The ultrastructure of the guinea pig rete testis

The chambers of the rete testis (RT) of guinea pig are lined by a simple epithelium, whose cells are squamous, cubical and columnar in shape. The epithelial cells with distinct shapes were counted and the quantitative analysis of the number of these cells showed relative predominance of cubical cells. The ultrastructural observations showed predominance of membrane interdigitations among the epithelial cells. These cells present common cytoplasmic organelles. The Golgi complex polarity is typical with observation of electronlucent vesicles on the Golgi cis face closely related to rough endoplasmic reticulum (ER) lamellae, mitochondria and large number of polysomes on the Golgi trans face. These related structures present in Golgi area of RT cells suggest secretory activity which maybe occurs in the RT epithelium. Endocytotic process also occurs in the RT and this function probably concerns the uptake of substances and resorption of seminiferous fluid. Apical cilia present in RT epithelium cells are related with fluid transport and perhaps with chemoreception. Presence of spermatozoa portions enclosed into the cytoplasm of some epithelium cells has been refferred to as spermatophagy. The RT complex is mainly supported by loose connective tissue, with collagen fibres and some Leydig cells. Leydig cells are adjacent to the network channels of the septal part of the RT and apparently are able to secrete inside the RT lumen.

Ultrastructural and morphometric analysis on the ovary of Wistar rats after chronic ethanol ingestion

In the present study, seventy-two adult rats (Rattus norvegicus albinus) aged three months were used. The animals were divided into two groups (control and alcoholic). The control group received a solid diet (Purina rat chow) and tap water ad libitum. The alcoholic group received the same solid diet and sugar-cane liquid (trade 51, 41° Gay Lussac - GL) diluted 30° GL. At the end or 90, 180 and 270 days of treatment, ten rats of each group were anaesthetized with ethyl ether and sacrificed. The ovaries were collected, fixed, included and submitted to analysis by both light and electron microscopy. The alcoholic group showed increase in the number of corpora lutea at both 180 and 270 days of treatment, atresic follicles at 270 days of treatment, decreased diameter of corpora lutea at 180 and 270 days of treatment, the granulosa layer of the antral follicles at 180 days of treatment, and gradual regression of the theca antral follicles. Furthermore, an increase in diameter and posterior regression of the antral follicle were observed, as well as vacuolation, increased lipid droplets in the granulosa cell at 90 days and in the theca at 180 and 270 days of treatment and gradually in the interstitial cell. The rats showed ovarian alterations after ingestion of alcohol. There was a correlation between exposure time to the drug and the injury observed.

Localization of myosin-Va in subpopulations of cells in rat endocrine organs

Myosin-Va is a Ca 2+/calmodulin-regulated unconventional myosin involved in the transport of vesicles, membranous organelles, and macromolecular complexes composed of proteins and mRNA. The cellular localization of myosin-Va has been described in great detail in several vertebrate cell types, including neurons, melanocytes, lymphocytes, auditory tissues, and a number of cultured cells. Here, we provide an immunohistochemical view of the tissue distribution of myosin-Va in the major endocrine organs. Myosin-Va is highly expressed in the pineal and pituitary glands and in specific cell populations of other endocrine glands, especially the parafollicular cells of the thyroid, the principal cells of the parathyroid, the islets of Langerhans of the pancreas, the chromaffin cells of the adrenal medulla, and a subpopulation of interstitial testicular cells. Weak to moderate staining has been detected in steroidogenic cells of the adrenal cortex, ovary, and Leydig cells. Myosin-Va has also been localized to non-endocrine cells, such as the germ cells of the seminiferous epithelium and maturing oocytes and in the intercalated ducts of the exocrine pancreas. These data provide the first systematic description of myosin-Va localization in the major endocrine organs of rat. © 2008 Springer-Verlag.

