Cycle Of Seminiferous Epithelium Research Articles

Spermatogenesis in the Microminipig.

The microminipig has considerable potential as an animal model to evaluate general toxicity; however, there are few studies on the male reproductive system, particularly regarding spermatogenesis. The objectives of the present study were to clarify the stages of the seminiferous epithelium cycle on the basis of spermiogenesis and to determine the duration of spermatogenesis in the microminipig. Eleven microminipigs from 6 to 9 months of age were used for histological analyses. Spermiogenesis and stages of the seminiferous epithelium cycle were classified according to the degree of acrosomal development as shown by the periodic acid-Schiff reaction. Three of the animals were intravenously injected with 5-bromo-2-deoxyuridine to determine the duration of spermatogenesis by immunohistochemistry. Spermiogenesis was classified into 15 steps according to the morphological development of the acrosome, nucleus, and flagellum. The seminiferous epithelium cycle was classified into 11 stages based on the steps of spermatid development and germ cell associations. The length of the seminiferous epithelium cycle and the overall spermatogenesis process in the microminipig were estimated to be approximately 9.1 and 40.9 days, respectively. The results indicate the potential application of the microminipig in the evaluation of testicular toxicity, such as spermatogenesis disruption, in general toxicity studies.

Duration of spermatogenesis and daily sperm production in the rodentProechimys guyannensis

The spiny rat (Proechimys guyannensis) is a neotropical rodent that is used in biomedical research, particularly research related to chronic resistance to epilepsy and infectious diseases. To our knowledge, there are few reports concerning the reproductive biology of this species. Therefore, besides providing basic biometric and morphometric data, in the present study we investigated testis function and spermatogenesis in adult spiny rats. The mean testis weight and gonadosomatic index obtained were 1.63 ± 0.2 g and 1.15 ± 0.1% respectively. Based on the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle were characterized. Stages VI and VII presented the highest frequencies (~17-19%), whilst stages II to V showed the lowest frequencies (~2-4%). The most advanced germ cell types labelled at 1 h or 20 days after BrdU injections were respectively preleptotene/leptotene spermatocytes at stage VII and elongated spermatids at stage III. The mean duration of one cycle was 7.5 ± 0.01 days and the entire spermatogenic process lasted 33.7 ± 0.06 days (~4.5 cycles). The seminiferous tubules (ST) occupied ~96 ± 1% of the testis parenchyma, whereas Leydig cells comprised only 1.5 ± 0.4%. The number of Sertoli cells (SC) per testis gram and the SC efficiency (spermatids/SC) were respectively 78 × 106 ± 11 × 106 and 7.9 ± 1. The daily sperm production per testis gram (spermatogenic efficiency; daily sperm production (DSP)/g/testis) was 78 × 106 ± 8 × 106. To our knowledge, this spermatogenic efficiency is among the highest found for mammals investigated to date and is probably related to the very short duration of spermatogenesis and the very high ST percentage and SC number obtained for this species.

Germ cells and the seminiferous epithelium cycle in the wild rodent Oxymycterus rufus (Rodentia: Cricetidae)

Oxymycterus rufus is a wild rodent that inhabits one of the world hotspots known as the Atlantic Rain Forest in Brazil. Due to the lack of reproductive data regarding such species, the present study aimed to describe O. rufus spermatogenic process through morphometrical and stereological analyses. To do so, testicular fragments of five sexually mature males were routinely processed for light and transmission electron microscopy. The results showed that each seminiferous epithelium cycle corresponded to 6.58 days, while the entire spermatogenic process lasted 29.61 days. The coefficient of spermatogonial mitoses was 5.64 and 2.79 spermatids were produced from each primary spermatocyte in pachytene. The spermatogenesis yield was 11.98 cells. The Sertoli cell index was 4.29, while its overall support capacity was 8.21 cells. The testicular sperm reserve was 183.5 x 10 6 cells, while 962.00 x 10 6 spermatids were found per gram of testis. The daily sperm production per testis and per gram of testis was 28.56 x 10 6 and 73.13 x 10 6 sperm, respectively. Therefore, the short duration of the seminiferous epithelium cycle along with rapid production of sperm indicates that O. rufus shows high efficiency in spermatogenesis.

