Effect of thyroid status and paraventricular area lesions on the release of thyrotropin-releasing hormone and catecholamines into hypophysial portal blood.

TRH is a potent stimulator of pituitary TSH release, but its function in the physiological regulation of thyroid activity is still controversial. The purpose of the present study was to investigate TRH and catecholamine secretion into hypophysial portal blood of hypothyroid and hyperthyroid rats, and in rats bearing paraventricular area lesions. Male rats were made hypothyroid with methimazole (0.05% in drinking water) or hyperthyroid by daily injections with T4 (10 micrograms/100 g BW). Untreated male rats served as euthyroid controls. On day 8 of treatment they were anesthetized to collect peripheral and hypophysial stalk blood. In euthyroid, hypothyroid and hyperthyroid rats plasma T3 was 1.21 +/- 0.04, 0.60 +/- 0.04, and 7.54 +/- 0.33 nmol/liter, plasma T4 50 +/- 3, 16 +/- 2, and 609 +/- 74 nmol/liter, and plasma TSH 1.58 +/- 0.29, 8.79 +/- 1.30, and 0.44 +/- 0.03 ng RP-2/ml, respectively. Compared with controls, hyperthyroidism reduced hypothalamic TRH release (0.8 +/- 0.1 vs. 1.5 +/- 0.2 ng/h) but was without effect on catecholamine release. Hypothyroidism did not alter TRH release, but the release of dopamine increased 2-fold and that of noradrenaline decreased by 20%. Hypothalamic TRH content was not affected by the thyroid status, but dopamine content in the hypothalamus decreased by 25% in hypothyroid rats. Twelve days after placement of bilateral electrolytic lesions in the paraventricular area plasma thyroid hormones and TSH levels were lower than in control rats (T3: 0.82 +/- 0.05 vs. 1.49 +/- 0.07 nmol/liter; T4: 32 +/- 4 vs. 66 +/- 3 nmol/liter; TSH: 1.08 +/- 0.17 vs. 3.31 +/- 0.82 ng/ml). TRH release in stalk blood in rats with lesions was 15% of that of controls, whereas dopamine and adrenaline release had increased by 50% and 40%, respectively. These results suggest that part of the feedback action of thyroid hormones is exerted at the level of the hypothalamus. Furthermore, TRH seems an important drive for normal TSH secretion by the anterior pituitary gland, and thyroid hormones seem to affect the hypothalamic release of catecholamines.

Control of primordial follicle recruitment by anti-Müllerian hormone in the mouse ovary.

The dimeric glycoprotein anti-Müllerian hormone (AMH) is a member of the transforming growth factor-beta superfamily of growth and differentiation factors. During male fetal sex differentiation, AMH is produced by Sertoli cells and induces degeneration of the Müllerian ducts, which form the anlagen of part of the internal female genital system. In females, AMH is produced by the ovary, but only postnatally. The function of AMH in the ovary is, however, still unknown. Female AMH null mice were reported to be fertile, with normal litter size, but this does not exclude a more subtle function for ovarian AMH. To investigate the function of AMH in the ovary, the complete follicle population was determined in AMH null mice, in mice heterozygous for the AMH null mutation, and in wild-type mice of different ages: 25 days, 4 months, and 13 months. In the present study we found that ovaries of 25-day- and 4-month-old AMH null females, compared to those of wild-type females, contain more preantral and small antral follicles. In addition, in 4- and 13-month-old AMH null females, smaller numbers of primordial follicles were found. Actually, in 13-month-old AMH null females, almost no primordial follicles could be detected, coinciding with a reduced number of preantral and small antral follicles in these females. In almost all females heterozygous for the AMH null mutation the number of follicles fell in between the numbers found in wild-type and AMH null females. In 4-month-old AMH null females serum inhibin levels were higher and FSH levels were lower compared to those in wild-type females. In contrast, inhibin levels were lower in 13-month-old AMH null females, and FSH levels were unchanged compared to those in wild-type females. Furthermore, the weight of the ovaries was twice as high in the 4-month-old AMH null females as in age-matched wild-type females. We conclude that AMH plays an important role in primordial follicle recruitment, such that more primordial follicles are recruited in AMH null mice than in wild-type mice; the mice heterozygous for the AMH null mutation take an in-between position. Consequently, the ovaries of AMH null females and those of females heterozygous for the AMH null mutation will show a relatively early depletion of their stock of primordial follicles. The female AMH null mouse may thus provide a useful model to study regulation of primordial follicle recruitment and the relation between follicular dynamics and ovarian aging.

