The present study was undertaken to characterize hormonal and morphological events in 4 day and 5 day rat cycles, in order to test 3 different theories concerning the etiological factors altering follicular phase length in mammals. Three rats were killed at each 2 h interval throughout each cycle type. In addition to organ weight measurements, serum levels of estradiol, progesterone, 20α-hydroxypreg-4-en-3-one, LH, FSH and prolactin were assessed in terminal samples. The beginning of uterine intraluminal fluid accumulation coincided with the decline in progesterone levels seen during diestrus, rather than with the rise in estrogen and was delayed 12 h in the 5 day cycle from the time of last estrus. The retention of this fluid during the evening of proestrus was 6 h longer in the 4 day cycling rat. Ovulation occurred earlier in the 5 day cycling rats than in those with a 4 day cycle. No significant differences in the gonadotropins or 20α-hydroxypreg-4-en-3-one were seen when comparing the 2 types of cycles. Estradiol rose during the night of diestrus I (the same time in the cycle) and was identical in both cycle types. Progesterone was not released on proestrus prior to the LH surge in either type of cycle. However, we did find that progesterone values were higher for a longer period of time throughout diestrus I in 5 day cycles. These data have led us to conclude that the 5 day cycle is due to a prolonged progesterone secretion during the metestrous and diestrous stages of the cycle.

Several studies have led to the concept that the length of the rat estrous cycle is dependent on the timing of estrogen (E) secretion. According to this hypothesis E secretion during the 5-day cycle is delayed. However, we have found LH and E levels in both 4 and 5 day cycles are identical. Therefore, even though E is critical for a cyclic gonadotropin release to occur it does not appear to be the factor, which under normal circumstances, determines cycle length. During the day of proestrus, progesterone (P) increase coincided, but did not precede the gonadotropin(s) surge in our study thus eliminating P as the normal physiological trigger of gonadotropin release. If P was the determining factor, one would expect to find 5-day P levels higher than 4-day cycle levels during the earlier stages of the cycle. Approximately 48 h following ovulation, on the day of diestrus, we found that the 5-day cycle did indeed have higher P levels. We propose that the regulation of P secretion during the early stages of the cycle controls the timing of the normal periodic gonadotropin release.

The purpose of this experiment was to determine whether surgical stress on the morning of proestrus would elicit an early release of gonadotropin from the pituitary. Animals exhibiting 5-day estrous cycles underwent bilateral sham-ovariectomy under ether anesthesia at 0800 h of proestrus. These animals had high levels of progesterone and estradiol following the surgery. These steroids were thought to be adrenal in origin, since animals adrenalectomized at 0800 h of proestrus had low progesterone levels and estradiol comparable to unoperated controls. Subsequently, the sham-operated animals showed high FSH but not LH values at 1300 h, prior to the normal critical period for gonadotropin release. By 1400, the LH surge had begun, and progesterone was again being released. Adrenalectomized and unoperated controls showed no increase in any steroid or gonadotropin measured before 1400 h. These findings suggest that stress-induced release of adrenal estradiol and progesterone, rather than some other consequence of the surgical procedure, during the morning of proestrus, can advance the onset of release of FSH, prior to LH. Ovariectomy at 0800 h proestrus led to a rapid and dramatic increase in FSH but not LH secretion by 4 h after surgery. By 6 h after ovariectomy FSH had increased to six times control values and LH had increased to twice control values. Estradiol remained at control values for 6 h following surgery but 20alpha-hydroxypreg-4-en-3-one (20alpha-OHP) dropped quickly to baseline values. It is possible that a reduction in circulating 20 alpha-OHP may be responsible for the increases in FSH prior to LH in this group, but the absence of other negative feedback factors from the ovary or adrenal may also be involved.

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.

The site and mechanism of action whereby barbiturates block the proestrous surge of LH and ovulation are unknown. The present study investigated the effects of pentobarbital (PB) on LH release in gonadectomized rats. Twenty—one days post—gonadectomy serial blood samples were withdrawn, via chronically implanted cannulae, at 30–min intervals from 0800–1100 or 1300–1600 hr. PB (30 mg/kg/ip) administration at 0830 hr significantly reduced the serum LH concentration of ovariectomized rats below the pre—drug control level at 0800 hr, and below the nearly constant level observed in saline injected controls between 0900 and 1030 hr. A significant rise in serum LH of ovariectomized rats occurred between 1400 and 1500 hr which was blocked by administration of PB at 1330–1345 hr. Castrated male rats did not show an afternoon rise in LH. PB lowered LH in males from 1400 to 1430 hr. The block of tonic LH release in males showed a shorter duration than in females, correlating with the shorter duration of anesthesia and the more rapid rate of disappearance of radioactivity from the serum after injection of 14C–PB in males. Our results indicate that: (1) PB reduces LH release independently of gonadal steroid action, and (2) a periodic neural timing center may exist for LH release in the ovariectomized female rat. (Endocrinology92: 1634, 1973)

The purpose of these experiments was to study some of the factors controlling the timing of mating behavior on the day of proestrus of the rat estrous cycle. In unoperated rats exhibiting 4-day cycles, the first appearance of the lordosis response to a male came after the onset (1400 hr) of the critical period for LH release; the average time was at 1641 hr. In unoperated 5-day rats half had shown the first lordosis by 1400; not only was the mean time of onset significantly earlier but the variance was significantly greater in the 5-day rat. Sham ovariectomy advanced the time of lordosis if performed early enough in the day of proestrus in both types of cyclers; ovariectomy also advanced lordosis except when performed at 0600 hr in 4-day rats, when the procedure blocked or delayed mating. Adrenalectomy delayed lordosis in both types of cyclers until after 1800 hr; combined ovariectomy and adrenalectomy blocked lordosis from appearing at all in significant numbers of animals of both cycle lengths. The combined surgery performed at 0600, followed by progesterone (2.5 mg) at 0800, elicited lordosis in 5-day cyclers, but not 4-day. Sham ovariectomy at 0600 hr in rats of either cycle length also advanced the time of LH discharge for ovulation, as deduced from barbital blockade experiments. It is concluded that the adrenal glands, presumably via progestin secretion, participate in the normal timing of mating behavior in the intact rat. Moreover, stressful surgical intervention can superimpose disturbances in this timing, as well as in the timing of LH release. (Endocrinology88: 325, 1971)