La surexpression de l'androgen-binding protein (ABP) provoque des modifications morphologiques et fonctionnelles dans le testicule de souris

The Sertoti cells (SC) of many species produce an androgen-binding protein (ABP) which is secreted into both the blood and lumen of the seminiferous tubule. In the latter, it is transported to the epididymis where is taken up by epithelial cells, and is thought to play a role in sperm maturation. In view of the importance of ABP, we thought it would be pertinent to make several transgenic mice (TM) lines bearing the rABP gene to unravel its role in male reproductive physiology. A 5.5 Kb rat genomic DNA clone was microinjected into the pronucleus of fertilized mouse ova which were subsequently implanted into the oviduct of pseudopregnant CD-1 female mice. Detection of TM was performed by Southern Blot and PCR analysis using respectively, a 32p labeled rABP cDNA probe and oligonucleotides recognizing exons 1 and 7 of rABP gene. Chromosomic localization of the transgene was carried out by fluorescent in situ hybridization (FISH) in metaphasic cells obtained from bone marrow of TM. rABP expression was analyzed by Northern blot and RT-PCR techniques in most tissues of heterozigote TM. In the testis, specific cell expression was determined by in situ hybridization (ISH) and protein localization by immunohistochemistry. ABP-binding activity was performed by the carbon dextran method and analysis of protein internalization by autohistoradiographic detection of a (3H) testosterone-ABP complex. DNA fragmentation was investigated by the TUNEL technique and by electrophoresis of total genomic DNA. Testicular and epididymal morphology was studied by light and electron microscopy. Two offspring carrying the rABP gene were identified by Southern blotting, and two lines of mice (designated ABP7 and ABP24) were generated by selective breeding of the male founders with normal B6D2F1 females. FISH analysis demonstrated a different chromosomal localization of the transgene in both lines. Both rABP transgenic pedigrees presented reduced fertility. Northern blot and RT-PCR studies showed overexpression of rABP mRNA in the testis. ovary and uterus in ABP24 and ABP7 transgenic lines. rABP mRNA was appropriately expressed in SC as demonstrated by ISH. DHT-sH binding of testicular homogenates was increased 10 fold in TM compared to controls. In adult testis of TM, some seminiferous tubules showed disorganization of the epithelium, increased number of SC, presence of vacuoles, germ cell meiotic arrest and germ cell degeneration. DNA fragmentation was demonstrated in germ cells during meiosis. rABP protein was localized in the intersticial space, and into some tubules, in SC and germ cells at different steps of maturation. rABP internalization was strongly increased in both germ and epididymal cells in TM. The present results reinforce the increasing evidence of the role of ABP during spermatogenesis, even though further experiments are required to unravel its definitive implication in testicular and epidididymal homeostasis.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

Testins are structurally related Sertoli cell proteins whose secretion is tightly coupled to the presence of germ cells

Previous studies from this laboratory have shown that Sertoli cell-enriched culture medium contained two immunologically and structurally related proteins designated CMB-22 and CMB-23 with Mr of 37,000 and 40,000, respectively. We have now demonstrated that both CMB-22 and CMB-23 are monomeric proteins with the following NH2-terminal amino acid sequences: CMB-22, NH2-TPDPSLDVEWNEWRTKHGKTYNMNEERLKR; CMB-23, NH2-XAPXPDPSLDVEXNEXRTK. These sequences are virtually identical except that CMB-23 has three extra NH2 terminus amino acids of X-A-P. Comparison of these sequences with those in the Protein Identification Resource revealed that they are unique proteins. CMB-22 and CMB-23 are highly concentrated in testes and their levels in this tissue increase with age. Studies using [35S]methionine incorporation and immunoprecipitation demonstrated that Sertoli cells synthesize and secrete these proteins in vitro. Because they seem not to have been isolated previously, are concentrated in and synthesized by the testes, and are structurally related, we propose that CMB-22 and CMB-23 be designated testin I and testin II, respectively. The distribution of these proteins in biological fluids were compared with those of testibumin and rat androgen binding protein (rABP), two other Sertoli cell proteins. The results suggest that testins, unlike testibumin and rABP, are not transported to the epididymis. Although the amount of testins secreted by Sertoli cells in vitro is similar to that of testibumin and rABP, the concentrations in testis and rete testis fluid are several orders of magnitude less than that of testibumin and rABP. These observations suggest that the secretion of these proteins in vivo might be suppressed by germ cells. The fact that 10 times more testins are secreted by tubules from immature rats than by those from adult rats and that there is an increase in the testicular content of testins following a single dose of busulfan, which depleted the germ cells from the seminiferous epithelium, supports this hypothesis. Thus, the secretion of testins by Sertoli cells appears to be tightly coupled to the presence of germ cells; there is an inverse relationship between the amount of testins in the testis and the number of germ cells. These results suggest that testins are unique testicular proteins that can be used to study Sertoli cell-germ cell interactions in the seminiferous epithelium.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.     

