211 PRODUCTION OF PROGESTERONE FROM CANINE CUMULUS CELLS MATURED IN VITRO WITH STEROIDS AND GONADOTROPIN

Abstract

The canine reproductive physiology is distinct from other mammals, especially concerning the role of progesterone in ovulation, oocyte maturation, and fertilisation. In vitro maturation of canine oocytes is challenging mostly due to the culture conditions required for this species and the lack of knowledge about the factors involved in meiotic resumption and complete maturation. To obtain more information about this physiological phenomenon, the present study was designed to investigate the ability of canine cumulus cells to secrete progesterone in the maturation medium during in vitro culture for up to 96 h. Ovaries were collected from 18 bitches at different reproductive status (follicular, luteal, and anestrous stages; various breeds, age 1–7 years) by routine ovariohysterectomy and sliced in PBS-polyvinyl alcohol (PVA) to release the cumulus‐oocyte complexes (COC). A total of 896 COC grade I (2 or more dense layers of cumulus cells and darkly pigmented cytoplasm) were selected for culture. Oocytes were randomly allocated in 2 media consisting of TCM-199 (Sigma Chemical Co., St. Louis, MO, USA) with antibiotics, 10% FBS, 2.2 mg mL^–1 sodium bicarbonate, and 20 μL mL^–1 pyruvic acid (control) and control + hormones (25 IU mL^–1 hCG (Sigma), 1 mg mL^–1 progesterone (Sigma), 1 mg mL^–1 estradiol (E2; Sigma). The COC were incubated at 38°C, 5% CO₂ in air and cultured for 24, 48, 72, and 96 h. Maturation medium for in vitro culture of COC was collected at 0, 24, 48, 72, and 96 h and checked for progesterone content by radioimmunoassay (RIA). Maturation rates (meiotic configuration) were also evaluated at each culture period by using Hoechst 33342 (Sigma). Polynomial regression analysis was applied to assess variation of progesterone concentration in the maturation medium during the 96 h of IVM. Progesterone was detected in the control and hormonal media; however, in the control medium the concentration was <0.5 ng mL^–1. In the hormonal medium, there was a decrease in progesterone concentration at 24 h in all groups (ranged from 461.8 to 587.2 ng mL^–1) followed by a progressive increase until 72 h in follicular group (1371.4 ng mL^–1) and until 96 h in luteal and anestrous groups (934.4 and 669.4 ng mL^–1, respectively). Maturation rates (metaphase II oocytes) were higher at 72 h and decreased at 96 h in all groups. The results of this study suggest that hormonal supplementation is necessary for canine oocyte to resume meiosis and to cumulus cells be capable of producing progesterone. Moreover, progesterone secretion by cumulus cells seems to be related to the reproductive status of the donor bitch once higher concentration of progesterone was detected in medium from follicular group and lower in anestrous group.