Detection of anti-m\l=u"\llerian activity in boar rete testis fluid

Transcription

1 Detection of anti-m\l=u"\llerian activity in boar rete testis fluid Nathalie Josso, J. Y. Picard, J. L. Dacheux and M. Courot Unité de Recherches de Génétique Médicale (INSERM), Hôpital des Enfants-Malades, Paris Cedex 15, and Vnstitut National de Recherches Agronomiques, Nouzilly, France Summary. Boar rete testis fluid was tested for its capacity to induce M\l=u"\llerian regression in 14\m=.\5-day-oldrat M\l=u"\llerianducts. Weak activity was present in crude RTF, but after gel filtration and 5-fold concentration, greater activity was detected in 1 out of 7 pools of the eluted fractions. The biologically active fraction (mol. wt \p=n-\310000) coincided with the elution of authentic labelled anti-m\l=u"\llerian hormone, obtained from bovine fetal testes. These results indicate that a small amount of anti-m\l=u"\llerianhormone is still synthesized in post-natal life. Introduction Regression of Miillerian ducts in males is mediated by a specific testicular factor (Jost, 1947). This anti-miillerian hormone has been shown to be a glycoprotein (Picard, Tran & Josso, 1978) synthesized by the non-germinal elements of the seminiferous tubules (Josso, 1973, 1974; Blanchard & Josso, 1974), probably the Sertoli cells. The ability of testicular expiants to induce regression of fetal rat Miillerian ducts placed next to them in organ culture is high during fetal life and progressively disappears in the perinatal period or shortly after birth (Picon, 1970; Josso, 1972; Donahoe, Ito, Price & Hendren, 1977). The cause of the irreversible disappearance of anti-miillerian activity from post-natal testicular tissue is not known. Josso (1972) concluded that anti-miillerian hormone was a fetal protein, synthesis of which is repressed after birth. Donahoe, Ito, Marfatia & Hendren (1976) and Donahoe et al (1977) related loss of testicular anti-miillerian activity to weaning, while Tran, Meusy-Dessolle & Josso (1977) and Picon, Picon, Chauffaux & Ktorza (1978) suggested the possiblity of a causal link between the disappearance of anti-miillerian activity from testicular tissue and formation of the blood-testis barrier. In the rat, anti-miillerian activity disappears from testicular tissue during the 3rd week after birth (Picon, 1970; Donahoe et al, 1976) at the time of appearance of tight junctions between adjacent Sertoli cells (Vitale, Fawcett & Dym, 1973). In the boar, anti-miillerian activity can no longer be detected in testicular tissue by the 10th week after birth (D. Tran,. Meusy-Dessolle &. Josso, unpublished observations). To investigate whether the anti-miillerian hormone is still synthesized by Sertoli cells in adult life but prevented from diffusing out of the adluminal compartment of seminiferous tubules, we have looked for anti-miillerian activity in the rete testis fluid of the boar. Materials and Methods adult boars cannulated as described by Rete testis fluid (RTF) was collected at 4 C from 3 Dacheux, O'Shea & Paquignon (1979) and was frozen in liquid nitrogen or used immediately. Anti-Miillerian activity of crude RTF was studied as previously described (Josso, Forest & Picard, 1975) after extensive dialysis against Eagle's minimum essential medium, and after gel

