO, USA and GenScript, Piscataway, NJ, USA.

Immunization procedures

All animal immunizations and tissue recoveries were performed under approval of the Animal Ethics Committee of the Wallaceville Animal Research Centre, Upper Hutt, New Zealand, in accordance with the 1999 Animal Welfare Act (Part 6), New Zealand, and by the Victoria University of Wellington Animal Ethics Committee. The experimental design for the immunization experiments is outlined in Fig. 1. For each proregion or peptide antigen, 3-month-old Balb/c female mice (n=30 per treatment) were immunized with a total of 0.4 mg conjugated antigen (i.p.) over 6 weeks (0.2 mg in Freund's complete adjuvant, with 2-weekly boosters of 0.1, 0.05, and 0.05 mg in a Span–Tween–Marcol adjuvant). Three separate immunization trials were undertaken, one trial for histological analyses (n=10 mice per treatment), and two trials for litter size analyses (data combined, n=20 mice per treatment). For histological analyses, ovaries were collected 1 week after the final booster. For litter size analyses, mice were allowed to mate during the 16-day period before immunization week 8, with the males being removed after mating.

Measurement of antibody titers by ELISA

Antisera from mice for litter size analyses were collected before immunization (pre), 2 days after immunization 3 (mid), and at killing (post). Antisera from mice for histological analyses were collected only after killing (post). To test sera (n=20–30 per treatment group) for reactivity to BMP15 and GDF9, wells of microtiter plates were coated at 4 °C overnight with 100 ng of unconjugated proregion protein or proregion peptide antigens in coating buffer (50 mmol/l carbonate–bicarbonate buffer, pH 9.6). GDF9 proregion protein was treated with 1% w/v SDS at 56 °C for 30 min before coating. Non-specific binding was blocked by incubation with 0.25 ml coating buffer containing 0.5% w/v gelatin for 30 min at room temperature. Wells were washed (three washes in PBS with 0.05% v/v Tween 20 for 3 min). Mouse sera were diluted 1:1000 in assay buffer (PBS with 0.05% v/v Tween 20 and 0.1% w/v gelatin), and 0.1 ml diluted sera incubated in each of the coated/washed wells of the microtiter plates for 2 h at 37 °C. The wells were washed and incubated with 0.1 ml HRP-conjugated goat anti-mouse immunoglobulin G (IgG) (1:10 000 dilution in assay buffer; Jackson ImmunoResearch Europe Ltd) for 1 h at 37 °C. Wells were washed and incubated with 0.1 ml of 50 mmol/l citrate buffer containing O-phenylenediamine (0.4 mg/ml; Sigma-Aldrich) and 0.04% v/v H2O2 for 30 min at room temperature in the darkness. Reactions were stopped by the addition of 0.05 ml of 2.5 mol/l H2SO4, and absorbance measured at 490 nm on a BioTek EL311 microplate autoreader (BioTek Instruments, Inc., Winooski, VT, USA). ELISA results were validated for multiplate assays with the coefficient of variation <6% between assay plates. Antisera from mice immunized with either BMP15 or GDF9 N-terminus proregion peptides were shown to specifically recognize the appropriate 293 HEK cell-expressed proregion and precursor promature proteins (McIntosh et al. 2008).

Histological analyses

Ovaries were collected from each treatment group, fixed in Bouin's fluid, serially sectioned at 5 μm, and stained with hematoxylin and eosin. The volume of one ovary per mouse (n=7–10 per group) was measured by the Cavalieri principle using the formula , where a, determined by point counting, is the cross-sectional area (μm2) of every tenth section and h is the distance (μm) between sections used to determine a. The number of CL was counted (n=9–10 mice per group, see Table 2) by drawing CL outlines of each ovary in every 50th slide using a projection microscope. Counting every 50th slide was valid since the minimum CL size was observed to be >250 μm. In mice, CL progress to form corpora albicans over four consecutive estrous cycles from the most recent cycle (Pedersen & Peters 1968). For the purposes of this paper, all corpora luteal structures regardless of age are referred to as CL. The ovulation cycle of origin of each CL was determined at higher magnification using an Olympus BH-2 microscope (Olympus New Zealand Ltd, Upper Hutt, New Zealand), according to the criteria: CL1, from the most recent cycle, with large luteal cells and significant number of visible blood vessel epithelial cells; CL2+3, from cycles 2 and 3, with smaller luteal cells than in CL1, less vascularization, and presence of macrophages; CL4, from cycle 4, with luteal cells compacted, membranes disintegrating, and increased number of macrophages (Fig. 3A). While CL1 and CL4 were easy to distinguish, it was not always possible to distinguish CL2 and CL3; therefore, in this study, the data for CL2 and CL3 were combined. The number of antral follicles (non-atretic, atretic, or abnormal) in ovaries of five mice per group was measured by counting antral follicles in every tenth section. Follicular stage was classified according to that proposed for mouse follicles (Pedersen & Peters 1968).

