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


The physiological role of activated hypothalamic N-methyl-d-aspartate (NMDA) receptors during the final phase of female sexual maturation was explored in the rat. The effects of administration of the specific non-competitive receptor antagonist MK-801 on the occurrence of first ovulation and on LH secretion were studied. Injections of MK-801 (0·1–0·2 mg/kg body wt, s.c.) were given once or twice daily, starting at 28 or 35 days of age and continuing up to the day of first ovulation, resulted in a significant delay of this ovulation. Rats that were treated daily with 0·2 mg MK-801/kg, starting on days 30 or 34 and continuing up to day 38, but not including the day of first pro-oestrus, also showed retarded first ovulation. No decrease in serum LH concentration, compared with control rats, could be detected in these rats.

Acute treatment with MK-801 (one or two injections of 0·2, or one injection of 0·5 mg/kg) given at 11.30 h (and 16.00 h) on the day of first pro-oestrus produced partial (1 × 0·2 mg/kg) or complete (2×0·2 and 1 × 0·5 mg/kg) blockade of first ovulation; blocked rats ovulated 1 day later. Serum LH concentrations at 16.00 h on the day of pro-oestrus were significantly decreased in all MK-801-treated groups compared with saline-injected control rats. At 19.00 and 22.00 h LH concentrations remained low in all non-ovulating MK-801-treated rats, but increased in the MK-801-treated rats that ovulated.

Thus chronic blockade of the NMDA receptors by the antagonist MK-801 delays but does not prevent first ovulation, whereas acute treatment blocks the pro-oestrous LH peak.

It was concluded that activation of NMDA receptors plays an important role both in tonic and preovulatory LH secretion during the onset of puberty in the female rat.

Journal of Endocrinology (1991) 131, 435–441

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H. M. A. Meijs-Roelofs, P. Kramer, W. A. van Cappellen and G. A. Schuiling


Subcutaneous injections of an LHRH antagonist (ALHRH; Org.30093) were administered to immature female rats. Neither a single high dose (50 μg) nor repeated daily doses of 5–30 μg ALHRH/day, administered between 28 and 38 days of age, influenced the age and body weight at the time of vaginal opening or first ovulation. If repeated daily doses of 2 × 10 μg ALHRH were given from 32 to 42 or from 37 to 47 days of age, first ovulation was delayed by 3·0 and 6·3 days respectively. Administration of 10 μg ALHRH at 09.00 h and again at 17.00 h on the day of first pro-oestrus was found to be sufficient to block the expected first ovulation in 36 out of 38 rats. This effect could be repeated by administering the same doses of ALHRH at pro-oestrus and again on the next day: ovulation was blocked in eight out of eight rats. A single dose of 10 μg ALHRH, administered on the morning of pro-oestrus, blocked ovulation in five out of twelve rats. Both the preovulatory LH and FSH surge, as measured at 16.00 h on pro-oestrus, were found to be inhibited by ALHRH treatment.

On the day after pro-oestrus no recruitment of new small antral follicles had occurred in rats with ovulatory blockade. Delayed ovulation took place 2–5 days after ALHRH injection at pro-oestrus; until 3 days after injection rats were able to ovulate their original preovulatory follicles, thereafter newly developed follicles ovulated and large ovarian cysts were found in the ovaries, next to fresh corpora lutea.

Chronic administration of two injections daily of 10 μg ALHRH from 34 days of age until the morning of first pro-oestrus had marginal effects on the timing of first pro-oestrus and on follicle dynamics.

It was concluded that with the ALHRH compound used, and in chronic as well as in acute experiments, first ovulation could only be delayed by its administration on the day of first pro-oestrus and that the effect was due to acute inhibition of the preovulatory gonadotrophin surge.

J. Endocr. (1987) 112, 407–415

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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

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A. I. Toorop, H. M. A. Meijs-Roelofs, P. Kramer and W. J. de Greef


Steroid concentrations were studied in ovarian tissue obtained by means of unilateral ovariectomy during a 10-day period preceding first ovulation. Furthermore, the levels of progesterone were measured in this period in the serum of simultaneous intact control rats.

Ovarian concentrations of testosterone and oestradiol were at about 70 and 15 fmol/mg ovary respectively from 10 to 4 days before first ovulation; they clearly increased to a maximum of 170 and 120 fmol/mg ovary during the last 1–3 days before first ovulation. Ovarian concentrations of 5α-reduced androgens (i.e. androsterone and 5α-androstane-3α, 17β-diol) were high (up to 2500 fmol/mg ovary) from 7 to 4 days before first ovulation, whereas low levels (500–750 fmol/mg ovary) were present during the last 3 days before ovulation. Both ovarian and serum concentrations of progesterone were constantly low (at 500–1000 fmol/mg ovary and at 17–32 nmol/l respectively) from 10 days to 1 day before first ovulation.