2 filtration on Ultrogel AcA 34, in 0-05 M-Tris-HCl ph 7-5, M-dithioerythritol and M-EDTA. The eluted fractions were pooled into 7 groups which were concentrated to a fifth of the initial volume of RTF by ultrafiltration on an Amicon (R) PM 30 membrane and dialysed against Eagle's minimum essential medium. After passage through a 0-45 pm Millipore filter, the medium to be tested was placed in the well of an organ culture dish containing the reproductive tracts of 14-5-day-old rat fetuses from which the gonads had been removed. The Miillerian ducts of rats of this age are suitable target organs for rat anti-miillerian hormone (Picon, 1969) and also for that produced by pig fetal testes (Tran et al, 1977). Twenty-two (22) rat reproductive tracts were exposed to crude RTF and 42 to the RTF eluate (6 tracts to each of the 7 groups of eluate fractions). Six control tracts were cultured in Eagle's minimum essential medium containing 30% boar plasma. After 3 days in culture, the tracts were fixed in Bouin's fluid, serially sectioned, mounted 1 section in 10 on coded slides and stained with PAS-haematoxylin. The degree of regression of Miillerian ducts on both sides (complete, incomplete or absent) was scored for each tract according to previously described criteria (Josso et al, 1975). Partly purified incubation medium produced by calf fetal testes incubated in the presence of tritiated fucose was applied to the column used for gel-filtration of RTF and the elution of radioactivity was monitored by scintillation counting. Picard et al. (1978) have shown that the main radioactive protein moiety remaining in the incubation medium after such partial purification co-purifies and co-precipitates with biological anti-miillerian activity and very probably represents the hormone itself. Calibration of the column and calculation of molecular weight values were performed as previously described (Picard et al, 1978). Results Low anti-miillerian activity was detected in crude RTF. Of the 22 rat reproductive tracts exposed to it, 18 showed evidence of incomplete Miillerian regression (PI. 1, Fig. 1) and the remaining 4 were normal. In the RTF eluate, high anti-miillerian activity (PI. 1, Fig. 2) was recovered in the pooled fractions containing molecules of molecular weight With the exception of 4 incompletely regressed tracts, no Miillerian regression was produced by the fractions of the other 6 pools (PI. 1, Fig. 3). Miillerian ducts were normal in the 6 control tracts exposed to 30% boar plasma. As shown in Text-fig. 1, the molecular weight of anti- Miillerian hormone from calf fetal testes was ± PLATE 1 Changes in the reproductive tract of a 14-5-day-old fetal rat after removal of the gonads and exposure in organ culture for 3 days to various substances. M, Miillerian duct; W, Wolffian duct. PAS, x375. Fig. 1. Exposure to crude boar RTF. The Miillerian duct is narrow and its cells are disorganized. Fibroblasts are accumulating between Wolffian and Miillerian ducts. Fig. 2. Exposure to Pool 4 of the eluted fractions from boar RTF submitted to gel filtration. Pool 4 was concentrated to a fifth of the volume of the RTF sample. The Miillerian duct has nearly disappeared. Fig. 3. Exposure to Pool 7 of the eluted fractions, concentrated to a fifth of the volume of the sample. The Miillerian duct is normal, the Wolffian duct is regressing. Wolffian ducts of the rat fetus become androgen-dependent on Day 16 (Price & Ortiz, 1965; Josso, 1970), and therefore regress in androgen-free media. However, fixation on Day 17 does not allow enough time for androgen deprivation to alter the histological appearance consistently.

3 PLATE 1

4 o co ( 0 5 < GO CO ( Relative elution volume 2 5 Text-fig. 1. Gel filtration experiments on an AcA 34 Ultrogel column cm, developed with 0-05 M-Tris-HCl ph 7-5, M-dithioerythritol and m-edta at a constant flow rate of 5 cm/h. (a) Optical density at 280 nm of the eluate from boar rete testis fluid(-) and radioactivity of the eluate from tritiated anti-miillerian factor obtained from calf fetal testes (-). (b) Biological activity of the eluate. The eluate was arbitrarily divided into 7 pools of fractions as indicated by the vertical lines. Regression of the Miillerian ducts in the rat reproductive tracts exposed to each pool was complete (#), incomplete (ß) or unaffected (O) (c) Calibration of the column by various protein markers: (1) a2-macroglobulin; (2) xanthine oxidase; (3) catalase; (4) immunoglobulin G; (5) caeruloplasmin; (6) lactate dehydrogenase; (7) a-fetoprotein; (8) phosphoglycerate kinase; (9) peroxidase. Discussion Since the anti-miillerian factor present in boar RTF has the same apparent molecular weight as that from bovine fetal testes, as assessed by gel filtration, it is likely that the same molecule in fetal testes and in RTF is responsible for Miillerian regression. The reason that anti-miillerian activity vanishes from post-natal testicular tissue, but is still detectable in RTF, may well lie in the modification of testicular architecture which occurs during maturation. On the one hand, differentiation of the successive layers of the blood-testis barrier restricts diffusion of Sertoli-cell products out of the seminiferous tubule and favours their exit via the rete testis, as shown by Tindall, Vitale & Means (1975) for androgen-binding protein. On the other hand, when the mitotic index of Sertoli cells falls, as it does from Day 30 after birth in the developing pig (Erickson, 1964), Sertoli cells are progressively outnumbered in the seminiferous epithelium by the proliferating germ cells. As recently discussed by Carreau, Drosdowsky & Courot (1979) for androgen-binding protein, this reduction of the percentage of Sertoli cells in testicular tissue could be responsible for the fall of testicular concentration of Sertoli cell products but would not affect their release into rete testis fluid.

