Myclobutanil-induced testis damage and apoptotic germ cell death through ER stress and autophagy in mouse testes.

A Sertoli Cell-Specific Knockout of Connexin43 Prevents Initiation of Spermatogenesis

Decision letter: The single-cell chromatin accessibility landscape in mouse perinatal testis development

BackgroundIn the testis, thyroid hormone (T3) regulates the number of gametes produced through its action on Sertoli cell proliferation. However, the role of T3 in the regulation of steroidogenesis is still controversial.MethodsThe TRαAMI knock-in allele allows the generation of transgenic mice expressing a dominant-negative TRα1 (thyroid receptor α1) isoform restricted to specific target cells after Cre-loxP recombination. Here, we introduced this mutant allele in both Sertoli and Leydig cells using a novel aromatase-iCre (ARO-iCre) line that expresses Cre recombinase under control of the human Cyp19(IIa)/aromatase promoter.FindingsWe showed that loxP recombination induced by this ARO-iCre is restricted to male and female gonads, and is effective in Sertoli and Leydig cells, but not in germ cells. We compared this model with the previous introduction of TRαAMI specifically in Sertoli cells in order to investigate T3 regulation of steroidogenesis. We demonstrated that TRαAMI-ARO males exhibited increased testis weight, increased sperm reserve in adulthood correlated to an increased proliferative index at P3 in vivo, and a loss of T3-response in vitro. Nevertheless, TRαAMI-ARO males showed normal fertility. This phenotype is similar to TRαAMI-SC males. Importantly, plasma testosterone and luteinizing hormone levels, as well as mRNA levels of steroidogenesis enzymes StAR, Cyp11a1 and Cyp17a1 were not affected in TRαAMI-ARO.Conclusions/SignificanceWe concluded that the presence of a mutant TRαAMI allele in both Leydig and Sertoli cells does not accentuate the phenotype in comparison with its presence in Sertoli cells only. This suggests that direct T3 regulation of steroidogenesis through TRα1 is moderate in Leydig cells, and that Sertoli cells are the main target of T3 action in the testis.

Immunolocalization of estrogen receptor alpha, estrogen receptor beta and androgen receptor in the pre-, peri- and post-pubertal stallion testis

Apical ectoplasmic specialization (ES) is a testis-specific hybrid cell/cell actin-based adherens junction and cell/matrix focal contact anchoring junction type restricted to the interface between Sertoli cells and developing spermatids. Recent studies have shown that laminin gamma3, restricted to elongating spermatids, is a putative binding partner of alpha 6beta 1-integrin localized in Sertoli cells at the apical ES. However, the identity of the alpha and beta chains, which constitute a functional laminin ligand with the gamma3 chain at the apical ES, is not known. Using reverse transcription-PCR and immunoblotting to survey all laminin chains in cells of the seminiferous epithelium, it was noted that alpha 2, alpha 3, beta1, beta2, beta3, and gamma3 chains were found in germ cells, whereas alpha 1, alpha 2, alpha 4, alpha 5, beta1, beta2, gamma1, gamma2, and gamma3 chains were found in Sertoli cells, implying that alpha 3 and beta3 are the plausible laminin chains restricted to germ cells that may be the bona fide partners of gamma3. To verify this postulate, recombinant proteins based on domain G of alpha 3 and domain I of beta3 and gamma3 chains were produced and used to obtain the corresponding specific polyclonal antibodies. Additional studies have demonstrated that the laminin alpha 3, beta3, and gamma3 chains indeed are restricted to germ cells at the apical ES, co-localizing with each other and with beta1-integrin. Furthermore, co-immunoprecipitation studies have confirmed the interactions among laminin alpha 3, beta3, and gamma3, as well as beta1-integrin. When the functional laminin ligand at the apical ES was disrupted via blocking antibodies, such as using anti-laminin alpha 3 or gamma3 IgG, this treatment perturbed adhesion between Sertoli and germ cells (mostly spermatids), leading to germ cell loss from the epithelium. More important, a transient disruption of the blood-testis barrier was also detected.

