Testicular ultrastructure and morphology of the seminal pathway in Prochilodus scrofa

The seminiferous tubules of Prochilodus scrofa present a coiled morphological arrangement with intertubular anastomoses and unrestricted spermatogonial distribution. The structural pattern of the seminiferous tubules is cystic, with cysts formed by cytoplasmic prolongations of Sertoli cells. Inside the cysts are observed different types of germ cells. The seminiferous tubules open individually on the ventral surface of the main testicular duct present in each testis. Each main testicular duct prolongs as a spermatic duct, fusing with the spermatic duct of the opposite side to form the common spermatic duct which opens into the urogenital papilla. The mature sperm cysts break and extravasate their content into the lumen of the seminiferous tubules from which the seminal fluid and the spermatozoa penetrate the main testicular duct, the spermatic duct and the common spermatic duct for semen ejaculation.

The ultrastructure of the pre-meiotic and meiotic stages of spermatogenesis in Plagioscion squamosissimus (Teleostei, Perciformes, Sciaenidae)

Spermatogenesis of 'corvina' P. squamosissimus starts from a stem cell that gives rise to germ cells. These cells are enveloped by Sertoli cells, forming cysts. The germ cells in the cysts are all at the same stage of development and are interconnected by cytoplasmic bridges. Spermatogonia are the largest germ cells. In the cysts, these cells differentiate into primary spermatogonia and secondary spermatogonia. The primary spermatogonia are isolated in the cyst and give rise to the secondary spermatogonia. After several mitotic divisions, they produce spermatocytes I, which can be identified by synaptonemal complexes in the nucleus. The spermatocytes I enter the first phase of meiosis to produce the spermatocytes II. These are not very frequently seen because they rapidly undergo a second phase of meiosis to produce spermatids.

Structural characterization of nuclear phenotypes during Scinax fuscovarius spermatogenesis (Anura, Hylidae)

In anuran amphibian Scinax fuscovarius, the spermatogenesis occurs in structures called seminiferous loculi, in which germ epithelium is organized in spermatocysts. Each cyst contains cells in the same stage of cytodifferentiation. Characteristics of each cellular type and their groups made the identification and differentiation of the germ lineage cells possible. In the basis of the epithelium there are the spermatogonia I, the biggest cells and always associated with the Sertoli cell. After the phase of mitotic proliferation, the cysts containing variable number of spermatogonia II are originated, quite smaller and with cellular boundaries a little distinct. After differentiation and growth in volume, the spermatocytes I appear, the nuclei of which are spherical and with different degrees of compaction of the nuclear material. Starting the meiotic process, the spermatocytes II are originated, which by means of the second meiotic division become haploid cells, the spermatids I. These two last spermatocysts are very similar. In this phase, the cells will go through a prominent process of differentiation until they form the spermatids II, which are elongated and begin to be organized in bundles supported by prominent Sertoli cells. With the process of spermiogenesis, spermatozoa appear, usually observed in compact bundles with tails turned to the lumen and their heads fitted in their support cells. In more advanced stages, the spermatozoa can be observed free in the locular lumen, ready to follow the spermatic path.

Diagnosis of 5α-reductase type 2 deficiency: Contribution of anti-Müllerian hormone evaluation

Aim: To evaluate anti-Müllerian hormone (AMH) levels in patients with clinical and molecular diagnosis of 5α-reductase 2 deficiency. Patients and methods: Data from 14 patients whose age ranged from 21 days to 29 years were analyzed according to age and pubertal stage. Sexual ambiguity was rated as Prader III in 11 patients. LH, FSH, testosterone (T), dihydrotestosterone (DHT) and AMH serum levels were measured in all but two patients, who had been previously submitted to gonadectomy; T and DHT were also measured in 20 age-matched controls. Results: Gonadotropin levels were normal in all but one patient who retained gonads (six of whom had reached puberty) and T/DHT ratio was elevated in all patients when compared to controls. All prepubertal patients had AMH levels < -1 SD for age, while most pubertal patients had AMH levels compatible with pubertal stage. Conclusions: Prepubertal patients with 5α-reductase 2 deficiency have AMH values in the lower part of the normal range. These data indicate that T does not need to be converted to DHT to inhibit AMH secretion by Sertoli cells. © Freund Publishing House Ltd., London.

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology - transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species. © Springer Science+Business Media B.V. 2008.

