Heteropicnotic chromatin and nucleolar activity in meiosis and spermiogenesis of Limnogonus aduncus (Heteroptera, Gerridae): a stained nucleolar organizing region that can serve as a model for studying chromosome behavior.

Males of Limnogonus aduncus were found to have the sex chromosome system X0 and chromosome number 2n = 23 (22A + X0). Testis cells were stained with lacto-acetic orcein and silver nitrate so that changes in the morphology and degree of staining of the heteropicnotic chromatin and the nucleolar material could be observed during meiosis and spermiogenesis. These structures share the same nuclear position and could be seen until almost the end of spermiogenesis. A chromosome region stained with silver nitrate was indicative of a nucleolar organizing region (NOR), which is rarely detected in Heteroptera with this technique. The NOR is located at one end of a single member of an autosome pair. The finding of this stained region enabled us to observe that the telomeric association of sister chromatids that characterizes the Heteroptera does not include the chromosome ends, where NORs are located; we also observed in anaphase that the chromosome end through which it is pulled to the pole is the one containing the NOR. Another observation was that the single nucleolar body present in the cells at anaphase never goes to the cell pole that does not receive the NOR. We conclude that L. aduncus is a good model for cytogenetic studies involving nucleolar activity and also may be useful for studying the mechanisms of activation and inactivation of kinetic activity at the chromosome ends. Although the chromosomes of Heteroptera are known to be holocentric, whether kinetic activity is restricted to one or involves both chromosome ends is still not well understood.

Organização celular dos testículos em Hylidae e leptodactylidae, no Pantanal (Estado do Mato Grosso do Sul, Brasil)

The objective of this work was to make a comparative analysis of germ cell organization at different stages of cellular differentiation in adult males of Dendropsophus nanus (Boulenger, 1889), Pseudis limellum (Cope, 1862), P. paradoxa (Linnaeus, 1758), and Scinax acuminatus (Cope, 1862), belonging to the family Hylidae; and Leptodactylus chaquensis (Cei, 1950) and L. podicipinus (Cope, 1862), belonging to the family Leptodactylidae, collected in the Pantanal and in Serra da Bodoquena, Mato Grosso do Sul State, Brazil. The testes were removed and fixed, dehydrated in a graded series of ethanol and embedded in methacrylate glycol resin (Historesin Leica®). The sections were stained by 1% toluidine blue and observed under light microscope. It was detected that all individual of the Hylidae family show, throughout the year, the presence of all germ cell types of spermatogenesis. However, all Leptodactylidae family individuals only show the presence of all germ cell types during the rainy season. The variations of characteristics in seminiferous epithelium organization, as well as the evident difference in the amount of spermatozoa inside the tubules, are evidence that the anurans in this work show different forms of spermatogenesis development throughout the year: the cycle is continuous for the Hylidae family, and discontinuous with explosive release of spermatozoa for the Leptodactylidae family.

Seleção de espermatozoides para ICSI

The application of assisted reproduction techniques has provided help to many men seeking to father a child, although the current success of these procedures remains suboptimal. Today some protocols allow sperm to be selected according to their ultrastructural morphology or surface molecular characteristics. On the other hand, successful human reproduction relies partly on the inherent integrity of sperm DNA. Therefore, it is now necessary to improve the safety of the sperm selection method. It is urgent to optimize procedures to isolate spermatozoa for ICSI with low risk of DNA damage. In recent years, two technologies have attracted the attention of specialists as methods capable of identifying a spermatozoon with low risk of DNA damage: Ultrastructural morphology sperm selection at high magnification and sperm head birefringence selection. This review analyses these two technologies. © Todos os direitos reservados a SBRA - Sociedade Brasileira de Reprodução Assistida.

Expression of acid phosphatase in the seminiferous epithelium of vertebrates

Acid phosphatases (AcPs) are known to provide phosphate to tissues that have high energy requirements, especially during development, growth and maturation. During spermatogenesis AcP activity is manifested in heterophagous lysosomes of Sertoli cells. This phagocytic function appears to be hormone-independent. We examined the expression pattern of AcP during the reproductive period of four species belonging to different vertebrate groups: Tilapia rendalli (Teleostei, Cichlidae), Dendropsophus minutus (Amphibia, Anura), Meriones unguiculatus (Mammalia, Rodentia), and Oryctolagus cuniculus (Mammalia, Lagomorpha). To demonstrate AcP activity, cryosections were processed for enzyme histochemistry by a modification of the method of Gömöri. AcP activity was similar in the testes of these four species. Testes of T. rendalli, D. minutus and M. unguiculatus showed an intense reaction in the Sertoli cell region. AcP activity was detected in the testes of D. minutus and O. cuniculus in seminiferous epithelium regions, where cells are found in more advanced stages of development. The seminiferous epithelium of all four species exhibited AcP activity, mainly in the cytoplasm of either Sertoli cells or germ cells. These findings reinforce the importance of AcP activity during the spermatogenesis process in vertebrates. © FUNPEC-RP.

