Obesogens and male fertility.

In the last decades, several studies evidenced a decrease in male fertility in developed countries. Although the aetiology of this trend in male reproductive health remains a matter of debate, environmental compounds that predispose to weight gain, namely obesogens, are appointed as contributors because of their action as endocrine disruptors. Obesogens favour adipogenesis by an imbalance of metabolic processes and can be found virtually everywhere. These compounds easily accumulate in tissues with high lipid content. Obesogens change the functioning of male reproductive axis, and, consequently, the testicular physiology and metabolism that are pivotal for spermatogenesis. The disruption of these tightly regulated metabolic pathways leads to adverse reproductive outcomes. Notably, adverse effects of obesogens may also promote disturbances in the metabolic performance of the following generations, through epigenetic modifications passed by male gametes. Thus, unveiling the molecular pathways by which obesogens induce toxicity that may end up in epigenetic modifications is imperative. Otherwise, a transgenerational susceptibility to metabolic diseases may be favoured. We present an up-to-date overview of the impact of obesogens on testicular physiology, with a particular focus on testicular metabolism. We also address the effects of obesogens on male reproductive parameters and the subsequent consequences for male fertility.

Testicular Metabolic Reprogramming in Neonatal Streptozotocin-Induced Type 2 Diabetic Rats Impairs Glycolytic Flux and Promotes Glycogen Synthesis.

Defects in testicular metabolism are directly implicated with male infertility, but most of the mechanisms associated with type 2 diabetes- (T2DM) induced male infertility remain unknown. We aimed to evaluate the effects of T2DM on testicular glucose metabolism by using a neonatal-streptozotocin- (n-STZ) T2DM animal model. Plasma and testicular hormonal levels were evaluated using specific kits. mRNA and protein expression levels were assessed by real-time PCR and Western Blot, respectively. Testicular metabolic profile was assessed by (1)H-NMR spectroscopy. T2DM rats showed increased glycemic levels, impaired glucose tolerance and hyperinsulinemia. Both testicular and serum testosterone levels were decreased, whereas those of 17ß-estradiol were not altered. Testicular glycolytic flux was not favored in testicles of T2DM rats, since, despite the increased expression of both glucose transporters 1 and 3 and the enzyme phosphofructokinase 1, lactate dehydrogenase activity was severely decreased contributing to lower testicular lactate content. However, T2DM enhanced testicular glycogen accumulation, by modulating the availability of the precursors for its synthesis. T2DM also affected the reproductive sperm parameters. Taken together these results indicate that T2DM is able to reprogram testicular metabolism by enhancing alternative metabolic pathways, particularly glycogen synthesis, and such alterations are associated with impaired sperm parameters.

High-energy diets: a threat for male fertility?

Male fertility is declining in developed countries, as well as in developing countries. External factors linked to lifestyle, such as eating disorders, negatively affect spermatogenesis, both at central and gonadal levels. The overconsumption of high-energy diets (HED) alters the functioning of the male reproductive axis and consequently affects the testicular physiology, disrupting its metabolism and bioenergetic capacity. Testicular metabolism presents unique characteristics, partly because of its cellular heterogeneity and to the specific functions that each cell type plays within the testicular environment. Disruption of the tightly regulated metabolic pathways leads to adverse reproductive outcomes, such as inefficient energy supply to germ cells, sperm defects or spermatogenesis arrest. Testicular metabolic alterations induced by HED intake may also lead to mitochondrial dysfunction, which is closely associated to reactive oxygen species (ROS) overproduction and oxidative stress. ROS easily target spermatozoa DNA and lipids, contributing to decreased sperm quality. Thus, understanding the detrimental effects of HED overconsumption on the pathways underlying testicular metabolism and sperm production is imperative; otherwise, one may favour a transgenerational amplification of subfertility. Herein, we present an up-to-date overview of the effects of HED on testicular metabolism, sperm parameters and the subsequent consequences for male fertility.

Transgenic overexpression of regucalcin leads to suppression of thapsigargin- and actinomycin D-induced apoptosis in the testis by modulation of apoptotic pathways.

