BIOLOGICAL MECHANISMS UNDERLYING THE REGULATION OF PORCINE OVARIAN FOLLICULAR DYNAMICS THROUGH GNRH-II AND ITS RECEPTOR

<b>Project Methods</b><br> Our laboratory has previously developed a line of swine with reduced endogenous GnRH-II receptor (GnRHR-II) levels. This GnRHR-II knockdown (KD) swine line will be utilized to elucidate the function of the GnRHR-II within the follicles of gilts via two objectives. In general, GnRHR-II KD (n = 15) and littermate control (n = 15) gilts will be monitored once daily for behavioral estrus beginning at 180 d of age. Once all females have exhibited their third behavioral estrus, they will be fed the progestogen, altrenogest (Matrix; 18 mg/d), for 14 d to synchronize estrus. At 8 h following final Matrix feeding (Day 0), gilts will be administered pregnant mare serum gonadotropin (1000 IU) to stimulate follicle growth. On Day 3, unilateral ovariectomy will be performed to collect the right ovary, providing medium follicles for experiments in Objective 1. At this time, indwelling jugular catheters (to be used in Objective 2) will also be surgically placed. After catheter placement, gilts will be administered 2 mL of Ovugel (triptorelin; 24 h) to induce ovulation,and twice daily detection of behavioral estruswill be performed. On Day13 of the estrous cycle, 10 mg prostaglandin-F2 alpha (Lutalyse) will be administered to induce luteolysis. Serial blood collections for Objective 2 will begin 72 h following Lutalyse administration, when medium follicles are prevalent, and continue until the onset of estrus. Gilts will be euthanized within 12 h of the onset of estrus, with the left ovarycontaining large (7 mm) folliclescollected to be used for experiments in Objective 2.Objective 1: Elucidate the role of GnRH-II and its receptor in 17ß-estradiol production by granulosa and theca cells of the porcine antral follicle.Experiment 1A. Determine the effect of GnRH analogue treatment on 17ß-estradiol secretion in granulosa cell cultures from GnRHR-II KD and control gilts.Granulosa cells will be collected from medium follicles (3-6.9 mm) of GnRHR-II KD (n = 15) and littermate control (n = 15) gilts, as they are the primary 17ß-estradiol producing cells. Isolated granulosa cells will be washed and plated at a density of 1.0 x 105 viable (trypan blue exclusion) cells/cm2. Media used to culture granulosa cellsis supplemented with androstenedione (1 μM) to provide an androgen substrate for aromatization to 17ß-estradiol.At plating, cells will be treated with D-ala6GnRH-II (1 μM; agonist), FSH (100 ng/ml; positive control) or vehicle. To determine if GnRH-II responsiveness is receptor mediated, treatments are given in the presence or absence of the GnRHR antagonist, SB-75 (1 μM; binds both GnRHR-I and -II). Media is collected immediately prior to treatment (0 h), and at 12, 24, 32, and 48 h post-treatment for analysis via 17ß-estradiol RIA. Media will be replenished, and treatment reapplied, at 24 h.Experiment 1B. Assess steroidogenic enzyme and GnRHR-II abundance, as well as GnRH-II concentrations, in theca and granulosa cells of GnRHR-II KD and control gilts.A portion of granulosa cells collected from medium follicles of GnRHR-II KD (n = 15) and littermate control (n = 15) gilts in Experiment 1A, as well as theca cells isolatedfrom the same follicles, will be snap frozen and stored at -80oC. We will perform immunoblots on protein isolated from theca and granulosa cells of transgenic and control gilts using antibodies directed against delta-4 steroidogenic enzymes (StAR, CYP11A1, 3ß-HSD, CYP17A1, 17ß-HSD, CYP19A1)and GnRHR-II. Glyceraldehyde-3-phospate dehydrogenase (GAPDH) will serve as the loading control. Moreover, GnRH-II concentrations will be measured in theca and granulosa cell homogenates via GnRH-II ELISA.Objective 2:Determine how GnRH-II and its receptor contribute to ovulation.Experiment 2A. Compare LH profiles of GnRHR-II KD and littermate control gilts stimulated with GnRH-II or 17ß-estradiol.Following Lutalyse administration (72 h), when medium follicles are prevalent, serial blood collections will be performed on GnRHR-II KD (n = 15) and littermate control (n = 15) gilts. After blood collection every 20 min for 2 h to establishbaseline concentrations, gilts (n = 5/treatment/line) will be injected I.M.with either: 1) D-ala6GnRH-II (150 ng/kg BW in DMSO); 2) estradiol benzoate (2 mg in sesame oil; positive control); or 3) vehicle (DMSO in sesame oil) based on previously determined doses. Upon treatment, blood will be collected every 10 min for 1 h, then every 20 min for 3 h, every 2 h for 8 h, and finally every 4 h until the onset of estrus. Serum will be assayed for LH by RIA, as the preovulatory LH surge (induced by rising concentrations of 17ß-estradiol)initiates the cascade of events leading to ovulation.Experiment 2B. Examine early indicators of the ovulatory cascade in large preovulatory follicles of GnRHR-II KD and littermate control gilts. Elevated LH receptor (LHR) expression and a shift from 17ß-estradiol to progesterone production in the preovulatory follicle are early indicators of ovulation. Transgenic (n = 15) and control (n = 15) gilts will be euthanized within 12 h following the onset of estrusfor the collection of large follicles. Follicular fluid will be aspirated, and theca and granulosa cells isolated. Immunoblotting will be performed on theca and granulosa cells from GnRHR-II KD and control gilts, with an antibody directed against the LHR. GAPDH will serve as the loading control. Progesterone concentrations in follicular fluid will be quantified by RIA.Statistical Analysis. Power calculations performed for planned outputs in the objectives indicate that n = 12 gilts per line will detect a 15% difference at a power of 0.86 (alpha = 0.05). We plan for 3 additional animals per line to account for potential failure of jugular catheters, as well as injury or illness. Thus, a total of 30 gilts will be required. Statistical analysis will be performed using SAS(9.4; Cary, NC). All data are examined for normality using the Shapiro-Wilk test prior to analysis. RIA: Hormone concentrations are analyzed with the MIXED procedure of SAS, with animal as the experimental unit; treatment, time, line, and their interactions as fixed effects; time as the repeated measure with gilt(trt*line) as the subject; and baseline concentrations as the covariate. Immunoblot and ELISA: Data will be analyzed with the MIXED procedure of SAS, with animal as the experimental unit, and line and cell type as fixed effects.