Cytogenetic evidence for de novo synthesis of rRNA and involvement of nucleolar material in the organization of cell structures during spermiogenesis of Chariesterus armatus (Heteroptera, Coreidae).

The nucleolar material of Chariesterus armatus was analyzed during spermiogenesis in cell preparations impregnated with silver nitrate. Nucleolar corpuscles were observed in spermatids at the beginning of the process, showing that this organoid is also maintained after meiosis. In addition, nucleoli were seen in the round spermatids connected to the X-chromosome (bearer of the nucleolar organizer in C. armatus), indicating de novo synthesis of nucleolar material. This differs from the reorganization of ribosomal granules, transported from meiotic spermatocytes to round spermatids, where they would support protein synthesis, which is reported for other species. We also observed connections of nucleolar corpuscles to the nuclear membrane regions where the tail and the acrosome will be formed, suggesting close involvement of the nucleolar material in the formation of these structures. In addition to the nucleolar bodies, we detected silver-positive structures, which will require new approaches to clarify their role. One of these structures, observed in the cytoplasm, appears to correspond to the chromatoid body, which has been found in several organisms, but is still poorly understood; another is a complex structure to which the tail appears to be connected. We conclude that C. armatus is an appropriate model for understanding not only the synthesis of rRNA in the spermiogenesis, but also the functional meaning of the close relationship of nucleolar material with other structures during this process.

Automatic classification of fish germ cells through optimum-path forest

The spermatogenesis is crucial to the species reproduction, and its monitoring may shed light over some important information of such process. Thus, the germ cells quantification can provide useful tools to improve the reproduction cycle. In this paper, we present the first work that address this problem in fishes with machine learning techniques. We show here how to obtain high recognition accuracies in order to identify fish germ cells with several state-of-the-art supervised pattern recognition techniques. © 2011 IEEE.

Cimetidine-induced vascular cell apoptosis impairs testicular microvasculature in adult rats

Cimetidine, an H2 receptor antagonist used for treatment of gastric ulcers, exerts antiandrogenic and antiangiogenic effects. In the testes cimetidine impairs spermatogenesis, Sertoli cells and peritubular tissue, inducing apoptosis in the myoid cells. Regarding the importance of histamine and androgens for vascular maintenance, the effect of cimetidine on the structural integrity of the testicular vasculature was evaluated. Adult male rats received cimetidine (CMTG) and saline (CG) for 50 days. The testes were fixed in buffered 4% formaldehyde and embedded in historesin and paraffin. In the PAS-stained sections, the microvascular density (MVD) and the vascular luminal area (VLA) were obtained. TUNEL method was performed for detection of cell death. Testicular fragments embedded in Araldite were analyzed under transmission electron microscopy. A significant decrease in the MVD and VLA and a high number of collapsed blood vessel profiles were observed in CMTG. Endothelial cells and vascular muscle cells were TUNEL-positive and showed ultrastructural features of apoptosis. These results indicate that cimetidine induces apoptosis in vascular cells, leading to testicular vascular atrophy. A possible antagonist effect of cimetidine on the H2 receptors and/or androgen receptors in the vascular cells may be responsible for the impairment of the testicular microvasculature.

Analysis of spermiogenesis like a tool in the study of the triatomines of the Brasiliensis subcomplex

The specific identification and systematic of triatomines have been based fundamentally on morphological observations. These organisms are classified into complexes and specific subcomplexes, principally for morphological parameters and geographical disposition. The use of cytogenetic analyzes has been represented as a tool in systematic and taxonomy of triatomines. Thus, the present work, through the analysis of spermiogenesis, aims to characterize this stage of spermatogenesis in triatomines little studied, and especially to compare it among the species Triatoma lenti and T. sherlocki, to assist in the diagnosis of differentiation of these insects. The presence of the heteropyknotic corpuscle is shown as a diagnostic tool to differentiate T. sherlocki and T. lenti, since it is absent in T. lenti. The analysis of the spermiogenesis in T. sherlocki also allowed us to address morphological differences between elongating cells, which were relatively smaller and more filamentous when compared to T lenti. Furthermore, the flagellum was observed in all stages of cell differentiation and elongation. This structure, which helps in the locomotion of the sperm, is hardly observed in cytogenetic analysis, especially throughout spermiogenesis. Thus, although other comparative approaches should be taken, this paper allowed emphasizing the analysis of spermiogenesis as an important cytotaxonomic tool that assists in the differentiation of morphologically related species, such as T. lenti and T. sherlocki. © 2013 Académie des sciences.

Gametogenesis and reproductive cycle of Melanorivulus aff. punctatus (Boulenger, 1895) (Cyprinodontiformes, Rivulidae) in Chapada dos Guimarães, Mato Grosso, Brazil

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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.