Recent evidence suggested the involvement of calcium-binding protein regucalcin (RGN) in testicular apoptosis. Herein, we investigated the role of RGN controlling apoptotic pathways in the testis by using a transgenic rat model overexpressing RGN (Tg-RGN). Seminiferous tubules (SeT) from Tg-RGN and their wild-type (Wt) counterparts were cultured ex vivo in presence or absence of apoptosis inducers thapsigargin (Thap, 10(-7) and 10(-6) m) and actinomycin D (Act D, 0.5 and 1 µg/mL). Expression levels of key regulators of apoptosis in SeT of Tg-RGN and Wt animals were determined by quantitative real-time PCR and Western blot analysis. Measurement of caspase-3 enzymatic activity was included as an end point of apoptosis. Tg-RGN SeT treated with 10(-6) m of Thap or 1 µg/mL of Act D showed a diminished enzymatic activity and gene transcription of caspase-3, along with increased mRNA and protein expression of antiapoptotic Bcl-2. Bcl-2/Bax (antiapoptotic/proapoptotic) protein ratio was also enhanced in these SeT. Although caspase-9 mRNA was increased in the SeT of Tg-RGN treated with Thap, no differences were observed at protein level, and no differences were also found on protein levels of apoptosis-inducing factor. mRNA expression of proapoptotic p53 and p21 was strongly decreased in Tg-RGN SeT treated with Thap (10(-6) m) or Act D (1 µg/mL). These findings demonstrated that RGN suppresses Thap- and Act D-induced apoptosis in SeT by modulating the expression and activity of key apoptotic and antiapoptotic factors. Moreover, results indicate that RGN overexpression protects germ cell from apoptosis induced by noxious stimuli, which could be a relevant mechanism for fertility preservation in situations of oncological treatments.

Molecular basis of bicarbonate membrane transport in the male reproductive tract.

Bicarbonate (HCO3⁻) membrane transport systems are crucial players in the physiology of several tissues. The molecular basis of HCO3⁻ membrane transport is of major physiological relevance since this ion is involved in the establishment of intracellular and extracellular ionic composition, osmolariy and pH. The membrane HCO3⁻ transporters are divided in two main families: solute carrier 4 (SLC4) and solute carrier 26 (SLC26), although HCO3⁻ concentration can also be regulated by the cystic fibrosis transmembrane regulator (CFTR). In most tissues the SLC4 family represents the majority of HCO3⁻ transporters members, which can be divided in two subgroups: the Na⁺-dependent and the Na⁺-independent transporters. The SLC26 family consists of ten members that can transport diverse ions besides HCO3⁻. In the male reproductive tract, HCO3⁻ transport occurs in several processes in order to assure a correct pursuance of the spermatogenetic event and spermatozoa capacitation, being also necessary for egg fertilization. Indeed, the formation of competent spermatozoa, the maintenance of an adequate ductal luminal milieu and spermatozoa capacitation are highly dependent of ionic balance and pH. Perturbations in these processes result in reduced male reproductive health and consequently male subfertility and/or infertility. Thus, it is imperative to understand HCO3⁻ transport dynamics in order to identify and counteract possible alterations related with reduced male fertility caused by pathological conditions. Herein, we will review the major families and subfamilies of HCO3⁻ membrane transport, discussing the molecular basis of HCO3⁻ transport in the male reproductive tract and its role in male-associated subfertility and/or infertility.

Sperm parameters and epididymis function in transgenic rats overexpressing the Ca2+-binding protein regucalcin: a hidden role for Ca2+ in sperm maturation?

Sperm undergo maturation acquiring progressive motility and the ability to fertilize oocytes through exposure to the components of the epididymal fluid (EF). Although the establishment of a calcium (Ca(2+)) gradient along the epididymis has been described, its direct effects on epididymal function remain poorly explored. Regucalcin (RGN) is a Ca(2+)-binding protein, regulating the activity of Ca(2+)-channels and Ca(2+)-ATPase, for which a role in male reproductive function has been suggested. This study aimed at comparing the morphology, assessed by histological analysis, and function of epididymis, by analysis of sperm parameters, antioxidant potential and Ca(2+) fluxes, between transgenic rats overexpressing RGN (Tg-RGN) and their wild-type littermates. Tg-RGN animals displayed an altered morphology of epididymis and lower sperm counts and motility. Tissue incubation with (45)Ca(2+) showed also that epididymis of Tg-RGN displayed a diminished rate of Ca(2+)-influx, indicating unbalanced Ca(2+) concentrations in the epididymal lumen. Sperm viability and the frequency of normal sperm, determined by the one-step eosin-nigrosin staining technique and the Diff-Quik staining method, respectively, were higher in Tg-RGN. Moreover, sperm of Tg-RGN rats showed a diminished incidence of tail defects. Western blot analysis demonstrated the presence of RGN in EF as well as its higher expression in the corpus region. The results presented herein demonstrated the importance of maintaining Ca(2+)-levels in the epididymal lumen and suggest a role for RGN in sperm maturation. Overall, a new insight into the molecular mechanisms driving epididymal sperm maturation was obtained, which could be relevant to development of better approaches in male infertility treatment and contraception.

