Reassessment of intra- and extra-ovarian expression of growth differentiation factor-9
Mammalian ovarian organogenesis is characterized temporally by oogonial mitosis, oogonial apoptosis, rescue of germ cells via interaction with somatic cells to form primordial follicles, and entrance into meiosis. Recent studies suggest that germ cell’s activity participate in this process via the production of local regulatory factors. Growth differentiation factor-9 (GDF-9), a novel transforming growth factor-family member, is expressed in ovaries of various species as a crucial factor in ovarian follicular development. Likewise, the expressional pattern of GDF-9 in nonovarian tissues has remained elusive, as current data continues to support the exclusive expression of GDF-9 within the mammalian ovary. Recently, our lab became interested in the pattern of expression associated with GDF-9 as it pertains to ovarian organogenesis and follicle formation in the neonatal hamster ovary due to the ability to obtain ovaries at specific stages of follicular organization. Further interests include investigating the possible expression of GDF-9 in nonovarian tissues. Consensus oligonucleotide primer pairs spanning at least one intron for GDF-9 were determined by analysis of the gene sequences for human, mouse, rat, bovine, and ovine for use in the hamster. mRNA for GDF-9 in ovarian and non-ovarian tissues was detected by RT/PCR using total RNA. PCR products were sequenced to determine the degree of homology for the gene relative to that of other species (mouse 91%, rat 90%, and human 84%). mRNA for GDF-9 was detected in ovarian samples for all days examined (Days 1, 3, 5, and 6-9 post delivery [Day 0]. This time frame reflects periods of active oogonial mitosis (Days 1 and 3), oogonial atresia (Day 5), and primordial follicle formation (Days 6-9). Translation of GDF-9 was observed prior to primordial follicle development (Day 5). For non-ovarian analysis mRNA, expression of GDF-9 was detected in several tissues (liver, kidney, spleen, and testis) at various stages of growth and development. Furthermore, white blood cells retrieved from adult hamsters revealed mRNA expression of this novel growth factor. Kidney perfusion experiments supported the idea that mRNA expression of GDF-9 in non-ovarian tissues could be the resultant of circulating white blood cells as a 35% reduction was seen in perfused samples. Immunological analyses of hamster ovarian sections detected expression of GDF-9 within the highly vascularized regions of the corpus luteum. In fact, re-investigations of early stage immunological analyses for GDF-9 suggest that mast cells are potential sites of GDF-9 protein expression. Reports suggest that GDF-9 is expressed exclusively in the ovary, specifically within the oocytes, and that expression of GDF-9 is not seen prior to primordial follicle formation. Our results conclude several revolutionary findings pertaining to ovarian and non-ovarian expression of GDF-9. Importantly we report that GDF-9 mRNA is expressed in the ovary prior to and during primordial follicle formation, and after ovarian senescence. And that GDF-9 is non-ovarian specific as previously indicated by scientific literature as we have localized non-ovarian transcription and translation of GDF-9 to white blood cells. Currently, only one type of immunologic cell, mast cells, has been identification to express GDF-9. Further individual white blood cell assays will be required to identify additional specific white blood cell expression of GDF-9.
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