Influence of scrotal bipartition on spermatogenesis yield and sertoli cell efficiency in sheep

Abstract With the objective to assess the effect of scrotal bipartition on spermatogenesis in sheep, the testes were used from 12 crossbred rams of sheep farms in the municipality of Patos, Paraíba, Brazil, distributed into two groups: GI with six rams with scrotal bipartition, and GII with six rams without scrotal bipartition. The testicular biometry was measured and the testes were collected, fixed in Bouin and fragments were processed to obtain histological slides. The spermatogenesis yield and the Sertoli cell efficiency was estimated by counting the cells of the spermatogenetic line at stage one of the seminiferous epithelium cycle and the Sertoli cells. The results were submitted to analysis of variance with the ASSISTAT v.7.6 program and the mean values were compared by the Student-Newman-Keuls test (SNK) at 5% significance. The testicular biometric parameters did not show statistical difference (p>0.05) between the groups. The meiotic, spermatogenetic and Sertoli cell efficiency were higher in bipartitioned rams (p<0.05), while the mitotic yield did not differ (p>0.05) between GI and GII. The results indicated that there is superiority in the spermatogenetic parameters of bi-partitioned rams, suggesting that these sheep present, as reported in goats, indication of better reproductive indices.

The Effect of Injection with Papaya (Carica Papaya L.) Seed Extract on Sperm Concentration and Spermatogenic Cells of Male Rats (Rattus norvegicus L.) Strain LMR.

So far men as a subject in family planning program had no priority, however recently men become a focus. Established mothodes for male contraception are through condom and vasectomy. Using condoms create psychological complaints, whereas vasectomy although very effective has often permanent effect. An other method of contraception is hormonal; besides that it is important to develop contraception using plants with antifertility effect such as papaya seed. Therefore, the aim of this research is to know the effect of extract papaya seeds on concentration and viability of sperms in vas deferens of male rat Strain LMR. This research was done using papaya seed extract, Bangka variety with 7 treatments, doses/kg/body weight, including 0 mg; 0.1 mg; 0.5 mg; 0.9 mg; 1.0 mg; 5.0 mg; 9.0 mg for times each treatment. Administration of papaya seed extract was performed by intramusculary injection for 20 days (1,5 seminiferous epithelium cycles). Investigation were done on 1) sperms concentration of vas deferens, 2) weight of testis, 3) seminiferous tubules diametric, 4) condition of spermatogenic cells. Injection with papaya seed extract for 20 days increased sperm concentration of vas deferens significantly (P 0,05)

The mechanism and control of Jagged1 expression in Sertoli cells.

The regulation of Sertoli cells by some hormones and signaling factors is important for normal spermatogenesis. Notch signaling is considered to be necessary for normal spermatogenesis in mouse. In this study, we revealed two new facts about Sertoli cells by western blotting experiments on different types of primary cells and microdissected tubules. The first is that Sertoli cells express the Jagged1 ligand in mice testes. The second is that the expression level of Jagged1 oscillates in the seminiferous epithelial cycle. Therefore, we inferred that Jagged1 in Sertoli cells contributes to the Notch signaling involved in spermatogenesis. Furthermore, we examined the regulation of Jagged1 expression and found that Jagged1 expression was suppressed by cAMP signaling and was promoted by TNF-α signaling in Sertoli cells. When cAMP and TNF-α were simultaneously added to Sertoli cells, Jagged1 expression was suppressed. Therefore, cAMP signaling dominates Jagged1 expression over TNF-α signaling. These results suggest that cAMP signaling may cause the periodicity of Jagged1 expression in the seminiferous epithelial cycle, and controlling Jagged1 expression by adding TNF-α or cAMP may contribute to normal spermatogenesis in vitro.