High-energy diets may induce a pre-diabetic state altering testicular glycolytic metabolic profile and male reproductive parameters.

Diabetes mellitus is a metabolic disorder that may arise from diet habits and is growing to epidemic proportions. Young male diabetic patients present high infertility/subfertility prevalence resulting from impaired reproductive function and poor semen quality. We aimed to evaluate the effects of a high-energy diet (HED) on glucose tolerance/insulin levels and correlate the observed effects on male reproductive function with overall testicular metabolism. After 1 month, HED fed rats showed increased glycaemic levels, impaired glucose tolerance and hypoinsulinaemia. Moreover, an imbalance of intratesticular and serum testosterone levels was observed, whereas those of 17ß-estradiol were not altered. High-energy diet also affected the reproductive parameters, with HED rats exhibiting a significant increase in abnormal sperm morphology. Glycolytic metabolism was favoured in testicles of HED rats with an increased expression of both glucose transporters 1 (GLUT1) and 3 (GLUT3) and the enzyme phosphofrutokinase 1. Moreover, lactate production and the expression of metabolism-associated genes and proteins involved in lactate production and transport were also enhanced by HED. Alanine testicular content was decreased and thus intratesticular lactate/alanine ratio in HED rats was increased, suggesting increased oxidative stress. Other energetic substrates such as acetate and creatine were not altered in testis from HED rats, but intratesticular glycine content was increased in those animals. Taken together, these results suggest that HED induces a pre-diabetic state that may impair reproductive function by modulating overall testicular metabolism. This is the first report on testicular metabolic features and mechanisms related with the onset of a pre-diabetic state.

Identification of androgen receptor variants in testis from humans and other vertebrates.

The androgen receptor (AR) is a ligand-activated transcription factor member of the nuclear receptor superfamily. The existence of alternatively spliced variants is well recognised for several members of this superfamily, most of them having functional importance. For example, several testicular oestrogen receptor variants have been suggested to play a role in the regulation of spermatogenesis. However, information on AR variants is mostly related to cancer and androgen insensitivity syndrome (AIS) cases. The objective of this study was to investigate the expression of AR variants in the testis from humans and other vertebrates. Four AR variants [ARΔ2(Stop) , ARΔ2(23Stop) , ARΔ3 and ARΔ4(120)] were identified in human testis. ARΔ2(Stop) and ARΔ3, with exon 2 or 3 deleted, respectively, were also expressed in human liver, lung, kidney and heart. In addition, ARΔ2(Stop) was expressed in rat and gilthead seabream testis, while an ARΔ3 was detected in African clawed frog testis. This is the first report revealing the existence of AR variants in the testis of evolutionarily distant vertebrate species and in nonpathological tissues. These data suggest the functional importance of these novel AR forms and demonstrate a complexity in AR signalling that is not exclusive of pathological conditions.

Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human Sertoli cells.

BACKGROUND: Sertoli cells metabolize glucose producing lactate for developing germ cells. As insulin regulates glucose uptake and its disturbance/insensitivity is associated with diabetes mellitus, we aimed to determine the effect of insulin deprivation in human Sertoli cell (hSC) metabolism and metabolism-associated gene expression. METHODS: hSC-enriched primary cultures were maintained in the absence/presence of insulin and metabolite variations were determined by (1)H-NMR. mRNA expression levels of glucose transporters (GLUT1, GLUT3), lactate dehydrogenase (LDHA) and monocarboxylate transporter (MCT4) were determined by RT-PCR. RESULTS: Insulin deprivation resulted in decreased lactate production and in decrease of glucose consumption that was completely reverted after 6h. Cells of both groups consumed similar amounts of glucose. In insulin-deprived cells, transcript levels of genes associated to lactate metabolism (LDHA and MCT4) were decreased. Transcript levels of genes involved in glucose uptake exhibited a divergent variation: GLUT3 levels were decreased while GLUT1 levels increased. Insulin-deprived hSCs presented: 1) altered glucose consumption and lactate secretion; 2) altered expression of metabolism-associated genes involved in lactate production and export; 3) an adaptation of glucose uptake by modulating the expression of GLUT1 and GLUT3. GENERAL SIGNIFICANCE: This is the first report regarding the effect of insulin-deprivation on hSC metabolism.

