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Fetal growth plays a role in programming of adult cardiometabolic disorders, which in men, are associated with lowered testosterone levels. Fetal growth and fetal androgen exposure can also predetermine testosterone levels in men, although how is unknown, because the adult Leydig cells (ALCs) that produce testosterone do not differentiate until puberty. To explain this conundrum, we hypothesized that stem cells for ALCs must be present in the fetal testis and might be susceptible to programming by fetal androgen exposure during masculinization. To address this hypothesis, we used ALC ablation/regeneration to identify that, in rats, ALCs derive from stem/progenitor cells that express chicken ovalbumin upstream promoter transcription factor II. These stem cells are abundant in the fetal testis of humans and rodents, and lineage tracing in mice shows that they develop into ALCs. The stem cells also express androgen receptors (ARs). Reduction in fetal androgen action through AR KO in mice or dibutyl phthalate (DBP) -induced reduction in intratesticular testosterone in rats reduced ALC stem cell number by ∼40% at birth to adulthood and induced compensated ALC failure (low/normal testosterone and elevated luteinizing hormone). In DBP-exposed males, this failure was probably explained by reduced testicular steroidogenic acute regulatory protein expression, which is associated with increased histone methylation (H3K27me3) in the proximal promoter. Accordingly, ALCs and ALC stem cells immunoexpressed increased H3K27me3, a change that was also evident in ALC stem cells in fetal testes. These studies highlight how a key component of male reproductive development can fundamentally reprogram adult hormone production (through an epigenetic change), which might affect lifetime disease risk.

Diabetes mellitus (DM) causes male infertility through the suppression of spermatogenesis and testosterone biosynthesis. The impact of DM on male reproduction has mainly been investigated in adulthood, therefore we aimed to study the developmental effects of DM, induced in early life, on testicular cell population and fertility. Neonatal (NDM) and prepubertal DM (PDM) were induced in immature rats by streptozotocin administration on day 1 or day 10, respectively. Germ (GCs) and somatic cells (Sertoli—SCs and Leydig cells—LCs) were counted in pubertal (25 day) and post-pubertal (45 day) rats in tandem with the measurement of serum testosterone levels and the protein expression of androgen receptor. Glucose levels were higher in PDM than in NDM. Incomplete spermatogenesis and reduced GC number were found in PDM but not in NDM. LC number, testosterone, and luteinizing hormone (LH) levels were differently altered by both types of DM with a pronounced negative impact of PDM. Protein expression of androgen receptor in SCs was altered only in PDM. Reduced sperm concentration and motility was found in both groups. Thus, our results provide new insights into different mechanisms of action of PDM and NDM on developing germ cells that involved disturbances in androgen production by Leydig cells and androgen action in Sertoli cells.

Introduction : Spermatozoa are rapidly changing cellular structures that are highly dependent on their interaction with the environment. These interactions cause fundamental changes in the spermatozoa’s cells and membrane. All physiological changes that a spermatozoon goes through are required for fertilization. One of the proteins that are essential for the physiological processes in the spermatozoon membrane is the testicular angiotensin-converting enzyme (tACE). In human ejaculated spermatozoa, tACE is found on sperm plasma membrane in the head, neck, and midpiece of the tail having an active role in the capacitation and acrosome reaction. Aim : Immuno-histochemical and fluorescent testing of the testicular isoform of the angiotensin-converting enzyme during spermiogenesis and acrosome membrane of spermatozoa. Materials and methods : Testis biopsies from infertile males have used immunohistochemical testing and fixed spermatozoa for the immunofluorescence assay of tACE. Results : The immunohistochemical test showed tACE expression during spermiogenesis and its participation in the stages of spermatid differentiation in the testis. The immunofluorescent test follows the manifestation of tACE in untreated, capacitated, and acrosome-reacting spermatozoa. In the process of capacitation and acrosome reaction, we found considerable dynamics accompanied by a change in the expression of tACE on the sperm membrane. Conclusions : tACE expression during spermiogenesis and its visualization in the acrosome region confirms the active role of the enzyme in the processes of maturation, capacitation, and acrosome reaction, which determines the enzyme as a reliable marker for the selection of quality spermatozoa in assisted reproduction.