Effect of Dietary Selenium and Vitamin E Supplementation on Testicular Morphology and Serum Testosterone Concentration in Goats Following Scrotal Insulation

Background: Heat directly applied to the testis has been providing information regarding the damage triggering mechanisms on spermatogenesis and possible treatments to prevent testicular changes. Testis submitted to heat-shock have inhibition of the local antioxidant defense mechanisms against lipid peroxidation and free radicals. Vitamin E and Selenium protect biological membranes against free radicals to prevent membrane lipid peroxidation. The current assay evaluated the effect of dietary supplementation with Selenium and Vitamin E on testicular parenchyma and testosterone levels of goats submitted to heat shock by scrotal insulation.Materials, Methods & Results: The effect of dietary selenium and vitamin E supplementation on testicular parameters and serum testosterone concentration was evaluated in goats subjected to scrotal insulation. The animals were randomly allocated into two groups (n = 6) to receive either a control diet (CO) or a diet supplemented with selenium and vitamin E (SE). The animals received supplementation for 120 days: 60 days prior to scrotal insulation, 18 days during scrotal insulation and 42 days after scrotal insulation. Orchiectomy was performed on three animals from each group, immediately after the end of scrotal insulation. The remaining animals were neutered at the end of the experimental period (120 days). Testicles were routinely processed and embedded in glycol methacrylate, stained with toluidine blue/1% sodium borate and evaluated qualitative and quantitatively. Serum testosterone concentrations were determined by enzyme immunoassay at the time of the orchiectomy. Scrotal circumference was greater (P < 0.05) in goats of the SE group (23.0 ± 1.00 cm) than those of the control group (20.0 ± 1.00 cm) at the end of the scrotal insulation period (Day 18). At the end of the experimental period (Day 42 post-scrotal insulation (PSI)), the seminiferous tubule diameter and seminiferous epithelium height were greater (P < 0.05) in the SE group than in control. Histological changes associated with testicular degeneration were detected after 18 days of scrotal insulation in the goats of the control group. The animals of SE group had some histological changes of seminiferous tubules but the majority of them had normal association of germ cells. Selenium and vitamin E supplementation did not seem to avoid testicular damage caused by scrotal insulation but accelerated testicular recovery after the removal of insulation. Testosterone serum levels were not changed in the animals submitted to scrotal insulation, with or without dietary supplementation with selenium and vitamin E.Discussion: In the current study, scrotal insulation for 18 days caused testicular degeneration in both groups. However, selenium and vitamin E supplementation were capable of maintaining the scrotal circumference on the 18th day of insulation in the SE group. Previous reports suggested that selenium and vitamin E could protect cell membranes against the harmful effects of reactive oxygen species. However, the histopathological changes and morphometric data observed in the both groups after 18 days of insulation demonstrated that supplementation with these antioxidants did not prevent the damage caused by heat stress. In turn, at 42 days after the removal of insulation, the tubular diameter and seminiferous epithelium height was greater in animals supplemented with selenium and vitamin E. In addition, the animals that received supplementation had most of seminiferous tubules with cell associations of the seminiferous epithelium cycle. Vitamin E and selenium may reduce testicle sensitivity to heat and thereby shorten the spermatogenesis recovery time by 10 to 20 days. Selenium plus vitamin E added to feed was unable to prevent the degeneration of the testicular parenchyma in these animals. Nonetheless, the supplementation with both antioxidants hastened the recovery of spermatogenesis after the thermal injury.