Pregnancy and litter size measurement

Five days after immunization 3, female mice were housed with male mice (5:1 ratio). The presence of copulatory plugs was checked daily as an evidence of mating, noting the possibility of false negative data due to potential loss of plugs before observation. After 16 days, males were removed. From 18 days after immunization 3 onward, females were checked twice daily for parturition, when pups were counted and removed. Mothers were then killed, and post-immunization antisera were collected.

Statistical procedures

When comparing antibody titers, unpaired Student's t-tests were performed between each of the treated groups and the control (KLH) group tested with the same antigen. To compare the ovarian effects and litter sizes between the treated groups and the control KLH group, the data were subjected to analysis of variance (ANOVA) and the Dunnett's post-hoc test. To test differences between immunization groups for rates of copulatory plugs, pregnancy, or number of litters, data were subjected to χ2 analysis.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the Royal Society of New Zealand Marsden Fund and a New Zealand Foundation for Research, Science and Technology Postdoctoral Fellowship.

Acknowledgements

We wish to thank Joan Bowen and Leanne Still, AgResearch Wallaceville, for their histological expertise and the Wallaceville Small Animal Breeding Unit, Upper Hutt, New Zealand for the supply and care of animals.

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  • Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG & Montgomery GW 2000 Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nature Genetics 25 279283. doi:10.1038/77033.

    • Search Google Scholar
    • Export Citation
  • Gilchrist RB, Lane M & Thompson JG 2008 Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human Reproduction Update 14 159177. doi:10.1093/humupd/dmm040.

    • Search Google Scholar
    • Export Citation
  • Gray AM & Mason AJ 1990 Requirement for activin A and transforming growth factor–beta 1 pro-regions in homodimer assembly. Science 247 13281330. doi:10.1126/science.2315700.

    • Search Google Scholar
    • Export Citation
  • Hanrahan JP, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell R & Galloway SM 2004 Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries). Biology of Reproduction 70 900909. doi:10.1095/biolreprod.103.023093.

    • Search Google Scholar
    • Export Citation
  • Haramoto Y, Tanegashima K, Onuma Y, Takahashi S, Sekizaki H & Asashima M 2004 Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region. Developmental Biology 265 155168. doi:10.1016/j.ydbio.2003.09.015.

    • Search Google Scholar
    • Export Citation
  • Hashimoto O, Moore RK & Shimasaki S 2005 Posttranslational processing of mouse and human BMP-15: potential implication in the determination of ovulation quota. PNAS 102 54265431. doi:10.1073/pnas.0409533102.

    • Search Google Scholar
    • Export Citation
  • Hill JJ, Davies MV, Pearson AA, Wang JH, Hewick RM, Wolfman NM & Qiu Y 2002 The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum. Journal of Biological Chemistry 277 4073540741. doi:10.1074/jbc.M206379200.

    • Search Google Scholar
    • Export Citation
  • Jiang MS, Liang LF, Wang S, Ratovitski T, Holmstrom J, Barker C & Stotish R 2004 Characterization and identification of the inhibitory domain of GDF-8 propeptide. Biochemical and Biophysical Research Communications 315 525531. doi:10.1016/j.bbrc.2004.01.085.

    • Search Google Scholar
    • Export Citation
  • Juengel JL & McNatty KP 2005 The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development. Human Reproduction Update 11 144161. doi:10.1093/humupd/dmh061.

    • Search Google Scholar
    • Export Citation
  • Juengel JL, Hudson NL, Heath DA, Smith P, Reader KL, Lawrence SB, O'Connell AR, Laitinen MP, Cranfield M & Groome NP 2002 Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep. Biology of Reproduction 67 17771789. doi:10.1095/biolreprod.102.007146.

    • Search Google Scholar
    • Export Citation
  • Laissue P, Christin-Maitre S, Touraine P, Kuttenn F, Ritvos O, Aittomaki K, Bourcigaux N, Jacquesson L, Bouchard P & Frydman R 2006 Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. European Journal of Endocrinology 154 739744. doi:10.1530/eje.1.02135.

