Postnatal sex reversal of the ovaries in mice lacking estrogen receptors alpha and beta.

Mice lacking estrogen receptors alpha and beta were generated to clarify the roles of each receptor in the physiology of estrogen target tissues. Both sexes of alphabeta estrogen receptor knockout (alphabetaERKO) mutants exhibit normal reproductive tract development but are infertile. Ovaries of adult alphabetaERKO females exhibit follicle transdifferentiation to structures resembling seminiferous tubules of the testis, including Sertoli-like cells and expression of Müllerian inhibiting substance, sulfated glycoprotein-2, and Sox9. Therefore, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual ERKO mutants, which indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary.

Characterization of gametogenetin 1 (GGN1) and its potential role in male fertility through the interaction with the ion channel regulator, cysteine-rich secretory protein 2 (CRISP2) in the sperm tail.

Cysteine-rich secretory protein 2 (CRISP2) is a testis-enriched protein localized to the sperm acrosome and tail. CRISP2 has been proposed to play a critical role in spermatogenesis and male fertility, although the precise function(s) of CRISP2 remains to be determined. Recent data have shown that the CRISP domain of the mouse CRISP2 has the ability to regulate Ca(2+) flow through ryanodine receptors (RyR) and to bind to MAP kinase kinase kinase 11 (MAP3K11). To further define the biochemical pathways within which CRISP2 is involved, we screened an adult mouse testis cDNA library using a yeast two-hybrid assay to identify CRISP2 interacting partners. One of the most frequently identified CRISP2-binding proteins was gametogenetin 1 (GGN1). Interactions occur between the ion channel regulatory region within the CRISP2 CRISP domain and the carboxyl-most 158 amino acids of GGN1. CRISP2 does not bind to the GGN2 or GGN3 isoforms. Furthermore, we showed that Ggn1 is a testis-enriched mRNA and the protein first appeared in late pachytene spermatocytes and was up-regulated in round spermatids before being incorporated into the principal piece of the sperm tail where it co-localized with CRISP2. These data along with data on RyR and MAP3K11 binding define the CRISP2 CRISP domain as a protein interaction motif and suggest a role for the GGN1-CRISP2 complex in sperm tail development and/or motility.

Anti-Müllerian hormone concentrations in the follicular fluid of the preovulatory follicle are predictive of the implantation potential of the ensuing embryo obtained by in vitro fertilization.

CONTEXT: The strong relationship between serum anti-Müllerian hormone (AMH) levels and the number of antral follicles supports the use of AMH measurements as a quantitative marker of the ovarian follicular status. Yet, it still is unclear whether the aptitude of an individual follicle to produce AMH reflects its reproductive competence. OBJECTIVE: This study examined the possible relationship between serum or follicular fluid (FF) AMH concentrations and the fate of the ensuing oocytes and embryos obtained by in vitro fertilization-embryo transfer conducted in monodominant follicle cycles. DESIGN AND SETTING: We conducted a prospective study at the University of Paris XI, Assistance Publique-Hôpitaux de Paris, Institut National de la Santé et de la Recherche Médicale U782. PATIENTS: Patients included 118 infertile in vitro fertilization-embryo transfer candidates. INTERVENTIONS: Concentrations of AMH, progesterone, and estradiol were measured in the serum on cycle d 3 and on the day of oocyte pickup (dOPU), and in FF. Cycles were sorted into three sets of three distinct groups according to whether serum d 3, serum dOPU, and FF AMH concentrations were 30th centile or below (low AMH), between the 31st and the 70th centiles (average AMH), or above the 70th centile (high AMH) of measurements. MAIN OUTCOME MEASURE: Clinical pregnancy and embryo implantation rates were assessed. RESULTS: Clinical pregnancy rates (5.7, 20.0, and 39.5%, respectively; P < 0.002) and embryo implantation rates (11.8, 30.8, and 65.4, respectively; P <0.001) were markedly different among the low, moderate, and high FF AMH groups but not among the serum (d 3 or dOPU) AMH groups. Fertilization rates and embryo morphology remained similar irrespective of AMH concentrations in the serum or in FF. Incidentally, FF AMH concentrations were negatively correlated with FF progesterone (r = -0.27; P <0.003) and FF estradiol (r = -0.21; P <0.02) concentrations. CONCLUSIONS: Concentrations of AMH in the FF, but not in the serum, constitute a useful follicular marker of embryo implantation and are negatively related to FF progesterone and estradiol concentrations.