Infection of SARS-CoV-2 causes severe pathological changes in mouse testis

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has affected more than 600 million people worldwide. Several organs including lung, intestine, and brain are infected by SARS-CoV-2. It has been reported that SARS-CoV-2 receptor angiotensin-converting enzyme-2 (ACE2) is expressed in human testis. However, whether testis is also affected by SARS-CoV-2 is still unclear. In this study, we generate a human ACE2 (hACE2) transgenic mouse model in which the expression of hACE2 gene is regulated by hACE2 promoter. Sertoli and Leydig cells from hACE2 transgenic mice can be infected by SARS-CoV-2 pseudovirus in vitro, and severe pathological changes are observed after injecting the SARS-CoV-2 pseudovirus into the seminiferous tubules. Further studies reveal that Sertoli and Leydig cells from hACE2 transgenic mice are also infected by authentic SARS-CoV-2 virus in vitro. After testis interstitium injection, authentic SARS-CoV-2 viruses are first disseminated to the interstitial cells, and then detected inside the seminiferous tubules which in turn cause germ cell loss and disruption of seminiferous tubules. Our study demonstrates that testis is most likely a target of SARS-CoV-2 virus. Attention should be paid to the reproductive function in SARS-CoV-2 patients.     

Localization and synthesis of entactin in seminiferous tubules of the mouse.

Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin.     

Beta-nerve growth factor influences the expression of androgen-binding protein messenger ribonucleic acid in the rat testis.

The effect of infusion of nerve growth factor (NGF) into the rat testis on the expression of androgen-binding protein (ABP) mRNA was studied. A major 1.7-kb and a minor 3.7-kb ABP mRNA were present at all stages of the seminiferous epithelium with maximal levels at stages VIII-XI and the lowest levels at stages IV-VI. Infusion of 15 ng/h of NGF with a mini-osmotic pump for 14 days resulted in a 2-fold increase of ABP mRNA as revealed by Northern blots, whereas the mRNA level of another Sertoli cell protein, urokinase-type plasminogen activator, remained unchanged. Image analysis of autoradiograms obtained by in situ hybridization of sections from treated testes showed a similar increase in APB mRNA compared to noninfused or PBS-infused testes. However, at the cellular level the labeling intensity for ABP mRNA over Sertoli cells of different stages of the seminiferous epithelium was the same in NGF-infused and control testes. This suggests that the increase of ABP mRNA in NGF-infused testes was caused by prolongation of stages VII-VIII with maximal ABP mRNA expression; the suggestion is supported by an increase of 30 percent in frequency of these stages in histological sections from NGF-infused testes.     

Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells.

The expression of the mRNA for SLF (the c-kit ligand), a product of the "steel" locus, has been investigated in postnatal mouse testis and homogeneous populations of testicular cells. The message was found expressed in postnatal mouse testis but not in germ cells. Studies on primary mouse Sertoli cell cultures from 18 day old mice show that Sertoli cells are the site of SLF mRNA expression in the seminiferous tubules. Treatment of Sertoli cell cultures with cAMP analogs led to a significant increase in the SLF mRNA levels.     

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.     

The effect of cryptorchidism and subsequent orchidopexy on testicular function in adult rats

Cryptorchidism for 28 or 10 days resulted in a severe disruption of spermatogenesis (assessed histologically or by fertility tests), Sertoli cell function (assessed by seminiferous tubule fluid production after efferent duct ligation, ABP levels, binding of 125I-labelled FSH to testis homogenates and serum FSH levels) and Leydig cell function (assessed by serum LH and testosterone levels, in-vitro testosterone production, binding of 125I-labelled hCG). Orchidopexy after 28 days of cryptorchidism resulted in a poor recovery of spermatogenesis since the majority of tubules were lined by Sertoli cells and a few spermatogonia. No recovery occurred in the indicators of Sertoli and Leydig cell function. Orchidopexy after 10 days of cryptorchidism also resulted in a poor recovery of spermatogenesis, with a few animals showing partial recovery after 6 months. No recovery occurred in seminiferous tubule fluid production but partial recovery occurred in ABP content and production rate. Serum FSH, LH levels and in-vitro testosterone production by the testis remained elevated and did not change from the values found during cryptorchidism. Fertility testing at 6 months revealed a small number of rats in which fertility was restored although the number of embryos was lower than in controls. In this group of animals there was a significant improvement in a number of indicators of Sertoli cell and Leydig cell function. These data provide further evidence to link the changes in Sertoli cell and Leydig cell function to the germ cell complement present in the testis.     

