Nina Atanassova

Permanent Effects of Neonatal Estrogen Exposure in Rats on Reproductive Hormone Levels, Sertoli Cell Number, and the Efficiency of Spermatogenesis in Adulthood*

This study aimed to identify the mechanism(s) for impairment of spermatogenesis in adulthood in rats treated neonatally with estrogens. Rats were treated (days 2-12) with 10, 1, or 0.1 microg diethylstilbestrol (DES), 10 microg ethinyl estradiol (EE), 10 mg/kg of a GnRH antagonist (GnRHa), or vehicle and killed in adulthood. DES/EE caused dose-dependent reductions in testis weight, total germ cell volume per testis, and Sertoli cell volume per testis. Sertoli cell number at 18 days of age in DES-treated rats was reduced dose dependently. GnRHa treatment caused changes in these parameters similar to those in rats treated with 10 microg DES. Plasma FSH levels were elevated (P < 0.001) to similar levels in all treatment groups regardless of differences in Sertoli cell number and levels of inhibin B; the latter reflected Sertoli cell number, but levels were disproportionately reduced in animals treated with high doses of DES/EE. Neonatal estrogen treatment, but not GnRHa, caused dose-dependent reductions (40-80%) in plasma testosterone levels in adulthood, but did not alter LH levels. Preliminary evidence suggests that the decrease in testosterone levels in estrogen-treated rats is not due to reduced Leydig cell volume per testis. GnRHa-treated rats exhibited a significant increase in germ cell volume per Sertoli cell and a reduction in germ cell apoptosis, probably because of the raised FSH levels. Despite similar raised FSH levels, rats treated with DES (10 or 1 microg) or EE (10 microg) had reduced germ cell volume/Sertoli cell and increased germ cell apoptosis, especially when compared with GnRHa-treated animals. The latter changes were associated with an increase in lumen size per testis, indicative of impaired fluid resorption from the efferent ducts, resulting in fluid accumulation in the testis. Rats treated neonatally with 0.1 microg DES showed reduced germ cell apoptosis comparable to that in GnRHa-treated animals. The changes in apoptotic rate among treatment groups occurred across all stages of the spermatogenic cycle. It is concluded that 1) neonatal estrogen treatment results in dose-dependent alterations in Sertoli cell numbers, germ cell volume, efficiency of spermatogenesis, and germ cell apoptosis in adulthood; 2) the relatively poor spermatogenesis in estrogen-treated animals is most likely due to altered testis fluid dynamics and/or altered Sertoli cell function; 3) as indicated by FSH (LH) and testosterone levels, the hypothalamic-pituitary axis and Leydig cells are probably more sensitive than the Sertoli cells to reprogramming by estrogens neonatally; and 4) elevated FSH levels in adulthood may improve the efficiency of spermatogenesis.

Androgen action via testicular peritubular myoid cells is essential for male fertility

Androgens are essential for normal spermatogenesis and male fertility, but how androgens exert this effect remains uncertain. Androgen receptors (ARs) are expressed in several testicular cell types, but continuing uncertainty exists over which cell type mediates androgen control of spermatogenesis. Androgen signaling via Sertoli cells (SCs) is essential for complete spermatogen‐esis, but the role for androgen signaling via peritubular myoid (PTM) cells is contentious. To address this controversy, we generated PTM‐specific AR‐knockout (PTM‐ARKO) mice in which gross reproductive development was normal, but all PTM‐ARKO males were azoospermic and infertile. Testis weight was reduced beyond puberty, and in adulthood there was an 86% reduction in germ cells, compared with wild‐type littermates. These changes were not explained by any deficits in testosterone, lutein‐izing hormone, or follicle‐stimulating hormone concentrations. SC function was impaired in PTM‐ARKO males, indicated by reduced seminiferous tubule fluid production and reduced expression of some androgen‐depen‐dent SC genes. Androgen action via PTM cells is therefore essential for normal testis function, spermatogenesis, and fertility in males. This study also provides the first direct evidence for the importance of androgen‐driven stromal‐epithelial interactions underpinning the regulation of spermatogenesis; PTM‐ARKO mice will enable identification of the new molecular pathways involved.— Welsh, M., Saunders, P. T. K., Atanassova, N., Sharpe, R. M., Smith, L. B. Androgen action via testicular peritu‐bular myoid cells is essential for male fertility. FASEB J. 23, 4218‐4230 (2009). www.fasebj.org

Effect of Neonatal Gonadotropin-Releasing Hormone Antagonist Administration on Sertoli Cell Number and Testicular Development in the Marmoset: Comparison with the Rat

