anti-Müllerian Hormone Production Research Articles

Impact of previous chemotherapy on ovarian production of estradiol, progesterone, and AMH: an in vitro controlled study

While numerous chemotherapy agents are known to cause ovarian follicle cell death, it is unknown whether chemotherapy has a detrimental effect on ovarian stromal cells. Our purpose was to assess the impact of chemotherapy on human ovarian stromal function by comparing in vitro estradiol (E2) and progesterone (P4) levels in ovarian cortical cultures from cancer patients who were and were not previously exposed to chemotherapy. To monitor follicle growth initiation in vitro, Anti-mullerian hormone (AMH) measurements were used.

Intrafollicular anti-mullerian hormone (AMH) levels positively correlate with follicle sensitivity to follicle stimulating hormone (FSH) and are elevated in polycystic ovary syndrome (PCOS)

Serum AMH levels predict ovarian reserve during gonadotropin therapy for in vitro fertilization (IVF), with elevated AMH production in PCOS from increased follicle number or altered granulosa cell function. To determine whether AMH predicts quantitative and qualitative aspects of follicle development during gonadotropin therapy for IVF, this study examines whether intrafollicular AMH levels positively correlate with 1) numbers of oocytes retrieved and 2) FSH-stimulated estradiol (E2) production by follicles of normoandrogenic ovulatory (NO) and PCOS patients.

Serum Anti-Mullerian Hormone Concentrations Are Not Altered by Acute Administration of Follicle Stimulating Hormone in Polycystic Ovary Syndrome and Normal Women

In the human ovary, expression of anti-Mullerian hormone (AMH) is detected primarily in granulosa cells of preantral and small antral follicles. This finding is consistent with the tight correlation between circulating AMH levels and the number of small antral follicles (2-5 mm) in normal and polycystic ovary syndrome (PCOS) women. In addition, the greater follicle count in PCOS is mirrored by significantly higher serum AMH levels compared with those of normal women. Despite the utility of AMH measurements in evaluating ovarian physiology and function, the regulation of AMH remains poorly understood. The objective was to determine whether gonadotropins acutely regulate serum AMH in women with PCOS and normal women. We conducted a prospective study to compare ovarian responses to FSH in two groups of women. The study was conducted in a General Clinical Research Center in a tertiary academic medical center. Women with PCOS (age, 18-35 yr; n = 16) and normal ovulatory controls (age, 18-35 yr; n = 11) were recruited for study. Serum samples were measured over a 24-h period after an iv injection of recombinant human FSH (150 IU). Serum AMH responses after FSH administration were measured. Basal serum AMH levels were markedly increased in women with PCOS compared with levels observed in normal women. After FSH injection, PCOS women failed to demonstrate changes in circulating AMH over 24 h. A similar lack of alteration in serum AMH was observed in normal women. These findings suggest that in PCOS and normal women, acute exposure to FSH does not appear to exert an effect on AMH production.

Clinical significance of serum concentration of anti-Müllerian hormone in obese women with polycystic ovary syndrome

In the human ovary, expression of anti-Müllerian hormone (AMH) is detected primarily in granulosa cells of preantral and small antral follicles. The aim of this study was to compare serum AMH measurements in obese women with polycystic ovary syndrome (PCOS) with those in obese normo-ovulatory women and to evaluate the role of AMH as a predictor of ovulation induction by clomiphene citrate compared to FSH. Sixty-eight obese women with PCOS were compared to 17 normo-ovulatory obese women. All women had a body mass index greater than 30 kg/m 2. Women with PCOS received clomiphene citrate (150 mg/day) for 5 days starting from day 3 of cycle and were subdivided into responsive and non-responsive groups. There was a significant difference in AMH concentration between women with PCOS and the control group ( P < 0.05) and also between women with PCOS who responded to clomiphene citrate and those who did not ( P < 0.01). A value of 1.2 ng/ml AMH could be used to predict response to clomiphene citrate in obese women with PCOS (sensitivity 71%, specificity 65.7%). AMH production increases in women with PCOS compared to controls. AMH measurement could also be useful in the prediction of ovarian response to clomiphene citrate.