Anti-Müllerian hormone attenuates the effects of FSH on follicle development in the mouse ovary.

Although ovarian follicle growth is under the influence of many growth factors and hormones of which FSH remains one of the most prominent regulators. Therefore, factors affecting the sensitivity of ovarian follicles to FSH are also important for follicle growth. The aim of the present study was to investigate whether anti-Müllerian hormone (AMH) has an inhibitory effect on follicle growth by decreasing the sensitivity of ovarian follicles to FSH. Furthermore, the combined action of AMH and FSH on ovarian follicle development was examined. Three different experiments were performed. Using an in vitro follicle culture system it was shown that FSH-stimulated preantral follicle growth is attenuated in the presence of AMH. This observation was confirmed by an in vivo experiment showing that in immature AMH-deficient females, more follicles start to grow under the influence of exogenous FSH than in their wild-type littermates. In a third experiment, examination of the follicle population of 4-month-old wild-type, FSH beta-, AMH-, and AMH-/FSH beta-deficient females revealed that loss of FSH expression has no impact on the number of primordial and preantral follicles, but the loss of inhibitory action of AMH on the recruitment of primordial follicles in AMH-deficient mice is increased in the absence of FSH. In conclusion, these studies show that AMH inhibits FSH-stimulated follicle growth in the mouse, suggesting that AMH is one of the factors determining the sensitivity of ovarian follicles for FSH and that AMH is a dominant regulator of early follicle growth.

Different effects of the antiprogesterone RU486 on progesterone secretion by the corpus luteum of rats with 4- and 5-day oestrous cycles.

Administration of the antiprogesterone RU486 (2 mg/day) for 14 days to rats with a 5-day reproductive cycle resulted in an increase in both ovarian and pituitary weight in contrast with rats with a 4-day oestrous cycle. Luteal progesterone production decreased earlier in 4-day than in 5-day cyclic rats. Treatment of 5-day cyclic rats with antiprogesterone from the day of metoestrus onwards resulted in the advancement of the preovulatory prolactin surge by 24 h. Progesterone production by the corpus luteum was, however, not affected, indicating that in 5-day cyclic rats the corpora lutea are still functionally active at the time of the preovulatory surge of prolactin. They become, therefore, stimulated both in size and progesterone production. In contrast, the corpora lutea in 4-day cyclic rats are functionally inactive at the time of the preovulatory surge of prolactin, and prolactin acts luteolytically. In conclusion, the advancement of the preovulatory surge of prolactin by 24 h accounts, at least in part, for the increase in ovarian weight in 5-day cyclic rats after treatment with antiprogesterone. The results of these experiments do not agree with a direct effect of the antiprogesterone RU486 on progesterone secretion by the corpus luteum.

Levels of dopamine and thyrotrophin-releasing hormone in hypophysial stalk blood during an oestrogen-stimulated surge of prolactin in the ovariectomized rat.

