Biomedical subjects
N Josso
Publications and source records attributed to N Josso.
Antimüllerian hormone: new perspectives for a sexist molecule.
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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.
Initiation of production of anti-müllerian hormone by the fetal gonad.
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Purification of testicular anti-Müllerian hormone allowing direct visualization of the pure glycoprotein and determination of yield and purification factor.
An improved method is described for the purification of anti-Müllerian hormone from incubation medium of bovine fetal testes, using RIA to optimize the yield at different steps. Proteins present in incubation medium are precipitated by ammonium sulphate at 30-45% saturation and subjected to ion-exchange chromatography on DEAE-Sepharose. The bulk of the hormone is eluted from the ion-exchanger by a 0.12 M concentration of NaCl. Purification is achieved by immunochromatography on a monoclonal antibody: usually, addition of extraneous protein to the eluting buffer is required, but can be dispensed with if a second, cumulative, immunochromatography is performed by pooling the eluates from the first procedure. AMH obtained by this procedure has been studied using Coomassie blue and immunoblotting, and compared with fucose-labelled AMH, which is recognized by the same monoclonal antibodies as the carrier of the anti-Müllerian biological activity. In the absence of reducing conditions, several multimers, from 145 000 to 235 000 in molecular weight, are present. Reduction of disulphide bond linkages results in the disappearance of the multimeric forms, and the appearance of a 72 000 monomer. Equivalence between RIA and weight units has been established in one experiment: one AMH unit corresponds to 15 micrograms of protein. Pure AMH isolated by this procedure is highly bioactive at a concentration of 200 mU/ml.
Preservation of gonadal function in true hermaphroditism.
Twelve documented cases of true hermaphroditism have been studied since 1965; there was histological evidence of both ovarian and testicular tissue in all cases. The clinical, anatomical, and therapeutic procedures are reviewed with special emphasis on gonadal morphology, histology, and function. Seven patients were reared as females, 5 as males; 6 are now postpubertal and 3 are more than 30 years old. Five underwent bilateral castration; in seven cases, only the discordant gonadal tissue has been removed so that these patients may have endogenous gonadal secretion in keeping with their sex of rearing.
Production of anti-Müllerian hormone: another homology between Sertoli and granulosa cells.
Anti-Müllerian hormone (AMH) has been detected by RIA in the follicular fluid of mature bovine ovaries and in incubation medium of bovine granulosa cells. Purification of AMH from two independent batches of follicular fluid was achieved with a yield of 11% and 15% respectively. Both ovarian and control testicular AMH produced near-complete regression of fetal rat Müllerian ducts exposed to it in culture at a final concentration of 200-300 mU/ml and were recognized by the same monoclonal and polyclonal antibodies. These findings indicate that adult mammalian granulosa cells are capable of producing immunoreactive and bioactive AMH at a rate apparently similar to that already demonstrated for mature Sertoli cells and add yet another item to the homologies reported between male and female somatic gonadal cells.
Anti-Müllerian hormone is not cytotoxic to human endometrial cancer in tissue culture.
To determine whether anti-Müllerian hormone (AMH) is the factor responsible for the cytotoxic effect of testicular preparations towards endometrial cancer, homogenates and incubation media of bovine fetal testes were fractionated by lectin affinity chromatography on wheat germ agglutinin and their cytotoxic effect tested on an endometrial cancer cell line, HEC-1, and on a control cell line of renal origin. Homogenate-derived testicular proteins proved more cytotoxic than incubation medium-derived ones, and the cytotoxic factor did not bind to the lectin of wheat germ, in contrast to AMH which did. Bioactive AMH purified by monoclonal antibody chromatography, at a concentration, determined by RIA, more than 20 times that present in the cytotoxic testicular preparations, had no effect upon the growth of the malignant endometrial cells. These results indicate that the testicular agent cytotoxic to malignant endometrial cells is a substance other than AMH.
Origin of anti-Müllerian hormone in bovine freemartin fetuses.