An inverse correlation was observed between the ovarian content of 5α-reduced androgens and that of oestradiol, which is in agreement with the existence of a prepubertal shift in pathways of steroid production; this shift seems to take place between 4 and 3 days before first ovulation.

Changes in ovarian steroids on the day of the first pro-oestrus were fully comparable to those at pro-oestrus in adults: relatively high levels of ovarian testosterone and oestradiol, reaching values of 339 and 711 fmol/mg ovary respectively during the morning, an increase in progesterone (from 986 to > 40 000 fmol/mg ovary) first observed at 14.00 h and a decline, observed at 17.00 h, in ovarian testosterone and oestradiol concentration, resulting in values of 9 and 3 fmol/mg ovary respectively at 21.00 h.

J. Endocr. (1984) 100, 281–286

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H. J. Sander, H. M. A. Meijs-Roelofs, P. Kramer and E. C. M. van Leeuwen


The presence and physiological significance of ovarian inhibin-like activity (ILA) in immature rats was studied by measuring ILA in ovarian homogenates and by studying the short-term changes in serum concentrations of FSH and LH after unilateral or bilateral ovariectomy. Inhibin-like activity in ovarian homogenates was estimated using an in-vitro bioassay system with dispersed rat anterior pituitary cells and subsequent measurement of FSH and LH levels in the spent pituitary cell medium. Inhibin-like activity, expressed in units/ovary, was undetectable in 13-day-old ovaries, was present in 18-day-old ovaries (5·8 units/ovary) and rose significantly to a value of 29·1 units/ovary on day 23. Another significant rise was seen between 28 and 33 days of age, with ILA reaching a value of 66·4 units/ovary, a value still well below that found in adult dioestrous ovaries (155·6 units/ovary). In the bioassay no systematic dose-dependent influence on LH secretion was found. Serum concentrations of FSH after unilateral or bilateral ovariectomy at 18 days of age showed no change at 5 h and a significant (P < 0·05) increase compared with sham-operated controls at 24 h after operation. At 23, 28 and 33 days of age significant increases in FSH concentration were seen at 5 and 8 h after both unilateral and bilateral ovariectomy. At 24 h after unilateral ovariectomy, FSH had returned to the control values in these rats, whereas after bilateral ovariectomy a further increase in FSH was seen. Concentrations of LH generally varied widely and, compared with the situation after sham-operation, did not show a systematic trend of change during the 24-h period after unilateral or bilateral ovariectomy.

It was concluded that ILA is present in immature rat ovaries from the age of 18 days and increases, to a still sub-adult value, until at least 33 days of age. A physiological role for ILA, as a short-term regulator of FSH secretion seems to be present from at least 23 days of age and may account for the decreasing FSH concentrations seen during the late-prepubertal period, when ovarian steroids alone cannot explain the low FSH levels measured.

J. Endocr. (1985) 107, 251–257

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


The effect on first ovulation of the massive reduction of the total pool of ovarian follicles during the infantile and late juvenile period was studied in rats. Treatment with an LH-releasing hormone antagonist (LHRH-A) during infancy (5 mg/kg body weight on days 6, 9, 12 and 15 of life) was combined with unilateral ovariectomy performed on either day 15 (early ULO) or 2–5 days before the expected day of first ovulation (late ULO). Rats were killed on the day of first or second oestrus, when blood was collected and the (remaining) ovaries were prepared for differential counting of follicles and corpora lutea. In addition, blood was sampled 8 h after ULO and the ovaries studied histologically in the group of rats which were unilaterally ovariectomized 2–5 days before first ovulation.

The time of first ovulation was not influenced by treatment with LHRH-A, early or late ULO, or a combination of LHRH-A treatment and ULO. Ovulation rate after LHRH-A treatment was decreased, but was still within the normal range in intact rats and in early ULO rats compared with saline-treated controls.

Serum FSH concentrations 8 h after ULO performed 2–5 days before first ovulation were similar in saline- and LHRH-A-treated rats (845 ± 59 and 801 ± 99 (s.e.m.) μg/l respectively) and had increased compared with intact controls (216 ± 15 μg/l).

Treatment with LHRH-A resulted in a reduction of more than 50% in healthy and atretic follicles, and late ULO reduced the number of healthy follicles even further. In saline-treated rats late ULO decreased the rate of atresia, but in LHRH-A-treated rats atresia was not reduced further by (late or early) ULO.

It is concluded that even after massive reduction of the pool of ovarian follicles by early LHRH-A treatment combined with late or early ULO, the timing of the first ovulation was normal and ovulation rates, although somewhat lower in some LHRH-A-treated rats, were within the normal range.

Journal of Endocrinology (1992) 135, 439–446

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


In late-prepubertal female rats passive immunoneutralization of endogenous inhibin was achieved by injection of inhibin antiserum. Effects on follicle population, timing of sexual maturation, ovulation rate at first and second oestrus and serum FSH levels were studied.