The changes in hypothalamic release of dopamine and thyrotrophin-releasing hormone (TRH) into the hypophysial portal vascular system during an oestrogen-stimulated surge of prolactin in ovariectomized rats were investigated. A single injection of 5 micrograms oestradiol benzoate resulted in a reliable increase in the plasma levels of prolactin during the afternoon 3 days later. Anaesthesia did not block this afternoon surge of prolactin, although its magnitude was only half of that of unanaesthetized rats. Before and during this surge, hypophysial stalk blood was collected into methanol to analyse the hypothalamic release of dopamine and TRH. Immunoreactive TRH in these methanolic extracts eluted as a single peak with the same retention time as authentic TRH on reverse-phase high performance liquid chromatography. In comparison to the morning values, levels of dopamine decreased and those of TRH increased in hypophysial stalk blood by 50 and 240% respectively. These data indicate that hypothalamic dopamine and TRH may be involved in the afternoon surge of prolactin. Daily treatment with parachlorophenylalanine, an inhibitor of serotonin synthesis, reduced the hypothalamic release of TRH by 50%, but did not prevent the afternoon surge of prolactin and TRH induced by oestradiol benzoate.

Effects of ovarian inhibin on pulsatile release of gonadotrophins and secretion of LHRH in ovariectomized rats: evidence against a central action of inhibin.

Intraperitoneal administration of bovine follicular fluid (bFF) decreased plasma concentrations of FSH in ovariectomized rats after 2-3 h, while plasma LH and prolactin were unaffected. In untreated ovariectomized animals the concentrations of these hormones were found to show pulsatile variations. Concomitant occurrence of peak values of LH and FSH was found in about 40% of the pulses. No pulses of FSH were observed after i.p. treatment with bFF or partly purified preparations of inhibin from bFF, but the pulsatile release of LH and prolactin remained similar. Infusion of bFF into the lateral ventricle of the brain did not alter the concentrations of FSH, whereas administration of bFF into the pituitary gland diminished the plasma concentrations of FSH. Anaesthesia (urethane plus xylazine) did not prevent the occurrence of the pulses of FSH and LH, but it reduced the pulse amplitude and clearance. During this anaesthesia, the concentrations of LHRH in the hypophysial stalk plasma decreased by 30% after administration of bFF, but did not alter after treatment with partly purified preparations of inhibin. It is concluded that the inhibin-like activity in bFF suppresses pulsatile FSH secretion in ovariectomized rats by an action on the pituitary gland, but has no effect on the pulsatile release of LH and prolactin.

Levels of LH-releasing hormone in hypophysial stalk plasma during an oestrogen-stimulated surge of LH in ovariectomized rats.

Treatment of ovariectomized rats with 50 micrograms oestradiol benzoate, followed by 20 micrograms oestradiol benzoate 3 days later, induced surges of LH and FSH on the day following the second injection with oestradiol benzoate. During this surge of gonadotrophins, which was not blocked by the anaesthetic required to collect hypophysial stalk blood, increased hypophysial stalk plasma levels of immunoreactive LHRH were noted. Furthermore, the levels of LHRH in hypophysial portal blood were found to fluctuate. Measurement of LHRH in a pool of portal plasma revealed similar results when determined by radioimmunoassay and by a sensitive in-vitro bioassay. To mimic the observed release of LHRH during the surge of gonadotrophins, LHRH was infused, either systemically or directly into a long portal vessel, into oestrogen-treated, ovariectomized rats which had their endogenous release of LHRH blocked by pentobarbitone. An infusion of LHRH into the jugular vein, resulting in peripheral levels of LHRH which were somewhat lower than those measured in hypophysial stalk plasma, caused a surge of FSH similar to that found in rats used for collection of hypophysial stalk blood. When compared with the values in the latter animals, however, the levels of LH became two to four times higher by this infusion of LHRH. When LHRH was infused directly into a long portal vessel to mimic the observed secretion rate of LHRH during the oestrogen-stimulated surge of gonadotrophins, then the surges of LH and FSH were lower than those observed in the rats used for collection of stalk blood.

Recombinant FSH-induced follicle development in immature rats treated with an LHRH antagonist:a direct effect of RU486 on follicular atresia.