Prenatal and lactation nicotine exposure affects Sertoli cell and gonadotropin levels in rats

Nicotine is largely consumed in the world as a component of cigarettes. It can cross the placenta and reach the milk of smoking mothers. This drug induces apoptosis, affects sex hormone secretion, and leads to male infertility. To investigate the exposure to nicotine during the whole intrauterine and lactation phases in Sertoli cells, pregnant rats received nicotine (2 mg/kg per day) through osmotic minipumps. Male offsprings (30, 60, and 90 days old) had blood collected for hormonal analysis (FSH and LH) and their testes submitted for histophatological study, analysis of the frequency of the stages of seminiferous epithelium cycle, immunolabeling of apoptotic epithelial cells (TUNEL and Fas/FasL), analysis of the function and structure of Sertoli cells (respectively using transferrin and vimentin immunolabeling), and analysis of Sertoli-germ cell junctional molecule (β-catenin immunolabeling). The exposure to nicotine increased the FSH and LH plasmatic levels in adult rats. Although nicotine had not changed the number of apoptotic cells, neither in Fas nor FasL expression, it provoked an intense sloughing of epithelial cells and also altered the frequency of some stages of the seminiferous epithelium cycle. Transferrin and β-catenin expressions were not changed, but vimentin was significantly reduced in the early stages of the seminiferous cycle of the nicotine-exposed adult rats. Thus, we concluded that nicotine exposure during all gestational and lactation periods affects the structure of Sertoli cells by events causing intense germ cell sloughing observed in the tubular lumen and can compromise the fertility of the offspring.

Disrupting Cyclin Dependent Kinase 1 in Spermatocytes Causes Late Meiotic Arrest and Infertility in Mice1

While cyclin dependent kinase 1 (CDK1) has a critical role in controlling resumption of meiosis in oocytes, its role has not been investigated directly in spermatocytes. Unique aspects of male meiosis led us to hypothesize that its role is different in male meiosis than in female meiosis. We generated a conditional knockout (cKO) of the Cdk1 gene in mouse spermatocytes to test this hypothesis. We found that CDK1-null spermatocytes undergo synapsis, chiasmata formation, and desynapsis as is seen in oocytes. Additionally, CDK1-null spermatocytes relocalize SYCP3 to centromeric foci, express H3pSer10, and initiate chromosome condensation. However, CDK1-null spermatocytes fail to form condensed bivalent chromosomes in prophase of meiosis I and instead are arrested at prometaphase. Thus, CDK1 has an essential role in male meiosis that is consistent with what is known about the role of CDK1 in female meiosis, where it is required for formation of condensed bivalent metaphase chromosomes and progression to the first meiotic division. We found that cKO spermatocytes formed fully condensed bivalent chromosomes in the presence of okadaic acid, suggesting that cKO chromosomes are competent to condense, although they do not do so in vivo. Additionally, arrested cKO spermatocytes exhibited irregular cell shape, irregular large nuclei, and large distinctive nucleoli. These cells persist in the seminiferous epithelium through the next seminiferous epithelial cycle with a lack of stage XII checkpoint-associated cell death. This indicates that CDK1 is required upstream of a checkpoint-associated cell death as well as meiotic metaphase progression in mouse spermatocytes.

From cyst to tubule: innovations in vertebrate spermatogenesis.

Although vertebrates share many common traits, their germline development and function exhibit significant divergence. In particular, this article focuses on their spermatogenesis. The fundamental elements that constitute vertebrate spermatogenesis and the evolutionary changes that occurred upon transition from water to land will be discussed. The life‐long continuity of spermatogenesis is supported by the function of stem cells. Series of mitotic and meiotic germ cell divisions are ‘incomplete’ due to incomplete cytokinesis, forming syncytia interconnected via intercellular bridges (ICBs). Throughout this process, germ cells are supported by appropriate microenvironments established primarily by somatic Sertoli cells. In anamniotes (fish and amphibians) spermatogenesis progresses in cysts, in which developing germ cell syncytia are individually encapsulated by Sertoli cells. Accordingly, Sertoli cells undergo turnover with germ cells that they nourish. This mode of cystic spermatogenesis is also observed in nonvertebrates as insects. In amniotes (reptiles, birds, and mammals), however, Sertoli cells do not turn over but comprise a persistent structure of seminiferous tubules. Sertoli cells nourish different stages of germ cells simultaneously in distinct regions of their surface. This function of Sertoli cells is spatiotemporally orchestrated, and the seminiferous epithelial cycle and spermatogenic wave make the seminiferous tubules a high‐throughput factory for sperm production. Furthermore, contrary to the organized differentiating cells, undifferentiated spermatogonia that comprise the stem cell compartment exhibit active motion over the basal layer of seminiferous tubules and the frequent breakdown of ICBs. Thus, amniote seminiferous tubules represent a typical facultative (or open) niche environment without a stem cell tethering anatomically defined niche. WIREs Dev Biol 2016, 5:119–131. doi: 10.1002/wdev.204For further resources related to this article, please visit the WIREs website.

