Article

Mouse Oocyte Mitogenic Activity Is Developmentally Coordinated throughout Folliculogenesis and Meiotic Maturation

The Reproductive Medicine Unit, Adelaide University, Adelaide, 5011, Australia.
Developmental Biology (Impact Factor: 3.64). 01/2002; 240(1):289-98. DOI: 10.1006/dbio.2001.0451
Source: PubMed

ABSTRACT Oocytes secrete soluble factors that regulate the growth and differentiation of follicular cells, including maintenance of the distinctive cumulus cell phenotype. This study determines whether the mitogenic activity of oocytes is developmentally regulated and examines the responsiveness of follicular cells to oocytes at different stages of follicular development. Prepubertal SV129 mice of varying ages were primed with 5 IU equine chorionic gonadotropin (eCG) and oocytes/zygotes collected either 46 h post-eCG (immature oocytes), 12 h after administration of 5 IU human CG (hCG; ovulated ova), or 12 h post-hCG and mating (zygotes). Mural granulosa cells (MGC) from antral follicles and GC from preantral follicles were cultured +/- denuded oocytes (DO) for 18 h, followed by a 6-h pulse of [(3)H]thymidine as an indicator of cellular DNA synthesis. Coculturing MGC with meiotically maturing oocytes led to a dose-dependent increase in [(3)H]thymidine incorporation (20-fold above control levels at 0.5 DO/microl). However, [(3)H] counts remained unchanged from control levels when cultured with meiotically incompetent DO from 11- to 15-day-old mice (3% germinal vesicle breakdown; GVB), irrespective of dose of DO or developmental status of GC (MGC or preantral GC). In some treatments, spontaneous meiotic resumption of competent oocytes was prevented by culturing with 5 microM milrinone, a selective inhibitor of oocyte-specific cyclic nucleotide phosphodiesterase. The mitogenic capacity of oocytes was found to decline during and after oocyte maturation. [(3)H]Thymidine incorporation in MGC was highest (11-fold above controls) when cultured with meiotically inhibited (milrinone-treated) GV DO, stimulated 5.5-fold by culture with maturing oocytes, 3-fold with ovulated ova, and unstimulated by zygotes. [(3)H]Thymidine incorporation in MGC was not altered by the dose of milrinone, either in the presence or absence of DO. Metaphase I marked the beginning of the decline in the capacity of oocytes to promote MGC DNA synthesis. These results demonstrate that the capacity of oocytes to promote proliferation of granulosa cells follows a developmental program, closely linked to oocyte meiotic status, increasing with the acquisition of meiotic competence and declining during and after oocyte maturation.