The reproductive cycle of the male sleep snake Sibynomorphus mikanii (Schlegel, 1837) from southeastern brazil

This study describes the male reproductive cycle of Sibynomorphus mikanii from southeastern Brazil considering macroscopic and microscopic variables. Spermatogenesis occurs during spring-summer (September-December) and spermiogenesis or maturation occurs in summer (December-February). The length and width of the kidney, the tubular diameter, and the epithelium height of the sexual segment of the kidney (SSK) are larger in summer-autumn (December-May). Histochemical reaction of the SSK [periodic acid-Schiff (PAS) and bromophenol blue (BB)] shows stronger results during summer-autumn, indicating an increase in the secretory activity of the granules. Testicular regression is observed in autumn and early winter (March-June) when a peak in the width of the ductus deferens occurs. The distal ductus deferens as well as the ampulla ductus deferentis exhibit secretory activities with positive reaction for PAS and BB. These results suggest that this secretion may nourish the spermatozoa while they are being stored in the ductus deferens. The increase in the Leydig cell nuclear diameter in association with SSK hypertrophy and the presence of sperm in the female indicate that the mating season occurs in autumn when testes begin to decrease their activity. The peak activity of Leydig cells and SSK exhibits an associated pattern with the mating season. However, spermatogenesis is dissociated of the copulation characterizing a complex reproductive cycle. At the individual level, S. mikanii males present a continuous cyclical reproductive pattern in the testes and kidneys (SSK), whereas at the populational level the reproductive pattern may be classified as seasonal semisynchronous. © 2012 Wiley Periodicals, Inc.

Cyp17a1 and cyp19a1 in the zebrafish testis are differentially affected by oestradiol

Oestrogens can affect expression of genes encoding steroidogenic enzymes in fish gonads. However, little information is available on their effects at the protein level. In this context, we first analysed the expression of key steroidogenic enzyme genes and proteins in zebrafish testis, paying attention also to other cell types than Leydig cells. Gene expression was analysed by quantitative PCR on fluorescence-activated cell-sorting fractions coupled or not to differential plating, while protein synthesis was studied by immunohistochemistry using specific antibodies against zebrafish Cyp17a1, Cyp19a1a and Cyp19a1b. Furthermore, we have evaluated the effect of oestrogen treatment (17β-oestradiol (E2), 10 nM) on the localization of these enzymes after 7 and 14 days of in vivo exposure in order to study how oestrogen-mediated modulation of their expression is linked to oestrogen effects on spermatogenesis. The major outcomes of this study are that Leydig cells express Cyp17a1 and Cyp19a1a, while testicular germ cells express Cyp17a1 and both, Cyp19a1a and Cyp19a1b. As regards Cyp17a1, both protein and mRNA seem to be quantitatively dominating in Leydig cells. Moreover, E2 exposure specifically affects only Leydig cell Cyp17a1 synthesis, preceding the disruption of spermatogenesis. The oestrogen-induced suppression of the androgen production capacity in Leydig cells is a major event in altering spermatogenesis, while germ cell steroidogenesis may have to be fuelled by precursors from Leydig cells. Further studies are needed to elucidate the functionality of steroidogenic enzymes in germ cells and their potential role in testicular physiology. © 2013 Society for Endocrinology.

Avaliação histopatológica e caracterização morfométrica testicular e epididimária em cães adultos sem raça definida (SRD)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Thyroid Hormone Stimulates the Proliferation of Sertoli Cells and Single Type A Spermatogonia in Adult Zebrafish (Danio rerio) Testis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Expressão imuno-histoquímica de KI-67, COX-2, MMP-9 e P53 nos tumores testiculares caninos

Pós-graduação em Ciência Animal - FMVA

Interpreting histopathology in the epididymis

While most of this Special Issue is devoted to the testis (which is where most drug and chemically induced toxicity of the male reproductive tract is identified), being able to recognize and understand the potential effects of toxicants on the epididymis is immensely important and an area that is often overlooked. The epididymis is the organ where the post-testicular sperm differentiation occurs, through a complex and still not completely understood sperm maturation process, allowing them to fertilize the oocyte. Also in the epididymis, sperm are stored until ejaculation, while being protected from immunogenic reaction by a blood-epididymis barrier. From a toxicologic perspective the epididymis is inherently complicated as its structure and function can be altered both indirectly and directly. In this review we will discuss the factors that must be considered when attempting to distinguish between indirect and direct epididymal toxicity and highlight what is currently known about mechanisms of epididymal toxicants, using the rat as a reference model. We identify 2 distinguishable signature lesions - one representing androgen deprivation (secondary to Leydig cell toxicity in the testis) and another representing a direct acting toxicant. Other commonly observed alterations will also be shown and discussed. Finally, we point out that many of the key functions of the epididymis can be altered in the absence of a detectable change in tissue structure. Collectively, we hope this will provide pathologists with increased confidence in identification of epididymal toxicity and enable more informed guidance as mechanism of action is considered.