Cell junctions in fish seminiferous epithelium

Similar to mammals, in fish the cellular interactions between Sertoli cells (SC) and germ cells (GC) in the seminiferous epithelium have important structural and functional roles. In this review, we give a brief summary of these interactions, in particular those on the cell junctions. Despite the scarcity of detailed empirical data, it appears that both basic types of adhesive junctions (actin- and intermediate filaments-related) are present between SC. However, the actin-related multifunctional junction known as the ectoplasmic specialization is seemingly present only in some cartilaginous fish. Conversely, SC in other fish species are joined by actin-related junctions similar to typical zonulae or puncta adherens found in other epithelia. Adhesive junctions are also found between SC and GC and between GC and GC, and due to their particular characteristics these junctions are known as desmosome-like junctions. In terms of intercellular communication, connexins and gap junctions have been shown to occur between SC in fish, and they may be involved in the coordination of the synchronous development of GC within the cysts. It is also possible that gap junctions may form an interconnected network between SC and GC within a cyst. Concerning the SC barrier, tight junctions between fish SC apparently form a functional barrier only in cysts containing haploid GC, and different from mammals, meiotic GC in fish are not shielded from the vascular system. In summary, although still not well investigated, cell-cell interactions in the seminiferous epithelium of fish seem to be crucial for GC development, and their disturbance, for example by changing environmental conditions, will probably affect GC survival and fertility. © Springer Science+Business Media B.V. 2008.

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.

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.

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.

Morfologia testicular e estrutura da espermatogênese em peixes ornamentais Tetra Negro, Gymnocorymbus ternetzi; Tetra Amarelo, Hyphessobrycon bifasciatus e Mato Grosso, Hyphessobrycon eques (Teleostei, Characiformes)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Puberdade em caititus (Tayassu tajacu): estudo da espermatogênese em diferentes faixas etárias

Esta pesquisa analisa o desenvolvimento da espermatogênese em caititus (Tayassu tajacu) e determina a idade em que atingem a puberdade, considerando-se a biometria testicular e dos túbulos seminíferos, a quantificação das células espermatogênicas, a descrição morfológica dos estádios do ciclo do epitélio seminífero (CES), a freqüência relativa com a qual aparecem no interior dos túbulos seminíferos e o rendimento geral da espermatogênese. No experimento, os animais foram divididos de acordo com a faixa etária, em cinco grupos, com três animais em cada grupo, sendo G1 (7 a 8 meses), G2 (9 a 10 meses), G3 (11 a 12 meses), G4 (13 a 14 meses) e G5 (15 a 16 meses). Os animais foram submetidos à cirurgia de orquiectomia, para obtenção das amostras testiculares, as quais foram fixadas em solução de Alfac por 24 horas e processadas histologicamente, onde cortes de 5 μm foram corados em Hematoxilina-Eosina. Tendo como base, o método da morfologia tubular, foi feita a quantificação dos tipos celulares corrigidos para seus diâmetros nucleares médios de 10 secções transversais de túbulos seminíferos no estádio 1 do CES para animais com a espermatogênese estabelecida e 20 secções transversais para os animais mais jovens, nos quais não havia atividade espermatogênica estabelecida; e também a classificação dos estádios do ciclo do epitélio seminífero, por meio da análise de 100 secções transversais de túbulos seminíferos. Os dados da biometria testicular, a saber, o peso, o comprimento e a largura, revelaram crescimento constante e gradual com diferenças estatísticas significativas (p<0,05) e alta correlação entre si. Os valores do diâmetro tubular apresentaram significância estatística do G1 ao G4, a partir deste, tiveram crescimento acelerado e contínuo, não havendo significância estatística (p >0,05). De acordo com as análises quantitativas e morfológicas das células espermatogênicas que compõem o epitélio germinativo, os grupos etários dos animais foram classificados nas fases seguintes: impúbere (G1), pré-púbere (G2), puberdade (G3), pós-puberdade 1 (G4) e pós-puberdade 2 (G5). A fase em que ocorreu o início da atividade reprodutiva, ou seja, a puberdade, foi determinada nos animais a partir de 11 meses de idade, onde ocorreu o maior crescimento no número de células espermatogênicas e uma correlação positiva com o peso testicular. Nessa fase, as células de Sertoli, apresentaram um decréscimo significativo (p<0,05). Na determinação da freqüência relativa dos estádios do ciclo do epitélio seminífero, foram observadas oito tipos de associações, segundo o método da morfologia tubular, onde o estádio com maior e menor freqüências foram o 1 e o 3, respectivamente. A fase pós-meiótica, apresentou maior freqüência e a fase meiótica, a menor, sendo estatisticamente significativa em relação às demais. A eficiência reprodutiva foi demonstrada por meio dos valores traduzidos pelas razões celulares entre as espermatogônias do tipo A e espermátides arredondadas, não tendo sido observado aumento significativo das espermátides arredondadas entre os grupos G3 ao G5 (p>0,05); e o índice das células de Sertoli apresentou diferença estatística significativa entre todas as possíveis comparações dos grupos etários do G3 ao G5 (p <0,05).

