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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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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. 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. 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

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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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J. Th. J. Uilenbroek, H. M. A. Meijs-Roelofs, P. J. A. Woutersen, P. Kramer, W. A. van Cappellen, L. A. Gribling-Hegge and W. J. de Greef


To determine whether the decrease in ovarian 5α-reduced androgen production before first ovulation might be caused by an increase in serum LH, prepuberal female rats were injected at 28–31 days of age with low doses of human chorionic gonadotrophin (hCG) (0·05–0·075 i.u., four times daily). This treatment resulted in ovulation of six to ten ova per rat on day 32 in all animals.

Treatment with hCG resulted in a gradual decrease in ovarian content and production (i.e. content in ovary and medium after 4 h of incubation) of 5α-dihydrotestosterone (DHT) and 5α-androstane-3α,17β-diol. The ovarian content of DHT and the production of 5α-androstane-3α,17β-diol decreased within 24 h after the first injection of hCG. Oestradiol content and production increased between 24 and 48 h after the start of treatment and was maximal on day 31 (day of pro-oestrus).

Activities of 5α-reductase and aromatase were measured in ovarian homogenates obtained on days 29–31. Activity of 5α-reductase in hCG-treated rats was lower than that in control rats on all days studied. Aromatase activity in hCG-treated rats increased between days 29 and 31.

It was concluded that multiple injections of low doses of hCG, which may induce ovulation, cause a decrease in 5α-reduced androgen production, which is probably due to a decrease in 5α-reductase activity. The subsequent increase in oestradiol production corresponds with an increase in aromatase activity. The results indicate that the decrease in 5α-reductase activity as observed in ovaries of spontaneously ovulating rats might be caused by the gradual increase in serum LH, which has been found to occur during the last week before first ovulation.

J. Endocr. (1985) 107, 113–119

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W A van Cappellen, H M A Meijs-Roelofs, P Kramer, E C M van Leeuwen, R de Leeuw and F H de Jong


The effects of a single injection of recombinant human FSH (rhFSH; Org32489) on ovulation rate and timing and on antral follicle growth were studied in adult 5-day cyclic rats. Rats injected at 1700 h on dioestrus-2 with a dose of 10 IU rhFSH showed, on average, no increase in ovulation rate on the day of expected oestrus. However, an additional, precocious ovulation resulting in a normal number of corpora lutea 13·3±0·4, n=6) was found to take place on the night after injection, i.e. dioestrus-3. No mating behaviour, as shown by the absence of vaginal plugs the next morning, was observed at this ovulation. Follicle counts showed a loss of large antral follicles due to ovulation and increased numbers of healthy small antral follicles at 17 and 41 h after injection, indicating a decrease of atresia of growing follicles as well as additional recruitment of new antral follicles. The endogenous serum FSH concentration on the subsequent day of oestrus (65 h after the rhFSH injection) as well as recruitment of small antral follicles were lower in the rhFSH-treated rats than in saline-treated controls. The ovulation at oestrus, 48 h after the precocious, rhFSH-induced ovulation showed large differences in the number of oocytes between the rats in one treatment group.

Similar results in terms of immediate ovulation induction were obtained by using a highly purified human urinary FSH preparation (i.e. metrodin). Furthermore, the direct induction of ovulation by rhFSH or metrodin could not be prevented by the injection of an LHRH antagonist.

It was concluded that rhFSH can induce acute ovulation in rats, and stimulates follicular development directly or indirectly through increased FSH levels after ovulation. It induces antral follicle growth and decreases early atresia in small antral follicles.

Journal of Endocrinology (1995) 144, 39–47

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


On the various days of the 5-day oestrous cycle of the rat, ovarian antral follicles were dissected out and grouped in five size classes. Four follicles of the same size class were homogenized jointly in medium, after which inhibin-like bioactivity, inhibin immunoreactivity and oestradiol-17β content were measured. In general, there was a significant correlation between immunologically and biologically active inhibin levels in the different size classes; overall correlation was 0·85 (n=87, P<0·00001). In the smallest antral follicles (classes 1 and 2) inhibin bioactivity was detected only during the first three days of the cycle. With increasing follicle size, inhibin bioactivity and immunoreactivity increased, with maximal activity present in the largest, i.e. preovulatory, follicles (class 5) during the last three days of the cycle (the day of oestrus denotes day 1 of the cycle). These results indicate that only follicles which reach the antral stage at oestrus, and are known to be recruited by the periovulatory FSH peak, acquire the potency to produce biologically active inhibin. This is the cohort of follicles from which selection of ovulatory follicles will normally take place.