Multiple connexins have been identified in testicular cells. Several lines of evidences indicate that, among them, connexin 43 (Cx43) may be unique for control of gonad development and spermatogenesis. To date, however, it is not known whether Cx43 is expressed in the fetal testis and what possible types of cellular interactions mediated by this connexin are critical to male fertility. In the present work, expression of Cx43 was investigated at various developmental ages in cryosections from mouse testis by using specific antibodies against Cx43. In serial or double-labeled sections, Cx43 localization was compared with immunocytochemical distribution of steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD), Mullerian inhibitory hormone (MIH), and germinal nuclear cell antigen (GCNA1), which are specific markers, respectively, of interstitial Leydig, Sertoli, and germinal cells. Sections were analyzed by fluorescence microscopy. We found that Cx43 immunofluorescence (IF) was uniformly distributed in the undifferentiated gonad at 11.5 days post coitus (dpc) and in cells of the mesonephric tubules. In the undifferentiated gonad, Cx43 was localized between primordial germ cells and somatic cells. At 12.5 dpc, when the gonad has undergone sexual differentiation, in the interstitium Cx43 was localized in Leydig cells and in the seminiferous cord it was localized between adjacent Sertoli cells. In Leydig and Sertoli cells, Cx43 labeling increased at 14.5, 16.5, and 18.5 dpc. From day 12.5 up to 18.5 dpc, Cx43 was also localized in cell borders between germinal and Sertoli cells. In conclusion, this study demonstrates that from the earliest stages of gonadal development, Cx43 is expressed in the principal cell types that participate in the control of male fertility. It also shows that Cx43 expression in Leydig and Sertoli cells increase during fetal life. Finally, it provides evidence that, throughout embryonic life, Cx43 forms gap junctions between Sertoli and germinal cells.

The natural history of endocrine function and spermatogenesis in Klinefelter syndrome: what the data show

The local production and action of an epidermal growth factor (EGF)-like substance within the seminiferous tubule was investigated as a potential mediator of cell-cell interactions. Peritubular (myoid) and Sertoli cells were isolated and cultured under serum-free conditions. Proteins secreted by Sertoli and peritubular cells were found to contain a component that bound to the EGF receptor in a RRA. Separation of secreted proteins by reverse phase chromatography fractionated a protein that contained EGF bioactivity in its activity to stimulate growth of an EGF-dependent cell line. Biochemical properties examined for both Sertoli and peritubular cell EGF activities were similar with each other, but distinct from murine EGF. Northern blot analysis with an EGF cDNA probe did not detect EGF gene expression in peritubular, Sertoli, or germ cells. The possible production of an EGF-like substance such as transforming growth factor-alpha (TGF alpha) was investigated with a molecular probe to human TGF alpha. Both peritubular and Sertoli cells contained a 4.5-kilobase mRNA species that hybridized in a Northern blot analysis with a human TGF alpha cRNA probe. An immunoblot with a TGF alpha antisera confirmed the production of TGF alpha by the detection of a protein in both Sertoli and peritubular cell secreted proteins. TGF alpha gene expression was not detected in freshly isolated germ cells. Scatchard analysis revealed the presence of high affinity EGF receptors on peritubular cells and the absence of such receptors on Sertoli or germ cells. TGF alpha was found to stimulate peritubular cell proliferation, but had no effect on Sertoli cell growth. The effects of hormones and TGF alpha on Sertoli cell function and differentiation were assayed through an examination of transferrin production by Sertoli cells. TGF alpha had no direct effect on transferrin production or the ability of hormones to influence Sertoli cells. However, the presence of peritubular cells in coculture with Sertoli cells allowed TGF alpha to stimulate transferrin production. TGF alpha was also found to have relatively rapid effects on peritubular cell migration and the promotion of colony formation in culture. Cocultures of peritubular and Sertoli cells also responded to TGF alpha by the formation of large clusters of cells. Observations demonstrate the local production of TGF alpha by Sertoli and peritubular cells, and action of TGF alpha on peritubular cells and, potentially, Sertoli cells. The local production and action of TGF alpha may have a critical role as a paracrine/autocrine factor involved in the maintenance of testicular function.

Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects

In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses

Thyroid hormones are important for growth and development of many tissues. Altered thyroid hormone status causes testicular abnormalities. For instance, juvenile hypothyroidism/neonatal transient hypothyroidism induces macroorchidism, increases testicular cell number (Sertoli, Leydig, and germ cells) and daily sperm production. Triiodothyronine (T3) receptors have been identified in sperm, developing germ cells, Sertoli, Leydig, and peritubular cells. T3 stimulates Sertoli cell lactate secretion as well as mRNA expression of inhibin-alpha, androgen receptor, IGF-I, and IGFBP-4. It also inhibits Sertoli cell mRNA expression of Müllerian inhibiting substance (MIS), aromatase, estradiol receptor, and androgen binding protein (ABP) and ABP secretion. T3 directly increases Leydig cell LH receptor numbers and mRNA levels of steroidogenic enzymes and steroidogenic acute regulatory protein. It stimulates basal and LH-induced secretion of progesterone, testosterone, and estradiol by Leydig cells. Steroidogenic factor-1 acts as a mediator for T3-induced Leydig cell steroidogenesis. Although the role of T3 on sperm, germ, and peritubular cells has not yet been completely studied, it is clear that T3 directly regulates Sertoli and Leydig cell functions. Further studies are required to elucidate the direct effect of T3 on sperm, germ, and peritubular cells.

In order to clarify the role of germ cells in the regulation of Sertoli cell secretions, three experimental models of germ cell depletion were used: hypodactyl rat mutation (testis weight [TW]: 55% less than controls), in utero busulfan treatment (TW: 88% less than controls), and neonatal experimental cryptorchidism (TW: 72% less than controls). The aim of this work was to compare the numbers of Leydig and Sertoli cells and the production of germ cells in each experimental model to the in vitro secretions of Leydig and Sertoli cells in conditioned media and to the hormonal serum profiles of the same animal in vivo. In the three models, serum levels of hypophyseal and testosterone hormones were significantly increased and decreased, respectively. In the absence of germ cells, the total length of seminiferous tubules, the total numbers of Sertoli and Leydig cells, and the daily production of germ cells were significantly diminished. The addition of both control and damaged seminiferous tubule culture media (STM: media conditioned by 10 cm of seminiferous tubules) to 10(6) control or damaged Leydig cells led to a further increase of testosterone production after ovine LH stimulation. However, expressed per Sertoli cell, testosterone production by control Leydig cells was reduced by addition of damaged STM as compared to addition of control STM, and similarly, the addition of control STM to damaged Leydig cells enhanced testosterone production more than did the addition of damaged STM. Secretions of transferrin per Sertoli cell in STM were reduced as compared to controls by the absence of germ cells but to a lesser extent than was production of spermatocytes and of spermatids. In conclusion, secretions by Sertoli cells of the paracrine factor involved in the control of testosterone production by Leydig cells and of transferrin are modified by germ cells.

Human infertility affects approximately 15% of couples at reproductive age worldwide. Male factors contribute up to 45% of infertility cases. Inflammatory conditions in the male genital system are responsible for about 10% of male infertility in developed countries, and the incidence of immunological infertility can be considerably higher in developing countries where medical cares and environment conditions are poor. In particular, immunological disorders in the testis freguently cause infertility because it is a organ where spermatogenesis carries out. The testis possesses a special immune environment because of its immunoprivileged status and innate immune system. Testicular immune privilege is essential for the protection of male germ cells from detrimental immune responses, whereas the local innate immune system plays a crucial role in the testicular defense against microbial infections. Most of testicular cells are involved in the regulation of testicular immune homeostasis. Disruption of testicular immune homeostasis may result in orchitis, one aetiological factor of male infertility. Multiple mechanisms cooperatively regulate testicular immune homeostasis. Recent studies have revealed important roles of Tyro3/Axl/Mer (TAM) receptor tyrosine kinases and pattern recognition receptors (PRRs) in regulating testicular immune environment. TAM receptors belong to one subfamily of receptor tyrosine kinases. The product of growth arrest-specific gene 6 (Gas6) is common ligand of TAM receptors. TAM and Gas6 are expressed in the testis. TAM triple knockout mice are male sterile and develop autoimmune orchitis. TAM receptors regulate testicular immune homeostasis via three mechanisms: (i) PRR-initiated innate immune responses in Sertoli and Leydig cells are inhibited by TAM/Gas6 signaling, which reduce inflammatory cytokine production in the testis; (ii) TAM receptors promote phagocytosis of apoptotic germ cells by Sertoli cells. The phagocytic clearance of apoptotic germ cells by Sertoli cells can remove germ cell antigens, thereby preventing endogenous immune response; (iii) TAM receptors favor systemic immune tolerance to male germ cells. Experimental autoimmune orchitis (EAO) is a testis-specific autoimmune inflammation in rodent animals after immunization with male germ cell antigens. Axl and Mer double knockout mice are susceptible to EAO induction, suggesting Axl and Mer cooperatively regulate the systemic immune tolerance to male germ cell antigens. Although the testis is an immunoprivileged organ, it can be infected by a broad spectrum of microbial pathogens. However, the testis usually recovers from microbial infections without evident inflammatory response, suggesting that the testis adopts an effective local innate defense system against invading microorganisms. Recognition of microbial pathogens by PRRs initiates innate immune responses, which is the first line of the host defense against invading microbes. Several subfamilies of PRRs are expressed in the testis. In particularly, major testicular cells, including Sertoli, Leydig, and male germ cells, express various PRRs. PRRs can be activated in testicular cells by pathogen molecular patterns, thereby initiating innate immune responses. These observations indicate that testicular cells are equipped with innate immune machinery and may be involved in the testicular defense against microbial infections. This article describes role of TAM receptors and PRRs in regulating immune environment in the testis, and speculates aspects of which need priorizing in future investigation.