Introduction: The testis is an immune privileged organ that provides a specific environment for germ cell development. Various factors responsible for inflammatory changes can lead to deterioration of the immune tolerant model found in the testis. As a result, the thickness of the proper membrane of seminiferous tubules changes and the process of spermatogenesis is disturbed. Aim: The purpose of the present study was to find the connection between the changes in the level of testis-specific isoform of angiotensin-converting enzyme (tACE) expression and the morphological changes of the seminiferous tubule wall of the testis in patients with infertility. Materials and methods: The study included 24 infertile men who underwent a testicular biopsy. Routine histological techniques, immunohistochemical reactions for tACE, α-smooth muscle actin, and morphometric analysis were performed to examine the biopsy preparations. Results: By using testicular biopsy to diagnose patients with infertility, a stage-specific pattern of the processes associated with thickened proper membrane of seminiferous tubules was established and a decreased or absent spermatogenic activity was observed. Conclusions: A significant increase in the proper membrane thickness of the seminiferous tubules in the testis was found in patients with infertility. This finding shows that the processes take place gradually over time, correlating with the degree of pathology, and that changes do not depend on the factors causing them. We also found that the degree of proper membrane thickening correlated with disturbances of spermatogenesis, using tACE expression as a marker for spermatogenic epithelium differentiation.

Testicular angiotensin converting enzyme (ACE) is known to play an essential role in the male reproduction and fertility. Data about tACE in cases of male infertility are quite scarce, and in this respect we aimed to study localization and distribution of tACE protein in the neck and mid-piece of spermatozoa from pathological samples in relation to sperm motility. The enzyme expression during capacitation and acrosome reaction was quantitatively assessed. In human ejaculated spermatozoa tACE is localized on sperm plasma membrane of the head, the neck and mid-piece of the tail. The immunoreactivity becomes stronger in capacitated spermatozoa followed by a decrease in acrosome reacted sperm. In different cases of semen pathology (oligozoospermia, asthenozoospermia and teratozoospermia) fluorescent signals in the neck and mid-piece are in punctate manner whereas in normozoospermia they were uniformly distributed. The expression area of tACE the neck and mid-piece was decreased in ejaculated and capacitated sperm from pathological semen samples compared to normospermia. Significant positive correlation was established between tACE area and progressive sperm motility, whereas with immotile sperm the correlation was negative. Our data suggest that proper distribution of tACE in the neck and mid-piece is required for normal sperm motility that could be used as a novel biomarker for male infertility.

The tamoxifen-inducible Cre system is a popular transgenic method for controlling the induction of recombination by Cre at a specific time and in a specific cell type. However, tamoxifen is not an inert inducer of recombination, but an established endocrine disruptor with mixed agonist/antagonist activity acting via endogenous estrogen receptors. Such potentially confounding effects should be controlled for, but >40% of publications that have used tamoxifen to generate conditional knockouts have not reported even the minimum appropriate controls. To highlight the importance of this issue, the present study investigated the long-term impacts of different doses of a single systemic tamoxifen injection on the testis and the wider endocrine system. We found that a single dose of tamoxifen less than 10% of the mean dose used for recombination induction, caused adverse effects to the testis and to the reproductive endocrine system that persisted long-term. These data raise significant concerns about the widespread use of tamoxifen induction of recombination, and highlight the importance of including appropriate controls in all pathophysiological studies using this means of induction.

Spermatogenesis is a complex process reliant upon interactions between germ cells (GC) and supporting somatic cells. Testicular Sertoli cells (SC) support GCs during maturation through physical attachment, the provision of nutrients, and protection from immunological attack. This role is facilitated by an active cytoskeleton of parallel microtubule arrays that permit transport of nutrients to GCs, as well as translocation of spermatids through the seminiferous epithelium during maturation. It is well established that chemical perturbation of SC microtubule remodelling leads to premature GC exfoliation demonstrating that microtubule remodelling is an essential component of male fertility, yet the genes responsible for this process remain unknown. Using a random ENU mutagenesis approach, we have identified a novel mouse line displaying male-specific infertility, due to a point mutation in the highly conserved ATPase domain of the novel KATANIN p60-related microtubule severing protein Katanin p60 subunit A-like1 (KATNAL1). We demonstrate that Katnal1 is expressed in testicular Sertoli cells (SC) from 15.5 days post-coitum (dpc) and that, consistent with chemical disruption models, loss of function of KATNAL1 leads to male-specific infertility through disruption of SC microtubule dynamics and premature exfoliation of spermatids from the seminiferous epithelium. The identification of KATNAL1 as an essential regulator of male fertility provides a significant novel entry point into advancing our understanding of how SC microtubule dynamics promotes male fertility. Such information will have resonance both for future treatment of male fertility and the development of non-hormonal male contraceptives.