Early postnatal methoxychlor exposure inhibits folliculogenesis and stimulates anti-Mullerian hormone production in the rat ovary

Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane; MXC] is a chlorinated hydrocarbon pesticide commonly used in the United States as a replacement for DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]. While MXC is a weak estrogenic compound, its more active, major metabolite [2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane; HPTE] shows estrogenic, anti-estrogenic, or anti-androgenic properties depending on the receptor subtype with which it interacts. Anti-Mullerian hormone (AMH) is a paracrine factor that suppresses initial follicle recruitment in the ovary. Studies have shown the effects of exposure to MXC on adult ovarian morphology and function. However, the effect of exposure to MXC at an early postnatal stage on pre-pubertal follicular development and ovarian AMH production has not been studied. Around postnatal day (P) 4, most of the primordial follicular assembly in rats is complete, and a large number of primordial follicles transition into the primary follicle stage, a process that is inhibited by estrogen. The objective of this study was to examine the effect of early postnatal (P3-P10) MXC exposure on ovarian morphology and size, follicle number, and AMH production in the pre-pubertal (P20) rat ovary and to investigate the effect of HPTE on AMH production in immature rat granulosa cells in vitro. Female rats were injected (s.c.) daily with vehicle (control) or 1, 10, 50, 100, or 500 mg MXC/kg per day (referred to here as 1MXC, 10MXC, and so forth.) between P3 and P10. On P20, uterine and ovarian weights were determined, ovarian histology was examined, and follicles were counted and classified into primordial, primary, secondary, pre-antral, or antral stages using the two largest serial sections at the center of the ovary. Ovarian AMH production was examined using immunohistochemistry and western blot analysis. The effect of HPTE (0.5-25 microM) on AMH production in cultured immature rat granulosa cells was determined by western blot analysis. Ovarian weight was reduced by 50, 100, and 500MXC (P < 0.01). MXC treatment inhibited folliculogenesis. Both 100 and 500MXC had a reduced number of antral follicles (P < 0.05) with a concomitant increase in pre-antral follicles (P < 0.05). Follicle numbers were not significantly affected by 1, 10, or 50MXC. Total follicle number and the number of primordial, primary, or secondary stage follicles were not significantly different in all treatment groups. Immunohistochemistry showed that MXC-treated ovaries had more AMH-positive follicles with stronger AMH immunostaining. Western blot analysis showed that AMH production was 1.6 +/- 0.2, 1.85 +/- 0.6, and 2.2 +/- 0.5 times higher in the 50, 100, and 500MXC ovaries as compared with the control ovaries respectively (P < 0.05). Granulosa cells treated with 1 or 5 microM HPTE had significantly greater AMH production (P < 0.05). These results demonstrate that MXC inhibits early ovarian development and stimulates AMH production directly in the rat ovary. In addition, HPTE was shown to stimulate AMH production in rat granulosa cells. Endocrine disruptors are widespread in the environment, and MXC represents a model endocrine disruptor due to the multiple actions of its metabolites. This study confirms that the endocrine disruptor MXC inhibits follicular development and demonstrates for the first time that MXC and HPTE directly stimulate AMH production in the ovary. This novel finding suggests that elevated AMH may play a role in MXC's inhibitory effect in the ovary.

Granulosa Cell Production of Anti-Müllerian Hormone Is Increased in Polycystic Ovaries