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Available from: Robert B Gilchrist, Aug 07, 2014
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    • "There are various paracrine factors involved in regulating follicle growth (McGrath et al., 1995; Dube et al., 1998; Gilchrist et al., 2001; Markstrom et al., 2002). Moreover, it has been suggested that the growth and maturation of intrafollicular oocytes in preantral follicles are significantly influenced by cell to cell interaction between oocytes and surrounding follicular cells (granulosa and theca cells) through paracrine methods (Eppig et al., 2002). "
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    ABSTRACT: This study was conducted to improve the yield of mature oocytes from in vitro-culture of ovarian primary follicles by optimizing follicle retrieval from neonatal mice of different ages. Primary follicles of 75 to 99 μm in diameter were collected daily from 7- to 14-day-old neonatal mice, and subsequently cultured in α-MEM medium. Number of primary follicles isolated, growth of the follicle during in vitro-culture and maturation of intrafollicular oocytes were monitored. Overall, mean number of preantral follicles per animal was improved from 10.7 to 88.7 as the age of follicle donors was increased from 7 to 14-day-old. Number of primary follicles was increased gradually up to 11-day-old (35.7 follicle per an animal), then reduced to 29 in 14-day-old (p = 0.0013). More follicles retrieved from 10-day-old or 11-day-old females maintained their morphological normality at the end of primary culture than the follicles retrieved from 9-day-old. Of those cultured, primary follicles retrieved from 11-day-old mice yielded largest larger number of early secondary follicles than the follicles retrieved from in the other ages (39 vs. 13 to 29%). More than 3.3-times increase (0.86 to 2.86; p<0.05) in an average number of mature oocytes per animal was observed in the group of 11-day-old, compared with 9-day-old. However, no difference was found in the percentage of primary follicles developing into the pseudoantral stage (21 to 30%; p = 0.5222) and in the percentage of oocytes mucified (32 to 39%; p = 0.5792). In conclusion, a positive correlation between retrieval time and follicle growth was detected, which influences the efficiency to derive mature oocytes by follicle culture.
    Asian Australasian Journal of Animal Sciences 05/2012; 25(5):629-34. DOI:10.5713/ajas.2010.10249 · 0.56 Impact Factor
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    • "Stimulation of Ccnd2 (Gilchrist et al., 2006) Stimulation of GC/CC DNA synthesis, cell number or follicle growth (Vanderhyden et al., 1992; Lanuza et al., 1998; Li et al., 2000; Gilchrist et al., 2001; Eppig et al., 2002; Brankin et al., 2003; Gilchrist et al., 2003b; Glister et al., 2003; Gilchrist et al., 2004; Hickey et al., 2005; Gilchrist et al., 2006) Interaction of OSFs with IGF-I (Lanuza et al., 1998; Li et al., 2000; Brankin et al., 2003; Gilchrist et al., 2003b; Hickey et al., 2005) Stimulation of CC Ar (Diaz et al., 2007) "
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    ABSTRACT: This chapter contains sections titled: Introduction Historical Background Localization and Specificity Structure and Genetic Diversity of Gdf9 and Bmp15 Signalling Mechanisms of Gdf9 and Bmp15 Roles of Oocyte-Secreted Factors Manipulation and Use in Reproductive Technologies Concluding Remarks References
    01/2012: pages (in press); Wiley Blackwell Publishing.
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    • "Mural granulosa and cumulus cell [ 3 H]-thymidine incorporation assay This bioassay was performed as previously described (Gilchrist et al., 2001, 2004, 2006). Briefly, isolated cumulus cell clumps were generated by mechanically denuding cumulus – oocyte complexes of their cumulus cells by vigorously pipetting complexes up and down with a P200 pipette. "
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    ABSTRACT: Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are two proteins selectively expressed in the oocyte which are essential for normal fertility. Both of these proteins are members of the transforming growth factor beta (TGF-β) superfamily and as such are produced as pre-proproteins, existing after proteolytic processing as a complex of the respective pro and mature regions. Previous work has shown that these two proteins interact both at the genetic and cellular signalling levels. In this study, our aim was to determine if the purified mature regions of GDF9 and BMP15 exhibit synergistic interactions on granulosa cells and to determine if such interactions are specific to these two proteins. We have used primary cultures of murine granulosa cells and [(3)H]-thymidine incorporation or transcriptional reporter assays as our readouts. We observed clear synergistic interactions between the mature regions of GDF9 and BMP15 when either DNA synthesis or SMAD3 signalling were examined. GDF9/BMP15 synergistic interactions were specific such that neither factor could be replaced by an analogous TGF-β superfamily member. The GDF9/BMP15 synergistic signalling response was inhibited by the SMAD2/3 phosphorylation inhibitor SB431542, as well as inhibition of the mitogen-activated protein kinase or rous sarcoma oncogene (SRC) signalling pathways, but not the nuclear factor kappa B pathway. In this study, we show that purified mature regions of GDF9 and BMP15 synergistically interact in a specific manner which is not dependent on the presence of a pro-region. This synergistic interaction is targeted at the SMAD3 pathway, and is dependent on ERK1/2 and SRC kinase signalling.
    Molecular Human Reproduction 09/2011; 18(3):121-8. DOI:10.1093/molehr/gar056 · 3.48 Impact Factor
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