This chapter describes the effects of forskolin and gonadotropins during the estrous cycle in perifused rat ovaries. It has been indicated that the perfusion system is suitable for studying factors controlling the release of irIα in vitro and that the effects of forskolin and gonadotropins vary depending on the cycle stage of the donor rat and presumably the microanatomy of the follicular pool. The effects of gonadotropins and forskolin in a more dynamic system monitor minute-to-minute changes in inhibin release. Additionally, the system permits the study of effects of potential regulators on ovaries containing intact follicles, including thecal cells and granulosa cells, and allows to study the effects of potential regulators on ovaries removed under varying physiological conditions. The stimulatory effects of gonadotropins and forskolin on inhibin release from cultured granulosa cells are well documented. However, serum levels of gonadotropins and inhibin and ovarian levels of mRNA for inhibin subunits change frequently during the 4-day rat estrous cycle. Thus, incubating granulosa cells with potential stimulants for 48 hours or longer does not necessarily reflect true physiological circumstances.

A heterologous radioimmunoassay developed to measure inhibin in rat plasma was validated and used to characterize changes in peripheral concentrations of immunoreactive inhibin (ir-inhibin) in relation to follicle stimulating hormone (FSH) concentrations during the estrous cycle of mares. The primary antiserum used in the assay was developed against a synthetic porcine inhibin α-subunit [(1-26)-Gly-Tyr] fragment. The same synthetic peptide was used for preparation of standards and tracer. Slopes of the dose-response curves for pooled estrus and diestrus mare plasma and equine follicular fluid were similar to the slopes for the porcine inhibin α-subunit standard curve and porcine follicular fluid dose-response curve. Twelve mares were bled once daily beginning when diameter of the largest follicle reached ≤25 mm and continuing until 3 days after the end of an interovulatory interval (ovulation=Day 0). Each of the 12 interovulatory intervals were normalized to the mean length of the interovulatory interval (22.2 days; range, 19 to 26). There was an effect of day for concentrations of ir-inhibin (P<0.001) and FSH (P<0.006). Significant mean changes were as follows: 1) ir-inhibin decreased between Days 0 and 1, whereas FSH increased between Days 0 and 5; and 2) ir-inhibin increased between Days 7 and 12, whereas FSH decreased between Days 11 and 14. Mean concentrations of ir-inhibin and FSH were negatively correlated (r=-0.548; P<0.002). In conclusion, mean peripheral concentrations of ir-inhibin and FSH were inversely related during the estrous cycle of mares.

We have measured changes in circulating immunoreactive (ir-) inhibin in male and female rats using an RIA with an antiserum raised against porcine inhibin alpha (1-26)-Gly-Tyr. The same synthetic peptide was used for standards and for the preparation of tracer. Serum ir-inhibin levels were significantly higher in intact female than in intact male rats (p less than 0.001). Immunoreactive inhibin was significantly reduced in both sexes 24 h after bilateral gonadectomy (p less than 0.0001). Unilateral ovariectomy (ULO) of female rats on metestrus caused a transient decrease in serum inhibin 8 h after surgery, but levels were not significantly different from those of sham-operated controls at later times after surgery. Increases in serum FSH and LH were observed for 8-18 h after ULO. Serum ir-inhibin levels were also measured on the early morning of estrus during the secondary FSH surge. At this time, ir-inhibin levels were low, while FSH levels were high and LH levels were low. These results show that serum ir-inhibin levels in rats are decreased at times when serum FSH levels are high.

Four- and 5-day cyclic mice housed with and without males, respectively, were autopsied at 10 a.m. on proestrus (P) or estrus (E) after no treatment, or after ovariectomy (Ovax) or sham Ovax at 10 a.m. or 4 p.m. on one of the preceding 3 days. Results indicate the ovarian must be in situ between 4 p.m. the day before P and 10 a.m. the day of P for a normal proestrous uterine weight increase to occur in both 4- and 5-day cyclic mice. For estrous cornification to occur, ovaries must be in situ between 10 a.m. and 4 p.m. on diestrus (D) in 4-day cyclic mice, and between 4 p.m. on D-I and 10 a.m. on D-II in 5-day cyclic mice. Sham Ovax tended to inhibit ovulation in both 4- and 5-day cyclic mice. The effects of both Ovax and sham Ovax on proestrous and estrous pituitary LH values were variable. ttiming of the feedback relationship between ovary and hypothalamus-pituitary in mice may be imprecise, leading to easy disruption of the estrous cycle in this species.

Many studies have suggested that rats exhibiting 4-dayestrous cycles may differ from those exhibiting 5-day estrous cycles in hormonal secretion rates. The present study was carried out to delineate the pattern of the ovulatory surge of LH secreted in these two types of cycles. During the day of proestrus, 1–3 serum samples per rat were collected by cardiac puncture, under ether anesthesia, at 30-min intervals, from 13.30 to 20.30 h. Serum LH was determined using the 0:0 RIA. No rat had values above 10 ng/ml before 14.30 h and individual animals showed a high rate of LH release for variable lengths of time. Serum LH values did not differ significantly between 4- and 5-day cycles over the times observed. At 13.30, pentobarbital, which has been reported to be less effective in blocking ovulation in the 5-day cyclic rat, lowered serum LH in both 4- and 5-day groups to 1–17 ng/ml over an identical time period. However, at 15.30 h the 4-day pentobarbital-treated animals had a significantly higher LH level. This may have been due to an effect of cardiac puncture at 15.00 h.

In prepubertal rats injected with PMSG an ovulatory surge of LH was secreted between the hours of 1400 and 1800 on the second day after PMSG. Administration of phenobarbital at 1100 prevented the LH surge and ovulation from occurring; however, if progesterone was injected 1 hr prior to the phenobarbital, most rats ovulated. No consistent evidence of an LH surge was seen in rats treated with progesterone and phenobarbital even though they ovulated.

Several variables were examined at nine time periods over a 24-hour period in rats following prolonged exposure to constant light (LL), compared with animals housed in an alternating light-dark (LD) environment. The data indicated: LL animals had lower ovarian weights and increased uterine weights, which were not dependent on the time of day of autopsy; pituitary LH content was lower in LL animals, and dependent on time of day only in LD rats; there were no significant differences in serum LH concentrations between the LL and LD animals, nor among times of blood collection; serum FSH concentrations varied significantly with time, but were not different between LL and LD animals. It is suggested that, in the absence of cyclic surges of LH, low tonic levels of serum LH can support estrogen secretion, resulting in higher uterine weight, and that fluctuating but low serum FSH levels may help maintain the follicular stimulation observed in LL animals.