Annual male reproductive activity and stages of the seminiferous epithelium cycle of the large fruit-eating Artibeus lituratus (Chiroptera: Phyllostomidae)

The large fruit-eating phyllostomid bat, Artibeus lituratus (Olfers, 1818), forearm 69-75 mm, body mass 66-82 g, has a diversified geographic distribution in the Neotropical region. Therefore it is subjected to different climatic conditions that affect its reproduction, leading to different reproductive strategies such as continuous reproduction, seasonal monoestry or seasonal bimodal polyestry. In this study we used morphometric and histological methods to analyze the annual reproductive activity of A. lituratus males in a population living in the Atlantic Forest, Southeastern Brazil. Testis mass, epididymis mass, gonadosomatic index, seminiferous tubule diameter, and Leydig cell nucleus diameter showed no significant differences (p > 0.05) in the two seasons (wet: October to March; dry: April to September). Additionally, the cauda epididymis was packed with sperm throughout the period of study. Our data indicate that in this population spermatogenic activity was continuous throughout the year. Slight variations in accumulated frequency of pre-meiotic, meiotic and post-meiotic stages of the seminiferous epithelium cycle were observed when compared to other bat species, probably due to species-specific characteristics.

Humanin protects against chemotherapy-induced stage-specific male germ cell apoptosis in rats.

Humanin (HN) has cytoprotective action on male germ cells after testicular stress induced by heat and hormonal deprivation. To examine whether HN has protective effects on chemotherapy-induced male germ cell apoptosis, we treated four groups of adult rats with (i) vehicle (control), (ii) HN, (iii) cyclophosphamide (CP); or (iv) HN+CP. To investigate whether the protective effects of HN on germ cells require the presence of Leydig cells, another four groups of rats were pre-treated with ethane dimethanesulfonate (EDS), a Leydig cell toxicant, to eliminate Leydig cells. After 3 days, when Leydig cells were depleted by EDS, we administered: (i) vehicle, (ii) HN, (iii) CP; or (iv) HN+CP to rats. All rats were killed 12 h after the injection of HN and/or CP. Germ cell apoptosis was detected by TUNEL assay and quantified by numerical count. Compared with control and HN (alone), CP significantly increased germ cell apoptosis; HN +CP significantly reduced CP-induced apoptosis at early (I-VI) and late stages (IX-XIV) but not at middle stages (VII-VIII) of the seminiferous epithelial cycle. Pre-treatment with EDS markedly suppressed serum and intratesticular testosterone (T) levels, and significantly increased germ cell apoptosis at the middle (VII-VIII) stages. CP did not further increase germ cell apoptosis in the EDS-pre-treated rats. HN significantly attenuated germ cell apoptosis at the middle stages in EDS pre-treated rats. To investigate whether HN has any direct effects on Leydig cell function, adult Leydig cells were isolated and treated with ketoconazole (KTZ) to block testosterone synthesis. HN was not effective in preventing the reduction of T production by KTZ in vitro. We conclude that HN decreases CP and/or EDS-induced germ cell apoptosis in a stage-specific fashion. HN acts directly on germ cells to protect against EDS-induced apoptosis in the absence of Leydig cells and intratesticular testosterone levels are very low.

