Throughout a period of pseudopregnancy the peripheral blood levels of progesterone, oestradiol-17 beta, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), as well as the size-distribution of ovarian antral follicles were estimated in the rat. The progesterone concentrations, as measured by a competitive protein-binding technique, exceeded metoestrous values (25 ng/ml plasma) from day 3 of pseudopregnancy onwards. The highest levels were found on days 6 and 8 (91 ng/ml). From day 8 onwards the levels decreased gradually but were still above metoestrous values on the day of pro-oestrus after pseudopregnancy. Concentrations of oestradiol-17 beta, as measured by radioimmunoassay, were within the range of those at metoestrus (about 5 pg/ml plasma) until day 10. Thereafter levels increased to a value of 57 pg/ml. Concentrations of FSH, measured by radioimmunoassay, were within the range of metoestrous values until day 10 (about 100 ngNIAMD-rat-FSH RP-1/ml serum), but declined to a level of 33 ng/ml on day 12. Concentrations of LH, measured by radioimmunoassay, were generally within the wide range of metoestrous values (9-60 ng NIAMD-rat-LH RP-1/ml serum), but concentrations found on days 4, 8 and 10 were significantly lower than those found on preceding or subsequent days. Histological determination of the number of follicles present in various volume-classes, showed an increase in antral follicles on days 1 and 2, comparable to the increase observed during metoestrus and dioestrus 1 of the normal cycle. There was no change in the follicles between days 3 and 10 and they resembled those of early dioestrus. Preovulatory growth had occurred by day 12. Injection of human chorionic gonadotrophin (HCG) on days 2, 4 or 6 showed that ovulation could be induced only in some of the larger follicles. On the basis of these results it is suggested that during pseudopregnancy the high progesterone levels present result in a decreased plasma LH level which is insufficient to cause full maturation of the follicles and to stimulate oestrogen secretion to the levels required for induction of an ovulatory surge of LH release.

Ovarian follicles (greater than or equal to 100 X 10(5) microns 3 or a mean diameter of greater than or equal to 275 microns) in adult rats were classified as non-atretic and atretic during the oestrous cycle and recorded in 5 volume classes. The atretic follicles were also categorized in several stages according to the progress of atresia. The degeneration of the entire granulosa wall until the induced changes in the oocyte took at least 24 h. Another 24 h elapsed before the oocyte became denuded. Therefore the % of atretic follicles, i.e. follicles in all stages of atresia, could not be used as indicator for the rate of atresia. The atretic portion in the follicle population greater than or equal to 100 X 10(5) microns 3 increased from early dioestrus 1 to early dioestrus 3, reached a plateau during dioestrus 3 and pro-oestrus, and declined at late oestrus to the level of early dioestrus 1. The sudden decrease in number of atretic follicles after late pro-oestrus was caused by the discard of many atretic follicles in the advanced stages due to various deformities as revealed by histological observation. By using the % of atretic follicles in the earliest stage as indicator of atretic rate, two waves of atresia were found affecting the population of antral follicles during their growth, the first at dioestrus 1 amounting to 15-20% and then at dioestrus 3, affecting 35% of the population. The present study also shows the extension of atresia in the various volume classes of follicles during the oestrous cycle. A pool of approximately 7 follicles in the smallest volume class was maintained after ovulation, grew further in the next cycle with a new cohort of 20 follicles, and seemed to provide the required number of follicles destined to ovulate. This suggests that the follicles that ovulate were already present at an antral stage in the preceding cycle and needed two cycles for their growth to ovulation.

Ovarian follicular development was studied in the rat during a 15-day period preceding first ovulation. Ovaries were obtained by unilateral ovariectomy performed at various ages and the rats were allowed to live until the day after first ovulation. The timing of this ovulation was compared with that in unoperated, paired control rats of the same age. For estimation of gonadotrophin levels, blood was taken from the paired control rats at the time when experimental rats were unilaterally ovariectomized. There was no evidence that unilateral ovariectomy had any influence on the timing of first ovulation. Therefore the ovaries obtained could be dated in relation to first ovulation, and follicular growth during the final prepubertal period could thus be studied in a genuine developmental sequence. Results revealed that follicular growth leading to first ovulation starts at +/- 8 days before this ovulation; follicular processes taking place are comparable to those found during the adult 5-day cycle but proceed more slowly. Gonadotrophin concentrations accompanying the follicular dynamics and measured at 11.00 h, showed a clear tendency for FSH concentrations to decrease with increasing age, i.e. approaching first ovulation. Concentrations of LH did not show a definite pattern and were generally low, although in some individual rats relatively high LH values ( greater than 100 micrograms/l) were found in the period of 5-3 days before first ovulation.

Events in the ovaries during the first spontaneous pro-oestrus were compared with those in the adult rat. The occurrence of first spontaneous pro-oestrus was determined from vaginal smears after surgical opening of the vagina. Pubertal rats showed a remarkable increase in ovarian weight from 10.00 to 15.00 h on the day of pro-oestrus, which was absent in the adults. They also showed an earlier and more pronounced interstitial oedema. Dispersion of cumulus cells, resumption of meiosis and ovulation also occurred slightly earlier in pubertal rats. A distinct difference was found in the growth rate of follicles of ovulatory size, i.e. follicles of larger than or equal to 500 X 10(5) mum3. At the first pro-oestrus these follicles showed a slower and less pronounced increase in size from 10.00 to 17.00 h compared with the adult follicles at this time, although this disparity was made up by a marked increase in growth from 17.00 to 24.00 h. The development of a new crop of follicles progressed similarly in the pubertal and adult animals. Follicular atresia seemed to progress more rapidly from 10.00 to 15.00 h in the pubertal animals compared with adults but from 15.00 h onwards no further differences were observed. The possible causal factors underlying the observed differences between the pubertal and adult animal are discussed.

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Ovarian steroid contents and serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin were measured during the days after first ovulation in rats unilaterally ovariectomized in late prepuberty. In addition, follicle counts were made at second estrus and second metestrus. During the cycle following first ovulation, ovarian estradiol contents in unilaterally ovariectomized (ULO) rats were significantly increased as compared to intact rats on the day of metestrus, on diestrus 1 and on second estrus. Ovarian progesterone was significantly increased on the days of metestrus, on diestrus 1, second proestrus and second estrus, but no differences were seen in ovarian androgen contents. After ULO there was an indication of an augmented FSH surge at the first and the second ovulation. Follicle counts revealed that the total number of healthy as well as of atretic antral follicles on the day of second estrus was significantly increased after ULO, due to increased numbers of the smallest antral follicles. At second metestrus the number of larger antral follicles (350-500 micron 3) and the total number of healthy antral follicles was higher after ULO. It is concluded that the compensatory process after ULO involved increased recruitment of small antral follicles. Activities in the remaining ovary were not simply doubled but a new hormonal balance was established.

Summary. The ovaries of pro-oestrous rats (pubertal and adult) and hamsters were explanted in toto at various times after the preovulatory gonadotrophin surge to obtain endogenously stimulated ovaries. Subnormal numbers of ovulations from pubertal and adult rat ovaries were found when incubation was started during the period from 17:00 to 24:00 h on the day of pro-oestrus. Normal numbers of ovulations only occurred from rat ovaries in vitro when incubation was started at 01:00 h, i.e. when ovulation had already begun in vivo. In contrast, full ovulation from hamster ovaries was observed in vitro after incubation at 22:00 h and later, although the ovulatory process had not started in vivo. This difference in the ability of rat and hamster follicles to ovulate in vitro could be due to a different role of the accumulation of tissue fluid for the mechanism of ovulation.