    • Search Google Scholar
    • Export Citation
  • Le Good JA, Joubin K, Giraldez AJ, Ben-Haim N, Beck S, Chen Y, Schier AF & Constam DB 2005 Nodal stability determines signaling range. Current Biology 15 3136. doi:10.1016/j.cub.2004.12.062.

    • Search Google Scholar
    • Export Citation
  • Liao WX, Moore RK, Otsuka F & Shimasaki S 2003 Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep. Journal of Biological Chemistry 278 37133719. doi:10.1074/jbc.M210598200.

    • Search Google Scholar
    • Export Citation
  • Makanji Y, Walton KL, Chan KL, Gregorevic P, Robertson DM & Harrison CA 2011 Generation of a specific activin antagonist by modification of the activin A propeptide. Endocrinology 152 37583768. doi:10.1210/en.2011-1052.

    • Search Google Scholar
    • Export Citation
  • McIntosh CJ, Lun S, Lawrence S, Western AH, McNatty KP & Juengel JL 2008 The proregion of mouse BMP15 regulates the cooperative interactions of BMP15 and GDF9. Biology of Reproduction 79 889896. doi:10.1095/biolreprod.108.068163.

    • Search Google Scholar
    • Export Citation
  • McNatty KP, Moore LG, Hudson NL, Quirke LD, Lawrence SB, Reader K, Hanrahan JP, Smith P, Groome NP & Laitinen M 2004 The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology. Reproduction 128 379386. doi:10.1530/rep.1.00280.

    • Search Google Scholar
    • Export Citation
  • McNatty KP, Lawrence S, Groome NP, Meerasahib MF, Hudson NL, Whiting L, Heath DA & Juengel JL 2006 Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants. Reproduction, Fertility, and Development 18 403412. doi:10.1071/RD05104.

    • Search Google Scholar
    • Export Citation
  • McNatty KP, Hudson NL, Whiting L, Reader KL, Lun S, Western A, Heath DA, Smith P, Moore LG & Juengel JL 2007 The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate. Biology of Reproduction 76 552560. doi:10.1095/biolreprod.106.054361.

    • Search Google Scholar
    • Export Citation
  • Myllymaa S, Pasternack A, Mottershead DG, Poutanen M, Pulkki MM, Pelliniemi LJ, Ritvos O & Laitinen MP 2010 Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice. Reproduction 139 587598. doi:10.1530/REP-09-0391.

    • Search Google Scholar
    • Export Citation
  • Palmer JS, Zhao ZZ, Hoekstra C, Hayward NK, Webb PM, Whiteman DC, Martin NG, Boomsma DI, Duffy DL & Montgomery GW 2006 Novel variants in growth differentiation factor 9 in mothers of dizygotic twins. Journal of Clinical Endocrinology and Metabolism 91 47134716. doi:10.1210/jc.2006-0970.

    • Search Google Scholar
    • Export Citation
  • Pedersen T & Peters H 1968 Proposal for a classification of oocytes and follicles in the mouse ovary. Journal of Reproduction and Fertility 17 555557. doi:10.1530/jrf.0.0170555.

    • Search Google Scholar
    • Export Citation
  • Sengle G, Ono RN, Sasaki T & Sakai LY 2011 Prodomains of transforming growth factor β (TGFβ) superfamily members specify different functions. Journal of Biological Chemistry 286 50875099. doi:10.1074/jbc.M110.188615.

    • Search Google Scholar
    • Export Citation
  • Shi M, Zhu J, Wang R, Chen X, Mi L, Walz T & Springer TA 2011 Latent TGF-[bgr] structure and activation. Nature 474 343349. doi:10.1038/nature10152.

  • Walton KL, Makanji Y, Wilce MC, Chan KL, Robertson DM & Harrison CA 2009 A common biosynthetic pathway governs the dimerization and secretion of inhibin and related transforming growth factor beta (TGFbeta) ligands. Journal of Biological Chemistry 284 93119320. doi:10.1074/jbc.M808763200.

    • Search Google Scholar
    • Export Citation
  • Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS & Dube JL 2001 Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Molecular Endocrinology 15 854866. doi:10.1210/me.15.6.854.

    • Search Google Scholar
    • Export Citation
  • Yeo CX, Gilchrist RB, Thompson JG & Lane M 2008 Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice. Human Reproduction 23 6773. doi:10.1093/humrep/dem140.

