http://repub.eur.nl/res/aut/624/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/9858/ 2002-01-01T00:00:00Z Recruitment of primordial follicles is essential for female fertility; however, the exact mechanisms regulating this process are largely unknown. Earlier studies using anti-Mullerian hormone (AMH)-deficient mice suggested that AMH is involved in the regulation of primordial follicle recruitment. We tested this hypothesis in a neonatal ovary culture system, in which ovaries from 2-d-old C57Bl/6J mice were cultured for 2 or 4 d in the absence or presence of AMH. Ovaries from 2-d-old mice contain multiple primordial follicles, some naked oocytes, and no follicles at later stages of development. We observed that in the cultured ovaries, either nontreated or AMH-treated, follicular development progressed to the same extent as in in vivo ovaries of comparable age, confirming the validity of our culture system. However, in the presence of AMH, cultured ovaries contained 40% fewer growing follicles compared with control ovaries. A similar reduction was found after 4 d of culture. Consistent with these findings, we noted lower inhibin alpha-subunit expression in AMH-treated ovaries compared with untreated ovaries. In contrast, expression of AMH ligand type II receptor and the expression of oocyte markers growth and differentiation factor 9 and zona pellucida protein 3 were not influenced by AMH. Based on the results, we suggest that AMH inhibits initiation of primordial follicle growth and therefore functions as an inhibitory growth factor in the ovary during these early stages of folliculogenesis. http://repub.eur.nl/res/pub/9773/ 2001-01-01T00:00:00Z 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-Mullerian 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. http://repub.eur.nl/res/pub/9546/ 2000-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/9201/ 1999-01-01T00:00:00Z The dimeric glycoprotein anti-Mullerian 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 Mullerian 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. http://repub.eur.nl/res/pub/8903/ 1998-01-01T00:00:00Z The clinical use of diethylstilbestrol (DES) by pregnant women has resulted in an increased incidence of genital carcinoma in the daughters born from these pregnancies. Also, in the so-called DES-sons abnormalities were found, mainly, the presence of Mullerian duct remnants, which indicates that fetal exposure to DES may have an effect on male sex differentiation. Fetal regression of the Mullerian ducts is under testicular control through anti-Mullerian hormone (AMH). In male mice, treated in utero with DES, the Mullerian ducts do not regress completely, although DES-exposed testes do produce AMH. We hypothesized that incomplete regression in DES-exposed males is caused by a diminished sensitivity of the Mullerian ducts to AMH. Therefore, the effect of DES on temporal aspects of Mullerian duct regression and AMH type II receptor (AMHRII) messenger RNA (mRNA) expression in male mouse fetuses was studied. It was observed that Mullerian duct regression was incomplete at E19 (19 days post coitum), upon DES administration during pregnancy from E9 through E16. Furthermore, analysis of earlier time points of fetal development revealed that the DES treatment had clearly delayed the onset of Mullerian duct formation by approximately 2 days; in untreated fetuses, Mullerian duct formation was complete by E13, whereas fully formed Mullerian ducts were not observed in DES-treated male fetuses until E15. Using in situ hybridization, no change in the localization of AMH and AMHRII mRNA expression was observed in DES-exposed male fetuses. The mRNA expression was quantified using ribonuclease protection assay, showing an increased expression level of AMH and AMHRII mRNAs at E 13 in DES-exposed male fetuses. Furthermore, the mRNA expression levels of Hoxa 11 and steroidogenic factor-1 (SF-1) were determined as a marker for fetal development. Prenatal DES exposure had no effect on Hoxa 11 mRNA expression, indicating that DES did not exert an overall effect on the rate of fetal development. In DES-exposed male fetuses, SF-1 showed a similar increase in mRNA expression as AMH, in agreement with the observations that the AMH gene promoter requires an intact SF-1 DNA binding site for time- and cell-specific expression, although an effect of DES on SF-1 expression in other tissues, such as the adrenal and pituitary gland, cannot be excluded. However, the increased expression levels of AMH and AMHRII mRNAs do not directly explain the decreased sensitivity of the Mullerian ducts to AMH. Therefore, it is concluded that prenatal DES exposure of male mice delays the onset of Mullerian duct development, which may result in an asynchrony in the timing of Mullerian duct formation, with respect to the critical period of Mullerian duct regression, leading to persistence of Mullerian duct remnants in male mice. http://repub.eur.nl/res/pub/8924/ 1998-01-01T00:00:00Z This study aimed to investigate the time course of disappearance of the mRNAs of the various subunits of inhibin in follicles which become atretic. An animal model was used in which atresia of preovulatory follicles could be studied in a chronological order. Injection of gonadotrophin-releasing hormone (GnRH) antagonist (20 microg) at the morning of pro-oestrus (P) blocked ovulation and the 10-12 preovulatory follicles became gradually atretic. A second injection was given the next day to prevent delayed ovulation. The rate of atresia could be delayed by simultaneous administration of a subovulatory dose of human chorionic gonadotrophin (hCG) (0.5 IU) and could be advanced by administration of a fivefold larger amount of GnRH antagonist. Functional activity of follicles becoming atretic was studied by measuring