5 However, these factors alone cannot account for the total disappearance of anti-miillerian activity from testicular tissue reported in the literature (Picon, 1970; Josso, 1972; Donahoe et al, 1976, 1977). During the bioassay used by these workers to demonstrate anti-miillerian activity, open sections of seminiferous tubules are in direct contact with the rat reproductive tracts, and should allow at least some opportunity for diffusion of tubular contents into the culture medium. Picon et al. (1978) found no re-activation of anti-miillerian capacity in 2 canine Sertoli-cell tumours in which tubular structure had been destroyed. The anti-miillerian activity of crude RTF is quite low compared to that of fetal pig testicular tissue which consistently produces total regression of all the Miillerian ducts exposed to it in organ culture (Tran et al, 1977). In the present experiments, total regression of Miillerian ducts was only obtained in two-thirds of the ducts exposed to RTF concentrated 5-fold by gel filtration. We conclude that Sertoli cells do continue to produce anti-miillerian hormone in post-natal life, albeit in low amounts. The cause of this change in the level of biosynthesis is not established at the present time; it could be due to the genetic programming of the cells or to a change in their hormonal environment. Further studies are needed to clarify this point. We are grateful to Miss Axelle Simon and Miss Claudine Pisselet for technical assistance, and to INSERM grant for financial support. We thank Dr Nicole Meusy-Dessolle for the gift of boar plasma. References Blanchard, M.G. & Josso, N. (1974) Source of the anti- Miillerian hormone synthesized by the fetal testis. Miillerian inhibiting activity of fetal bovine Sertoli cells in tissue culture. Pediat. Res. 8, Carreau, S., Drosdowsky, M.A. & Courot, M. (1979) Age-related effects on androgen-binding protein (ABP) in sheep testis and epididymis. Int. J. Androl. 2, Dacheux, J.L., O'Shea, T. & Paquignon, J.M. (1979) Effects of osmolality, bicarbonate and buffer on the metabolism and motility of testicular, epididymal, and ejaculated spermatozoa of boars. J. Reprod. Fert. 55, Donahoe, P.K., Ito, Y., Marfatla, S. & Hendren, W.H., III (1976) The production of Miillerian inhibiting substance by the fetal, neonatal and adult rat. Biol. Reprod. 15, Donahoe, P.K., Ito, Y., Price, J.M. & Hendren, W.H., III (1977) Miillerian inhibiting substance activity in bovine fetal, newborn and prepubertal testes. Biol. Reprod. 16, Erickson, B.H. (1964) Effects of neonatal gamma irradiation on hormone production and spermatogenesis in the testis of the adult pig. /. Reprod. Fert. 8, Josso, N. (1970) Action de la testosterone sur le canal de Wolff du foetus de rat en culture organotypique. ArchsAnat. microsc. Morph. exp. 59, Josso, N. (1972) Evolution of the Müllerian-inhibiting activity of the human testis. Effect of fetal, peri-natal and post-natal human testicular tissue on the Miillerian duct of the fetal rat in organ culture. Biol. Neonate 20, Josso, N. (1973) In vitro synthesis of Müllerian-inhibit ing hormone by seminiferous tubules isolated from the calf fetal testis. Endocrinology 93, Josso, N. (1974) Müllerian-inhibiting activity of human fetal testicular cells deprived of germ cells by in vitro irradiation. Pediat. Res. 8, Josso, N., Forest, M.G. & Picard, J.Y. (1975) Müllerianinhibiting activity of calf fetal testes: relationship to testosterone and protein synthesis. Biol. Reprod. 13, Jost, A. (1947) Recherches sur la differentiation sexuelle de l'embryon de lapin. III. Rôle des gonades foetales dans la différenciation sexuelle somatique. Archs Anat. microsc. Morph. exp. 36, Picard, J.Y., Tran, D. & Josso, N. (1978) Biosynthesis of labelled anti-müllerian hormone by fetal testes. Evi dence for the glycoprotein nature of the hormone and for its di-sulfide bonded structure. Mol. cell. Endocr. 12, Picon, R. (1969) Action du testicule foetal sur le développement in vitro des canaux de Müller chez le rat. Archs Anat. microsc. Morph. exp. 58, Picon, R. (1970) Modifications, chez le rat, au cours du développement du testicule, de son action inhibitrice sur les canaux de Müller in vitro. C. r. hebd. Séanc. Acad. Sci., Paris D 271, Picon, R., Plcon, L., Chattaux, S. & Ktorza, A. (1978) Effects of canine fetal testes and testicular tumors on Müllerian ducts. Biol. Reprod. 18, Price, D. & Ortiz, E. ( 1965) The role of fetal androgen in sex differentiation in mammals. In Organogénesis, pp Eds R. L. De Haan & H. Ursprung. Holt, Rinehart Winston, New York. Tlndall, DJ., Vitale, R. & Means, A.R. (1975) Androgen binding protein as a biochemical marker of forma tion of the blood-testis barrier. Endocrinology 97, Tran, D., Meusy-Dessolle, N. & Josso, N. (1977) Anti- Müllerian hormone is a functional marker of fetal Sertoli cells. Nature, Lond. 269, Vitale, R., Fawcett, D.W. & Dym, M. (1973) The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment. Anat. Ree. 176, Received 12 February 1979