    • Search Google Scholar
    • Export Citation
  • Yoshino O, McMahon HE, Sharma S & Shimasaki S 2006 A unique preovulatory expression pattern plays a key role in the physiological functions of BMP-15 in the mouse. PNAS 103 1067810683. doi:10.1073/pnas.0600507103.

    • Search Google Scholar
    • Export Citation

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  • View in gallery

    Experimental timeline for the immunization experiments. Mice were immunized four times with KLH-conjugated peptides or protein, or KLH as a control (n=30 per treatment group). Each treatment group was divided into two experiments: 1) where ovaries from ten mice per treatment group were collected a week after the final immunization for follicle and CL counts, and 2) where 20 mice per treatment group were allowed to breed and the number of pups born per litter were counted. In experiment 1, visible CL were linked to the last four ovulation cycles before ovary collection. Mating in experiment 2 was therefore allowed only during the same time period of these four ovulation cycles, in order to link the number of CL counted to fertility from the same cycles. Sera were collected after immunization for experiment 1, and pre-, mid-, and post-immunization for experiment 2.

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    Mouse BMP15 and GDF9 proregion peptides for immunization. N- and C-terminus proregion peptides of mouse BMP15 and GDF9 were selected so that a) C-terminus peptides encompassed a conserved tyrosine residue (underlined) that results in human infertility when mutated in human BMP15 (Di Pasquale et al. 2004); b) N-terminus peptides encompassed a hydrophobic motif (underlined) thought to bind to a mature protein sequence that is also the bone morphogenetic protein receptor, type II (serine/threonine kinase) (BMPR2) binding site of the mature protein (Walton et al. 2009).

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    Number of corpora lutea (CL) according to ovulation cycle of CL origin. (A) Morphological classification of CL into groups to identify the ovulation cycle from which the CL developed. CL1, CL from the most recent ovulation cycle 1; CL2+3, combined CL from cycles 2 and 3 before the most recent ovulation; and CL4, the oldest visible CL from cycle 4. Scale=50 μm. (B) Mean number of CL of mice within each immunization group, according to CL group, ±s.e.m. **P<0.01 and ***P<0.001 compared with KLH control groups.

  • Anderson SB, Goldberg AL & Whitman M 2008 Identification of a novel pool of extracellular pro-myostatin in skeletal muscle. Journal of Biological Chemistry 283 70277035. doi:10.1074/jbc.M706678200.

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  • Brown MA, Zhao Q, Baker KA, Naik C, Chen C, Pukac L, Singh M, Tsareva T, Parice Y & Mahoney A 2005 Crystal structure of BMP-9 and functional interactions with pro-region and receptors. Journal of Biological Chemistry 280 2511125118. doi:10.1074/jbc.M503328200.

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  • Di Pasquale E, Beck-Peccoz P & Persani L 2004 Hypergonadotropic ovarian failure associated with an inherited mutation of human bone morphogenetic protein-15 (BMP15) gene. American Journal of Human Genetics 75 106111. doi:10.1086/422103.

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  • Di Pasquale E, Rossetti R, Marozzi A, Bodega B, Borgato S, Cavallo L, Einaudi S, Radetti G, Russo G & Sacco M 2006 Identification of new variants of human BMP15 gene in a large cohort of women with premature ovarian failure. Journal of Clinical Endocrinology and Metabolism 91 19761979. doi:10.1210/jc.2005-2650.

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  • Dixit H, Rao LK, Padmalatha VV, Kanakavalli M, Deenadayal M, Gupta N, Chakrabarty B & Singh L 2006 Missense mutations in the BMP15 gene are associated with ovarian failure. Human Genetics 119 408415. doi:10.1007/s00439-006-0150-0.

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  • Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N & Matzuk MM 1996 Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383 531535. doi:10.1038/383531a0.

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  • Elvin JA, Clark AT, Wang P, Wolfman NM & Matzuk MM 1999a Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Molecular Endocrinology 13 10351048. doi:10.1210/me.13.6.1035.

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  • Elvin JA, Yan C, Wang P, Nishimori K & Matzuk MM 1999b Molecular characterization of the follicle defects in the growth differentiation factor 9-deficient ovary. Molecular Endocrinology 13 10181034. doi:10.1210/me.13.6.1018.

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  • Eppig JJ, Wigglesworth K & Pendola FL 2002 The mammalian oocyte orchestrates the rate of ovarian follicular development. PNAS 99 28902894. doi:10.1073/pnas.052658699.