Seminal plasma anti-Müllerian hormone level correlates with semen parameters but does not predict success of testicular sperm extraction (TESE).

AIM: To assess seminal plasma anti-Müllerian hormone (AMH) level relationships in fertile and infertile males. METHODS: Eighty-four male cases were studied and divided into four groups: fertile normozoospermia (n = 16), oligoasthenoteratozoospermia (n = 15), obstructive azoospermia (OA) (n = 13) and non-obstructive azoospermia (NOA) (n = 40). Conventional semen analysis was done for all cases. Testicular biopsy was done with histopathology and fresh tissue examination for testicular sperm extraction (TESE) in NOA cases. NOA group was subdivided according to TESE results into unsuccessful TESE (n = 19) and successful TESE (n = 21). Seminal plasma AMH was estimated by enzyme linked immunosorbent assay (ELISA) and serum follicular stimulating hormone (FSH) was estimated in NOA cases only by radioimmunoassay (RIA). RESULTS: Mean seminal AMH was significantly higher in fertile group than in oligoasthenoteratozoospermia with significance (41.5 +/- 10.9 pmol/L vs. 30.5 +/- 10.3 pmol/L, P < 0.05). Seminal AMH was not detected in any OA patients. Seminal AMH was correlated positively with testicular volume (r = 0.329, P = 0.005), sperm count (r = 0.483, P = 0.007), sperm motility percent (r = 0.419, P = 0.021) and negatively with sperm abnormal forms percent (r = -0.413, P = 0.023). Nonsignificant correlation was evident with age (r = -0.155, P = 0.414) and plasma FSH (r = -0.014, P = 0.943). In NOA cases, seminal AMH was detectable in 23/40 cases, 14 of them were successful TESE (57.5%) and was undetectable in 17/40 cases, 10 of them were unsuccessful TESE (58.2%). CONCLUSION: Seminal plasma AMH is an absolute testicular marker being absent in all OA cases. However, seminal AMH has a poor predictability for successful testicular sperm retrieval in NOA cases.

[Anti-mullerian hormone and its role in the regulation of ovarian function. Review of the literature]. / Le rôle de l'hormone anti-muillerienne dans la régulation du fonctionnement de l'ovaire. Revue de la littérature.

Anti-mullerian hormone, also called AMH, belongs to the large family of transforming growth factor P. Its role in the sexual differentiation of male fetus is now well known. Recently, AMH has been demonstrated to play an important role in the ovarian function. In fact, AMH seems to regulate the kinetics of follicular development, inhibiting the follicular recruitment and the follicular growth. Thus, this intra-gonadic cybernin could be a decisive determinant of the rapidity of follicular pool exhaustion. Today, some experimental data from the literature suggest that AMH could be a reliable marker of ovarian reserve. This review summarizes the present knowledge about AMH and its role in physiology but also in ovarian pathology.

Modification of photosynthetic regulation in tomato overexpressing glutathione peroxidase.

To investigate the function of glutathione peroxidase (GPX) in plants, we produced transgenic tomato plants overexpressing an eukaryotic selenium-independent GPX (GPX5). We show here that total GPX activity was increased by 50% in transgenic plants, when compared to control plants transformed with the binary vector without the insert (PZP111). A preliminary two-dimensional electrophoretic protein analysis of the GPX overexpressing plants showed notably a decrease in the accumulation of proteins identified as rubisco small subunit 1 and fructose-1,6-bisphosphate aldolase, two proteins involved in photosynthesis. These observations, together with the fact that in standard culture conditions, GPX-overexpressing plants were not phenotypically distinct from control plants prompted us to challenge the plants with a chilling treatment that is known to affect photosynthesis activity. We found that upon chilling treatment with low light level, photosynthesis was not affected in GPX-overexpressing plants while it was in control plants, as revealed by chlorophyll fluorescence parameters and fructose-1,6-biphosphatase activity. These results suggest that overexpression of a selenium-independent GPX in tomato plants modifies specifically gene expression and leads to modifications of photosynthetic regulation processes.