There has been renewed interest in anti-Müllerian hormone (AMH) because of its role in the ovary. Data on its actions are sparse, but it appears to inhibit follicle growth. Interestingly, serum AMH is two to three times higher in women with polycystic ovary (PCO) syndrome than women with normal ovaries. We examined the production of AMH by cells from a range of follicle sizes from normal ovaries and compared this with production by ovulatory and anovulatory (anov) PCOs. Granulosa cells (GCs) and theca and follicular fluid (ff) were isolated from intact follicles. Cells were cultured for 48 h +/- FSH or LH, and AMH was measured in ff and cell-conditioned media (CM). AMH levels in ff and GC-CM ranged from 42 to 2240 and 0.025 to 1.7 ng/ml, respectively, and were low or undetectable in ff and GC-CM from follicles greater than 9 mm, luteinized cells, and theca and stroma. The mean level of AMH was four times higher in GC-CM from ovulatory PCOs [mean (range) 1.56 (0.025-7)] and 75 times higher from anovPCO [21.4 (17.2-43 ng/ml)] than normal ovaries [0.37 (0.025-1.7)]. Neither LH nor FSH had an effect on AMH production by GCs from normal ovaries, but in cells from PCOs, FSH significantly decreased AMH, and in contrast, LH increased AMH. The reduction of AMH in follicles greater than 9 mm from normal ovaries appears to be an important requirement for the selection of the dominant follicle. AMH production per GC was 75 times higher in anovPCOs, compared with normal ovaries. This increase in AMH may contribute to failure of follicle growth and ovulation seen in polycystic ovary syndrome.

P-725: Correlation of antimullerian hormone levels with primordial follicle count in human ovary

Previous studies indicated the anti mullerian hormone (AMH) is expressed in preantral follicles and it might be a marker of ovarian reserve. However, there is no direct evidence tying AMH production to primordial follicles in humans. In the human ovary, AMH expression begins at 36 weeks of gestation. Our objective was to investigate if AMH levels directly correlate with primordial follicle numbers in human adult ovarian tissue using an in vitro model. In vitro investigation of AMH production from adult ovarian cortical pieces and fetal ovarian tissue Ovarian cortical pieces from 9 cancer patients undergoing ovarian cryopreservation for fertility preservation and human fetal ovarian tissue from abortuses of 24 weeks gestation were included in the study. 2x2 mm fetal and 5x5 mm adult ovarian pieces were either fixed, paraffin embedded, and serially sectioned to determine baseline follicle density (primordial follicle/mm2) on every fifth section, or cultured in alpha-MEM medium supplemented with 20 % fetal bovine serum and 300-mIU/mL recombinant FSH for seven days. Half of the culture medium was removed every day and stored at - 80 0C until assayed for AMH levels by ELISA. Primordial follicle density was re-evaluated after the 7-day culture. Primordial follicle density was determined as the mean of follicle counts/mm2 in every fifth section. Mean ± [SE] age of the adult patients from whom the tissues were obtained was 27.5 ± 2.7 (range 18-36). The mean primordial follicle density at baseline and after culture was 29.5 ± 3.1 and 3.12 ± 1.3 in fetal ovaries, and 9.31 ± 2.6 and 5.83 ± 2.1 in adult patients, respectively. There were no growing follicles in fetal ovarian tissue before and after culture. In adult tissue 98.2% of follicles were at primordial stage before culture but no growing follicles were seen after the 7-day culture period. In fetal ovarian tissue, 100% of follicles were in primordial stage before and after culture. As expected, AMH was undetectable in fetal ovarian cultures. In adult ovarian tissue cultures, both AMH level on culture day 7 and age correlated with post-culture follicle counts (p<0.01, Fig-1). We provided the first evidence linking the in vitro AMH production to primordial follicle density in human ovarian tissue.

Anti-Müllerian hormone: clinical insights into a promising biomarker of ovarian follicular status

In contrast to most hormonal biomarkers of the follicular status, anti-Müllerian hormone (AMH) is exclusively produced by the granulosa cells of a wide range of follicles (primary to the early antral stages), presumably FSH-independently and with little susceptibility to disorders of antral follicle growth during the luteal–follicular transition. This paper summarizes the authors’ clinical research on the role of AMH as a marker of ovarian functioning. It shows that the relationship between antral follicle counts and serum AMH concentrations is stronger than that observed with FSH, inhibin B and oestradiol on day 3, and that intercycle reproducibility of AMH measurements is better than the latter parameters. In addition, peripheral AMH concentrations decline during ovarian stimulation, thus confirming that maturing follicles loose progressively their ability to produce AMH. Indeed, follicular fluid (FF) AMH concentrations in small antral follicles are 3-fold as high as AMH in pre-ovulatory follicles. Further, human chorionic gonadotrophin-driven luteinization additionally curtails follicular AMH production. Finally, AMH production measured in FF from individual follicles is increased in women having normal follicular counts and responsiveness to ovarian stimulation. Together, these data reinforce the soundness of AMH measurements as a quantitative and possibly qualitative marker of granulosa cell activity and health.

