Open Access Te Herenga Waka-Victoria University of Wellington
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Effects of Species Differences on Oocyte Regulation of Granulosa Cell Proliferation

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posted on 2021-11-12, 13:09 authored by Lin, Jia Yi

The role of oocytes in regulating ovulation quota between species is not fully understood. In humans, sheep, and rodents the oocyte-derived growth factors, bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9) have profound effects on ovarian follicular development and ovulation quota. The aim of these studies was to compare the ability of oocytes from sheep (low ovulation-rate) and rat (poly-ovulator) to stimulate radiolabelled ³H-thymidine uptake by granulosa cells (GC) both within and between the two species, and to assess species differences of GDF9 and BMP15 in co-incubations. For these experiments, oocytes denuded of cumulus-cells (DO) were co-incubated with a fixed number of GC from either species. Rat or sheep DO stimulated ³H-thymidine uptake by GC from the same species (P<0.005). Sheep oocytes also stimulated ³H-thymidine uptake by rat GC (P<0.001) but not vice versa. To investigate this further, oocytes and GC were co-incubated with monoclonal antibodies specific to GDF9 or BMP15 or to a hydatids antigen (control). Both sheep and rat oocyte stimulation of ³H-thymidine uptake by GC was inhibited with the GDF9 antibody (P<0.05) but not the control antibody, irrespective of the species of GC. Sheep DO stimulation of rat GC was also inhibited using an antibody to BMP15 (P<0.05). However, when using the BMP15 antibody to block the effects of rat DO on rat GC, no inhibition of ³H-thymidine uptake was observed (P=0.988). The molecular forms of GDF9 and BMP15 protein in oocyte lysates and spent media were examined by Western blotting under reducing conditions. For both species, GDF9 protein was present in the mature form in both the lysate and the spend media. For sheep oocytes, BMP15 protein was present as pro-mature and monomeric mature forms in the lysate and in the spent media, whereas from rats pro-mature forms were detected in the oocyte lysate but there was minimal evidence for the mature form in the spent media. The mRNA levels of GDF9, BMP15 and several cumulus cell (CC) genes were measured at 8h intervals over a 24h incubation period. For both sheep and the rat, GDF9 and BMP15 mRNA expression levels were highly correlated (R²=0.99). In the rat, the relative mean expression level of Gdf9 mRNA was four times higher than Bmp15 mRNA, whereas in sheep the relative mean expression ratio of GDF9 to BMP15 was approximately one. For these studies, two types of incubations were performed, namely COC alone or co-incubations of DO with GC. In co-incubations of DO with GC, the relative GDF9 and BMP15 mRNA levels did not change significantly over time, whereas incubations of COC showed that the relative levels of GDF9 and BMP15 mRNA declined significantly during the incubation period. The CC genes (FSHR, LHR, KITL, CX43, AROM and CYCD2) were measured during the 24h incubation period for the COC but not the DO experiment. In rats, none of the CC genes showed any changes in mRNA levels over time except Kitl, where levels at 8h and 16h mRNA levels were significantly lower compared to those at 0h and 24h. In sheep, there were no significant mRNA changes in any of the CC genes except AROM. The level of AROM mRNA reduced to near zero by 8h and remained at low levels at both 16h and 24h. These findings suggest that, under the incubation conditions used, that the gene expression levels for most CC genes were maintained but that the oocyte growth factors alone were unable to maintain AROM gene expression in sheep. In conclusion, major differences were observed in GDF9 and BMP15 expression and regulation between rats and sheep. The lower expression levels of Bmp15 mRNA and protein are likely the reason why rat DO failed to stimulate ³H-thymidine uptake by sheep GC. The evidence suggests that oocyte-derived GDF9 is sufficient to stimulate ³H-thymidine incorporation and presumably DNA synthesis in rat GC whereas in sheep both BMP15 and GDF9 are required. These findings raise the possibility that in species with a low ovulation rate phenotype there is a higher level of GDF9 mRNA and protein synthesis and that BMP15 is a major factor in restricting ovulation quota in mammals.


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Date of Award



Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains Copyright

Degree Discipline

Cell and Molecular Bioscience

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level


Degree Name

Doctor of Philosophy

Victoria University of Wellington Item Type

Awarded Doctoral Thesis



Victoria University of Wellington School

School of Biological Sciences


McNatty, Ken; Juengel, Jenny