Estradiol and regulation of anti-Müllerian hormone, inhibin-A, and inhibin-B secretion: analysis of small antral and preovulatory human follicles' fluid.

CONTEXT: In ovaries surgically removed for fertility preservation, hormone concentrations in fluid from small antral follicles were determined. Levels were compared with those found in preovulatory follicular fluid. OBJECTIVE: The objective of this study is to measure intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-A, inhibin-B, estradiol, and progesterone. SETTING: The study was set in a university hospital. PATIENTS: Patients were 22 women suffering from a cancer disease and 16 women undergoing assisted reproduction. INTERVENTIONS: Fluid from 35 follicles (diameter, 3-8 mm) was included and compared with that of 32 preovulatory follicles. MAIN OUTCOME MEASURES: The main outcome measures were intrafollicular concentrations of the measured hormones and their possible correlation. RESULTS: Concentrations of AMH in small antral follicles were almost three orders of magnitude higher than in follicle fluid of preovulatory follicles, 790 +/- 95 vs. 1.17 +/- 0.14 ng/ml (mean +/- sem), respectively. There was a significant negative correlation between estradiol and AMH in fluid from small antral follicles, whereas inhibin-A and inhibin-B were correlated positively with estradiol concentrations. Progesterone showed a similar correlation to levels of AMH but only in fluid of preovulatory follicles. CONCLUSIONS: The high expression of AMH in granulosa cells of small antral follicles actually translates into very high follicle fluid AMH concentrations. This most likely explains the correlation between serum AMH levels and the number of small antral follicles as previously demonstrated. The negative correlation between estradiol and AMH suggests that FSH down-regulates AMH expression. Thus, the microenvironment of the follicle shows profound changes with developmental stage and highlights the importance of studies to understand the mechanisms that regulate follicular growth and development during antral stages of development.

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 tumor of the ovary.

Adult granulosa cell tumor (GCT) of the ovary is oftentimes a hormonally active, stromal cell neoplasm that is distinguished by its ability to secrete sex steroids such as estrogen. Patients may present with vaginal bleeding caused by endometrial hyperplasia or uterine cancer as a result of prolonged exposure to tumor-derived estrogen. In addition, GCT is a vascular tumor that may occasionally rupture and result in abdominal pain, hemoperitoneum, and hypotension, mimicking an ectopic pregnancy in younger patients. GCT is usually associated with a mass on pelvic examination that is subsequently confirmed on ultrasonography. Surgery is required for definitive tissue diagnosis, staging, and tumor debulking. In older women, a total abdominal hysterectomy and bilateral salpingooophorectomy are typically performed. In women of childbearing age, a more conservative unilateral salpingo-oophorectomy may be performed, assuming that careful staging reveals that the disease has not extended outside of the involved ovary and that a concomitant uterine cancer has been excluded. Survival of patients with GCT is generally excellent because most patients present with early-stage disease, although certain high-risk patient groups may be identified. Stage is the most important prognostic factor, with a higher risk of relapse being associated with stages II through IV disease. In addition, patients with stage I disease associated with features such as large tumor size, high mitotic index, or tumor rupture may also be at higher risk in some series. The value of postoperative adjuvant therapy for high-risk patients has not been investigated by prospective randomized trials, which are difficult to perform because of the rarity of this tumor. Nonetheless, the use of adjuvant chemotherapy or radiation has sometimes been associated with prolonged disease-free survival in patients with high-risk features. Because of the propensity of GCT to recur years after initial diagnosis, prolonged surveillance with serial physical examination and serum tumor markers such as estradiol and inhibin is reasonable.

[Anti-Mullerian hormone: a gonadal hormone with multiple diagnostics resources].

Anti-Mullerian hormone (AMH) is a gonadal hormone synthesized by granulose cells of the ovary and Sertoli cells of the testis. Anti-Mullerian hormone is used to facilitate the evaluation of intersex disorders and as a marker in some ovarian tumors or ovarian reserve assessment in the infertility cases. Serum levels of AMH hold objective information, which is useful in the clinical practice. Therefore it is necessary to decimate the normal and the abnormal levels of AMH.

A model system for studying inhibin.

