In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.

Members of the Pumilio (also called PUF) gene family belong to a class of highly conserved developmental regulators that are present in both flies and humans. Much is known about the function of Pumilio genes in invertebrate development, in particular their role as stem cell factors required for maintenance and/or self-renewal of germline stem cells in Drosophila and Caenorhabditis elegans. It remains unknown whether Pumilio genes are also required for development in mammals; however, several lines of evidence suggest similar functions based on extensive sequence homology, similar RNA-binding properties to their invertebrate counterparts and well-documented interactions with germ cell factors required for fertility. Here we report characterization of a gene trap mutation that disrupts the mouse Pumilio-2 (Pum2) gene. Our data confirm that Pumilio-2 is expressed most abundantly in germ cells with the highest expression in undifferentiated gonocytes and spermatogonia. Furthermore, the mutation in Pum2 results in significantly smaller testes although the mutants are otherwise viable and fertile. In addition, we observed no stronger reproductive defects on a genetic background homozygous for a Pum2 null mutation and heterozygous for a Dazl mutation than Pum2 mutant alone. Thus, as in C. elegans where single members of the Pumilio gene family are dispensable for reproductive development and viability, this individual member of the Pumilio gene family in mice is also not essential for reproduction or viability.

SummaryOuter dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a wide-spread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein-protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice.

Anti-Mullerian hormone (Amh) is expressed in the granulosa cells of growing and preovulatory follicles in the mouse ovary where it acts to decrease responsiveness to follicle stimulating hormone (FSH) and plays a role in inhibiting primordial follicle recruitment. Recently, co-culture of isolated oocytes and granulosa cells has demonstrated that Amh expression is up-regulated in the presence of oocytes and, at preantral stages, this effect is dependent upon close contact. In Sertoli cells, Amh expression is regulated by several transcription factors including SOX9, SF1, GATA4, WT1, and DAX1, which, with the exception of SOX9, are also expressed in granulosa cells where GATA4 is known to up-regulate Amh expression antagonised by FOG2. Here, we demonstrate that Sox8, which is closely related to Sox9 and encodes the protein SOX8 which can transactivate Amh, is expressed in the postnatal mouse ovarian follicle but is not co-expressed with Amh in the granulosa cells of preantral follicles. Sox8 expression was found only in the oocytes of preantral follicles and in the oocytes, cumulus cells, and mural granulosa cells of preovulatory follicles. Also, increased expression of Amh in granulosa cells co-cultured with oocytes was not associated with increased mRNA levels of the transcription factors Sf1, Gata4, Wt1, Dax1, or Fog2. These findings reveal Sox8 expression in the ovarian follicle and show that oocyte regulation of Amh expression is not due to oocyte regulation of Sf1, Gata4, Wt1, Dax1, or Fog2 expression in granulosa cells.