Inactivating Mutations of the Human Luteinizing Hormone Receptor in Both Sexes

The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproductive physiology. Over the past 15 years, several homozygous or compound heterozygous loss-of-function mutations in the LHCGR gene have been described in males and females. In genetic males, mutations in LHCGR were associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a phenotypic spectrum. Patients with the severe form of LH resistance have predominantly female external genitalia and absence of secondary sex differentiation at puberty. Patients with milder forms have predominantly male external genitalia with micropenis and/or hypospadias or only infertility without ambiguity. The undermasculization is associated with low basal, as well as human CG-stimulated, testosterone levels and elevated LH levels after pubertal age, without abnormal step-up in testosterone biosynthesis precursors. The testes have only slightly reduced size but mature Leydig cells are absent or scarce (Leydig cell hypoplasia). Genetic females with inactivating LHCGR mutations have female external genitalia, spontaneous breast and pubic hair development at puberty, and normal or late menarche followed by oligoamenorrhea and infertility. Estradiol and progesterone levels are normal for the early to midfollicular phase, but do not reach ovulatory or luteal phase levels. Serum LH levels are high whereas follicle-stimulating hormone levels are normal or only slightly increased. Pelvic ultrasound has demonstrated a small or normal uterus and normal or enlarged ovaries with cysts. The inactivating mutations of the LHCGR have provided important insights into distinct physiological roles of LH in reproduction of both sexes.

Neuropeptide Y acts within the rat testis to inhibit testosterone secretion

The factors that influence Leydig cell activity currently include peptides such as neuropeptide Y (NPY). In this work we investigated the ability of this compound, injected directly into the testes of adult male rats, to alter testosterone (T) release into the general circulation. At a 5μg/kg dose administered 1h prior to challenge with human chorionic gonadotropin (hCG, 1.0 U/kg, iv), NPY significantly (P<0.01) blunted the T response to this gonadotropin. The inhibitory effect of NPY was observed in animals pretreated with an antagonist to gonadotropin-releasing hormone or not, indicating that the decrease in plasma T found was most likely independent of pituitary luteinizing hormone. However, testicular levels of steroidogenic acute regulatory (STAR) protein or translocator protein (TSPO) in the Leydig cells did not exhibit consistent changes, which suggested that other mechanisms mediated the blunted T response to hCG. We therefore used autoradiography and immunohistochemistry methodologies to identify NPY receptors in the testes, and found them primarily located on blood vessels. Competition studies further identified these receptors as being Y(1), a subtype previously reported to modulate the vasoconstrictor effect of NPY. The absence of significant changes in STAR and TSPO levels, as well as the absence of Y(1) receptors on Leydig cells, suggest that NPY-induced decreases in T release is unlikely to represent a direct effect of NPY on these cells. Rather, the very high expression levels of Y(1) found in testicular vessels supports the concept that NPY may alter gonadal activity, at least in part, through local vascular impairment of gonadotropin delivery to, and/or blunted T secretion from, Leydig cells.

An examination of the characteristics, concentration and distribution of androgen receptor in rat testis during sexual maturation

Previously reported androgen receptor concentrations in rat testis and testicular cell types have varied widely. In the studies reported here a nuclear exchange assay was established in rat testis in which exchange after 86 hours at 4$\sp\circ$C was greater than 85% complete and receptor was stable. Receptor concentration per DNA measured by exchange declined between 15 and 25 days of age in the rat testis, then increased 4-fold during sexual maturation. Proliferation of germ cells which had low receptor concentration appeared to account for the early decline in testicular receptor concentration, whereas increase in receptor number per Sertoli cell between 25 and 35 days of age contributed to the later increase. Increase in Leydig cell number during maturation appeared to account for the remainder of the increase due to the high receptor concentration in these cells. Detailed studies showed that other possible explanations for changes in receptor number (e.g. shifts in receptor concentration between the cytosol and nuclear subcellular compartments or changes in the affinity of the receptor for its ligands) were not likely.^ Androgen receptor dynamics in testicular cells showed rapid, specific uptake of ($\sp3$H) -testosterone that was easily blocked by unlabeled testosterone (RA of 7 nM in both cell types), and medroxyprogesterone acetate (RA of 28 and 16 nM in Sertoli and peritubular cells, respectively), but not as well by the anti-androgens cyproterone acetate (RA of 116 and 68 nM) and hydroxyflutamide (RA of 300 and 180 nM). The affinity of the receptor for the ligand dimethylnortestosterone was similar in the two cell types (K$\rm\sb{d}$ values of 0.78 and 0.71 nM for Sertoli and peritubular cells) and was virtually identical with the affinity of the whole testis receptor (0.89 nM). Medroxyprogesterone acetate and testosterone significantly increased nuclear androgen receptor concentration relative to untreated controls in Sertoli and peritubular cells, whereas hydroxyflutamide and cyproterone acetate did not. Despite the different embryological origins of peritubular and Sertoli cells, their responses to both androgens and anti-androgens were similar. In addition, these studies suggest that peritubular cells are as likely as Sertoli cells to be primary androgen targets. ^