We report here a sensitive model for studying inhibin-like activity in crude ovine testicular extracts (CoTE). The administration of CoTE in 25-100 mg amounts to 35-day-old immature male rats, orchidectomized just prior to use, resulted in the prevention of a rise in plasma FSH levels, seen 10 h post-treatment. In a second model, CoTE was injected at 1000 h of day 35 to a group of rats that was castrated 12-24 h prior to injection, and the animals were sacrificed 6 h later; plasma FSH levels were found to be significantly suppressed. CoTE, administered subcutaneously, both in multiple doses and as a single injection, was found to be equally effective. A single injection suppressed FSH levels within 3-6 h while LH levels were unaffected. The suppressive effect was dose-dependent, reaching a maximum value at doses of 100 mg CoTE and above. FSH levels could not be suppressed below the tonic level, either by the administration of a single large dose of CoTE, or by repetitive injections of maximal doses of CoTE at 3 h intervals. The suppression in FSH levels caused by a single injection of CoTE waned with time and totally disappeared by 36h. CoTE was prepared by the heat treatment (55 C for 30 min) of an aqueous extract of ovine testis, followed by centrifugation and ether extraction of the supernatant. The aqueous layer was then dialyzed and lyophilized. The lyophilized material was found to be free of testosterone, as measured by a specific radioimmunoassay, indicating that the active factor is a heat-stable, lyophilizable, nondialyzable material, free of contamination by testosterone and probably by other steroids.

A monoclonal antibody against bovine anti-Müllerian hormone.

Anti-Müllerian hormone (AMH) was partially purified from incubation medium of calf fetal testes and injected into a BALB/c mouse, whose splenocytes were fused with Sp2/Ag8 myeloma cells. Hybridomas were screened for specific antibody production by double antibody precipitation of labeled AMH, which was obtained by incubating fetal calf testes in the presence of tritiated fucose and submitting the medium to the standard procedure of purification. In spite of the extremely low concentration of AMH in the preparation used for immunization, three hybridomas gave positive results in the screening assay. One was cloned and grown in mice. The monoclonal antibody purified from ascites fluid abolished anti-Müllerian activity of partially purified AMH, whether or not the immune complex was removed from solution by a second, antimouse immunoglobulin antibody. The monoclonal antibody also blocked anti-Müllerian activity of calf but not rat fetal testes. Our results indicate that the monoclonal antibody is species specific and is directed towards the antigenic determinant responsible for biological activity.

Detection of Müllerian inhibiting substance in biological samples by a solid phase sandwich radioimmunoassay.

Müllerian inhibiting substance (MIS) is a 140,000-dalton glycoprotein responsible for regression of Müllerian ducts in a male embryo. It has recently been demonstrated that MIS inhibits the growth of tumors in vivo and in vitro. In this study, we have constructed a sensitive, solid phase sandwich RIA using monoclonal antibodies raised to bovine MIS. The amount of MIS detected was based on the protein concentration of Green 3, the most purified fraction of MIS available. The assay could detect 20 ng Green 3 or 0.14 pmol in physiological samples. There was no evidence of cross-reactivity of the antibodies raised to bovine MIS with chicken, rat, mouse, or human MIS.

Cellular localization of müllerian inhibiting substance in the developing rat ovary.

The ontogeny of Müllerian Inhibiting Substance (MIS) production was studied in the immature developing rat ovary using immunohistochemistry and bioassay. In a graded organ culture assay, in which regression of the Müllerian duct of the 14 1/2-day rat fetus was used as a measure of bioactivity, MIS could not be detected in ovarian fragments from fetal rats. After birth, however, MIS bioactivity first became detectable at 4 days of age. Fragments from ovaries of rats 7 days of age and older contained moderate levels of MIS activity which remained detectable throughout the prepubertal period, although extreme individual variability was characteristic. A rabbit polyclonal antibody against human recombinant MIS was used to localize MIS in rat ovarian tissue. Avidin-biotin enhanced immunoperoxidase staining could not detect MIS in the 15-day fetal or 1 day postnatal ovary, where no follicular growth was noticed. In ovaries from rats 1 week or older, where follicular growth was apparent, MIS could be localized specifically and exclusively in the cytoplasm of granulosa cells. MIS was found more in the innermost layers of granulosa cells than in the peripheral layers in preantral follicles. In antral follicles, MIS was found predominantly in the cumulus oophorus cells and periantral cells. In these developing ovaries, MIS could not be found in follicles with features of atresia.

