Gonadotropin suppression in men leads to a reduction in claudin-11 at the Sertoli cell tight junction.

Are Sertoli cell tight junctions (TJs) disrupted in men undergoing hormonal contraception? Localization of the key Sertoli cell TJ protein, claudin-11, was markedly disrupted by 8 weeks of gonadotropin suppression, the degree of which was related to the extent of adluminal germ cell suppression. Sertoli cell TJs are vital components of the blood-testis barrier (BTB) that sequester developing adluminal meiotic germ cells and spermatids from the vascular compartment. Claudin-11 knockout mice are infertile; additionally claudin-11 is spatially disrupted in chronically gonadotropin-suppressed rats coincident with a loss of BTB function, and claudin-11 is disorganized in various human testicular disorders. These data support the Sertoli cell TJ as a potential site of hormonal contraceptive action. BTB proteins were assessed by immunohistochemistry (n = 16 samples) and mRNA (n = 18 samples) expression levels in available archived testis tissue from a previous study of 22 men who had undergone 8 weeks of gonadotropin suppression and for whom meiotic and post-meiotic germ cell numbers were available. The gonadotropin suppression regimens were (i) testosterone enanthate (TE) plus the GnRH antagonist, acyline (A); (ii) TE + the progestin, levonorgestrel, (LNG); (iii) TE + LNG + A or (iv) TE + LNG + the 5α-reductase inhibitor, dutasteride (D). A control group consisted of seven additional men, with three archived samples available for this study. Immunohistochemical localization of claudin-11 (TJ) and other junctional type markers [ZO-1 (cytoplasmic plaque), β-catenin (adherens junction), connexin-43 (gap junction), vinculin (ectoplasmic specialization) and β-actin (cytoskeleton)] and quantitative PCR was conducted using matched frozen testis tissue. Claudin-11 formed a continuous staining pattern at the BTB in control men. Regardless of gonadotropin suppression treatment, claudin-11 localization was markedly disrupted and was broadly associated with the extent of meiotic/post-meiotic germ cell suppression; claudin-11 staining was (i) punctate (i.e. 'spotty' appearance) at the basal aspect of tubules when the average numbers of adluminal germ cells were <15% of control, (ii) presented as short fragments with cytoplasmic extensions when numbers were 15-25% of control or (iii) remained continuous when numbers were >40% of control. Changes in localization of connexin-43 and vinculin were also observed (smaller effects than for claudin-11) but ZO-1, β-catenin and β-actin did not differ, compared with control. Claudin-11 was the only Sertoli cell TJ protein investigated, but it is considered to be the most pivotal of constituent proteins given its known implication in infertility and BTB function. We were limited to testis samples which had been gonadotropin-suppressed for 8 weeks, shorter than the 74-day spermatogenic wave, which may account for the heterogeneity in claudin-11 and germ cell response observed among the men. Longer suppression (12-24 weeks) is known to suppress germ cells further and claudin-11 disruption may be more uniform, although we could not access such samples. These findings are important for our understanding of the sites of action of male hormonal contraception, because they suggest that BTB function could be ablated following long-term hormone suppression treatment. National Health and Medical Research Council (Australia) Program Grants 241000 and 494802; Research Fellowship 1022327 (to R.I.M.) and the Victorian Government's Operational Infrastructure Support Program. None of the authors have any conflicts to disclose. Not applicable.

McCabe MJ ，Tarulli GA ，Laven-Law G ，Matthiesson KL ，Meachem SJ ，McLachlan RI ，Dinger ME ，Stanton PG ... -  《-》

Integrity of the blood-testis barrier in healthy men after suppression of spermatogenesis with testosterone and levonorgestrel.

Ilani N ，Armanious N ，Lue YH ，Swerdloff RS ，Baravarian S ，Adler A ，Tsang C ，Jia Y ，Cui YG ，Wang XH ，Zhou ZM ，Sha JH ，Wang C ... -  《-》

Effects of testosterone and levonorgestrel combined with a 5alpha-reductase inhibitor or gonadotropin-releasing hormone antagonist on spermatogenesis and intratesticular steroid levels in normal men.