Age-related changes in the function of the pituitary-gonadal axis in a sterile male rat mutant (hd/hd)

Testicular growth is depressed in the genetically sterile male rat (hd/hd) relative to its LE phenotype littermates (by 50% and 73% at 27 and 90 days of age, respectively). Within the hd/hd testis, both the tubular and seminiferous tubule tissues are affected by the mutation. In addition, there is significantly less germ cell production from the primary spermatocyte stage of spermatogenesis onwards and the total number of Sertoli cells observed is less. In the intertubular tissue, the total volume and the total number of Leydig cells per testis is significantly less, but the mean volume of an average Leydig cell is not modified. The serum gonadotropin levels are higher in the hd/hd rat, whereas from 40 days of age onwards the level of testosterone is lower. The FSH and LH binding affinity constants are unchanged by the mutation; however, the total number of FSH binding sites per 10(6) Sertoli cells is lower while that of LH per 10(6) Leydig cells is greater. Indeed, it is likely that the lesser concentration of serum testosterone in the hd/hd rat is a result of a smaller number of Leydig cells since their individual function is not modified. The testicular androgen binding protein (ABP) content and the ABP output towards the epididymis are lower as a consequence of both a lesser number and an altered function of the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sertoli and granulosa cell-specific Cre recombinase activity in transgenic mice

We have established transgenic mice expressing the Cre recombinase under the control of the anti-Müllerian hormone (AMH) gene promoter. Cre activity and specificity were evaluated by different means. In AMH-Cre mice, expression of the Cre recombinase mRNA was confined to the testis and ovary. AMH-Cre mice were crossed with reporter transgenic lines and the offspring exhibited Cre-mediated recombination only in the testis and the ovary. In male, histochemical analysis indicated that recombination occurred in every Sertoli cells. In female, Cre-mediated recombination was restricted to granulosa cells, but the protein was not evenly active in every cells. From these results, we conclude that potentially, this transgenic line possessing AMH promoter-driven expression of the Cre recombinase is a powerful tool to delete genes in Sertoli cells only, in order to study Sertoli cell gene function during mammalian spermatogenesis.     

Hereditary defects in both germ cells and the blood-testis barrier system in as-mutant rats: evidence from spermatogonial transplantation and tracer-permeability analysis

The rat mutant allele as is located on chromosome 12. Homozygous (as/as) males show arrested spermatogenesis, mainly at the pachytene spermatocyte stage. It is not clear whether this defective spermatogenesis is caused by a failure in a somatic cell component that supports spermatogenesis or in the germ cell itself. Spermatogonial transplantation was performed to identify the genetically defective site in the as/as testis. In experiment 1, germ cells collected from as/as testes were transplanted into the testes of immunodeficient mice and normal rats. In experiment 2, normal rat germ cells were transplanted into as/as testes. The results of experiment 1 showed arrest of spermatogenesis at the pachytene spermatocyte stage, accompanied by a characteristic morphological feature, i.e., the formation of inclusion-like bodies in the cytoplasm, in both rat and mouse recipients. These results revealed the intrinsic effect of the mutant gene(s) on germ cells. In experiment 2, no restoration of spermatogenesis was detected in the recipient testes despite thorough histological examination. These results suggest that defects in a somatic cell component in as/as testes prevent the donor germ cells from colonizing and regaining their spermatogenetic ability. When the seminiferous epithelium of the as/as testis was examined by electron microscopy, no morphological abnormalities, including the formation of ectoplasmic specializations between adjacent Sertoli cells, were observed in the somatic cell components. However, when cytochrome c was applied as a tracer material, it penetrated the tight junctions between the Sertoli cells, indicating dysfunction of the blood-testis barrier in the as/as testis. The lack of restoration of spermatogenesis in the as/as testis after transplantation of normal germ cells may have been caused by the unfavorable environment in the seminiferous epithelium resulting from the incomplete barrier system between adjoining Sertoli cells. The gene(s) at the as locus may have a role in both germ cell differentiation and the establishment of the blood-testis barrier.     

