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The migratory and matrix-invading capacities of the cumulus–oocyte complex have been shown to be important for the ovulatory process. In metastatic cancers, these capacities are due to increased expression of proteases, however, there is limited information on protease expression in the cumulus–oocyte complexes. The present study examined cumulus–oocyte complex expression of plasmins, matrix metalloproteases, and A Disintegrin and Metalloproteinase with Thrombospondin Motifs family members in the rat and human. In the rat, human chorionic gonadotropin (hCG) administration increased cumulus–oocyte complex expression of Mmp2, Mmp9, Mmp13, Mmp14, Mmp16, Adamts1, and the protease inhibitors Timp1, Timp3,and Serpine1by 8–12 h. This ovulatory induction of proteases in vivo could be mimicked by forskolin and ampiregulin treatment of cultured rat cumulus–oocyte complexes with increases observed in Mmp2, Mmp13, Mmp14, Mmp16, Mmp19, Plat, and the protease inhibitors Timp1, Timp3,and Serpine1.Comparison of expression between rat cumulus–oocyte complexes and granulosa cells at the time of ovulation showed decreased Mmp9and increased Mmp13, Mmp14, Mmp16, Adamts1, Timp1, and Timp3expression in the cumulus–oocyte complexes. In human, comparison of expression between cumulus and granulosa cells at the time of in vitro fertilization retrieval showed decreased MMP1, MMP2, MMP9, and ADAMTS1, while expression of MMP16, TIMP1,and TIMP3were increased. Treatment of expanding rat cumulus–oocyte complexes with a broad spectrum matrix metalloproteases inhibitor, GM6001, significantly reduced the migration of cumulus cells in vitro. These data provide evidence that multiple proteases and their inhibitors are expressed in the cumulus–oocyte complex and play an important role in imparting the migratory phenotype of the cumulus–oocyte complex at the time of ovulation.Summary SentenceMultiple proteases and their inhibitors are induced in the cumulus–oocyte complex (COC) during the periovulatory period and potentially play an important role in imparting the migratory phenotype of the COC at the time of ovulation.

The luteinizing hormone (LH) surge induces paracrine mediators within the ovarian follicle that promote ovulation. The present study explores neurotensin (NTS), a neuropeptide, as a potential ovulatory mediator in the mouse ovary. Ovaries and granulosa cells (GCs) were collected from immature 23-day-old pregnant mare serum gonadotropin primed mice before (0 h) and after administration of human chorionic gonadotropin (hCG; an LH analog) across the periovulatory period (4, 8, 12, and 24 h). In response to hCG, Ntsexpression rapidly increased 250-fold at 4 h, remained elevated until 8 h, and decreased until 24 h. Expression of Nts receptors for Ntsr1remained unchanged across the periovulatory period, Ntsr2was undetectable, whereas Sort1expression (also called Ntsr3) gradually decreased in both the ovary and GCs after hCG administration. To better understand Ntsregulation, inhibitors of the LH/CG signaling pathways were utilized. Our data revealed that hCG regulated Ntsexpression through the protein kinase A (PKA) and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways. Additionally, epidermal-like-growth factor (EGF) receptor signaling also mediated Ntsinduction in GCs. To elucidate the role of NTS in the ovulatory process, we used a Ntssilencing approach (si-Nts) followed by RNA-sequencing (RNA-seq). RNA-seq analysis of GCs collected after hCG with or without si-Nts identified and qPCR confirmed Ell2, Rsad2, Vps37a,and Smtnl2as genes downstream of Nts. In summary, these findings demonstrate that hCG induces Ntsand that Ntsexpression is mediated by PKA, p38MAPK, and EGF receptor signaling pathways. Additionally, NTS regulates several novel genes that could potentially impact the ovulatory process.Ntsexpression increased 250-fold 4 h after hCG in mouse ovaries and is regulated through classical hCG signaling pathways. Using siRNA followed by RNAseq, Ell2, Rsad2, Vps37a, and Smtnl2were identified as NTS downstream targets in granulosa cells.

Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitroPanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation.