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H. M. A. Meijs-Roelofs and P. Kramer


The effects on sexual maturation of the opioid receptor antagonists naloxone and naltrexone were studied in the female rat. Neonatal treatment (days 1–10) with either naloxone (2·5 mg/kg at 6-h intervals) or naltrexone (20 or 50 mg/kg per day) did not advance sexual maturation as judged by age and body weight at vaginal opening and first ovulation. After treatment with naltrexone (20 mg/kg) first ovulation occurred 2·3 days earlier than in saline-treated control rats but this could be attributed to a growth-stimulating effect of naltrexone; the effect was not observed with 50 mg/kg. An effect of neonatal treatment with naloxone on serum LH levels was seen at 23 days of age (155±36 (s.e.m.) vs 14±4 μg LH/1 in controls, P<0·01), but not at 29 or 33 days of age, at 2 days before first ovulation nor at first pro-oestrus. There were no differences in the number of ova released at first oestrus, nor in the length of the first cycle. Neonatal treatment with naltrexone (50 mg/kg per day) did not alter the response to treatment with human chorionic gonadotrophin at 28–31 days of age: ovulation of a mean of 7·3 ova was induced on day 32 in both naltrexone- and saline-treated rats. Naltrexone treatment (four daily injections of 20 mg/kg at 2-h intervals) at 28–32 days of age advanced first ovulation by 4·4 days in about 40% of the rats.

Short-term effects of naloxone and naltrexone treatment on LH secretion were studied in 15-day-old rats and a clear increase in serum LH concentration was seen 15 min after injection; LH levels were similar with the various doses of antagonist used (2·5 and 20 mg naloxone/kg; 2·5, 20 and 50 mg naltrexone/kg). At 30 min after injection LH levels were still maximal if the 2·5 mg/kg dose of either antagonist was used, whereas with the higher doses a significant (P<0·01) decrease in the level of LH was observed, indicating a longer-lasting effect of the lower (2·5 mg/kg) dose of antagonist on LH secretion. No differences in effectiveness were seen between the supposedly short-lasting opioid receptor antagonist naloxone and the supposedly long-lasting antagonist naltrexone.

It was concluded that neonatal treatment with the opioid antagonists naloxone and naltrexone did not specifically influence sexual maturation. The antagonists clearly increased LH secretion in short-term experiments. Treatment with naltrexone at 28–32 days of age advanced first ovulation in 40% of the rats, a result that is in agreement with a possible opiatergic influence on the gradually increasing LH release during the final phase of sexual maturation.

The effects of the opioid antagonist naltrexone on the LH release mechanism did not parallel its reported long-lasting effects on nociceptive response, no long-lasting increase of LH release was seen, and the timecourse of the LH response was the same using the antagonists naloxone and naltrexone.

J. Endocr. (1988) 117, 237–243

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The involvement of the adrenal gland in the release of gonadotrophins and the onset of puberty in female rats was studied. Two and four days after adrenalectomy (ADX) on either day 5 or 10 after birth, a significant decrease in the concentration of FSH was found; 4 days after ADX on either day 15 or 20, FSH concentrations had increased significantly compared with sham-operated and/or intact controls. However, in the rats adrenalectomized on day 15 or 20, the body weights were lower than in control rats. Relative uterine weights (mg/100 g body wt) in adrenalectomized rats never differed from those of control rats.

A delay in the time at which vaginal opening and the first oestrus occurred was found in rats adrenalectomized at 20 or 25 days of age; however this delay was accompanied in these rats by a retardation in the gain in body weight. It is argued that the effects of ADX on both the release of gonadotrophins and the onset of puberty are primarily, and presumably exclusively, due to the effects on general bodily development (expressed in body weight). The lack of effect of ADX on uterine weight supports the hypothesis that 'oestrogen-like' products from the adrenal gland are not biologically active as oestrogens.

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The effect was studied of five daily injections of 50 μg of either 5α-androstane-3α,17β-diol (3α-androstanediol) or its 3β-epimer (3β-androstanediol), starting on day 22 of life, on sexual maturation in female rats. No difference was found in the age and body weight at first oestrus between oil-treated rats and rats treated with either 3α- or 3β-androstanediol. The only difference observed between these groups consisted of the occurrence of a 'pinhole' vaginal opening a few days before oestrus in 50% of the steroid-treated rats; in oil-treated rats immediate full vaginal opening and first oestrus coincided in ten out of 12 rats.

Different effects were obtained when the higher dose of 100 μg daily was used; effects were dissimilar in rats treated with 3α- and 3β-androstanediol. If administration of the higher dose of 3β-androstanediol was started on day 22 and continued until the day of full vaginal opening and first oestrus, a significant delay of this first oestrus, preceded by a few days of a 'pinhole' type of vaginal opening, was observed. After administration of the higher dose of 3α-androstanediol a 'pinhole' type of vaginal opening, accompanied by dioestrous-like vaginal smears, was also found, but oestrus did not occur during the period when injections were given. After the injections were stopped on day 45, first oestrus developed within 6 days in all rats.

The previous findings of others that administration of 3β-androstanediol to the immature female rat may induce precocious puberty (i.e. precocious vaginal opening and first ovulation) were not confirmed in the present study. Our results indicate that high doses of free 3α-androstanediol, and to a lesser degree 3β-androstanediol, may even delay first ovulation in the rat. A possible interference of 3α-androstanediol with the triggering of the first ovulatory gonadotrophin peaks is discussed.

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The maturation of the inhibitory feedback action of oestrogen on FSH secretion in the immature female rat was studied from 5 days of age until after the first ovulation. To study the role of the oestrogen binding alpha-foetoprotein (AFP) which is present in the blood of young animals, the effects of various doses of oestradiol and of the synthetic oestrogen R2858 (11β-methoxy-17-ethynyl-oestradiol), which is not bound by AFP, were compared in ovariectomized rats.

A rise in the serum concentration of FSH within 2 days of ovariectomy was first observed in rats ovariectomized at 8 days of age. Between 8 and 28 days of age the rise in FSH after ovariectomy could be prevented by oestrogen injections in such a way that the resulting FSH concentration amounted to 50% of that in ovariectomized control rats. This was achieved with a constant dose of 0·00015 μg R2858/100 g body weight, whereas the dose of oestradiol needed decreased from 0·05 to 0·01 μg/100 g body weight indicating an increased sensitivity to the feedback action of oestradiol. After day 28, sensitivity to the feedback action of both R2858 and oestradiol decreased progressively up to the time of the first ovulation.

In contrast to results at earlier ages, none of the doses of either oestrogen was capable of maintaining near-physiological concentrations of FSH after 20 days of age.

It is concluded that the apparent increase in sensitivity to the feedback action of oestradiol occurring before 28 days of age reflects the disappearance of AFP from the blood, whereas the subsequent decrease in sensitivity is independent of AFP. Moreover, it is concluded that up to about 20 days of age oestradiol could be, though not necessarily is, the sole ovarian factor involved in regulating FSH secretion, whereas at later ages additional steroids and/or factors must be involved.

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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 (> 100 μg/l) were found in the period of 5–3 days before first ovulation.

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H. M. A. Meijs-Roelofs, P. Kramer, and P. Osman


Precocious first ovulation, preceded by an endogenous preovulatory LH surge, could be predictably induced in immature female rats by administering repeated injections of human chorionic gonadotrophin (hCG). Administration of a dose of 0·05–0·075 i.u. hCG, four times a day from day 28 to day 31 of age resulted in a highly constant ovulatory response: at 4·0±0·0 days after the start of treatment 7·7±0·3 (n = 15) ova were found. Use of a higher dose of hCG (0·1 i.u.) resulted in lower numbers of ova (5·6±0·4, n = 7; P<0·005) whereas use of a lower dose of hCG (0·025–0·038 i.u.) resulted in a less constant timing of the induced ovulation at 5·4±0·2 days after the start of treatment (n = 7; P<0·0005). In animals treated with the dose of 0·05–0·075 i.u. hCG, a positive correlation was found between body weight at the start of treatment and the number of ova released (r = 0·75, n = 25; P<0·001).

Ovarian follicle dynamics were studied on the various days of hCG treatment (dose 0·05–0·075 i.u.) and compared with the follicle changes that take place after electrical stimulation of the hypothalamus, performed on day 28, a treatment known to result in first ovulation 4–5 days later. In both groups a decrease in the number of the smallest and the middle-sized antral follicles as compared with their respective controls was seen, whereas numbers of follicles in the largest, 'ovulatable' size classes gradually increased. The pattern was more conspicuous in the hCG-treated group, presumably related to greater constancy in timing of the ovulatory response in this group.

The present data support the view that endogenous changes in LH secretion during late prepuberty (which have been found to take place) play a significant role in stimulating late-prepubertal follicle growth and the ensuing first ovulation.

J. Endocr. (1985) 106, 61–66

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The inhibitory action on FSH secretion of combined oestradiol and progesterone treatment of ovariectomized, immature rats was studied at various ages. At all ages studied (13–35 days) an additional inhibitory action of progesterone, if combined with oestradiol, could be found as compared with the effect of oestradiol alone. Until 20 days of age, the rise in serum FSH concentration as measured 2 days after ovariectomy could be completely prevented by administration of 0·05 μg oestradiol/100 g body weight or by administration of a lower dose of oestradiol (0·01–0·025 μg) combined with progesterone (0·5–1·5 mg/100 g body weight). After 20 days neither oestradiol nor the combined oestradiol/progesterone treatment resulted in an FSH concentration similar to that found in intact rats. However, the lowest FSH concentrations were reached by using combinations of oestradiol and progesterone.

Using progesterone alone, FSH concentration in ovariectomized rats was significantly reduced between 18 and 30 days of age, but not before or after this period.

Taken together with data on uterine weight and serum concentrations of progesterone, these findings suggest that (1) both oestradiol and progesterone exert an age-dependent role in regulating FSH secretion in the immature female rat, and (2) amounts of oestradiol and progesterone capable of maintaining, in ovariectomized rats, uterine weights not different from those in intact rats will maintain near-physiological concentrations of FSH before but not after day 20. Thus, ovarian factors other than oestradiol and progesterone must be involved in the regulation of FSH secretion in the female rat after 20 days of age.

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H. M. A. Meijs-Roelofs, P. Kramer, and H. J. Sander

Concentrations of LH in the serum were estimated in rats bled either once or twice during a 15-day period preceding first ovulation. In rats bled once (between 09.00 and 17.00 h) serum concentrations showed little change between 15 and 9 days before first ovulation and averaged 16 μg/l (days −15 to −9). A shift in LH level, to a mean of 31 μg/l, was seen on day −8, whereafter LH concentrations increased gradually. Basal LH values of < 10 μg/l were only found until day −4.

The finding that LH values increased with age was confirmed by data from rats bled twice with an interval of ≥ 3 days between bleedings. Furthermore, both in rats bled twice at 11.00 h and in rats bled twice at 15.00 h LH concentrations were significantly higher in the second sample.

Both morning (11.00 h) and afternoon (15.00 h) LH concentrations in rats bled once also indicated a rise in LH concentrations with age but it became apparent that only morning values showed a shift in LH concentration (from ∼ 15 to ∼42 μg/l) from day −9 to day −8. In contrast, mean afternoon values showed a gradual increase from day −15 on. From day −8 on the number of rats with LH values ≥50 μg/l increased, and from day −5 on they were more frequent at 15.00 than at 11.00 h.

Thus a clear increase in LH secretion, most likely of a pulsatile nature, was found in the female rat approaching puberty. The correlation in time and possible functional relationship with late-prepubertal follicular growth is discussed.

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A possible role of 5α-androstane-3α,17β-diol (3α-androstanediol) in the control of FSH secretion was studied at various ages in ovariectomized rats. In the rat strain used, vaginal opening, coincident with first ovulation, generally occurs between 37 and 42 days of age. If 3α-androstanediol alone was given as an ovarian substitute, an inhibitory effect on FSH release was evident with all three doses tested (50, 100, 300 μg/100 g body wt) between 13 and 30 days of age; at 33–35 days of age only the 300 μg dose caused some inhibition of FSH release. Results were more complex if 3α-androstanediol was given in combined treatment with oestradiol and progesterone. Given with progesterone, 3α-androstanediol showed a synergistic inhibitory action on FSH release between 20 and 30 days of age. However, when 3α-androstanediol was combined with oestradiol a clear decrease in effect, as compared to the effect of oestradiol alone, was found between 20 and 30 days of age. Also the effect of combined oestradiol and progesterone treatment was greater than the effect of combined treatment with oestradiol, progesterone and 3α-androstanediol. At all ages after day 20 none of the steroid combinations tested was capable of maintaining FSH levels in ovariectomized rats similar to those in intact rats.

It is concluded that 3α-androstanediol might play a role in the control of FSH secretion in the immature rat, but after day 20 the potentially inhibitory action of 3α-androstanediol on FSH secretion is limited in the presence of oestradiol.

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H. M. A. Meijs-Roelofs, W. A. van Cappellen, E. C. M. van Leeuwen, and P. Kramer


The effects of the suppression of the high gonadotrophin concentrations normally present by the end of the second week of life on ovarian follicle dynamics were studied in immature rats. Gonadotrophins were suppressed by treatment with an LHRH antagonist (LHRH-A; Org. 30276) on days 6, 9, 12 and 15, and the total population of ovarian follicles was studied at 15 and 28 days, on the day of first oestrus and on the day of oestrus at or following 90 and 300 days of age. Primordial follicles were counted and growing follicles were counted and measured. In rats treated with LHRH-A, follicle recruitment into the growing pool was clearly diminished; the number of growing follicles was significantly (P<0·01) lower up to the day of first oestrus and the pool of primordial follicles was significantly (P<0·05) larger at 15 and 28 days. Ovarian weights were significantly lower in rats treated with LHRH-A until at least 90 days of age. However, on the day of oestrus at or after 90 and 300 days of age, there were no differences in either the pool of primordial follicles or the pool of growing follicles between rats treated with LHRH-A and control rats. There was also no difference between groups in the number of fresh corpora lutea at these ages. It was concluded that the early peak in gonadotrophin concentrations in immature rats causes substantial recruitment of follicles into the growing pool. Thus, the number of follicles entering the growing pool is not solely dependent upon the size of the pool of primordial follicles but is clearly influenced by the level of circulating gonadotrophins.

In contrast, the large gonadotrophic stimulation that normally takes place during the second and third week of life is neither a prerequisite for functional sexual maturation nor for later cyclic function. Shortly before the time of first ovulation a tight control of follicle dynamics is established which is largely independent of previous gonadotrophin concentrations and follicle dynamics.

Journal of Endocrinology (1990) 124, 247–253