To investigate whether the progesterone antagonist RU486 has a direct effect on ovarian function, it was administered to immature female rats rendered hypogonadotrophic by administration of an LHRH antagonist and in which follicle development was stimulated by recombinant human FSH (recFSH). In the first experiments the effects of LHRH antagonist and recFSH on follicle growth were evaluated. Female rats of 22 days of age were injected with an LHRH antagonist (Org 30276; 500 micrograms/100 g body weight) every other day. This treatment resulted in a tenfold decrease in serum LH concentrations and a twofold decrease in serum FSH concentrations at day 30 and caused a reduction in the number and size of antral follicles. Treatment with recFSH (Org 32489) twice daily from day 26 for 4 days in a total dose ranging from 5 to 20 IU/animal increased the number and size of antral follicles in a dose-related manner and resulted after 20 IU recFSH in a tenfold increase in the concentration of inhibin in serum and ovaries at day 30. Once it was established that LHRH antagonist treatment in immature rats could be used to study the effects of gonadotrophins or steroids on follicle function, this animal model was used to study the effects of RU486 on the ovary. RU486 was administered (twice daily for 4 days, 1 mg/injection) to LHRH antagonist-treated rats in which follicular growth and differentiation were stimulated by 10 IU recFSH or by 10 IU recFSH plus 0.5 IU human chorionic gonadotrophin (hCG). RU486 had no effect on circulating levels of LH and FSH, but stimulated follicular atresia both in rats treated with recFSH alone and in rats treated with recFSH and hCG. Inhibin concentrations both in serum and ovaries were significantly increased after hCG treatment. RU486, however, did not increase inhibin in the rats treated with recFSH and in those treated with recFSH and hCG. In summary, the present study has demonstrated that (1) immature rats treated with an LHRH antagonist can be used to study the effects of gonadotrophins and steroids on follicular function and (2) RU486 has a direct stimulatory effect on follicular atresia.

Inhibition of induction of 20 alpha-hydroxysteroid dehydrogenase activity in rat corpora lutea in vitro by the progesterone antagonist RU486.

To determine if the antiprogestagen RU486 has a direct effect on luteal progesterone secretion, whole corpora lutea or dispersed luteal cells were incubated in the presence of RU486. Whole corpora lutea, isolated from rats at day 5 of pseudopregnancy, were incubated individually in hormone-free medium. The concentrations of progesterone and 20 alpha-dihydroprogesterone in the medium plus corpus luteum was measured before and after 24 h of incubation. In the absence of RU486 the concentration of 20 alpha-dihydro-progesterone increased, while that of progesterone remained unchanged. In the presence of RU486 (230 microM) the concentration of both progesterone and 20 alpha-dihydro-progesterone was increased. Dispersed luteal cells were incubated for 24 h in the presence of various amounts of RU486. In the absence and in the presence of 0.2 and 2.3 microM RU486 a high ratio between 20 alpha-dihydro-progesterone and progesterone was found, while in the presence of 23 microM RU486 the concentrations of progesterone and 20 alpha-dihydro-progesterone were equal. 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD) activity measured in luteal homogenates started to increase between 6 and 12 h of incubation. This increase could be prevented after incubation of the corpora lutea in the presence of 23 or 230 microM RU486 for 24 hrs. It is concluded that the progesterone antagonist RU486 can have a direct effect on luteal progesterone production. RU486 prevents the increase in 20 alpha-HSD activity that normally occurs during in vitro incubation. However, since these effects in vitro can only be obtained with high concentrations of RU486, it is unlikely that this antiluteolytic effect plays a role after injection of RU486 in vivo.

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)

Significance of oestradiol for follicular development in hypogonadotrophic immature rats treated with FSH and hCG.