Influence of 5α-dihydrotestosterone and 17ß-estradiol on human Sertoli cells metabolism.

Sertoli cells metabolize glucose, converting it to lactate that is used by developing germ cells for their energy metabolism. Androgens and oestrogens have metabolic roles that reach far beyond reproductive processes. So, the main purpose of this study was to examine the effect of sex steroid hormones on metabolite secretion/consumption in human Sertoli cells. Human Sertoli cell-enriched primary cultures were maintained in a defined medium for 50 h and glucose, pyruvate, lactate and alanine variations were determined using (1) H-NMR spectra analysis, in the absence or presence of 100 nm 17ß-estradiol (E(2) ) or 100 nm 5α-dihydrotestosterone (DHT). The mRNA expression levels of glucose transporters, lactate dehydrogenase and monocarboxylate transporters were also determined using semi-quantitative RT-PCR. Cells cultured in the absence (control) or presence of E(2) consumed the same amounts of glucose at similar rates during the 50 h. During the first 15 h of treatment with DHT, glucose consumption and glucose consumption rate were significantly higher. Nevertheless, DHT-treated cells secreted a significantly lower amount of lactate than control and E(2) -treated cells. Such a decrease was concomitant with a significant decrease in lactate dehydrogenase A mRNA levels after 50 h treatment in DHT-treated groups. Finally, alanine production was significantly increased in E(2) -treated cells after 25 h treatment, which indicated a lower redox/higher oxidative state for the cells on those conditions. These results support the existence of a relationship between sex steroid hormones action and energy metabolism, providing the first assessment of androgens and oestrogens as metabolic modulators of human Sertoli cells.

Regucalcin is broadly expressed in male reproductive tissues and is a new androgen-target gene in mammalian testis.

Regucalcin (RGN) is a calcium (Ca(2)(+))-binding protein which regulates intracellular Ca(2)(+) homeostasis by modulating the activity of enzymes regulating Ca(2)(+) concentration and enhancing Ca(2)(+)-pumping activity. Several studies have described the pivotal role of proper Ca(2)(+) homeostasis regulation to spermatogenesis and male fertility. Recently, RGN was identified as a sex steroid-regulated gene in prostate and breast; however, a possible role of RGN in spermatogenesis has not been examined. In this study, the expression and localization of RGN in rat and human testis, and other rat reproductive tissues was analyzed. Moreover, we studied whether RGN protein was present in seminiferous tubule fluid (STF). Finally, we examined the effect of 5α-dihydrotestosterone (DHT) on the expression of Rgn mRNA in rat seminiferous tubules (SeT) cultured ex vivo. The results presented in this study show that RGN is expressed in Leydig and Sertoli cells, as well as in all types of germ cells of both rat and human testis. RGN is also expressed in rat prostate, epididymis, and seminal vesicles. Moreover, RGN protein is present in rat STF. The results also demonstrate that Rgn expression is age dependent in rat testis, and is upregulated by the non-aromatizable androgen DHT in rat SeT cultured ex vivo. Taken together, these findings indicate that Rgn is a novel androgen-target gene in rat testis and that it may have a role in male reproductive function, particularly in the control of spermatogenesis.

Sex steroids and spermatogenesis in the African catfish (Clarias gariepinus).

Little is known about the physiological regulations of spermatogenesis of the African catfish (Clarias gariepinus), a species used as an experimental model for two decades. After a brief introduction to the cystic type of spermatogenesis in fish, the role of androgens and estrogens will be discussed, leading to a conclusion that androgens are required for spermatogenesis while their mode of action is poorly understood. In the cystic mode of spermatogenesis in fish, the Sertoli cells are formed cystic, in the somniferous lobules enclosing a single germ cell clone, providing suitable experimental models to address questions of relevance for vertebrate spermatogenesis in general.

Developmental ontogeny of prolactin and prolactin receptor in the sea bream (Sparus aurata).