Lack of AMH Response to EFORT Suggests That AMH Production Is Gonadotropin-Independent in Adult Women

In women, anti-Müllerian hormone (AMH), a glycoprotein that belongs to the TGFβ superfamiliy, is produced by ovarian granulosa cells from ovarian follicles not only at the selectable stage but also at earlier stages, when follicles are barely FSH-sensitive. Whereas, in adult men, FSH administration has been shown to upregulate testicular AMH production in absence of testosterone (Young et al., 2005), direct data on the possible regulation of follicular AMH production by pituitary gonadotropins still is lacking. Hence, in an effort to challenge the hypothesis that ovarian AMH production in FSH-independent in adult women, we measured serum AMH dynamics after FSH administration in the early follicular phase, according to a modified exogenous FSH ovarian reserve test (EFORT) (Fanchin et al., 1994). Prospective study. We studied 29 normo-ovulating, infertile patients. On cycle day 3 (BL), after blood samplings were obtained for serum AMH, E2, and inhibin B measurements, they received 300 IU of recombinant FSH, SC, (FSH group, n=14) or served as untreated controls (Control group, n=15). Twenty-four hours later (t24), patients underwent similar hormonal measurements as at BL. Changes in hormonal levels were expressed in % of increase from BL to t24 and differences in serum hormone levels between groups were assessed by using the Wilcoxon signed-rank test. FSH administration increased significantly serum E2 (182%, ranges: 38%-484%, P< 0.0001) and inhibin B (95%, ranges: 0%-150%, P< 0.0001), but not AMH (0.2%, ranges: -12.8%-19.8%) levels in the FSH group. Serum levels of all 3 hormones remained steady in the control group. These results indicate that FSH administration on cycle day 3 does not exert significant, short-term consequences in serum AMH levels, but induce a remarkable increase in serum E2 and inhibin B levels. The present data provide direct documentation that AMH production by functional granulosa cells probably is FSH-independent in adult women.

Per-follicle measurements indicate that anti-müllerian hormone secretion is modulated by the extent of follicular development and luteinization and may reflect qualitatively the ovarian follicular status

To investigate the possible influence of follicular maturation and luteinization on anti-müllerian hormone (AMH) secretion and the relationship between per-follicle AMH levels, ovarian follicular status, and responsiveness to controlled ovarian hyperstimulation (COH). Prospective study. University hospital in France. Thirty seven in vitro fertilization/embryo transfer candidates undergoing COH. On the day of oocyte retrieval, serum samples and follicular fluids from two small (8-12 mm in diameter) and two large (16-20 mm in diameter) follicles were collected for AMH, E2, and progesterone (P4) measurements. Per-follicle AMH levels. Small follicles secreted AMH levels that were approximately three times as high as large follicles. Follicular fluid AMH and P4 levels were negatively correlated to each other both in small and large follicles. Per-follicle AMH levels in both follicular classes were positively correlated with antral follicle count on cycle day 3 before COH and with growing follicle (> or =12 mm) count and oocytes retrieved, but negatively correlated with FSH requirement. Both final follicular maturation and luteinization interfere with granulosa cell AMH production. The relationship between intrafollicular AMH content, the surrounding follicular status, and ovarian response to COH indicates that peripheral AMH levels reflect not only follicle count but also per-follicle AMH production.