    • Search Google Scholar
    • Export Citation
  • Galloway SM, McNatty KP, Cambridge LM, Laitinen MP, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG & Montgomery GW 2000 Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner. Nature Genetics 25 279283. doi:10.1038/77033.

    • Search Google Scholar
    • Export Citation
  • Gilchrist RB, Lane M & Thompson JG 2008 Oocyte-secreted factors: regulators of cumulus cell function and oocyte quality. Human Reproduction Update 14 159177. doi:10.1093/humupd/dmm040.

    • Search Google Scholar
    • Export Citation
  • Gray AM & Mason AJ 1990 Requirement for activin A and transforming growth factor–beta 1 pro-regions in homodimer assembly. Science 247 13281330. doi:10.1126/science.2315700.

    • Search Google Scholar
    • Export Citation
  • Hanrahan JP, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell R & Galloway SM 2004 Mutations in the genes for oocyte-derived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries). Biology of Reproduction 70 900909. doi:10.1095/biolreprod.103.023093.

    • Search Google Scholar
    • Export Citation
  • Haramoto Y, Tanegashima K, Onuma Y, Takahashi S, Sekizaki H & Asashima M 2004 Xenopus tropicalis nodal-related gene 3 regulates BMP signaling: an essential role for the pro-region. Developmental Biology 265 155168. doi:10.1016/j.ydbio.2003.09.015.

    • Search Google Scholar
    • Export Citation
  • Hashimoto O, Moore RK & Shimasaki S 2005 Posttranslational processing of mouse and human BMP-15: potential implication in the determination of ovulation quota. PNAS 102 54265431. doi:10.1073/pnas.0409533102.

    • Search Google Scholar
    • Export Citation
  • Hill JJ, Davies MV, Pearson AA, Wang JH, Hewick RM, Wolfman NM & Qiu Y 2002 The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum. Journal of Biological Chemistry 277 4073540741. doi:10.1074/jbc.M206379200.

    • Search Google Scholar
    • Export Citation
  • Jiang MS, Liang LF, Wang S, Ratovitski T, Holmstrom J, Barker C & Stotish R 2004 Characterization and identification of the inhibitory domain of GDF-8 propeptide. Biochemical and Biophysical Research Communications 315 525531. doi:10.1016/j.bbrc.2004.01.085.

    • Search Google Scholar
    • Export Citation
  • Juengel JL & McNatty KP 2005 The role of proteins of the transforming growth factor-{beta} superfamily in the intraovarian regulation of follicular development. Human Reproduction Update 11 144161. doi:10.1093/humupd/dmh061.

    • Search Google Scholar
    • Export Citation
  • Juengel JL, Hudson NL, Heath DA, Smith P, Reader KL, Lawrence SB, O'Connell AR, Laitinen MP, Cranfield M & Groome NP 2002 Growth differentiation factor 9 and bone morphogenetic protein 15 are essential for ovarian follicular development in sheep. Biology of Reproduction 67 17771789. doi:10.1095/biolreprod.102.007146.

    • Search Google Scholar
    • Export Citation
  • Laissue P, Christin-Maitre S, Touraine P, Kuttenn F, Ritvos O, Aittomaki K, Bourcigaux N, Jacquesson L, Bouchard P & Frydman R 2006 Mutations and sequence variants in GDF9 and BMP15 in patients with premature ovarian failure. European Journal of Endocrinology 154 739744. doi:10.1530/eje.1.02135.

    • Search Google Scholar
    • Export Citation
  • Le Good JA, Joubin K, Giraldez AJ, Ben-Haim N, Beck S, Chen Y, Schier AF & Constam DB 2005 Nodal stability determines signaling range. Current Biology 15 3136. doi:10.1016/j.cub.2004.12.062.

    • Search Google Scholar
    • Export Citation
  • Liao WX, Moore RK, Otsuka F & Shimasaki S 2003 Effect of intracellular interactions on the processing and secretion of bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9. Implication of the aberrant ovarian phenotype of BMP-15 mutant sheep. Journal of Biological Chemistry 278 37133719. doi:10.1074/jbc.M210598200.

    • Search Google Scholar
    • Export Citation
  • Makanji Y, Walton KL, Chan KL, Gregorevic P, Robertson DM & Harrison CA 2011 Generation of a specific activin antagonist by modification of the activin A propeptide. Endocrinology 152 37583768. doi:10.1210/en.2011-1052.