The expression of PRL and its receptor (PRLR) were characterised during sea bream embryonic and larval development, by semi-quantitative and quantitative RT-PCR, respectively, until 46 days post-hatch (DPH). Immunocytochemistry with antisera specific for sea bream PRLR was carried out with larval sections from hatching up to 46 DPH. A single transcript of PRL (1.35 Kb) and PRLR (2.8 Kb) identical to the transcripts previously characterised in adult tissue, are present in sea bream embryos and larvae. PRL expression is first detectable at neurula and in all samples collected thereafter. The lowest levels of PRL mRNA are detected in sea bream embryos up until neurula when expression starts to increase. The maximal levels of PRL expression were detected at 24 DPH. PRLR transcripts first appear at 12h post-fertilisation (0.002 rho mol/microg total larvae RNA) (blastula) and increase significantly during gastrulation (0.245 rho mol/microg total larvae RNA) reaching a maximum at 2 DPH (0.281 rho mol/microg total larvae RNA). After hatching a significant reduction in PRLR expression is observed which reaches a minimum at 4 DPH (0.103 rho mol/microg total larvae RNA), gradually increasing thereafter. Immunocytochemistry revealed the presence of PRLR in early post-hatching stages of larvae in tissues derived from all three germ layers.

Testosterone inhibits 11-ketotestosterone-induced spermatogenesis in African catfish (Clarias gariepinus).

Male fish produce 11-ketotestosterone as a potent androgen in addition to testosterone. Previous experiments with juvenile African catfish (Clarias gariepinus) showed that 11-ketotestosterone, but not testosterone, stimulated spermatogenesis, whereas testosterone, but not 11-ketotestosterone, accelerated pituitary gonadotroph development. Here, we investigated the effects of combined treatment with these two types of androgens on pituitary gonadotroph and testis development. Immature fish were implanted for 2 wk with silastic pellets containing 11-ketotestosterone, testosterone, 5alpha-dihydrotestosterone, or estradiol-17beta; cotreatment groups received 11-ketotestosterone in combination with one of the other steroids. Testicular weight and pituitary LH content were higher (two- and fivefold, respectively) in the end control than in the start control group, reflecting the beginning of normal pubertal development. Treatment with testosterone or estradiol-17beta further increased the pituitary LH content four- to sixfold above the end control levels. This stimulatory effect on the pituitary LH content was not modulated by cotreatment with 11-ketotestosterone. However, the stimulatory effect of 11-ketotestosterone on testis growth and spermatogenesis was abolished by cotreatment with testosterone, but not by cotreatment with estradiol-17beta or 5alpha-dihydrotestosterone. Also, normal pubertal testis development was inhibited by prolonged (4 wk) treatment with testosterone. The inhibitory effect of testosterone may involve feedback effects on pituitary FSH and/or on FSH receptors in the testis. It appears that the balanced production of two types of androgens, and the control of their biological activities, are critical to the regulation of pubertal development in male African catfish.

Gonadotropins, their receptors, and the regulation of testicular functions in fish.

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.

Steroid hormones stimulate gonadotrophs in juvenile male African catfish (Clarias gariepinus).

In juvenile African catfish (Clarias gariepinus), the pituitary LH content strongly increased after the beginning of spermatogonial proliferation. We hypothesized that a signal of testicular origin is involved in stimulating the gonadotrophs. We investigated the effects of castration and sex steroid treatment on gonadotrophs in juvenile males by quantifying LH production and release and LH subunit transcript levels and by examining gonadotroph morphology and proliferation. Castration reduced but did not abolish the maturation-associated elevation in pituitary LH content. Treatment with testosterone but not with 11-ketotestosterone, an otherwise potent androgen in fish, reversed the castration-induced decrease of pituitary LH levels. An increased pituitary LH content was accompanied by an increased number of cytologically mature gonadotrophs. However, no evidence was found for gonadotroph proliferation, so that quiescent gonadotrophs may have become activated. Although 11-ketotestosterone treatments had no effect in castrated males, this androgen attenuated gonadotroph activation in intact males. Because androgen production in juvenile catfish is downregulated by treatment with 11-ketotestosterone, its inhibitory effects on gonadotrophs in gonad-intact males may be due to suppression of Leydig cell testosterone production, which appears to be a limiting factor for the activation of catfish gonadotrophs. Aromatizable androgens may have opposite effects on fish (stimulatory) and mammalian (inhibitory) gonadotrophs.

Cloning, characterization, and tissue distribution of prolactin receptor in the sea bream (Sparus aurata).