AKAP9, a Regulator of Microtubule Dynamics, is Essential for Remodeling of the Blood‐Testis Barrier

The blood‐testis barrier (BTB) is formed by tight, adherens and gap junctions between adjacent Sertoli cells of the seminiferous tubules. The BTB undergoes extensive remodeling during the seminiferous epithelial cycle of spermatogenesis to allow the transport of preleptotene spermatocytes from the basal compartment to the adluminal compartment for further development. The actin cytoskeleton serves unique structural and supporting roles in this process, but little is known about the role of microtubules and their regulators in BTB remodeling. The cAMP responsive scaffold protein AKAP9 has been shown to regulate microtubule dynamics. We demonstrate that conditional and inducible deletion of AKAP9 in mice after the initial formation of the BTB leads to infertility despite evidence of germ cell meiosis. AKAP9 deletion results in marked alterations in the integrity of microtubules in Sertoli cells, mislocalization of gap and tight junctional molecules at the BTB and a progressive loss of barrier integrity despite normal localization of F‐actin at the BTB. These changes were accompanied by an arrest of meiosis and spermiogenesis. Unexpectedly, barrier integrity and meiosis were restored in older AKAP9 deficient animals. Yet, significant defects in spermiogenesis persisted suggesting a role for AKAP9 in the transport of developing spermatids across the adluminal compartment during spermiogenesis. These results suggest that AKAP9 is critical for both dynamic restructuring of the BTB during the epithelial cycle and subsequent spermiogenesis.

Seminiferous Epithelium Cycle in Bombina orientalis

ABSTRACTThe purpose of the present study was to examine the seminiferous epithelium cycle of Bombina orientalis using a light microscope. The cycle was divided into a total of 10 stages, according to the morphological characteristics of the cells. The spermatogenetic cells included primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatid and sperm. At stage I, the primary spermatogonia was located closer to basal lamina of the seminiferous tubule without spermatocyst formations. Especially at the stage II, the secondary spermatogonia were located in the spermatocyst. The primary and secondary spermatocytes were found from stages III to VI. The secondary spermatocytes were smaller in size than the primary spermatocytes, but they had thicker nucleoplasm and smaller nuclei. The round-shaped, early sperm cells were formed in stage VII, and further divided at stage VIII to have more concentrated nucleoplasm before division to matured sperm cells. At stage X, the matured sperm cells emerged from the spermatocyst. Considering the above results, this study presented the special characteristics in the generation and type of sperm formation. The germ cell formation occurred in various stages, like the perspectives of Franca et al (1999), ultimately, providing taxonomically useful information.

Seminiferous Epithelium Cycle in Bombina orientalis.

The purpose of the present study was to examine the seminiferous epithelium cycle of Bombina orientalis using a light microscope. The cycle was divided into a total of 10 stages, according to the morphological characteristics of the cells. The spermatogenetic cells included primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatid and sperm. At stage I, the primary spermatogonia was located closer to basal lamina of the seminiferous tubule without spermatocyst formations. Especially at the stage II, the secondary spermatogonia were located in the spermatocyst. The primary and secondary spermatocytes were found from stages III to VI. The secondary spermatocytes were smaller in size than the primary spermatocytes, but they had thicker nucleoplasm and smaller nuclei. The round-shaped, early sperm cells were formed in stage VII, and further divided at stage VIII to have more concentrated nucleoplasm before division to matured sperm cells. At stage X, the matured sperm cells emerged from the spermatocyst. Considering the above results, this study presented the special characteristics in the generation and type of sperm formation. The germ cell formation occurred in various stages, like the perspectives of Franca et al (1999), ultimately, providing taxonomically useful information.

HGF Modulates Actin Cytoskeleton Remodeling and Contraction in Testicular Myoid Cells.