Androgens influence transcription of their target genes through the activation of the androgen receptor (AR) that subsequently interacts with specific DNA motifs in these genes. These DNA motifs, called androgen response elements (AREs), can be classified in two classes: the classical AREs, which are also recognized by the other steroid hormone receptors; and the AR-selective AREs, which display selectivity for the AR. For in vitro interaction with the selective AREs, the androgen receptor DNA-binding domain is dependent on specific residues in its second zinc-finger. To evaluate the physiological relevance of these selective elements, we generated a germ-line knockin mouse model, termed SPARKI (SPecificity-affecting AR KnockIn), in which the second zinc-finger of the AR was replaced with that of the glucocorticoid receptor, resulting in a chimeric protein that retains its ability to bind classical AREs but is unable to bind selective AREs. The reproductive organs of SPARKI males are smaller compared with wild-type animals, and they are also subfertile. Intriguingly, however, they do not display any anabolic phenotype. The expression of two testis-specific, androgen-responsive genes is differentially affected by the SPARKI mutation, which is correlated with the involvement of different types of response elements in their androgen responsiveness. In this report, we present the first in vivo evidence of the existence of two functionally different types of AREs and demonstrate that AR-regulated gene expression can be targeted based on this distinction.

Regulation of blood flow through the testicular microvasculature by vasomotion is thought to be important for normal testis function as it regulates interstitial fluid (IF) dynamics which is an important intra-testicular transport medium. Androgens control vasomotion, but how they exert these effects remains unclear. One possibility is by signalling via androgen receptors (AR) expressed in testicular arteriole smooth muscle cells. To investigate this and determine the overall importance of this mechanism in testis function, we generated a blood vessel smooth muscle cell-specific AR knockout mouse (SMARKO). Gross reproductive development was normal in SMARKO mice but testis weight was reduced in adulthood compared to control littermates; this reduction was not due to any changes in germ cell volume or to deficits in testosterone, LH or FSH concentrations and did not cause infertility. However, seminiferous tubule lumen volume was reduced in adult SMARKO males while interstitial volume was increased, perhaps indicating altered fluid dynamics; this was associated with compensated Leydig cell failure. Vasomotion was impaired in adult SMARKO males, though overall testis blood flow was normal and there was an increase in the overall blood vessel volume per testis in adult SMARKOs. In conclusion, these results indicate that ablating arteriole smooth muscle AR does not grossly alter spermatogenesis or affect male fertility but does subtly impair Leydig cell function and testicular fluid exchange, possibly by locally regulating microvascular blood flow within the testis.

Catecholamines play functional roles in the mature and developing mammalian testis but the cell types responsible for their local synthesis are still controversially discussed. Here, we demonstrate that four enzymes involved in the biosynthesis of catecholamines, namely, tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DBH) and phenylethanolamine- N-methyltransferase (PNMT), are expressed in Leydig cells of the human testis. Tyrosine hydroxylase, the key enzyme of the biosynthesis of catecholamines, was localized to Leydig cells both at the transcript level (by RT-PCR analyses and by in situ hybridization assays) and at the protein level (by immunoblotting and by immunohistochemistry). The other enzymes were also demonstrated in Leydig cells by RT-PCR and immunohistochemical analyses. The presence of TH, AADC, DBH, and PNMT in human Leydig cells was found, in addition, by immunohistochemical approaches carried out on sections from prenatal human testes. Thus, the present study identifies the Leydig cells as the presumed sites of catecholamine production in both the mature and fetal human testes and further supports the previously recognized neuroendocrine characteristics of this cell type.