Testicular Anti-Müllerian Hormone Secretion Is Stimulated by Recombinant Human FSH in Patients with Congenital Hypogonadotropic Hypogonadism

Serum anti-Mullerian hormone (AMH), a prepubertal Sertoli cell marker, declines during puberty as an early sign of testicular testosterone (T) production. When T synthesis or action is impaired, serum AMH is abnormally high in the first months after birth and at puberty but normal between these two periods. We postulated that FSH might be responsible for AMH up-regulation in the absence of androgen inhibition. To test this hypothesis, we administered recombinant human (rh) FSH to eight patients aged from 18-31 yr with untreated congenital hypogonadotropic hypogonadism. This situation is ideal to study the effect of FSH on AMH production because it avoids interference by endogenous gonadotropins and T. The patients received daily sc injections of 150 IU rhFSH for 1 month, followed in seven of them by a combined treatment of rhFSH plus human chorionic gonadotropin (hCG; 1500 UI im, twice a week) for 2 months. Gonadotropins, T, AMH, and inhibin B were measured in plasma before treatment every 10 d during rhFSH treatment and every month during combined rhFSH and hCG treatments. All hormones were at prepubertal levels before treatment. Although LH and T did not vary, AMH and inhibin B levels gradually increased after 20 d of FSH administration. However, in contrast to rhFSH alone, the combined rhFSH plus hCG stimulation of the testis dramatically suppresses the secretion of AMH and induced a modest but significant reduction of circulating inhibin B levels. We conclude that FSH stimulates AMH production in the testis when it is at a prepubertal stage. In addition, the decrease of serum AMH during combined rhFSH and hCG testicular stimulation is in agreement with the concept that during pubertal development and in adult life, the suppressive effect of LH-driven testicular androgens outweighs the stimulating effect of FSH on AMH production by Sertoli cells. Finally, the hCG-induced decrease in inhibin B suggests that in humans, as previously demonstrated in monkeys, testicular T is also able to inhibit inhibin B secretion.

Per-follicle measurements indicate that anti-Müllerian hormone secretion is modulated by the extent of follicular development and luteinization and may reflect qualitatively the ovarian follicular status

In adult women, anti-Müllerian hormone (AMH) expression in granulosa cells is, at least in part, regulated by the stage of follicular growth, with predominant production by preantral and early antral follicles. Whereas the ability of preovulatory follicles to secrete AMH is reduced (Baarends et al. Endocrinology, 1995), the consequences of follicular luteinization on AMH production are less documented. Also, high peripheral AMH levels reflect increased number of early antral follicles (Fanchin et al. Fertil. Steril., 2003), but whether they also reflect increased per-follicle AMH production remains unclear. Hence, we decided to investigate the possible modulating role of follicular maturation and luteinization on AMH secretion and the possible relationship between per-follicle AMH levels and ovarian follicular status and responsiveness to controlled ovarian hyperstimulation (COH). Prospective study. We studied 37 normo-ovulating IVF-ET candidates undergoing COH. On the day of oocyte retrieval, 36 h after hCG administration, serum samples and follicular fluids (FFs) from 2 large (16–20 mm in diameter) and 2 small (8–12 mm in diameter) follicles were collected for serum and FF AMH, E2, and progesterone (P4) measurements. Sizeable AMH levels were detected both in serum and FFs. Hormonal contents were similar between two small and between two large follicles in the same patient and were averaged. Small follicles secreted approximately 3 times as high AMH levels as large follicles (P < 0.0001), but less P4 (P < 0.0001), and similar E2 levels. FF AMH and P4 levels correlated negatively both in small (r=-0.61; P < 0.0001) and large (r=-0.36; P < 0.03) follicles. In large follicles, FF AMH levels correlated positively with the number of early antral follicles on cycle day 3 before COH (r= 0.58; P < 0.0002), growing follicles (beyond 11 mm; r= 0.45; P < 0.0005) and oocytes retrieved (r= 0.48; P < 0.0003), and negatively with FSH requirement (r= -0.48; P < 0.0003). AMH production by granulosa cells is modulated by the degree of follicular development, with a hefty gradient from small to large follicles, and luteinization, as indicated by the inverse relationship between FF AMH and P4 levels. The remarkable correlation between intra-follicular AMH content, baseline ovarian follicular status, and response to COH indicates that peripheral AMH measurements may reflect not only follicle count but also per-follicle AMH production. Further insights into this sensitive issue may contribute to refine the interpretation of serum AMH levels not only as a quantitative but also as a qualitative marker of ovarian follicular status.