The presence of the HGF/Met system in the testicular myoid cells was first discovered by our group. However, the physiological role of this pathway remains poorly understood. We previously reported that HGF increases uPA secretion and TGF-β activation in cultured tubular fragments and that HGF is maximally expressed at Stages VII–VIII of the seminiferous epithelium cycle, when myoid cell contraction occurs. It is well known that the HGF/Met pathway is involved in cytoskeletal remodeling; moreover, the interaction of uPA with its receptor, uPAR, as well as the activation of TGF-β have been reported to be related to the actin cytoskeleton contractility of smooth muscle cells. Herein, we report that HGF induces actin cytoskeleton remodeling in vitro in isolated myoid cells and myoid cell contraction in cultured seminiferous tubules. To better understand these phenomena, we evaluated: (1) the regulation of the uPA machinery in isolated myoid cells after HGF administration; and (2) the effect of uPA or Met inhibition on HGF-treated tubular fragments. Because uPA activates latent TGF-β, the secretion of this factor was also evaluated. We found that both uPA and TGF-β activation increase after HGF administration. In testicular tubular fragments, HGF-induced TGF-β activation and myoid cell contraction are abrogated by uPA or Met inhibitor administration.

Hierarchical differentiation competence in response to retinoic acid ensures stem cell maintenance during mouse spermatogenesis.

Stem cells ensure tissue homeostasis through the production of differentiating and self-renewing progeny. In some tissues, this is achieved by the function of a definitive stem cell niche. However, the mechanisms that operate in mouse spermatogenesis are unknown because undifferentiated spermatogonia (Aundiff) are motile and intermingle with differentiating cells in an ‘open’ niche environment of seminiferous tubules. Aundiff include glial cell line-derived neurotrophic factor receptor α1 (GFRα1)+ and neurogenin 3 (NGN3)+ subpopulations, both of which retain the ability to self-renew. However, whereas GFRα1+ cells comprise the homeostatic stem cell pool, NGN3+ cells show a higher probability to differentiate into KIT+ spermatogonia by as yet unknown mechanisms. In the present study, by combining fate analysis of pulse-labeled cells and a model of vitamin A deficiency, we demonstrate that retinoic acid (RA), which may periodically increase in concentration in the tubules during the seminiferous epithelial cycle, induced only NGN3+ cells to differentiate. Comparison of gene expression revealed that retinoic acid receptor γ (Rarg) was predominantly expressed in NGN3+ cells, but not in GFRα1+ cells, whereas the expression levels of many other RA response-related genes were similar in the two populations. Ectopic expression of RARγ was sufficient to induce GFRα1+ cells to directly differentiate to KIT+ cells without transiting the NGN3+ state. Therefore, RARγ plays key roles in the differentiation competence of NGN3+ cells. We propose a novel mechanism of stem cell fate selection in an open niche environment whereby undifferentiated cells show heterogeneous competence to differentiate in response to ubiquitously distributed differentiation-inducing signals.

Classification of the spermatogenic cycle, seasonal changes of seminiferous tubule morphology and estimation of the breeding season of the large Japanese field mouse (Apodemus speciosus) in Toyama and Aomori prefectures, Japan.