Long-term effects of perinatal androgenization on reproductive parameters of male rat offspring androgenization and male rat reproduction

It is known that during sex differentiation, fetal androgens are critical determinants of the male phenotype. Although testosterone is necessary for normal development of male sexual behavior, perinatal androgen treatment can result in disruption of normal male sexual reproduction. Pregnant Wistar rats were administered either corn oil (vehicle) or testosterone propionate at 0.2 mg/kg from gestational day 12 until the end of lactation and the reproductive function of male offspring was evaluated at 90 (adulthood) and 270 (middle age) days of age. Perinatal androgenization in the rat provoked a reduction in sperm production and reserves in adulthood that did not affect fertility and did not persist at more advanced ages, as shown by the results at post-natal day 270. If perinatal androgenization promotes similar effects in humans of reproductive age, the results of the present work can impact male reproduction health, given the less efficient spermatogenesis and lower sperm reserves in the human epididymis, compared to rodents. © Georg Thieme Verlag KG Stuttgart. New York.

Annual reproductive cycle of males of the flat-faced fruit-eating bat, Artibeus planirostris (Chiroptera: Phyllostomidae)

Artibeus planirostris is an endemic species of Phyllostomid bat from the Neotropical region. Some studies have indicated that it exhibits seasonal bimodal polyestry; however, others postulate that it may be able to produce young at any time during the year. Thus, the aim of this study was to evaluate the annual variations in testicular and epididymal parameters of this species in southeast Brazil and try to understand how the reproduction of this species is regulated in this environment. Sixty mature male specimens, collected between June 2009 and May 2010, were submitted to morphometric and immunohistochemical analysis. Our study showed that A. planirostris presented a continuously active pattern of spermatogenesis throughout the year, presenting spermatozoa inside its cauda epididymis in all months, but with two pronounced peaks of spermatogenic production, one in September and other in February. We propose that the males developed these two peaks in order to produce sufficient sperm for the reproduction in a harem system and to synchronize with the female reproductive cycle, which had a bimodal polyestric pattern. Control of this variation is directly linked to the expression of the androgen receptor (AR) in Sertoli cells and to serum testosterone levels, which appear to synchronize to establish these two peaks. In the months preceding the two peaks, the testis have a higher expression of the AR, which possibly stimulates the increase in PCNA, and drives a gradual increase in the testicular parameters. Taken together the results suggest that if sperm storage happens in this species, it is of short duration. © 2013 Elsevier Inc.

Reproductive biology of two macrourid fish, Nezumia aequalis and Coelorinchus mediterraneus, inhabiting the NW Mediterranean continental margin (400-2000m)

Nezumia aequalis and Coelorinchus mediterraneus are abundant species on the upper and lower continental slopes, respectively, in the Mediterranean Sea. A study on the reproductive strategy of the two species was conducted on the Catalan margin (NW Mediterranean). The reproductive cycle of both species was investigated using visual analyses of gonads and histological screening. The shallower species, N. aequalis, showed continuous reproduction with a peak of spawning females in winter months. In contrast, the deeper-living species, C. mediterraneus, showed semi-continuous reproduction with a regression period during the spring. Juveniles of N. aequalis were present in all seasons, but most abundant in the spring. Only two juveniles of C. mediterraneus were found. Both species had asynchronous oocyte development. The average fecundity of N. aequalis was 10,630 oocytes per individual, lower than known for the same species in the Atlantic Ocean. The fecundity of C. mediterraneus was measured for the first time in this study, with an average of 7693 oocytes per individual. Males of both species appear to have semi-cystic spermatogenesis. © 2013 Elsevier Ltd.

The relationships between scrotal surface temperature, age and sperm quality in stallions

In horses, spermatogenesis normally occurs at an average intratesticular temperature of 35. °C; therefore, mechanisms for testicular thermoregulation are essential. Measuring the scrotal surface temperature by thermography is one of the methodologies used to evaluate the effectiveness of testicular thermoregulation. The objective of this study was to determine the relationship between the control of scrotal surface temperature and sperm quality in horses of different ages. In total, 24 Quarter Horse stallions were divided into three groups: YS (young stallions), AS (adult stallions) and OS (old stallions). Initially, we calculated the testicular volume (TV) and evaluated various aspects of the semen (sperm kinetics, plasma membrane integrity and sperm morphology) for all the animals. We also evaluated rectal temperature (RT), body surface temperature (BST,) and average scrotal surface temperature in the testicular region (SST) before (M0) and after sun exposure (M1). Differences were observed (p<0.05) between the RT and BST before and after sun exposure in all three groups. However, there were no differences (p>0.05) in the SST values at these two time points, thus demonstrating the efficiency of the mechanisms for testicular thermoregulation. The SST was similar (p>0.05) among all three groups. Based on these results, we conclude that fertile stallions of different age groups are able to maintain SST and measuring the heat radiating from the scrotum using a digital infrared thermographer. We can also conclude that measuring the heat radiating from the scrotum using a digital infrared thermographer is a practical and efficient tool for monitoring SST in horses. © 2013 Elsevier B.V.