The prolactin receptor (PRLR) was cloned and its tissue distribution characterized in adults of the protandrous hermaphrodite marine teleost, the sea bream (Sparus aurata). An homologous cDNA probe for sea bream PRLR (sbPRLR) was obtained by RT-PCR using gill mRNA. This probe was used to screen intestine and kidney cDNA libraries from which two overlapping clones (1100 and 2425 bp, respectively) were obtained. These clones had 100% sequence identity in the overlapping region (893 bp) and were used to deduce the complete amino acid sequence of sbPRLR. The receptor spans 2640 bp and encodes a protein of 537 amino acids. Features characteristic of PRLR, two pairs of cysteines, WS box, hydrophobic transmembrane domain, box 1, and box 2, were identified and showed a high degree of sequence identity to PRLRs from other vertebrate species. SbPRLR is 29 and 32% identical to tilapia (Oreochromis niloticus) and goldfish (Carassius auratus) PRLRs, respectively. In the sea bream two PRLR transcripts of 2.8 and 3.2 kb were detected in the intestine, kidney, and gills and a single transcript of 2.8 kb was detected in skin and pituitary by Northern blot. Spermiating gonads (more than 95% male tissue; gonado-somatic index of 0.6) contained, in addition to the 2.8-kb transcript, three more transcripts of 1.9, 1.3, and 1.1 kb. RT-PCR, which is a far more sensitive method than Northern blot, detected PRLR mRNA in gills, intestine, brain, pituitary, kidney, liver, gonads, spleen, head-kidney, heart, muscle, and bone. Immunohistochemistry using specific polyclonal antibodies raised against an oligopeptide from the extracellular domain of sbPRLR detected PRLR in several epithelial tissues of juvenile sea bream, including the anterior gut, renal tubule, choroid membrane of the third ventricle, saccus vasculosus, branchial chloride cells, and branchial cartilage.

Androgen-induced changes in Leydig cell ultrastructure and steroidogenesis in juvenile African catfish, Clarias gariepinus.

The present report focuses on the mechanism(s) involved in the steroid-induced decrease of androgen production in immature African catfish testes that was observed in previous studies. Juvenile animals were implanted with Silastic pellets containing different 11-oxygenated androgens (11-ketotestosterone, KT; 11 beta-hydroxyandrostenedione, OHA; 11-ketoandrostenedione, KA), testosterone (T) or estradiol-17 beta (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were either incubated with increasing concentrations of catfish luteinizing hormone (LH), or incubated with [3H]-pregnenolone ([3H]-P5) or [3H]-androstenedione ([3H]-A). Tissue fragments were also prepared for the quantitative assessment of Leydig cell morphology. Most of the parameters studied were not affected significantly by implantation of E2. Implantation of all androgens inhibited both the basal and the LH-stimulated androgen secretory capacity in vitro. This was associated with a reduced size of the Leydig cells and loss of half of their mitochondria. The studies on the metabolism of tritiated steroid hormones indicated that steroidogenic steps prior to 11 beta-hydroxylation, probably C17-20 lyase activity, were affected by all androgens. Although the effects of 11-oxygenated androgens and T on Leydig cells were mostly similar, previous work showed that only the 11-oxygenated androgens stimulated spermatogenesis, suggesting that distinct mechanisms of action are used by 11-oxygenated androgens and T. These mechanisms, however, seem to merge on the same target(s) to impair Leydig cell androgen production. Such a negative feedback mechanism may be of relevance in the context of the decline in androgen secretion per milligram testis tissue that accompanies the first wave of spermatogenesis in pubertal African catfish.

Sex steroids and the initiation of puberty in male African catfish (Clarias gariepinus).

The effects of sex steroids on spermatogenesis and testicular androgen secretion were studied in juvenile (spermatogonia present in testes) African catfish. Fish were implanted with Silastic pellets containing 11-ketotestosterone (KT), 11beta-hydroxyandrostenedione (OHA), androstenetrione (OA), androstenedione (A), testosterone (T), 5alpha-dihydrotestosterone (DHT), or estradiol-17beta (E2). Control groups received steroid-free pellets. Two weeks later, testis tissue fragments were incubated with African catfish luteinizing hormone (LH) and the amount of OHA secreted in vitro (the main androgen produced by African catfish testes) was quantified. Tissue fragments were then fixed for histological analysis of spermatogenesis. Treatment with KT, OHA, and OA stimulated testicular growth and spermatogenesis (spermatocytes and spermatids were found), whereas T, DHT, A, or E2 had no such effects. All steroids, except for DHT and E2, reduced OHA secretion in the absence and presence of LH to approximately 10% of the control values. Previous studies have shown that KT, OHA, and OA have little effect on circulating LH levels in juvenile male African catfish, so that these androgens probably had direct effects on the testis. Inasmuch as OHA, OA, and KT have largely similar effects and because OHA and OA are converted to KT in vivo, we suggest that KT is physiologically the most relevant androgen for the initiation of spermatogenesis in African catfish.