A simple and rapid in vitro bioassay for inhibin.

This report describes a quantitative bioassay for inhibin which can be used to monitor purification and establish the physiological role of this hormone in spermatogenesis and folliculogenesis. Anterior pituitary cells from adult male Sprague-Dawley rats were dispersed and precultured for 18 h before the addition of inhibin-containing materials to the culture medium. After a further 72 h in culture, the medium was removed by aaspiration, and the cells were lysed releasing their intracellular hormone into RIA buffer. Cell content of FSH was reduced in inhibin-containing preparations, but without changes in LH, GH, and PRL concentrations. The inhibin dose-response curve, based on the inhibition of FSH in 15 experiments using an ovine testicular lymph preparation as a standard, had indices of precision between -0.032 and -0.098, and Finney's g ranged from 0.003--0.025. The interassay variability ranged from 15.0--16.9%. The assay had a practical capacity of 300--400 wells, which permitted the measurement of dose-response curves of 15 or more unknowns with quadruplicate wells per dose. The potency of unknown preparations was calculated with reference to the inhibin standard, which had a designated potency of 1 U/mg. Preparations showing nonparallelism were excluded. This assay presented advantages over those described previously, since it showed specificity of response to FSH and accommodated a large number of samples without loss of precision or reproducibility. It is therefore suitable for measurement of the inhibin-like activity in some biological fluids.

An inverse relationship exists between seminal plasma inhibin and serum follicle-stimulating hormone in man.

Inhibin concentrations were measured in 109 seminal plasma samples obtained from 32 normal subjects, 51 infertile patients with either azoospermia or oligospermia, and 20 patients 2-8 months post vasectomy. The infertile group included 14 azoospermic patients with raised peripheral plasma FSH levels (6.8-30.2 IU/liter) and 17 azoospermic patients in whom FSH levels were normal. Only 6 of the 20 patients with oligospermia had raised FSH levels. Seminal plasma inhibin was measured in individual samples using a quantitative in vitro rat anterior pituitary cell culture bioassay in which FSH cell anterior pituitary cell culture bioassay in which FSH cell content was measured after 72 h of incubation with the inhibin-containing material. Biopotencies were determined using combined multiple parallel line assays with reference to an inhibin standard with a potency of 1 U/mg. The concentrations of inhibin in normal seminal plasma were 31.4 +/- 3.0 U/ml, which contrasted with the low levels found in azoospermic patients with high plasma FSH levels. Of these, seven had undetectable inhibin levels (less than 2.5 U/ml) and seven had values ranging from 4.2-8.5 U/ml. These concentrations were significantly lower than those in azoospermic patients, in whom FSH was not raised (18.9 +/- 2.2 U/ml). Seminal plasma inhibin levels post vasectomy were 16.9 +/- 2.3 U/ml and were not significantly different from those measured in azoospermic-normal FSH patients. Peripheral plasma FSH levels were expressed as a function of seminal plasma inhibin concentrations (r = -0.736; P less than 0.001; excluding those patients with vasal obstruction). These findings show that inhibin-like activity in seminal plasma is reduced in infertile men with raised peripheral plasma FSH levels, and that a reciprocal inverse relationship exists between serum FSH and seminal plasma inhibin concentrations.