Follicle-stimulating hormone increases testicular Anti-Mullerian hormone (AMH) production through sertoli cell proliferation and a nonclassical cyclic adenosine 5'-monophosphate-mediated activation of the AMH Gene.

Anti-Müllerian hormone (AMH) production by testicular Sertoli cells is high before puberty and can be further induced by FSH. Our objective was to delineate the mechanisms by which FSH stimulates AMH production. Assay of serum AMH levels and histological morphometric analysis in prepubertal FSH-deficient transgenic mice showed that serum AMH and testicular mass were decreased owing to reduced Sertoli cell number. All parameters resumed normal values in mice treated with recombinant FSH. We also analyzed the ability of FSH and the factors involved in its signaling pathway to activate AMH transcription by transfecting AMH promoter-luc reporter constructs of different lengths in a prepubertal Sertoli cell line. Our results showed that FSH activates AMH transcription via adenylate cyclase, cAMP, and protein kinase A but involving a nonclassical cAMP-response pathway requiring nuclear factor-kappaB and activating protein 2 binding sites, which lie more than 1.9 kb upstream of the AMH transcription start site. This is the first report showing the importance of distant sequences in the regulation of AMH expression. We conclude that prepubertal testicular AMH production is increased by FSH stimulation through Sertoli cell proliferation and an enhancement of AMH gene transcription.

Antimüllerian hormone in patients with hypogonadotropic hypogonadism.

Antimullerian hormone (AMH) is produced by immature Sertoli cells until pubertal maturation. At puberty, elevation of serum testosterone correlates with a decrease in serum AMH. To further investigate the hormonal control of AMH secretion, serum AMH levels were measured in 20 normal men (20-60 yr), in 12 patients (19-30 yr) with congenital hypogonadotropic hypogonadism (CHH), and in 18 patients (19-65 yr) with acquired hypogonadotropic hypogonadism (AHH) either untreated or during testosterone or human chorionic gonadotropin (hCG) therapy. Mean serum AMH levels in normal adult men were low (20+/-4.9 pmol/L). In untreated CHH patients, mean serum AMH levels were significantly higher than in normal men (292+/-86 pmol/L, P < 0.001) and were similar to those previously reported in prepubertal boys. In men with AHH, mean serum AMH levels were also significantly increased (107+/-50 pmol/L; P < 0.01) when compared with healthy men but were less than in men with CHH. In addition, in 10 patients treated for prostate cancer, a modest but significant increase of serum AMH (from 11.4 +/-5.7 pmol/L to 49+/-9.9 pmol/L; P < 0.01) was observed 12 months after suppression of the gonadal axis with the GnRH agonist Triptorelin (3.75 mg IM once a month). Plasma testosterone (T) and serum AMH levels were measured at baseline and at 3 and 6 months in 10 HH patients (6 CHH and 4 AHH) treated with hCG (1500 IU/twice weekly for 6 months) and in 8 HH (4 CHH and 4 AHH) patients treated with T (T enanthate 250 mg/3 weeks for 6 months). hCG treatment induced an increase of plasma T (from 1.0+/-0.7 to 11+/-2.4 and 19+/-4.8 nmol/L, at 3 and 6 months respectively) associated with a dramatic decrease of serum AMH (from 314+/-93 to 56+/-30 and 17+/-4.3 pmol/L). The similar increase in plasma T levels (from 1.4+/-1.0 to 15.6+/-4.2 and 23+/-6.2 ng/mL) obtained with exogenous T induced a lesser decrease of serum AMH (from 221+/-107 pmol/L to 114+/-50 and 66+/-17 pmol/L, at 3 and 6 months respectively). In conclusion, high plasma AMH levels in CHH patients are related to the absence of pubertal maturation of Sertoli cells. The high AMH levels in AHH and its increase after Triptorelin-induced gonadotropin deficiency suggest that the suppression of AMH is a reversible phenomenon. Finally, the inhibition of AMH production by Sertoli cells is induced by intratesticular T.