Rats were injected with antiserum, (non-immune) control serum from castrated sheep (castrated serum) or their IgG fractions, or with saline on day 33 or 3 or 2 days (days −3/−2) before the expected day of first ovulation, day 38·5±0·2 (n = 70). Blood was collected from different subgroups at 8, 24 and 48 h, and at first and second oestrus after injection. At necropsy, ovaries were histologically prepared for differential counting of follicles (48 h and first oestrus) and counting of corpora lutea (CL; first and second oestrus) as an index of ovulation rate.

Results from rats injected with either serum or its IgG fraction were not different, as was the case when rats were injected with either castrated serum or saline. Thus, results from groups treated with antiserum and antiserum IgG were combined and labelled 'antiserum', and the castrated serum, castrated serum IgG and saline-treated groups were combined and labelled 'control'. The activity of inhibin-neutralizing antibodies in the circulation of antiserum-treated rats was reduced by 43% between 8 h and second oestrus after injection, as determined by the binding of purified bioactive radioiodinated 31 kDa bovine inhibin.

After antiserum injection on day 33, more healthy antral follicles (vol. > 100 × 105 μm3, diameter > 260 μm) were present in the ovaries at 48 h (70·6 vs 54·4; P < 0·05) and at first oestrus (73·1 vs 50·8; P < 0·05) if first oestrus was reached within 5 days, but numbers were not different if first oestrus was more than 5 days after injection (52·6 vs 50·8). The number of CL after injection of antiserum on day 33 was increased at first oestrus compared with control (13·4±0·5, n = 30, vs 10·0±0·2, n = 40; P<0·001), an effect that was even more clearly present in antiserum-injected rats ovulating within 5 days (14·4±0·7, n = 20; P < 0·001).

Rats injected with antiserum at days −3/−2 showed a doubling of ovulation rate at first oestrus when compared with control animals (21·5±0·8, n = 12, vs 10·5±0·2, n = 15; P < 0·001). No differences in the number of CL was seen at second oestrus. Age and body weight on the day of first ovulation were not influenced by antiserum treatment.

Serum FSH was significantly (P < 0·01) increased at 8 h after antiserum injection on either day 33 or on days −3/−2 to a level of 250 and 800% of control levels respectively.

Thus, injection with inhibin–neutralizing antiserum into prepubertal female rats resulted, through an increase in serum FSH concentration 8 h after injection, in the growth of additional numbers of healthy antral follicles. Supranormal ovulation rate occurred if antiserum injections were given within the last 5 days before first ovulation, with a maximal ovulation rate after injection on days −3/−2. The data support the view that, in the immature female rat during the last 5 days before the day of first ovulation, inhibin is (through its regulation of serum FSH levels) progressively involved in the control of follicle growth and ovulation rate.

Journal of Endocrinology (1991) 130, 289–296

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Luteinizing hormone, FSH, prolactin, oestradiol and progesterone concentrations were measured by radioimmunoassay in the blood of female rats from 7 days before the first ovulation (the day of first ovulation is designated day 0) until the day after the second ovulation. Serum LH concentrations varied between 29 and 59 ng NIAMDD-rat-LH RP-1/ml from days −7 to −2. On the day preceding first ovulation (day − 1) a steep increase in serum LH was found between 13.00 h and 15.00 h reaching a maximal value of 2100 ng/ml at 17.00 h. Thereafter LH levels decreased to undetectable values (< 15 ng/ml). Serum FSH concentrations from days −7 to −2 varied between 108–154 ng NIAMDD-rat-FSH RP-1/ml; increased concentrations were found on the afternoon of day − 1 and on the morning thereafter (first oestrus) with a maximal value of 1150 ng/ml at 19.00 h on day − 1. On the day preceding the second ovulation (day +4) LH and FSH peaks were again found.

Prolactin concentrations on the days preceding first ovulation were generally low. A small rise was found nearing first ovulation (12·7 ng/ml, day −6; 23·9 ng/ml, day −2). On day − 1 a peak (118 ng/ml) was found at 15.00 h, similar to that found on day +4 in adult pro-oestrous rats.

Plasma progesterone concentrations also reached a peak value (50 ng/ml) on day − 1, at 17.00 h, preceded by concentrations between 3–5 ng/ml from days −6 to −2. Progesterone concentrations after the first ovulation showed a pattern characteristic of the adult cycle.

Plasma oestradiol levels were undetectable (< 10 pg/ml) from days −6 to −2; a clearly increased oestradiol concentration was found on day −1, reaching a value of 98 pg/ml. One and two days before the second ovulation, oestradiol levels rose again.

The differences and similarities between pubertal and adult pro-oestrous hormone peaks are discussed.