The large Japanese field mouse, Apodemus speciosus, is a potential indicator of environmental stress, but this function has not been confirmed by histological studies. Since environmental stress affects the reproductive function of mice, we determined the reproductive characteristics of this species at two locations: Toyama (36°35ʹN, 137°24ʹE) and Aomori (40°35ʹN, 140°57ʹE). Mice were captured during May–November (n=119) and July–November (n=146) at these locations, respectively. We classified the breeding season from the numbers of pregnant females and young, in addition to the spermatogenic cycle and seasonal changes in seminiferous tubule morphology of males. Testicular weight was measured, and seminiferous tubule morphology was examined histologically. Fourteen stages were found in the seminiferous epithelium cycle based on acrosome formation and spermatid head morphology. At both locations, the breeding season peaked from late summer to early autumn and possibly in spring. Spermatogenic activity was classified into 4 periods from June to November: resting around June and October–November; resumptive around July; active around August; and degenerative around September. During the resting period, the seminiferous tubules consisted of Sertoli cells, spermatogonia and spermatocytes. Spermatogenesis began during the resumptive period, and spermatids were observed. During the active period, active spermatogenesis and a broad lumen were observed. During the degenerative period, spermatogenesis ended, and Sertoli cells, spermatogonia, spermatocytes and degenerating exfoliated round spermatids were observed. This study provides scientific information about the testicular histopathological evaluations of the large Japanese field mouse for its use as an index species of environmental pollution.

The immunoexpression of androgen receptor, estrogen receptors alpha and beta, vanilloid type 1 receptor and cytochrome p450 aromatase in rats testis chronically treated with letrozole, an aromatase inhibitor.

The function of testis is under hormonal control and any disturbance of hormonal homeostasis can lead to morphological and physiological changes. Therefore the aim of the study was to investigate the expression of androgen and estrogen receptors (AR, ERs), vanilloid receptor (TRPV1), cytochrome P450 aromatase (P450arom), as well as apoptosis of cells in testis of adult rats chronically treated with letrozole (LT), a non-steroidal aromatase inhibitor, for 6 months. The testicular tissues were fixed in Bouin's fixative and embedded in paraffin. Immunohistochemistry with monoclonal antibodies (abs) against AR, ERa, P450arom, and polyclonalabs against ERβ, TRPV1, caspase-3 was applied. Long-lasting estradiol deficiency, as an effect of LT treatment, produced changes in the morphology of testis and altered the expression of the studied receptors in cells of the seminiferous tubules and rate of cell apoptosis. The immunostaining for AR was found in the nuclei of Sertoli cells and the cytoplasm of spermatogonia and spermatocytes in III-IV stages of the seminiferous epithelium cycle. The intensity of staining for P450arom was lower in the testis of LT-treated rats as compared to control animals. The immunofluorescence of ERα and ERβ was observed exclusively in the nuclei of Leydig cells of LT-treated rats. There were no changes in localization of TRPV1, however, the intensity of reaction was stronger in germ cells of the seminiferous epithelium after LT treatment. The apoptosis in both groups of animals was observed within the population of spermatocytes and spermatids in II and III stages of the seminiferous epithelium cycle. In testis of LT-treated rats the immunoexpression of caspase-3 was additionally found in the germ cells in I and IV stages, and Sertoli, myoid and Leydig cells. In conclusion, our results underline the important role of letrozole treatment in the proper function of male reproductive system, and additionally demonstrate that hormonal imbalance can produce the morphological abnormalities in testis.

Testis Morphometry and Stages of the Seminiferous Epithelium Cycle in an Epididymal Sperm-storing Neotropical Vespertilionid,Myotis levis(Chiroptera)

Yellowish myotis, Myotis levis, is a seasonal, epididymal sperm-storing Neotropical vespertilionid. In the dry season, males show simultaneous testis regression and sperm storage in cauda epididymis, enabling them to mate during this season. In this study, we investigated seasonal variations in body mass, diameter and height of seminiferous tubules and nuclei of Leydig cells in a population of southeastern Brazil. We also determined the frequencies of the stages of the seminiferous epithelium cycle (SEC) of mature individuals of this population. Body mass and diameter of Leydig cell nuclei showed no significant differences between dry and rainy seasons and stages of annual reproductive cycle; however, all other morphometric parameters varied significantly. The relative cumulative frequency of pre-meiotic stages of the SEC (1-3) was 51%, of meiotic stage (4) was 2% and of post-meiotic stages (5-8) was 47%. We confirmed that the yellowish myotis presents seasonal sperm production as revealed by testis regression and epididymal sperm storage during the dry season.