Abstract:
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.