Administration of hCG (0.5 i.u.) to immature female rats that were made hypogonadotrophic by injections of an LHRH-antagonist at 22, 24, 26 and 28 days of age and in which follicular development was induced by FSH (10 i.u., given from day 26 until day 29) stimulates follicles to grow to their preovulatory size with a low amount of atresia. To investigate whether this effect of hCG on follicular growth is due to stimulation of oestradiol production, intraovarian concentrations of oestradiol were suppressed by an aromatase inhibitor and oestradiol action was blocked by an oestrogen antagonist; both of these were administered from day 25 until day 29. By day 30 this treatment had resulted in an increase in the percentage of atretic follicles. In turn, oestradiol benzoate (100 micrograms or 1000 micrograms day-1), given from day 26 until day 29 in rats treated with 10 i.u. FSH, resulted in (1) an increase in the total number of antral follicles, (2) an increase in follicular size and (3) a decrease in the percentage of atretic follicles. Although the administration of 1000 micrograms oestradiol benzoate can mimic the effects of 0.5 i.u. hCG on follicular growth to a large extent, the number of follicles with a diameter > 575 microns in rats treated with FSH plus oestrogen was smaller than that in rats treated with FSH plus hCG (10 +/- 2 and 24 +/- 2, respectively). Furthermore, a single injection of 10 i.u. hCG given on day 29 was used to induce ovulation: fewer ova were found in rats treated with FSH plus 1000 micrograms oestrogen than in rats treated with FSH plus hCG (15.2 +/- 2.1 and 46.5 +/- 1.6, respectively). From these studies, it is concluded that the effects of hCG on follicular growth and atresia can be largely attributed to the mitotic and anti-atretic effect of oestradiol. However, part of the effect of hCG cannot be explained on the basis of oestradiol action.

Apoptotic and proliferative changes during induced atresia of pre-ovulatory follicles in the rat.

Atresia, a degenerative process through which many follicles are removed from the growing pool, involves apoptotic changes in the follicular granulosa cells. To identify histochemical markers of early stages of atresia, an in-vivo rat model was used which allowed the study of atresia of pre-ovulatory follicles in a synchronized and chronological order. By blocking the pre-ovulatory luteinizing hormone surge with a gonadotrophin-releasing hormone (GnRH) antagonist, ovulation of the pre-ovulatory follicles is prevented, after which these follicles became atretic. The first morphological sign of atresia (pyknotic granulosa cell nuclei) was found 27 h after injection of GnRH antagonist. Since the pre-ovulatory follicles gradually become atretic in a synchronous fashion, this model provided an opportunity to study and define markers of future atresia in pre-ovulatory follicles. Atresia involves apoptosis of granulosa cells, and therefore internucleosomal DNA fragmentation was examined. Using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelling (TUNEL) assay it was found that the first sign of internucleosomal DNA fragmentation in granulosa cells of pre-ovulatory follicles was detectable 24 h after GnRH antagonist treatment. In order to find an upstream marker of atresia, the 5-bromo-deoxyuridine (BrdU) labelling index was used as a measure of proliferation. Already at 14 h after GnRH antagonist treatment, when morphological signs of atresia were not yet present, a clear decrease in BrdU labelling index was found in the granulosa cells.

A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the müllerian duct.

The activin and TGF-beta type II receptors are members of a separate subfamily of transmembrane receptors with intrinsic protein kinase activity, which also includes the recently cloned TGF-beta type I receptor. We have isolated and characterized a cDNA clone (C14) encoding a new member of this subfamily. The domain structure of the C14-encoded protein corresponds with the structure of the other known transmembrane serine/threonine kinase receptors. It also contains the two inserts in the kinase domain that are characteristic for this subfamily. Using in situ hybridization, C14 mRNA was detected in the mesenchymal cells located adjacent to the müllerian ducts of males and females at day 15 (E15) of embryonic development. Marked C14 mRNA expression was also detected in the female gonads. In female E16 embryos, the C14 mRNA expression pattern remained similar to that in E15 embryos. However, in male E16 embryos C14 mRNA was detected in a circular area that includes the degenerating müllerian duct. The expression of C14 mRNA was also studied using RNase protection assays. At E15 and E16, C14 mRNA is expressed in the female as well as in the male urogenital ridge. However, at E19, a high C14 mRNA level in the female urogenital ridge contrasts with a lack of C14 mRNA in the male urogenital ridge. This correlates with the almost complete degeneration of the müllerian ducts in male embryos at E19. C14 mRNA expression was also detected in embryonic testes at E15, E16 and E19 using RNase protection assays, but at much lower levels than those found in the developing ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)