257 Awardee Talk: Pursuit of Novel Factors that Regulate Ovarian Follicular Function

As follicles grow, theca cells (TC) and granulosa cells (GC) must proliferate with minimal differentiation while thecal vascularization increases so that follicles do not prematurely ovulate or luteinize before the oocyte is fully mature. In the early 2000s we used Affymetrix microarrays to discover several unique genes involved in ovarian follicular development. Thrombospondin and fibroblast growth factor (FGF) 2 receptor genes were stimulated by IGF1 in porcine GC. We compared GC gene expression in bovine cystic versus normal follicles and discovered several novel genes including Indian hedgehog protein (IHH), FGF9, brain ribonuclease (BRB), and G protein-coupled receptor 34 (GPR34), leading to identification of roles for these proteins in ovarian follicular development. During the past 10 years, follow-up TC microarray and mechanistic studies have identified FGF9 control of cell cycle proteins, tight junction proteins, and microRNA 221 (MIR221), and that the mitogenic and steroidogenic responses to the major trophic hormones of the ovary (including IGF1, LH and FSH) are altered by overexpression of MIR221 in GC. In addition, we discovered that: 1) FGF9 stimulates GC and TC mitosis while inhibiting steroidogenesis; 2) FGF9 induces E2 transcription factor (E2F)-1, E2F-8 and cyclin D1 (CCND1), and that both IGF1 and vascular endothelial growth factor-A (VEGFA) synergize with FGF9 to further induce E2F8 and CCND1 mRNA; 3) FGF9 induces the nuclear protein UHRF1; and 4) an E2F inhibitor blocks the stimulatory and inhibitory effects of FGF9 on GC proliferation and steroidogenesis, respectively, and down-regulates UHRF1 mRNA and up-regulates VEGFA mRNA. Thus, aberrant production of FGF9 and the factors it induces/inhibits may lead to vascular dysfunction and ovarian disorders such as ovarian cysts. With additional research, knowledge about these newly identified factors may be used to help the livestock industry improve reproductive efficiency via new treatments for estrous synchronization, superovulation and cystic ovaries.

Analysis of the Reversible Impact of the Chemodrug Busulfan on Mouse Testes

Spermatogenesis is a process within the testis that leads to the production of spermatozoa. It is based on a population of spermatogonial stem cells, which have the capacity to self-renew and to differentiate throughout life to ensure the functions of reproduction are maintained. Male fertility disorders are responsible for half of the cases of infertility in couples worldwide. It is well known that cancer treatments are associated with reversible or irreversible fertility disorders. Busulfan (Bu) is an alkylating agent that significantly inhibits spermatogenesis. The present study relied on a combination of in vivo and in vitro approaches as well as RNAseq analysis to characterize the effects of Bu, in which mouse testes were used as a model. An in silico analysis revealed that many of the Bu-modulated genes are potentially regulated by the SIN3 Transcription Regulator Family Member A (SIN3A) and E2F Transcription Factor (E2F) families of transcription factors. The results demonstrate that the deregulated genes function in processes related to the cell cycle, DNA repair, and cell death mechanisms, including the Tumor Protein 53 (TP53) pathway. This reinforces the role of the TP53 signaling pathway as a major player in Bu effects. In addition, Bu altered the patterns of mRNA accumulation for various genes in undifferentiated spermatogonia. This work provides significant insight into the kinetics and impacts of busulfan, which could pave the way for developing strategies to minimize the impact of chemodrugs and, thus, could lead to germ cell lineage regeneration following anticancer treatments.

Single mage gene in the chicken genome encodes CMage, a protein with functional similarities to mammalian type II Mage proteins

In mammals, the type II melanoma antigen (Mage) protein family is constituted by at least 10 closely related members that are expressed in different tissues, including the nervous system. These proteins are believed to regulate cell cycle withdrawal, neuronal differentiation, and apoptosis. However, the analysis of their specific function has been complicated by functional redundancy. In accordance with previous studies in teleosts and Drosophila, we present evidence that only one mage gene exists in genomes from protists, fungi, plants, nematodes, insects, and nonmammalian vertebrates. We have identified the chicken mage gene and cloned the cDNA encoding the chick Mage protein (CMage). CMage shares close homology with the type II Mage protein family, and, as previously shown for the type II Mage proteins Necdin and Mage-G1, it can interact with the transcription factor E2F-1. CMage is expressed in specific regions of the developing nervous system including the retinal ganglion cell layer, the ventral horn of the spinal cord, and the dorsal root ganglia, coinciding with the expression of the neurotrophin receptor p75 (p75NTR) in these regions. We show that the intracellular domain of p75NTR can interact with both CMage and Necdin, thus preventing the binding of the latter proteins to the transcription factor E2F-1, and facilitating the proapoptotic activity of E2F-1 in N1E-115 differentiating neurons. The presence of a single mage gene in the chicken genome, together with the close functional resemblance between CMage and Necdin, makes this species ideal to further analyze signal transduction through type II Mage proteins.