Anti-Müllerian hormone in early human development

Anti-Müllerian hormone (AMH) is a glycoprotein produced by immature Sertoli cells and responsible for the regression of Müllerian ducts in male fetuses. The ontogeny of the hormone in early human development was investigated. While no detectable AMH could be found in female fetal serum, in males, the mean ± S.E.M. AMH serum concentration was 40.5 ± 3.9 ng/ml from 19 to 30 weeks ( n = 13), and 28.4 ± 6.1 ng/ml from 30 weeks to term ( n = 9). The latter value is significantly different from the mean AMH concentration in serum from boys aged 2 months to 2 years (43.1 ± 3.7), suggesting that AMH production is sluggish during the perinatal period. The serum AMH concentration of a 46,XX male fetus was in the normal range for males. Using in situ hybridization, AMH transcripts were detected in the testicular tissue of all fetuses from 8 weeks onwards, but not in fetal ovaries nor in the yet undifferentiated gonadal tissue of a 7-week-old fetus bearing maledetermining DNA sequences. Together, these data indicate that AMH is a reliable marker for the presence of functional testicular tissue and, as such, may be helpful for the diagnosis of fetal sex, particularly in the presence.of sex chromosome abnormalities.

ANTI-MULLERIAN HORMONE IN EARLY HUMAN DEVELOPMENT

Anti-Mullerian hormone (AMH), also called Mullerian-inhibiting substance or factor, is a dimeric glycoprotein, produced by immature Sertoli cells, and responsible for Mullerian regress ion in male fetuses. To study the ontogeny of AMH production in the male against human recombinant AMH, the AMH concentration in 21 samples of amniotic fluid and 44 samples of fetal serum, initially collected for cytogenetical analysis in fetuses with sonographic abnormalities. No AMH was detectable in amnitotic fluid, whatever the fetal sex. Mean ± SEM AMH concentration was 40.5 ± 3.9 ng/ml in the serum of male fetuses from 19 to 30 weeks, and 28.4 ± 6.1 ng/ml in older ones. The AMH concentration in the serum of an male XX fetus, aged 24 weeks, 48.3 ng/ml, was the only biological indicator of fetal sex. No AMH was detectable in female serum at any time, allowing easy discrimination between male and female samples, even during the perinatal period, when mean scrum AMH concentration is decreased, compared to that of infants aged 2 months to 2 years (43.1 ± 3.7? P>0.05). AMH production in early fetal life was studied by in-situ hybridization, using AMH-specific sense and antisense riboprobes. AMH transcripts were detected in the Sertoli cells of fetuses aged 8 weeks or older, but not in ovarian tissue. Negative results were also found in the sexually undifferentiated gonadal tissue of one 7-week-old fetus, with detectable DMA SRY-specific sequences, confirming that AMH expression in the testis begins only after seminiferous tubule differentiation.

Immunocytochemical detection of anti-müllerian hormone in Sertoli cells of various mammalian species including human.

An immunocytochemical method, based on the use of a polyclonal antibody raised against purified bovine anti-Müllerian hormone (AMH), was used to detect AMH in Sertoli cell cytoplasm of various mammalian species, including human. Immunopurification of antiserum by AMH-affinity chromatography, although not mandatory, leads to better results and increased sensitivity. In human testicular tissue, AMH is detectable up to 6 years of age. In rats, AMH production is initiated at 13 days post coitum, peaks between 15 and 17 days, and is no longer detectable 1 week after birth. The reaction is strongest in Sertoli cells of calves, sheep, goats, and pigs, species characterized by a high degree of development of the rough endoplasmic reticulum. It is fainter in human, rat, rabbit, and cat Sertoli cells, in which the rough endoplasmic reticulum is not as abundant. This correlation is not unexpected, in view of the localization of reaction product in this cytoplasmic organelle. Preliminary results indicate that there may be a relationship between the amount of immunoreactive AMH present in testicular biopsies of intersex patients and the degree of regression of the Müllerian duct on the ipsilateral side. This may help to elucidate whether persistence of Müllerian ducts results from lack of testicular production of AMH or from peripheral resistance of the Müllerian primordia to the hormone.