    • Search Google Scholar
    • Export Citation
  • McIntosh CJ, Lun S, Lawrence S, Western AH, McNatty KP & Juengel JL 2008 The proregion of mouse BMP15 regulates the cooperative interactions of BMP15 and GDF9. Biology of Reproduction 79 889896. doi:10.1095/biolreprod.108.068163.

    • Search Google Scholar
    • Export Citation
  • McNatty KP, Moore LG, Hudson NL, Quirke LD, Lawrence SB, Reader K, Hanrahan JP, Smith P, Groome NP & Laitinen M 2004 The oocyte and its role in regulating ovulation rate: a new paradigm in reproductive biology. Reproduction 128 379386. doi:10.1530/rep.1.00280.

    • Search Google Scholar
    • Export Citation
  • McNatty KP, Lawrence S, Groome NP, Meerasahib MF, Hudson NL, Whiting L, Heath DA & Juengel JL 2006 Meat and Livestock Association Plenary Lecture 2005. Oocyte signalling molecules and their effects on reproduction in ruminants. Reproduction, Fertility, and Development 18 403412. doi:10.1071/RD05104.

    • Search Google Scholar
    • Export Citation
  • McNatty KP, Hudson NL, Whiting L, Reader KL, Lun S, Western A, Heath DA, Smith P, Moore LG & Juengel JL 2007 The effects of immunizing sheep with different BMP15 or GDF9 peptide sequences on ovarian follicular activity and ovulation rate. Biology of Reproduction 76 552560. doi:10.1095/biolreprod.106.054361.

    • Search Google Scholar
    • Export Citation
  • Myllymaa S, Pasternack A, Mottershead DG, Poutanen M, Pulkki MM, Pelliniemi LJ, Ritvos O & Laitinen MP 2010 Inhibition of oocyte growth factors in vivo modulates ovarian folliculogenesis in neonatal and immature mice. Reproduction 139 587598. doi:10.1530/REP-09-0391.

    • Search Google Scholar
    • Export Citation
  • Palmer JS, Zhao ZZ, Hoekstra C, Hayward NK, Webb PM, Whiteman DC, Martin NG, Boomsma DI, Duffy DL & Montgomery GW 2006 Novel variants in growth differentiation factor 9 in mothers of dizygotic twins. Journal of Clinical Endocrinology and Metabolism 91 47134716. doi:10.1210/jc.2006-0970.

    • Search Google Scholar
    • Export Citation
  • Pedersen T & Peters H 1968 Proposal for a classification of oocytes and follicles in the mouse ovary. Journal of Reproduction and Fertility 17 555557. doi:10.1530/jrf.0.0170555.

    • Search Google Scholar
    • Export Citation
  • Sengle G, Ono RN, Sasaki T & Sakai LY 2011 Prodomains of transforming growth factor β (TGFβ) superfamily members specify different functions. Journal of Biological Chemistry 286 50875099. doi:10.1074/jbc.M110.188615.

    • Search Google Scholar
    • Export Citation
  • Shi M, Zhu J, Wang R, Chen X, Mi L, Walz T & Springer TA 2011 Latent TGF-[bgr] structure and activation. Nature 474 343349. doi:10.1038/nature10152.

  • Walton KL, Makanji Y, Wilce MC, Chan KL, Robertson DM & Harrison CA 2009 A common biosynthetic pathway governs the dimerization and secretion of inhibin and related transforming growth factor beta (TGFbeta) ligands. Journal of Biological Chemistry 284 93119320. doi:10.1074/jbc.M808763200.

    • Search Google Scholar
    • Export Citation
  • Yan C, Wang P, DeMayo J, DeMayo FJ, Elvin JA, Carino C, Prasad SV, Skinner SS, Dunbar BS & Dube JL 2001 Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Molecular Endocrinology 15 854866. doi:10.1210/me.15.6.854.

    • Search Google Scholar
    • Export Citation
  • Yeo CX, Gilchrist RB, Thompson JG & Lane M 2008 Exogenous growth differentiation factor 9 in oocyte maturation media enhances subsequent embryo development and fetal viability in mice. Human Reproduction 23 6773. doi:10.1093/humrep/dem140.

    • Search Google Scholar
    • Export Citation
  • Yoshino O, McMahon HE, Sharma S & Shimasaki S 2006 A unique preovulatory expression pattern plays a key role in the physiological functions of BMP-15 in the mouse. PNAS 103 1067810683. doi:10.1073/pnas.0600507103.

    • Search Google Scholar
    • Export Citation