Müllerian Inhibiting Substance lowers testosterone in luteinizing hormone-stimulated rodents

Müllerian Inhibiting Substance (MIS) expression is inversely proportional to the serum concentration of testosterone in males after birth and in vitro studies have shown that MIS can lower testosterone production by Leydig cells. Also, mice overexpressing MIS exhibited Leydig cell hypoplasia and lower levels of serum testosterone, but it is not clear whether this is a result of MIS affecting the development of Leydig cells or their capacity to produce testosterone. To examine the hypothesis that MIS treatment will result in decreased testosterone production by mature Leydig cells in vivo, we treated luteinizing hormone (LH)-stimulated adult male rats and mice with MIS and demonstrated that it can lead to a several-fold reduction in testosterone in serum and in testicular extracts. There was also a slight decrease in 17-OH-progesterone compared to the more significant decrease in testosterone, suggesting that MIS might be regulating the lyase activity of cytochrome P450c17 hydroxylase/lyase (Cyp17), but not its hydroxylase activity. Northern analysis showed that, in both MIS-treated rats and mice, the mRNA for Cyp17, which catalyzes the committed step in androgen synthesis, was down-regulated. In rats, the mRNA for cytochrome P450 side-chain cleavage (P450scc) was also down-regulated by MIS. This was not observed in mice, indicating that there might be species-specific regulation by MIS of the enzymes involved in the testosterone biosynthetic pathway. Our results show that MIS can be used in vivo to lower testosterone production by mature rodent Leydig cells and suggest that MIS-mediated down-regulation of the expression of Cyp17, and perhaps P450scc, contributes to that effect.

Aromatase (Cyp19) expression is up-regulated by targeted disruption of Dax1

DAX-1 [dosage-sensitive sex reversal, adrenal hypoplasia congenita (AHC) critical region on the X chromosome, gene 1] is an orphan nuclear receptor that represses transcription by steroidogenic factor-1 (SF-1), a factor that regulates expression of multiple steroidogenic enzymes and other genes involved in reproduction. Mutations in the human DAX1 gene (also known as AHC) cause the X-linked syndrome AHC, a disorder that is associated with hypogonadotropic hypogonadism also. Characterization of Dax1-deficient male mice revealed primary testicular defects that included Leydig cell hyperplasia (LCH) and progressive degeneration of the germinal epithelium, leading to infertility. In this study, we investigated the effect of Dax1 disruption on the expression profile of various steroidogenic enzyme genes in Leydig cells isolated from Dax1-deficient male mice. Expression of the aromatase (Cyp19) gene, which encodes the enzyme that converts testosterone to estradiol, was increased significantly in the Leydig cells isolated from mutant mice, whereas the expression of other proteins (e.g., StAR and Cyp11a) was not altered. In in vitro transfection studies, DAX-1 repressed the SF-1-mediated transactivation of the Cyp19 promoter but did not inhibit the StAR or Cyp11a promoters. Elevated Cyp19 expression was accompanied by increased intratesticular levels of estradiol. Administration of tamoxifen, a selective estrogen-receptor modulator, restored fertility to the Dax1-deficient male mice and partially corrected LCH, suggesting that estrogen excess contributes to LCH and infertility. Based on these in vivo and in vitro analyses, aromatase seems to be a physiologic target of Dax-1 in Leydig cells, and increased Cyp19 expression may account, in part, for the infertility and LCH in Dax1-deficient mice.