Matthiesson KL ，Stanton PG ，O'Donnell L ，Meachem SJ ，Amory JK ，Berger R ，Bremner WJ ，McLachlan RI ... -  《JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM》

Novel male hormonal contraceptive combinations: the hormonal and spermatogenic effects of testosterone and levonorgestrel combined with a 5alpha-reductase inhibitor or gonadotropin-releasing hormone antagonist.

Matthiesson KL ，Amory JK ，Berger R ，Ugoni A ，McLachlan RI ，Bremner WJ ... -  《JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM》

Establishment, maintenance and functional integrity of the blood-testis barrier in organotypic cultures of fresh and frozen/thawed prepubertal mouse testes.

Can the spatio-temporal formation of an intact blood-testis barrier (BTB), which is essential for the progression of spermatogenesis, be reproduced in cultures of fresh or frozen/thawed prepubertal mouse testes? Organotypic cultures allow the establishment and maintenance of major BTB components and the formation of a functional BTB in mouse testicular tissues. In vitro maturation of prepubertal testicular tissues is a promising approach to restore fertility in adult survivors of childhood cancer. Although gametes can be successfully obtained from prepubertal mouse testes in organotypic cultures, the spermatogenic yield remains low compared to in vivo controls. Mouse testicular tissues were frozen using controlled slow freezing (CSF) or solid surface vitrification (SSV) procedures. A total of 158 testes (fresh n = 58, CSF n = 58 or SSV n = 42) from 6 to 7 days postpartum (dpp) mice were cultured at 34°C in basal medium (α-MEM, 10% KnockOut Serum Replacement, 5 μg/ml gentamicin) at a gas-liquid interphase (under 20% O2), with or without 10-6 M retinol, for 9, 16 and 30 days. In addition, 32 testes from 6-7, 15-16, 22-23 and 36-37 dpp mice were used as in vivo controls. The mRNA levels of BTB genes (Claudin 3, Claudin 11, Zonula occludens 1 and Connexin-43), germ cell-specific genes (Sal-like protein 4, Kit oncogene, Stimulated by retinoic acid gene 8, Synaptonemal complex protein 3, Transition protein 1 and Protamine 2), markers of Sertoli cell immaturity/maturity (anti-Mullerian hormone, androgen receptor, cyclin-dependent kinase inhibitor 1b) and the androgen-regulated gene Reproductive homeobox 5 (Rhox5) were measured by quantitative RT-PCR (RT-qPCR). The localization of BTB proteins in seminiferous tubules was studied by immunohistochemistry and spermatogenic progression was evaluated histologically. The integrity of the BTB was assessed using a biotin tracer. Modest differences in Claudin 11 (Cldn11), Zonula occludens 1 (Zo-1), Connexin-43 (Cx43) transcript levels and in the localization of the corresponding proteins were found between in vitro cultures of fresh or frozen/thawed testes and in vivo controls (P < 0.05). However, a 32-77-fold decrease in Claudin 3 (Cldn3) mRNA levels and a lack of CLDN3 immunolabelling in 36-44% of seminiferous tubules were observed in 30-day organotypic cultures (P < 0.05). Although Sertoli cell maturation and the completion of a full spermatogenic cycle were achieved after 30 days of culture, meiotic and postmeiotic progression was altered in cultured testicular tissues (P < 0.05). Moreover, an increased BTB permeability and a decreased expression of Rhox5 were observed at the end of the culture period in comparison with in vivo controls (P < 0.05). Completion of spermatogenesis occurred in vitro in seminiferous tubules with an intact BTB, and in those expressing or lacking CLDN3. None. Further studies will be needed to determine whether the expression of other BTB components is altered and to decipher the reason for lower Cldn3 and Rhox5 mRNA levels in organotypic cultures. This work contributes to a better understanding of the molecular mechanisms occurring in in vitro matured prepubertal testes. The organotypic culture system will have to be developed further and optimized for human tissue, before potential clinical applications can be envisaged. This work was supported by Rouen University Hospital, Ligue contre le Cancer (to L.D.), and co-supported by European Union and Région Normandie (to A.O.). Europe gets involved in Normandie with European Régional Development Fund (ERDF). The authors declare that they have no conflict of interest.

Rondanino C ，Maouche A ，Dumont L ，Oblette A ，Rives N ... -  《-》