Abstract The primary purpose of this study was to establish whether Sertoli cells proliferate in the neonatal period in the marmoset monkey (Callithrix jacchus) and whether administration of a long-acting GnRH antagonist (GnRHa) during this phase induced any transient or permanent effects on Sertoli cell number or on any other aspect of testicular development. Male marmoset co-twins (n = 9) were treated during Weeks 1–14 with either vehicle or GnRHa. Four sets of co-twins were examined at Weeks 18–22 (start of infancy) and 5 sets in adulthood (92+ wk), and Sertoli cell number was determined using either the nucleator or optical disector methods; other testicular morphometric analyses (e.g., germ cell volume, Leydig cell volume) used standard point-counting. Data for the marmoset were compared with that obtained in similarly treated rats. Sertoli cell number in marmosets treated neonatally with GnRHa was reduced by 35% compared with that of controls at Weeks 18–22 but was comparable to control values in adulthood. However, seminiferous epithelium volume was reduced significantly in adult marmosets treated neonatally with GnRHa, and there was a tendency for reduced germ cell volume per Sertoli cell. In the same animals, there was significant expansion of the interstitium and an increase in Leydig cell volume per testis when compared with co-twin controls; a similar increase in Leydig cell volume was evident in adult rats treated neonatally with GnRHa. Comparison of Sertoli cell numbers in 6 infantile (18–24 wk) and 10 adult marmosets showed that adult numbers of Sertoli cells were present by the start of infancy but, unlike rats, marmosets were still able to replicate Sertoli cells beyond this period. However, marmoset Sertoli cells supported only ∼20% of the germ cell volume supported by rat Sertoli cells, indicative of poor efficiency of spermatogenesis, as shown previously in the human. This finding, together with the demonstration of a temporal pattern of Sertoli cell replication similar to that in the human, supports the use of marmosets as a model for human male testicular development and function.

Loss of androgen receptor binding to selective androgen response elements causes a reproductive phenotype in a knockin mouse model

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.

Fetal programming of adult Leydig cell function by androgenic effects on stem/progenitor cells

Significance Men are defined by androgens (testosterone), which drive fetal masculinization (male development) and puberty and maintain masculinity in adulthood, including sex drive, erectile function, and fertility. Moreover, Western cardiometabolic diseases are all associated with lowered testosterone levels in men. Therefore, influences on testosterone levels in adulthood have pervasive importance for masculinity and health. Our study shows, for the first time, to our knowledge, that testosterone levels during fetal masculinization can (re)program adult testosterone levels through effects on stem cells, which develop into adult Leydig cells (the source of testosterone) after puberty. These stem cells are present in fetal testes of humans and animals, and using the latter, we show how these cells are reprogrammed to affect adult testosterone levels. 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.

KATNAL1 regulation of sertoli cell microtubule dynamics is essential for spermiogenesis and male fertility

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.

Androgen action via testicular arteriole smooth muscle cells is important for Leydig cell function, vasomotion and testicular fluid dynamics.

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.

Androgen Regulation of Stage-Dependent Cyclin D2 Expression in Sertoli Cells Suggests a Role in Modulating Androgen Action on Spermatogenesis

Abstract Regulation of spermatogenesis involves stage-dependent androgen action on Sertoli cells, but the pathways involved are unclear. We assessed if cyclin D2 could play a role. In rats, Sertoli cell nuclear, stage-dependent immunoexpression of cyclin D2 switched on after Day 10 and persisted through Day 35, but disappeared by adulthood. However, ethane dimethane sulfonate (EDS)-induced testosterone withdrawal in adult rats for 6 days induced stage-dependent cyclin D2 immunoexpression in Sertoli cells, with highest expression at stages IX-XII and nondetectable at stages VI–VIII (opposite that for androgen receptor [AR] immunoexpression). In EDS-treated rats, a single injection of testosterone but not of estrogen reversed this change in 4 h, and testosterone administration from the time of EDS treatment prevented expression of cyclin D2 in Sertoli cells. The EDS-induced changes in cyclin D2 immunoexpression were matched by changes in expression of Ccnd2 (cyclin D2) mRNA in isolated stage-dissected tubules. Treatment of adult rats with flutamide induced stage-dependent cyclin D2 immunoexpression in Sertoli cells within 18 h, and confocal microscopy revealed that immunoexpression of AR and cyclin D2 were mutually exclusive within individual seminiferous tubules in these animals. Sertoli cell-selective ablation of the AR in mice using Cre/loxP technology also resulted in stage-dependent Sertoli cell cyclin D2 immunoexpression. Downstream from cyclin D2 action is retinoblastoma 1 (RB1), a tumor suppressor protein, immunoexpression of which paralleled stage-dependent AR expression in Sertoli cells; RB1 stage specificity disappeared after EDS treatment. These results point to a non-cell cycle role for cyclin D2 and RB1 in mature Sertoli cells in the stage-dependent mechanisms regulated by AR expression and androgen action.

Catecholamine-synthesizing enzymes in the adult and prenatal human 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 β-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.

Bradykinin stimulates prepubertal rat germ cell proliferation in vitro.

The effect of bradykinin on germ cell proliferation was studied by using in vitro organ cultures of testicular fragments from 3.5- and 4.5-day-old rats in the presence of 3H-thymidine. Bradykinin in tested concentrations of 10(-8), 10(-6) and 10(-4) M manifested a significant stimulation of rat prespermatogonial cell proliferation. The percentage of labeled germ cells increased up to 3-fold in comparison with the control value especially in 3.5-day-old rat testicular explants. Two kininase inhibitors: phosphoramidon and captopril were also used in combination with bradykinin. The percentage of labeled germ cells was slightly increased as compared to the samples treated with bradykinin only. Experiments with two agonists and two antagonists of B1 and B2 receptors for kinins showed that the rat prespermatogonial cell proliferation response to bradykinin is probably mediated via B2 receptors. The present findings suggest that bradykinin may be an important local testicular factor with paracrine or autocrine role in the regulation of spermatogonial cell proliferation and germ cell number.