PIP: Inhibin concentrations were measured in 109 seminal plasma samples obtained from 32 normal subjects, 51 infertile patients with either azoospermia or oligospermia, and 20 patients 2-8 months postvasectomy. The infertile group included 14 azoospermic patients with raised peripheral plamsa FSH levels (6.8-30.2 IU/liter) and 17 azoospermic patients in whom FSH levels were normal. Only 6 of the 20 patients with oligospermia had raised FSH levels. Seminal plasma inhibin was measured in individual samples using a quantitive in vitro rat anterior pituitary cell culture bioassay in which FSH cell content was measured after 72 hours of incubation with the inhibin-containing material. Biopotencies were determined using combined multiple parallel line assays with reference to an inhibin standard with a potency of 1 U/mg. The concentrations of inhibin in normal seminal plasma were 31.4 +or- 3.0 U/ml, which contrasted with the low levels found in azoospermic patients with high plasma FSH levels. Of these, 7 had undetectable inhibin levels (2.5 U/ml) and 7 had values ranging from 4.2-8/5 U/ml. These concentrations were significantly lower than those in azoospermic patients, in whom FSH was not raised (18.9 +or- 2.2 U/ml). Seminal plasma inhibin levels postvasectomy were 16.9 +or- 2.3 U/ml and were not significantly different from those measured in azoospermic normal FSH patients. Peripheral plasma FSH levels were expressed as a function of seminal plasma inhibin concentrations (r= -0.736, P0.001; excluding those patients with vasal obstruction). These findings show that inhibin-like activity in seminal plasma is reduced in infertile men with raised peripheral plasma FSH levels, and that a reciprocal inverse relationship exists between serum FSH and seminal plasma inhibin concentrations.

Biochemical analysis of bovine testicular anti-Müllerian hormone.

Direct biochemical analysis has been applied to bovine testicular anti-Müllerian hormone (AMH), purified from incubation medium of bovine fetal testes by immunochromatography on a monoclonal antibody. The hormone contains a high proportion of hydrophobic amino acids and 13.5% carbohydrate. The oligosaccharide composition suggests that both N- and O-glycosidically linked chains are present. The molecular extinction coefficient is 3.27 +/- 0.06. One RIA unit, defined as the amount of hormone released by 1 g fetal bovine testicular tissue incubated during 4 h, corresponds to 3.06 +/- 0.17 microgram protein.

Immunocytochemical localization of Mullerian inhibiting substance in the rough endoplasmic reticulum and Golgi apparatus in Sertoli cells of the neonatal calf testis using a monoclonal antibody.

Mullerian Inhibiting Substance (MIS) has been localized in the Sertoli cells of the neonatal calf testis using preembedding immunoperoxidase techniques and a monoclonal antibody which almost completely blocks the biological activity of MIS. Both the peroxidase-labeled antibody method using a peroxidase-conjugated F(ab')2 fragment of IgG as a second antibody and the unlabeled antibody peroxidase-antiperoxidase (PAP) method using Fab fragments of the PAP complex were employed. With both methods, MIS was demonstrated within the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus. In the Golgi, MIS was concentrated in the transmost cisternae especially at their peripheral expansions. This study indicates that MIS is synthesized in the RER and transported to the Golgi apparatus, presumably for glycosidation, before secretion from Golgi derived vacuoles.

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.

Expression levels of Mullerian-inhibiting substance, GATA4 and 17alpha-hydroxylase/17,20-lyase cytochrome P450 during embryonic gonadal development in two diverse breeds of swine.

Sexual differentiation and early embryonic/fetal gonad development is a tightly regulated process controlled by numerous endocrine and molecular signals. These signals ensure appropriate structural organization and subsequent development of gonads and accessory organs. Substantial differences exist in adult reproductive characteristics in Meishan (MS) and White Composite (WC) pig breeds. This study compared the timing of embryonic sexual differentiation in MS and WC pigs. Embryos/fetuses were evaluated on 26, 28, 30, 35, 40 and 50 days postcoitum (dpc). Gonadal differentiation was based on morphological criteria and on localization of GATA4, Mullerian-inhibiting substance (MIS) and 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450(c17)). The timing of testicular cord formation and functional differentiation of Sertoli and Leydig cells were similar between breeds. Levels of GATA4, MIS and P450(c17) proteins increased with advancing gestation, with greater levels of MIS and P450(c17) in testes of MS compared with WC embryos. Organization of ovarian medullary cords and formation of egg nests was evident at similar ages in both breeds; however, a greater number of MS compared with WC embryos exhibited signs of ovarian differentiation at 30 dpc. In summary, despite breed differences in MIS and P450(c17) levels in the testis, which may be related to Sertoli and Leydig cell function, the timing of testicular differentiation did not differ between breeds and is unlikely to impact reproductive performance in adult boars. In contrast, female MS embryos exhibited advanced ovarian differentiation compared with WC embryos which may be related to the earlier reproductive maturity observed in this breed.