Secretogranin II is a novel periovulatory gene that is induced by human chorionic gonadotropin in human and rodent granulosa cells

Patrick R. Hannon 1 ● Linah Al-Alem 1 ● Katherine L. Rosewell 1 ● James W. Akin 2 ● Thomas E. Curry, Jr. • Department of Obstetrics & Gynecology, University of Kentucky ● Bluegrass Fertility Center, Lexington, KY

The events that occur in the ovary following the luteinizing hormone (LH) surge leading to ovulation are paramount for fertility, so much so that defects in ovulation are the leading cause of female infertility. Thus, there is an urgent need to elucidate the mechanisms that control ovulation, including a more thorough understanding of the mediators of ovulation. Evidence from our laboratory suggests that secretogranin II (SCG2) is a potential mediator of ovulation across species. In other tissues, SCG2 is involved in hormone packaging and secretion or is cleaved to peptides that promote leukocyte migration and angiogenesis. These processes are vital for ovulation; however, the characterization and regulation of SCG2 in ovarian function and ovulation is unstudied. Our hypothesis is that the LH surge increases SCG2 levels in human and rodent granulosa cells, via classic LH receptor signaling pathways, to serve as a mediator of ovulation. To test this, women undergoing tubal sterilization were recruited into this study and monitored by transvaginal ultrasound for 2-3 menstrual cycles. The dominant follicle was surgically excised at the preovulatory phase or women were given 250μg hCG and dominant follicles were collected at one of the three time intervals after hCG: early ovulatory (12-18h), late ovulatory (18-34h) and postovulatory (44-70h). Granulosa cells were isolated and processed for mRNA analysis or whole follicles were processed for immunohistochemistry. Our results indicated that hCG significantly increased the mRNA levels of SCG2 in early (15 fold increase), late (19 fold increase), and postovulatory (15 fold increase) granulosa cells when compared to the preovulatory granulosa cells in vivo (n=3-6 patients; p≤0.05). In the whole follicle in vivo, SCG2 protein staining was rather prominent in the theca and stroma across the periovulatory period but increased dramatically in the granulosa cells with hCG treatment. In order to examine the regulation of SCG2 induction, a validated model of cultured granulosa-lutein cells (GLCs) from IVF patients was utilized. GLCs were collected on the day of oocyte retrieval and were allowed to acclimate in culture for 6 days to regain LH/hCG responsiveness. The GLCs were then treated with vehicle control (DMSO), AG1478 (5µM; EGF receptor antagonist), hCG (1 IU), and hCG+AG1478 for 0, 6, 12, and 24h. Similar to the results in vivo, hCG significantly increased the mRNA levels of SCG2 at the 6 (9 fold increase) and 12h (8 fold increase) timepoints when compared to controls; however, the mRNA levels of SCG2 in the hCG+AG1478 groups were significantly reduced by 50% when compared to the hCG group (n=7-10 patients; p≤0.05). To further test our hypothesis in the rodent, the well-characterized immature rodent PMSG/hCG model was utilized. Following 0, 4, 8, 12, and 24h of hCG treatment, ovaries were removed, and follicles were punctured to collect granulosa cells for qPCR analysis in vivo. In mouse granulosa cells, hCG significantly increased the mRNA levels of Scg2 at 4, 8, and 12h when compared to the 0h group (n=5-6 mice; p≤0.05). In rat granulosa cells, hCG significantly increased the mRNA levels of Scg2 at 8 and 12h when compared to the 0h group (n=5-6 rats; p≤0.05). These data are the first to demonstrate that SCG2 is induced by hCG in the human and rodent periovulatory granulosa cell and that EGF receptor signaling partially mediates the hCG-induced increase in human periovulatory SCG2 expression. Supported by P01HD071875, HD057446, and the Lalor Foundation Postdoctoral Fellowship.

Postdoc Category

• 1st Place ($700): Dr. Lindsey R. Conroy – Markey Cancer Center – “Visualization of lung tumor microenvironmental glycogen by next generation digital pathology”

• 2nd Place ($500): Dr. Jacqueline Rivas - Microbiology, Immunology & Molecular Genetics – “Enhancing responses to immunotherapy in chronic lymphocytic leukemia”

• 3rd Place ($300): Dr. Felicia Michael (not pictured) - Spinal Cord and Brain Injury Research Center – “Optimization of mitochondrial transplantation via engineered erodible hydrogels”

Graduate Student Category

• 1st Place: Lyndsay E. Young - Molecular and Cellular Biochemistry – “In situ analysis of microenvironmental glycogen in Ewing’s sarcoma patient samples by mass spectrometry imaging”

• 2nd Place: Jenni Ho - Toxicology and Cancer Biology – “Extracellular vesicles released after cranial radiation contain protein markers of neuronal injury and induce immune activation”

• 3rd Place (Tie): Na Ding - Toxicology and Cancer Biology – “Elucidating peroxiredoxin IV secretion via unconventional protein secretory pathway”; Caitlin Miller - Toxicology and Cancer Biology – “Adaptation of redox state in cancer cells that survive radiation”

Postdoc Category

• First Place ($700): Meenakshi Banerjee- Department of Molecular and Cellular Biochemistry, “Endocytosis of Human Immunodeficiency Virus-1 (HIV-1) activates platelets”

• Second Place ($500): Ivan Vechetti - Department of Physiology, “Exercise-induced exosomal myomiR regulation of adipocyte metabolism”

• Third Place ($300): Brad Hubbard - Spinal Cord and Brain Injury Research Center, “Acute mitochondrial impairment underlies prolonged cellular dysfunction after repeated mild traumatic brain injuries”

Postdoc Category

• First Place ($700): Ramon Sun - "Ubiquitin-dependent nuclear glycogenolysis results in transcriptional activation"

• Second Place ($500): Hisashi Sawada - "LRP1 deletion in smooth muscle cells of the outer aortic media promotes angiotensin II-induced thoracic aortic aneurysm"

• Third Place ($300): Rakshamani Tripathi- "Abl and Arg mediate cysteine cathepsin secretion to facilitate melanoma invasion and metastasis"

Postdoc Category

• 1st Place ($700): Dr. Patrick Hannon - “Secretogranin II is a novel periovulatory gene that is induced by human chorionic gonadotropin in human and rodent granulosa cells”, Department of Obstetrics & Gynecology

• 2nd Place ($500): Dr. Nicolás Cifuentes-Muñoz - “Imaging the replication and assembly of the human metapneumovirus (HMPV) genome”, Department of Molecular & Cellular Biochemistry

• 3rd Place ($300): Dr. Wen Wen - “Ethanol induces ER-stress and the expression of mesencephalic astrocyte-derived neurotrophic factor in neuroblastoma cells”, Department of Pharmacology & Nutritional Sciences