Características reprodutivas de garanhões mantidos sob luz natural ou contínua, em ambiente tropical

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

Endocrine disrupter action in ruminants: A study of the effects of Ipomoea carnea in adult male goats

Ipomoea carnea is a toxic plant in Brazil and other tropical countries that often poisons livestock; its primary toxin is swainsonine. Some substances that interact with the endocrine system have been called endocrine disruptors (EDs). Considering swainsonine's mode of action, it is feasible to hypothesize that this compound could act as an ED. Bisphenol A (BPA), an estrogen-mimic, is considered a classical ED in rodents. This study aimed to evaluate the possible ED actions of I. cameo and BPA in male goats. Seventeen adult male goats were divided into three homogeneous groups: control (C, n = 5); IC (n = 6, received 5.0 g/kg body weight of freshly harvested I. cornea per day), and BPA (n = 6, received 25.0 mg/kg body weight of BPA per day). The experimental period was 120 days. During the experiment, blood samples were collected at 0, 30, 60, 90 and 120 days for biochemical and hormonal evaluations. On the same days, semen samples were collected for andrological evaluation, and scrotal circumference and testicular consistency were determined. The males were castrated on day 121, and fragments of testicle and epididymis were collected for histopathological evaluation. A decrease in serum T3 and T4 was observed in the IC group as well as an increase in the number of sperm with morphological alterations. In the BPA group, reduced serum 14 and a decreased percentage of sperm with plasma membrane integrates were observed. A histopathological examination revealed the vacuolar degeneration of Sertoli cells and areas of laminar patterns of calcium deposits in the IC group and vacuolar degeneration in the rete testis in the BPA group. These results indicate that both I. cameo and BPA are potential EDs in goats. This study emphasized the susceptibility of livestock to ED actions. We also demonstrated the action of I. cameo acting as EDs in the endocrine and reproductive systems. (C) 2014 Elsevier B.V. All rights reserved.

Ultrastructural features of spermatogenesis in Melanorivulus punctatus (Cyprinodontiformes: Rivulidae)

Fish belonging to the family Rivulidae possess one of the most complex reproductive systems. Rivulus, a genus of freshwater fish in the Rivulidae family, was recently reclassified into five genera, including Melanorivulus. Its type species, M. punctatus, is widely distributed and probably represents a species complex. The ultrastructure of sperm has been broadly used in systematics, and we hereby describe the ultrastructural features of spermatogenesis in M. punctatus. Ten M. punctatus males were collected from the reservoir of Parque Estadual da Quineira, municipality of Chapada dos Guimardes, Mato Grosso, Brazil, and prepared for analysis by light microscopy and transmission electron microscopy. M. punctatus undergoes cystic spermatogenesis. Its cysts consist of groups of germ cells that are in synchronous development and are surrounded by cytoplasmic projections of Sertoli cells. With the breakdown of the cysts, the spermatozoa are released and their maturation is completed in the duct, where part of the cytoplasmic material is discarded through the vesicles. The mature spermatozoon is characterized by a spherical head with homogeneously condensed chromatin, a symmetric midpiece consisting of a pair of perpendicular centrioles, a ring of mitochondria, several vesicles, and one flagellum medial to the nucleus. Early stages of spermatogenesis show no peculiarities; however, in spermiogenesis, we observed that the spermatids remain interconnected by cytoplasmic bridges and have pockets of residual cytoplasm. The sperm is of the aquasperm type and is similar to that observed in the members of the family Rivulidae. The spermatozoa have a single flagellum that consists of a classic axoneme (9 +2), as found in most groups of fish, despite the lateral extensions. (c) 2014 Elsevier Ltd. All rights reserved.

Male Gonadal Differentiation and the Paedomorphic Evolution of the Testis in Teleostei

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