Sertoli Cells Maintain Leydig Cell Number and Peritubular Myoid Cell Activity in the Adult Mouse Testis

The Sertoli cells are critical regulators of testis differentiation and development. In the adult, however, their known function is restricted largely to maintenance of spermatogenesis. To determine whether the Sertoli cells regulate other aspects of adult testis biology we have used a novel transgenic mouse model in which Amh-Cre induces expression of the receptor for Diphtheria toxin (iDTR) specifically within Sertoli cells. This causes controlled, cell-specific and acute ablation of the Sertoli cell population in the adult animal following Diphtheria toxin injection. Results show that Sertoli cell ablation leads to rapid loss of all germ cell populations. In addition, adult Leydig cell numbers decline by 75% with the remaining cells concentrated around the rete and in the sub-capsular region. In the absence of Sertoli cells, peritubular myoid cell activity is reduced but the cells retain an ability to exclude immune cells from the seminiferous tubules. These data demonstrate that, in addition to support of spermatogenesis, Sertoli cells are required in the adult testis both for retention of the normal adult Leydig cell population and for support of normal peritubular myoid cell function. This has implications for our understanding of male reproductive disorders and wider androgen-related conditions affecting male health.     

In vivo analysis of phagocytosis of apoptotic cells by testicular Sertoli cells

Sertoli cells, a somatic cell type present within the seminiferous tubules of testes, are responsible for the phagocytic elimination of apoptotic spermatogenic cells. We here established an in vivo assay system that enables us to quantitatively analyze Sertoli cell phagocytosis of apoptotic cells in testes of live mice. Apoptotic cells were injected into the seminiferous tubules of spermatogenic cell-depleted mice, and the occurrence of phagocytosis by Sertoli cells was examined by histochemically analyzing testis sections or dispersed testicular cells. We reproducibly observed similar levels of phagocytosis in either examination, and the ratio of Sertoli cells that engulfed injected apoptotic cells was almost the same between the two examinations. These results indicated that a quantitative in vivo assay system was established using the seminiferous tubules of live mice as 'test tubes.' We then determined the requirements for Sertoli cell phagocytosis of apoptotic cells using this assay. For this purpose, apoptotic cells were injected together with various phagocytosis inhibitors, and the extent of phagocytosis by Sertoli cells was determined. The results revealed that Sertoli cells phagocytose apoptotic cells in a manner dependent on class B scavenger receptor type I (SR-BI) of Sertoli cells and phosphatidylserine exposed at the surface of target cells, as previously observed in vitro using primary cultures of dispersed rat testicular cells. Furthermore, the amount of SR-BI in Sertoli cells increased after injection of apoptotic cells into the seminiferous tubules, suggesting a positive feedback regulation of the expression of this phagocytosis receptor.     

Mice that express enzymatically inactive cathepsin L exhibit abnormal spermatogenesis

The finding of large, stage-specific changes in secretion of procathepsin L by rat Sertoli cells has led to the hypothesis that this proenzyme promotes the survival, replication, or differentiation of spermatogenic cells. Experiments described herein used a mouse model to test this hypothesis. To prove that mice are appropriate for this purpose, we first demonstrate that mature mouse Sertoli cells express cathepsin L mRNA in the same stage-specific manner as rat Sertoli cells and they also secrete procathepsin L. To test whether catalytically active cathepsin L is required for normal spermatogenesis, we examined the testes of 110- to 120-day-old furless mice, which express catalytically inactive cathepsin L. Morphologic examination of testes of furless mice revealed both normal and atrophic seminiferous tubules. Enumeration of atrophic tubules in furless and control mice demonstrates that lack of functional cathepsin L results in a 12-fold increase in seminiferous tubule atrophy. To determine whether lack of functional cathepsin L affects the production of male germ cells in apparently normal, nonatrophic tubules, we compared numbers in control and furless mice of preleptotene spermatocytes, pachytene spermatocytes, and round spermatids per Sertoli cell. Results demonstrate that the lack of functional cathepsin L causes a 16% reduction in formation of preleptotene spermatocytes and a 25% reduction in differentiation of these cells into pachytene spermatocyte. These results suggest that procathepsin L either directly or indirectly has two distinct functions in the testis. This proenzyme prevents atrophy of seminiferous tubules and promotes the formation of preleptotene spermatocytes and the differentiation of these meiotic cells into pachytene spermatocytes.