Secretion of anti-Müllerian hormone by immature bovine Sertoli cells in primary culture, studied by a competition-type radioimmunoassay: lack of modulation by either FSH or testosterone

Secretion of anti-Müllerian hormone (AMH) by immature bovine Sertoli cells in primary culture was studied through a competition-type RIA employing a polyclonal antibody and 125I-labelled purified AMH. This RIA is approximately 10 times more sensitive than the solid-phase two-site monoclonal antibody-based RIA described previously. Biosynthesis and secretion of AMH by cultured Sertoli cells require approximately 48 h, are not influenced by FSH or testosterone and steadily decrease over a one-week period of culture. Cyclic AMP response to FSH stimulation is normal in cultured cells. Whether the factors responsible for the extinction of AMH production in vitro are in any way related to those operating during normal maturation, which lead to repression of AMH biosynthesis in adult Sertoli cells, is not known at the present time and deserves further study.

18 NEITHER TESTOSTERONE NOR FSH ARE RESPONSIBLE FOR DECREASED PRODUCTION OP AMH BY BOVINE SERTOLI CELLS IN PRIMARY CULTURE

Production of anti-Müllerian hormone (AMH) by Sertoli cells is maximal during the fetal and neonatal period, and tapers off in the course of postnatal development. To determine the factors responsible for the repression of AMH secretion after birth, Sertoli cells, isolated from immature calves, were plated in an hormonally defined medium and cultured in the presence of 0.1 mM MIX. AMH was assayed in the culture medium using a competition-type RIA capable of detecting 1.5 ng. The proportion of neosynthesized AMH was calculated by determining the amount of immunoreactive AMH unaffected by cycloheximide treatment in conditions where 92 % of protein neosynthesis is inhibited. AMH production by Sertoli cells, expressed per 24 hrs and per 106 cells, fell from 90 ng on the day following plating to 1.5 at day 7. The proportion of AMH synthesized after day 1 was 35 % on day 2 and 88 % on day 3. FSH, 2.5 μg/ml, and testosterone, 2 μM, were added to the culture medium on days 2 and 3, immunoreactive AMH was measured in the culture medium at day 3. Mean daily production of AMH by 106 Sertoli cells was 28.6±2.6 ng in control cultures, 24.6±2.8 in FSH-treated ones and 26.6±3.4 after testosterone treatment. Differences were not significant by analysis of variance. In the same conditions, FSH increased cyclic AMP production approximately tenfold. Further studies are in progress to determine which factors are essential to the continued production of AMH by Sertoli cells in vitro.

Use of monoclonal antibody techniques to study the ontogeny of bovine anti-Müllerian hormone.

Monoclonal antibodies against bovine anti-Müllerian hormone (AMH) were used to study the hormone in cattle. Anti-Müllerian activity of testicular tissue, immunoreactive testicular AMH, serum AMH concentration and AMH production by incubated testicular tissue were detectable from 42 days, i.e. at the time of seminiferous tubule differentiation, and peaked between 50 and 80 days, when the Müllerian ducts regress in the male fetus. All the values stabilized at a lower level until 30 days after birth and then slowly decreased. At 18 months, only traces of AMH immunoreactivity were detectable in testicular tissue and serum concentration and AMH production by incubated testicular tissue were negligible; the main source of AMH in the adult animal was the rete testis fluid. Study of the disappearance rate of AMH from the serum of castrated calves gave a half-life of approximately 2 days for bovine AMH.