oestradiol production after incubation of individual follicles for 4 h. Follicles isolated 24 h after the first injection of GnRH antagonist (P+24) already secreted significantly less oestradiol in vitro than follicles isolated at pro-oestrus, although they were morphologically not different from pro-oestrous follicles. Follicles isolated at P+24 from hCG-treated rats secreted more oestradiol compared with follicles from rats not treated with hCG. In contrast, follicles isolated at P+24 from rats that were given a fivefold larger amount of GnRH antagonist secreted less oestradiol. Once this model was validated, temporal changes in inhibin subunit mRNAs in follicles undergoing atresia were measured by in situ hybridization and RNase protection assay. In situ hybridization showed abundant alpha- and betaA-subunit mRNA in the whole granulosa layer of preovulatory follicles at P and P+24, while betaB-subunit mRNA was restricted to the antral layer and cumulus. At P+48 the amount of alpha- and betaA-subunit mRNA had declined and was restricted to the cumulus, whereas betaB-subunit mRNA was absent. In the atretic follicles present at P+72 and P+96, mRNAs of all three inhibin subunits were absent. Administration of 0.5 IU hCG delayed the decline in the amount of alpha, betaA and betaB mRNA in preovulatory follicles at P+48. RNase protection assay of inhibin subunits in isolated follicles revealed no changes between P and P+24. However, at P+48, the mRNAs of alpha- and betaA-subunits were decreased. Expression of the mRNA of betaB-subunit declined gradually from P to P+48. The present study demonstrates that in follicles which are becoming atretic, mRNAs of alpha- and betaA-subunits decline simultaneously with the appearance of pycnotic cells in the granulosa layer, while betaB-subunit mRNA declines earlier, simultaneously with the decrease in the ability to secrete oestradiol in vitro. http://repub.eur.nl/res/pub/8658/ 1997-01-01T00:00:00Z The objective of this study was to set up a superovulation protocol in adult cyclic rats by using recombinant human follicle stimulating hormone (rhFSH; Org32489). Good results were obtained by treatment with decreasing doses of rhFSH (2.5 to 0.5 IU) during the dioestrus period. The number of corpora lutea (CL) found in rats treated with this protocol was 43.5 +/- 3.4; this is more than three times the number in saline-treated control rats (13.0 +/- 0.4). Fertilization of oocytes after superovulation was as good as after normal ovulation in terms of number of 2-cell stage embryos found 2 days after mating. The absolute number of implantations was twice the number observed in saline-treated control rats (23.3 +/- 1.8 versus 10.6 +/- 0.5); therefore the number of implantations per CL was lower in superovulated rats. The serum concentrations of luteinizing hormone (LH), endogenous FSH and oestradiol-17beta were decreased during rhFSH treatment, while the inhibin serum concentration was increased. The progesterone serum concentration was increased on the days of pro-oestrus and oestrus after treatment. No difference was observed in the testosterone serum concentration. Pretreatment with 10 IU rhFSH at oestrus before giving the decreasing doses of rhFSH during dioestrus reduced the ovulatory response. Finally, treatment with a constant low dose of rhFSH instead of a decreasing dose of rhFSH did not result in spontaneous ovulation. However, ovulation induction by means of a human chorionic gonadotrophin bolus resulted in superovulation in six out of eight rats. It is concluded that superovulation in cyclic rats can be achieved using rhFSH treatment. However, it was found that the type of rhFSH regimen was very important to achieve appropriate stimulation. The optimal protocol was treatment with decreasing doses of rhFSH during dioestrus. The oocytes retrieved could be fertilized as well as oocytes of saline-treated control rats. The results also indicate that treatment with higher doses of rhFSH might induce a desensitization for FSH and LH. http://repub.eur.nl/res/pub/15063/ 1987-03-01T00:00:00Z One hundred and one patients on haemodialysis, 21 patients on peritoneal dialysis and 30 healthy controls received a trivalent split vaccine containing 15 micrograms haemagglutinin of a recent influenza A-H3N2, influenza A-H1N1 and influenza B strain, respectively. Antibody production after four weeks was determined by the haemagglutination-inhibition test and expressed as response rate, protection rate and overall mean fold increase. The patients on haemodialysis revealed a diminished seroresponse, as compared to patients on peritoneal dialysis and controls. For influenza A-H3N2, this was less distinct than for the other two antigens. In patients on haemodialysis the protection rate was 66% against the A-H3N2 vaccine component (versus 85% in controls, not significant), but only 25% against A-H1N1 and 27% against B (versus 84 and 77% in controls, p less than 0.001). Duration of haemodialysis up to eight years did not affect seroresponse. Patients on haemodialysis who were primed for influenza A-H1N1 in the period 1947-1957, reacted markedly better to the A-H1N1 vaccine component than subjects of other priming periods. A booster injection of the same vaccine dosage four weeks after the first immunization, performed in 98 patients on haemodialysis, was of little value: it had virtually no effect with regard to influenza A-H1N1 and influenza B, and showed, though significantly better, still poor results for A-H3N2. The differences in seroresponse between the A-H3N2 and A-H1N1 vaccine component suggest a major defect of primary, and a minor defect of secondary humoral response in patients on haemodialysis. The consequences for vaccine policy in these patients are discussed. http://repub.eur.nl/res/pub/15065/ 1987-02-01T00:00:00Z