In contrast to inhibin, follicular oestradiol-17β concentrations were negligible until the last days of the cycle when oestradiol-17β was present in follicles larger than class 2; levels increased with increasing follicle size and a maximal level was found in preovulatory follicles at pro-oestrus.

It is concluded that (1) there is a good correlation between follicular content of inhibin-like bioactivity and inhibin immunoreactivity and (2) there are differences between patterns of follicular levels of inhibin immunoreactivity and oestradiol-17β during the ovarian cycle: follicles destined to ovulate start to produce inhibin in volume classes 1 and 2 and only thereafter also contain oestradiol-17β. Follicles entering classes 1 and 2 at dioestrus-2 and -3 or at pro-oestrus do not produce inhibin and become atretic.

Journal of Endocrinology (1995) 146, 323–330

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


Ovulation rate, follicle growth, serum FSH and oestradiol concentrations were studied after a single intraperitoneal injection of inhibin antiserum in 5-day-cyclic rats. Control rats received (non-immune) serum from castrated sheep or saline. Rats were injected at 10.00 h on dioestrus-1 (D1), i.e. the day following the day of oestrus, or at 17.00 h on dioestrus-2 (D2). The ovaries were excised at necropsy 48 h after injection, or at first or second oestrus after injection. After routine histology fresh corpora lutea were counted and/or differential follicle counts were made.

Results from rats injected with either (non-immune) serum from castrated sheep or with saline were not different and were therefore combined to form the control group. The activity of inhibin-neutralizing antibodies in the circulation of antiserum-treated rats was reduced by approximately 39% between 8 h and second oestrus after injection, as determined by the binding of purified bioactive radioiodinated 31 kDa bovine inhibin.

Rats were injected on D1 and killed at first oestrus. The number of fresh corpora lutea was significantly higher in antiserum-treated rats than in controls (13·9±0·4 vs 11·8±0·4; P < 0·05). Other rats injected on D1 were killed either 48 h or at the second oestrus after injection. Blood was collected 8, 16, 24 and 48 h and at first and second oestrus after injection. At 48 h after injection differential follicle counts showed that the ovaries of antiserum-treated rats contained approximately 32 more healthy follicles and 11 fewer atretic follicles than controls (both P < 0·05 vs control; data for follicles with volume > 100 × 105μm3 and diameter > 260 μm). The ovaries of the antiserum-treated group collected at second oestrus contained more corpora lutea than controls (17·5±0·5 vs 13·6±0·4; P < 0·001). Serum FSH levels at 8, 16, 24 and 48 h after antiserum injection were elevated (P < 0·05). Overall oestradiol levels in antiserum-treated rats were increased from 8 to 24 h and at first oestrus (P < 0·05) as compared with control rats. Further rats were injected on D2 and necropsied at first or second oestrus which caused ovulation rate to almost double at first oestrus (antiserum 23·7±1·4 vs control 12·4±0·4; P < 0·01), while at second oestrus there was no difference between antiserum-treated and control rats.

The rise in FSH level after injection of antiserum on D1 caused follicle recruitment in addition to that normally occurring on the morning of oestrus (36 h earlier) and reduced atresia, resulting in a moderately increased ovulation rate on the first and second oestrus after injection. If the interval between antiserum injection and the next oestrus was shortened (injection on D2), ovulation rate was doubled, while on the next oestrus (second) there was no difference compared with controls. It is concluded that inhibition is progressively involved in the control of follicle growth and ovulation rate via its effect on serum FSH levels during the oestrus cycle of the rat.

Journal of Endocrinology (1991) 130, 297–303