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I A Klaij, M A Timmerman, P Kramer, H M A Meijs-Roelofs and F H de Jong


Age-related short-term effects of hemicastration on testicular weight, serum FSH, immunoreactive inhibin, LH and testosterone, testicular levels of inhibin subunit mRNA expression, and bioactive and immunoreactive inhibin were studied in rats of 8, 15 and 22 days of age. Hemicastration led to an increased weight of the remaining testis after 24 h in 8- and 15-day-old rats, but not in 22-day-old rats. Serum FSH levels were elevated in all hemicastrated rats after 8 h. However, serum immunoreactive inhibin levels were decreased only after 72 h in 8-day-old rats and after 24 h in 15- and 22-day-old rats. Inhibin α-subunit mRNA expression was increased in the testes of hemicastrated rats of 8 and 15 days of age, whereas inhibin βB-subunit mRNA expression was elevated in the testes of 15-day-old rats but not in those of 8- and 22-day-old rats. The increase in α-subunit mRNA content per testis was caused by an increased concentration and increased testicular weight, whereas the increase in βB-subunit mRNA in the remaining testis parallelled the increased testicular weight, indicating that different mechanisms play a role in the regulation of these mRNAs. In 22-day-old rats, a transiently decreased expression of inhibin βB-subunit mRNA was observed 8 h after hemicastration. The increased inhibin α- and βB-subunit mRNA expression in 8- and 15-day-old rats did not result in increased testicular bioactive and immunoreactive inhibin content of the remaining testis, whereas in 22-dayold rats an increased immunoreactive inhibin content of the remaining testis was observed. These data indicate that efficiency of translation, post-translational modifications or transport from the testis play an important role in determining the final testicular content of inhibin.

In conclusion, the response of the remaining testis and the role of inhibin in the regulation of the pituitary-testis axis after unilateral castration depend on the age at which the animals are hemiorchidectomized.

Journal of Endocrinology (1994) 141, 143–151

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


In order to relate various prepubertal events in a group of 95 late prepubertal female rats, the following data were obtained during the last 10 days before the day of first ovulation: (1) amounts of ovarian inhibin-like activity (ILA) in some animals (n=47); (2) size and numbers of healthy (antral) follicles with a volume ≥100× 105 μm3 (or diameter ≥260 μm) present per ovary in their litter-mates (n=48); (3) serum FSH concentrations in both groups.

Rats were unilaterally ovariectomized to obtain an ovary for either estimation of ILA content or for histological procedures and counting of follicles. At the time of unilateral ovariectomy they were bled to obtain serum for estimation of FSH concentrations. Rats were kept until the day after the day of first ovulation to determine the time-interval between the day of unilateral ovariectomy and first ovulation. They were studied between 10 and 1 days (days −10 to − 1, maturational age) before first ovulation. In addition, adult cyclic rats were bilaterally ovariectomized on different days of the oestrous cycle for estimation of ovarian ILA content.

The amount of ovarian ILA was estimated in steroid-free ovarian cytosols using an in-vitro bioassay system with dispersed anterior pituitary cells and subsequent measurement of FSH and LH in the spent medium.

The amount of ovarian ILA was about 83 units/ ovary from days −10 to −5, and subsequently increased (P < 0·005) to reach a maximum on day − 1, the day of pro-oestrus (213 units/ovary). Inhibin-like activity in adult rat ovaries at pro-oestrus amounted to 374 units/ovary. A significant relationship was found between ovarian ILA content and total volume of follicles of classes III–V (≥350 × 105 μm3) (r= 0·9683, P<0·005) except for the period between days −7 and −5 when this volume increased earlier than did the ILA content.

The total volume of all follicles ≥ 100 × 105 μm3 was steady from days −10 to −7. On day −6 this volume increased, mainly as a result of an increase of total volume of class II follicles. Thereafter, the total volume of follicles in classes III–V started to increase and was maximal on day −1, while the total volume of follicles in classes I plus II decreased and reached a minimum on day −1.

The serum FSH concentration declined between days −10 and −1 from 400 to 100 μg/l (P<0·001); the presence of follicles of classes III–V was always associated with FSH concentrations ≤200 μg/l (P<0·005). The presence of class I and II follicles was not related to FSH concentrations. This suggested that mainly follicles of classes III–V contribute to ovarian ILA.

The present data show that in immature rats ovarian ILA content increases towards the day of first pro-oestrus, as it does later during pro-oestrus in adult cyclic rats. Inhibin-like activity seems to be produced mainly by follicles of classes III–V which are present in the ovaries during the last 5 days preceding first ovulation. In this same period FSH concentrations are kept within narrow limits (<200 μg/l) as is the case during the adult cycle. Thus, ILA probably plays a role in the fine regulation of FSH secretion.

J. Endocr. (1986) 111, 159–166