The origin of AMH responsible for Müllerian duct regression in bovine freemartins has been reinvestigated, using a sensitive RIA for this hormone. Between 50 and 80 days, Müllerian duct regression occurs simultaneously in males and freemartins. Both twins exhibited high and positively correlated serum AMH concentrations, whereas gonadal in-vitro production of AMH and biological anti-Müllerian activity were detectable at a low level only in 2 out of 13 freemartins. In the gonads of approximately half the freemartins after 80 days, seminiferous tubules differentiated and the gonads produced AMH, but the output was very low compared to that of the male twin. These data suggest that regression of Müllerian duct in freemartins is essentially mediated by AMH produced by the testes of the male twin.
Incomplete androgen insensitivity syndrome: partially masculinized genitalia in two patients with absence of androgen receptor in cultured fibroblasts.
In male pseudo-hermaphroditism due to end organ unresponsiveness, partial androgen insensitivity can usually be related to low but detectable dihydrotestosterone (DHT) binding activity in cultured sexual skin fibroblasts. We have studied two patients with partial virilization and total absence of androgen receptors in cultured fibroblasts. A lack of DHT binding could be related to a selection of a fibroblast strain with no androgen receptor activity, to insufficient sensitivity of the method in detecting very low receptor concentrations or to an instability of the receptor or the androgen-receptor complex. However, these observations raise the question of masculinization of external genitalia with undetectable androgen receptor.
Persistence of Müllerian ducts in male pseudohermaphroditism, and its relationship to cryptorchidism.
Twenty-two cases of male pseudohermaphroditism with persistence of Müllerian duct derivatives were reviewed. In 12 cases of mixed gonadal dysgenesis and five cases of dysgenetic male pseudohermaphroditism, testosterone-dependent steps of sex differentiation were also impaired, and testicular dysgenesis was prominent, even in the younger age group. The capacity of testes to inhibit the ipsilateral Müllerian duct was correlated with testicular descent: it is suggested that testicular dysgenesis explains both the functional and topographical testicular abnormalities observed in these male pseudohermaphrodites. Five other patients were cryptorchid but externally normally virilised, persistence of Müllerian derivatives representing their only abnormality of sex differentiation. Testicular structure was usually normal or showed changes attributable to long-standing cryptorchidism. It is suggested that in these patients, persistence of Müllerian derivatives is due to an inborn error of metabolism, affecting the binding of anti-Müllerian hormone to its receptor and that failure of testicular descent is caused by mechanical restraint by the abdominal Müllerian organs.
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.
[Persistence of Mullerian ducts in the male rabbit by passive immunization against anti-mullerian hormone during fetal life].
A female Rabbit immunized against bovine anti-Müllerian hormone (AMH) was mated and gave birth to 3 litters, containing a total of 13 males. Ten of these presented with Müllerian derivatives, of variable development. Testicular position and structure, Wolffian development and virilization of the urogenital sinus were apparently normal in males with persistent Müllerian ducts.
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.
A sensitive radioimmunoassay for bovine anti-Müllerian hormone, allowing its detection in male and freemartin fetal serum.
Two monoclonal antibodies have been used to set up a solid-phase RIA for bovine anti-Müllerian hormone (bAMH). One AMH unit is defined as the amount released by 1 g of bovine fetal testicular tissue during a 4 h incubation period. Calibration curves were prepared using aliquots of a standard 500 ml pool of incubation medium, containing 200 AMH mU/ml, diluted either in 50% pig testes incubation medium, 5% horse serum, 10% female calf fetal serum or pure female calf fetal serum. Linearization of the calibration curves was achieved through "logit-log" transformation, all four lines were parallel. Within and between-assay variability were less than 5%. The RIA is at least 600 times more sensitive than the bioassay for anti-Müllerian activity and can detect AMH in male and freemartin fetal serum.
Localization of anti-Müllerian hormone in the rough endoplasmic reticulum of the developing bovine sertoli cell using immunocytochemistry with a monoclonal antibody.
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