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Derek A Heath, Janet L Pitman and Kenneth P McNatty

Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are oocyte-secreted factors with demonstrable effects on ovarian follicular development and ovulation rate. However, the molecular forms of BMP15 and GDF9 produced by oocytes remain unclear. The aims herein, using Western blotting (WB) procedures with specific monoclonal antibodies (mabs), were to identify the molecular forms of BMP15 and GDF9 synthesised and secreted by isolated ovine (o) and bovine (b) oocytes in vitro. The mabs were known to recognise the biological forms of BMP15 or GDF9 since they had previously been shown to inhibit their bioactivities in vitro and in vivo. Using recombinant variants of oBMP15 and oGDF9, including a cysteine mutant form of oBMP15 (S356C) and a human (h) BMP15:GDF9 heterodimer (cumulin), it was established that the mabs were able to identify monomeric, dimeric, promature and higher-molecular-weight forms of BMP15 and GDF9 and cumulin (GDF9 mab only). After using non-reducing, reducing and reducing + cross-linking conditions, the major oocyte-secreted forms of o and b BMP15 and GDF9 were the cleaved and uncleaved monomeric forms of the promature proteins. There was no evidence for dimeric or heterodimeric forms of either mature BMP15 or GDF9. From in silico modelling studies using transforming growth factor beta (TGFB), activin or BMP crystal templates, and both present and previously published data, a model is proposed to illustrate how the monomeric forms of BMP15 and GDF9 may interact with their type II and type I cell-surface receptors to initiate the synergistic actions of these growth factors.

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Kenneth P McNatty, Derek A Heath, Zaramasina Clark, Karen Reader, Jennifer L Juengel and Janet L Pitman

Ewes heterozygous for combinations of the Inverdale (FecXI; I+), Booroola (FecB; B+) and Woodlands (FecX2W; W+) mutations have ovulation rates higher than each mutation separately. The aims of the experiments described herein were to examine the ovarian phenotypes in I+B+ and I+B+W+ ewes and to compare these with the appropriate ++ (controls), I+ and BB animals available for this study. The mean ± s.e.m. ovulation rates in the ++ (n = 23), I+ (10), I+B+ (7), I+B+W+ (10) and BB (3) animals were 1.8 ± 0.1, 2.5 ± 0.2, 6.6 ± 1.0, 9.6 ± 0.9 and 9.7 ± 0.9 respectively. The maximum number of granulosa cells per follicle in the ++ and I+ genotypes was accumulated after exceeding 5 mm diameter, whereas in I+B+, I+B+W+ and BB animals, this was achieved when follicles reached >2–3 mm. The number of putative preovulatory follicles, as assessed from those with LH-responsive granulosa cells, 24 h after the induction of luteolysis, was higher (P < 0.01) in the I+B+ and I+B+W+ compared to the ++ and I+ genotypes. The median follicular diameters of these follicles in the ++, I+, I+B+, I+B+W+ and BB genotypes were 6, 5, 3, 3 and 3 mm respectively. The total number of granulosa cells in the putative preovulatory follicles when added together, and total mass of luteal tissue, did not differ between the genotypes. Thus, despite large ovulation rate differences between animals with one or more fecundity genes, the total cell compositions over all preovulatory follicles and corpora lutea, when added together, are similar to that from the one or two such follicles in the wild types.

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Jia Yi Lin, Janet L Pitman-Crawford, Adrian H Bibby, Norma L Hudson, C Joy McIntosh, Jennifer L Juengel and Kenneth P McNatty

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate 3H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R 2=0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.

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Jia Yi Lin, Janet L Pitman-Crawford, Adrian H Bibby, Norma L Hudson, C Joy McIntosh, Jennifer L Juengel and Kenneth P McNatty

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Zaramasina L Clark, Derek A Heath, Anne R O’Connell, Jennifer L Juengel, Kenneth P McNatty and Janet L Pitman

Ewes with single copy mutations in GDF9, BMP15 or BMPR1B have smaller preovulatory follicles containing fewer granulosa cells (GC), while developmental competency of the oocyte appears to be maintained. We hypothesised that similarities and/or differences in follicular maturation events between WT (++) ewes and mutant ewes with single copy mutations in BMP15 and BMPR1B (I+B+) are key to the attainment of oocyte developmental competency and for increasing ovulation rate (OR) without compromising oocyte quality. Developmental competency of oocytes from I+B+ animals was confirmed following embryo transfer to recipient ewes. The microenvironment of both growing and presumptive preovulatory (PPOV) follicles from ++ and I+B+ ewes was investigated. When grouped according to gonadotropin-responsiveness, PPOV follicles from I+B+ ewes had smaller mean diameters with fewer GC than equivalent follicles in ++ ewes (OR = 4.4 ± 0.7 and 1.7 ± 0.2, respectively; P < 0.001). Functional differences between these genotypes included differential gonadotropin-responsiveness of GC, follicular fluid composition and expression levels of cumulus cell-derived VCAN, PGR, EREG and BMPR2 genes. A unique microenvironment was characterised in I+B+ follicles as they underwent maturation. Our evidence suggests that GC were less metabolically active, resulting in increased follicular fluid concentrations of amino acids and metabolic substrates, potentially protecting the oocyte from ROS. Normal expression levels of key genes linked to oocyte quality and embryo survival in I+B+ follicles support the successful lambing percentage of transferred I+B+ oocytes. In conclusion, these I+B+ oocytes develop normally, despite radical changes in follicular size and GC number induced by these combined heterozygous mutations.