Abstract
The formation of primordial follicles involves the interaction between the oocytes and surrounding somatic cells, which differentiate into granulosa cells. Estradiol-17ß (E) promotes primordial follicle formation in vivo and in vitro; however, the underlying mechanisms are poorly understood. The expression of an ERBB3-binding protein 1 (EBP1) is downregulated in 8-day old hamster ovaries concurrent with the increase in serum estradiol levels and the formation of primordial follicles. The objectives of the present study were to determine the spatio-temporal expression and putative E regulation of EBP1 in ovarian cells during perinatal development with respect to primordial follicle formation. Hamster EBP1 nucleic acid and amino acid sequences were more than 93% and 98% similar, respectively, to those of mouse and human, and contained nucleolar localization signal, RNA-binding domain and several phosphorylation sites. EBP1 protein was present in somatic cells and oocytes from E15, and declined in oocytes by P1 and in somatic cells by P5. Thereafter, EBP1 expression increased through P7 with a transient decline on P8 primarily in interstitial cells. EBP1 mRNA levels mirrored protein expression pattern. E treatment on P1 and P4 upregulated EBP1 expression by P8 whereas E treatment on P4 downregulated it by 72 h suggesting a compensatory upregulation due to E pretreatment. Treatment with an FSH-antiserum, which suppressed primordial follicle formation, prevented the decline in EBP1 levels, and the effect was reversed by E treatment. Therefore, the results provide the first evidence that EBP1 may play an important role in mediating the effect of E in the differentiation of somatic cells into granulosa cells during primordial follicle formation.
Highlights
Undifferentiated somatic cells surrounding the egg nests differentiate into pregranulosa cells, and invade the egg nest to encircle individual oocytes forming primordial follicles
MO), human holotransferrin, selenium and bovine insulin were purchased from Collaborative research (Bedford, MA), Falcon non-tissue culture inserts and plates, solvents for histology, Western blotting supplies and other fine chemicals were purchased from Thermo-Fisher and GE Biosciences (Pittsburgh, PA) and, plastic embedding medium was from Electron Microscopy Sciences (Hatfield, PA), RNA extraction and quantitative RT-PCR chemicals were from Qiagen (Valencia, CA), primers and fluorescence-labeled probes were from Eurofins (Huntsville, AL), the polyclonal ERBB3 binding protein 1 (EBP1) antibody was purchased from Millipore Corporation (Billerica, MA), HRP-conjugated and DyLight fluorescence-tagged second antibodies were from Jackson Immunoresearch (West Grove, PA)
The results of this study provide the first evidence that EBP1, an ERBB3 binding protein, is expressed in perinatal ovary cells and the expression is downregulated in ovarian somatic cells with their differentiation into early granulosa cells of primordial follicles
Summary
Undifferentiated somatic cells surrounding the egg nests differentiate into pregranulosa cells, and invade the egg nest to encircle individual oocytes forming primordial follicles. Pregranulosa cells become granulosa cells once primordial follicles are formed, and this represents the first critical morphogenetic process in folliculogenesis. The regulation of primordial follicle morphogenesis is not well understood. E as well as ESR1 and ESR2 levels increase during primordial follicle formation in vivo [13,14,15]. E promotes primordial follicle formation in vivo and in vitro. Proteomic analysis of developing hamster ovaries has identified ERBB3 binding protein 1 (EBP1) to be significantly downregulated in hamster ovaries on postnatal day 8 (P8) when morphologically distinct primordial follicles can be first identified [16]
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