Use of a GnRH antagonist in conjunction with low amplitude, high frequency LH pulses to induce follicular growth without an LH surge and ovulation in ewes.

Abstract

The present study was undertaken to develop an experimental sheep model which could be used to investigate the abnormal follicle growth that is associated with the absence of the LH surge. On Day 10 of the oestrous cycle, 16 ewes were treated with an analogue of prostaglandin (cloprostenol; PG) and blood sampled every 4 h thereafter to determine the normal timing of the preovulatory LH surge. Three oestrous cycles later, all ewes were simultaneously treated with PG and a gonadotrophin-releasing hormone (GnRH) antagonist ([Ac-DNal1, D4C1Phe2, DTrp3, DArg6, DAla10] GnRH.HOAc; 50 micrograms kg-1 subcutaneously). Group 1 ewes (n = 6) received no further treatment. Group 2 ewes (n = 5) were additionally treated for a total of 7 days, starting at the time of PG injection, with purified ovine luteinising hormone (LH; preparation P 3 R3-5; equivalent to 1.25 micrograms NIH-oLH-S26) administered i.v. over a 2 min period. For the first 24 h, LH was given at 3 h intervals for 12 h, then every 2 h for 12 h, and thereafter hourly for 6 days. Group 3 ewes (n = 5) were treated as Group 2 but at 72 h received an additional antagonist injection (50 micrograms kg-1 subcutaneously). Mean values of LH from 24 to 96 h were significantly lower in untreated controls and Group 1 than in the other two groups (0.58 +/- 0.2 ng ml-1 and 0.55 +/- 0.2 ng ml-1 vs. 1.63 +/- 0.5 and 1.68 +/- 0.6 ng ml-1, respectively; P < 0.01). After treatment with PG alone in the untreated control group, the preovulatory LH surge began in all ewes at 59.9 +/- 2.8 h after PG. All Group 1 ewes also had an LH surge but the period from PG injection to the onset of the surge was 124 +/- 17.3 h (range 96-152 h). Only two of the Group 2 ewes had an LH surge (at 160 and 168 h, respectively) and no surge was detected in Group 3 ewes. In Group 1, mean values of follicle-stimulating hormone (FSH; 0.78 +/- 0.07 ng ml-1) were not affected by treatment with antagonist alone; however, in the two groups receiving exogenous LH pulses there was a marked decrease in FSH concentrations during the period 24-96 h. Progesterone concentrations increased 9 days after PG treatment in five out of six ewes in Group 1. In Group 2, there was evidence of a variable luteinisation response, but in Group 3 progesterone remained less than 0.08 ng ml-1 throughout. Endoscopy 112-115 h after PG confirmed that none of the 16 antagonist-treated ewes had ovulated; an event normally expected approximately 80 h after PG in sheep. The experimental protocol of Group 3 provides the basis for a model which will enable examination of the long-term functional capacity of ovarian follicles which have not been exposed to an LH surge.