The temporal appearance of seasonal changes in numbers of Leydig, Sertoli, and germ cells was evaluated to determine if seasonally increased daily spermatozoan production might be preceded by changes in numbers of either of two somatic testicular cells. A significant increase in numbers of spermatogonia and Sertoli cells preceded the significant increase in number of Leydig cells in the approaching breeding season. Seasonal changes in parenchymal weight and in numbers of Sertoli cells, Leydig cells, and germ cells were maximal in May and June. Numbers of A or B spermatogonia in June were 2.4 to 2.5 times the number present in January. During the same time period, numbers of other germ cells, as well as Leydig cells and Sertoli cells, were increased by 1.5 to 1.9 times. The magnitude of change between January and March (first time period that the change was significant) was greater for A spermatogonia (1.7-fold) than for other cell types (1.3-fold to 1.5-fold). Hence, the need to accommodate more spermatogonial progeny might cause increased testicular size and number of somatic cells, including Sertoli cells. Season did not influence the rate of degeneration between A and B spermatogonia. However, in the breeding season, the conversion of B spermatogonia to primary spermatocytes was reduced. The lack of a seasonal difference in the ratio of primary spermatocytes per Sertoli cell was consistent with a limited capacity of individual Sertoli cells to accommodate primary spermatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

From fetal life to adulthood, the testis evolves through maturational phases showing specific morphologic and functional features in its different compartments. The seminiferous cords contain Sertoli and germ cells, surrounded by peritubular cells, and the interstitial tissue contains Leydig cells and connective tissue. Sertoli cells secrete anti-Müllerian hormone (AMH), whereas Leydig cells secrete androgens. In the fetal and early postnatal testis, Leydig cells actively secrete androgens. Sertoli cells are morphologically and functionally immature--e.g., they secrete high levels of AMH--and germ cells proliferate by mitosis but do not enter meiosis. During infancy and childhood, Leydig cells regress and testosterone secretion declines dramatically. Sertoli cells remain immature and spermatogenesis is arrested at the premeiotic stage. At puberty, Leydig cells differentiate again, and testosterone concentration increases and provokes Sertoli cell maturation--e.g., down-regulation of AMH expression--and germ cells undergo meiosis, the hallmark of adult spermatogenesis driving to sperm production. An intriguing feature of testicular development is that, although testosterone production is as active in the fetal and early postnatal periods as in puberty, Sertoli cells and spermatogenesis remain immature until pubertal onset. Here, we review the ontogeny of the androgen receptor expression in the testis and its impact on Sertoli cell maturation and the onset of pubertal spermatogenesis. We show that the absence of androgen receptor expression in Sertoli cells underlies a physiological stage of androgen insensitivity within the male gonad in the fetal and early postnatal periods.