Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. II. Progesterone receptor expression in luteinizing granulosa cells

Abstract

The events in granulosa cells that are initiated by the midcycle LH surge during luteinization of the primate follicle are poorly defined. This study was designed 1) to determine whether an ovulatory dose of hCG can induce progesterone receptors (PR) in macaque granulosa cells, and if so, 2) to begin titrating gonadotropin requirements for PR expression and progesterone production by luteinizing granulosa cells. Rhesus monkeys were treated with human FSH and LH for up to 9 days to stimulate the growth of multiple follicles. The next day, animals (n = 4-5/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 Mg GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800,1100, and 1400 h); or 5) two injections of 50 μg GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h). Granulosa cells obtained by follicle aspiration 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 or 10 from animals receiving no ovulatory stimulus were processed for indirect immunocytochemistry using a monoclonal antibody to human PR (JZB39). Specific staining for PR, determined by comparing cells incubated with PR antibody vs. a nonspecific antibody, was undetectable in granulosa cells from monkeys without an ovulatory stimulus. In contrast, the majority (64 ± 5%) of cells from hCG-treated animals stained intensely for PR. In the GnRH/GnRHa groups, granulosa cells from only one animal (i.e. one GnRH-3 monkey) showed positive staining for PR. During 24-h culture in Ham's F-10 medium containing 10% monkey serum, basal progesterone production by cells from the hCG-treated group (2163 nmol/L·8 × 104 cells) was higher than that by cells from the no ovulatory stimulus/GnRH-1/GnRH-3/GnRHa groups (60, 111, 194, and 332 nmol/L, respectively). However, granulosa cells from the hCG-treated group were less responsive to hCG in vitro in terms of enhanced progesterone production (2 times control levels) than cells from the other four groups (up to 30 times control levels). This study provides direct evidence that an ovulatory dose of hCG induces PR expression in granulosa cells of luteinizing follicles during stimulated cycles in rhesus monkeys. However, repeated injections of GnRH/GnRHa that produced surge levels (>100 ng/mL) of endogenous LH for up to 14 h failed to induce PR expression or progesterone production by granulosa cells. Thus, an extended LH surge more typical of that in the normal menstrual cycle (48-50 h) may be necessary for PR expression and luteinization of granulosa cells in primate follicles.