College of Agriculture & Natural Resources
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Item OVARIAN STROMAL CELLS IMPROVE SURVIVAL, BUT NOT GROWTH, IN PRE- AND EARLY ANTRAL FELINE FOLLICLES(2024) Marks, Batsheva Naomi; Keefer, Carol; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ovarian stromal cells act as crucial support and regulators for in vivo folliculogenesis; however, less is known about their effect on in vitro grown follicles. The objective of this study was to investigate the impact of ovarian stromal cell co-culture or conditioned medium (CM) on survival and development of cat pre-, early, and antral follicles in vitro. Ovaries were obtained from cats older than six months (n = 3), then enzymatically digested to release stromal cells. The ovarian stromal cells were allowed to grow to confluency in a T75 flask, before being cryopreserved for long term storage in liquid nitrogen. Cells were thawed one week prior to follicular culture onset, and passaged once before CM collection. CM was subsequently removed 24 - 48 hours after feeding, and stored at -80C until used. Ovarian follicles were mechanically isolated from cats older than six months (n = 23 cats, 155 follicles), encapsulated in 0.5% alginate hydrogel. The isolated follicles were then divided into five treatment groups (control, ovarian stromal cell co-culture, 20% CM, 50% CM, and 100% CM in Endothelial Cell Growth Medium), and classified based on initial diameter as preantral (224.4 + 4.7 m), early antral (394.8 + 7.4 m), or antral (592.2 + 18.8 m). Culture subsequently lasted for 13 days, and survival and growth of the follicles were evaluated on Days 0, 4, 6, 8, 11 and 13. At the end of culture, follicles were assessed via qRT-PCR for expression of CYP19A, FSHR, and GDP9 to further quantify development. Statistical analysis was done in R software. Follicles in 100% CM had higher survival up to Day 11 of culture as compared to other treatment groups (Cox proportional hazards model, p < 0.01). Initial stage also influenced survival, with antral follicle survival significantly lower than that of pre- and early antral follicles (p < 0.0001). However, no differences in growth were detected across the treatment groups, nor across initial size classifications (Kruskal-Wallis test, p > 0.05). Post culture qRT-PCR analysis of the three selected genes showed upregulation of CYP19A in 50% CM follicles compared to the control (ANOVA, p < 0.05). However, there were no differences in CYP19A expression between the control and other treatment groups, or in GDF9 and FSHR expression among culture groups (p > 0.05). In summary, the findings demonstrated that conditioned medium collected from primary culture of ovarian stromal cells improves in vitro survival and modulates CYP19A expression of isolated cat follicles. Further research to identify paracrine factors present in conditioned medium will elucidate the roles of ovarian stromal cells pertaining to follicle survival during in vitro folliculogenesis.Item INVESTIGATING SPERM MATURATION DURING EPIDIDYMAL TRANSIT IN THE DOMESTIC CAT FOR THE DEVELOPMENT OF ASSISTED REPRODUCTIVE TECHNIQUES(2017) Rowlison, Tricia Marie; Ottinger, Mary Ann; Comizzoli, Pierre; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Understanding the physiological processes involved in the sperm maturation process is critical to elucidating mechanisms underlying male infertility. Maturation of the spermatozoa during epididymal transit is associated with the integration of specific proteins and acquisition of functionality. Many underlying processes are unclear thereby making it challenging to develop applications for assisted reproductive technologies. The goal of this research was to understand key phases in the sperm maturation process associated with functional effects of protein incorporation via epididymosome secretion on sperm cell motility and fertilization. Specific objectives were to (1) identify essential proteins that contribute to sperm centrosome maturation throughout epididymal transit, (2) characterize the transfer of key factors via epididymosomes between the epididymis and maturing sperm cells and (3) demonstrate the impact of such mechanisms on the acquisition of motility and fertilizing ability by the spermatozoa. Using the domestic cat model (Felis catus), we demonstrated that the secreted epididymal vesicles termed epididymosomes supply critical proteins to the developing spermatozoa and examined the effects of exposure of these vesicles on motility and ability to successfully fertilize an oocyte. Exposure of immature sperm cells to epididymosomes in vitro resulted in the significant incorporation of cenexin, critical for the maturation of the sperm centrosome, which then enables proper pronuclear syngamy and formation of the first mitotic spindle following oocyte penetration. Furthermore, this research focused on assessing the impact of malformed spermatozoa at the centrosome on fertilization success in the domestic cat. Findings revealed that improper maturation of the centrosome may be a source of infertility in this species as well as in wild felids. In summary, this research has provided insights into the sperm cell maturation process which will enhance the use of assisted reproductive techniques and reproductive success of many species including the human and wild felids.