Ubiquitin protein E3 ligase ASB9 suppresses proliferation and promotes apoptosis in human spermatogonial stem cell line by inducing HIF1AN degradation

BACKGROUND: Spermatogonial stem cells (SSCs) are critical for sustaining spermatogenesis. Even though several regulators of SSC have been identified in rodents, the regulatory mechanism of SSC in humans has yet to be discovered. METHODS: To explore the regulatory mechanisms of human SSCs, we analyzed publicly available human testicular single-cell sequencing data and found that Ankyrin repeat and SOCS box protein 9 (ASB9) is highly expressed in SSCs. We examined the expression localization of ASB9 using immunohistochemistry and overexpressed ASB9 in human SSC lines to explore its role in SSC proliferation and apoptosis. Meanwhile, we used immunoprecipitation to find the target protein of ASB9 and verified its functions. In addition, we examined the changes in the distribution of ASB9 in non-obstructive azoospermia (NOA) patients using Western blot and immunofluorescence. RESULTS: The results of uniform manifold approximation and projection (UMAP) clustering and pseudotime analysis showed that ASB9 was highly expressed in SSCs, and its expression gradually increased during development. The immunohistochemical and dual-color immunofluorescence results displayed that ASB9 was mainly expressed in nonproliferating SSCs. Overexpression of ASB9 in the SSC line revealed significant inhibition of cell proliferation and increased apoptosis. We predicted the target proteins of ASB9 and verified that hypoxia-inducible factor 1-alpha inhibitor (HIF1AN), but not creatine kinase B-type (CKB), has a direct interaction with ASB9 in human SSC line using protein immunoprecipitation experiments. Subsequently, we re-expressed HIF1AN in ASB9 overexpressing cells and found that HIF1AN reversed the proliferative and apoptotic changes induced by ASB9 overexpression. In addition, we found that ABS9 was significantly downregulated in some NOA patients, implying a correlation between ASB9 dysregulation and impaired spermatogenesis. CONCLUSION: ASB9 is predominantly expressed in human SSCs, it affects the proliferation and apoptotic process of the SSC line through HIF1AN, and its abnormal expression may be associated with NOA.

The role of retinoic acid in the commitment to meiosis / 亚洲男科学杂志(英文版)

Male meiosis is a complex process whereby spermatocytes undergo cell division to form haploid cells. This review focuses on the role of retinoic acid (RA) in meiosis, as well as several processes regulated by RA before cell entry into meiosis that are critical for proper meiotic entry and completion. Here, we discuss RA metabolism in the testis as well as the roles of stimulated by retinoic acid gene 8 (STRA8) and MEIOSIN, which are responsive to RA and are critical for meiosis. We assert that transcriptional regulation in the spermatogonia is critical for successful meiosis.

Aspectos da biologia reprodutiva de Boa constrictor constrictor: um estudo histológico dos testículos nos períodos reprodutivos de quiescência e máxima atividade / Boa constrictor constrictor reproductive biology aspects: the testes histological study in the reproductive periods of quiescence and maximal activity

As serpentes pertencem ao segundo maior grupo dentro dos répteis, podendo apresentar sazonalidade quanto à espermatogênese, com produção descontínua ou contínua. O presente trabalho tem como objetivo caracterizar aspectos da biologia reprodutiva de Boa constrictor constrictor com base nos achados histológicos dos testículos nos períodos de máxima atividade (período de gametogênese) e quiescência. Os testículos de dois espécimes de Boa c. constrictor (7767 e 11752) foram cortados a uma espessura de 3µm em micrótomo, corados com azul de toluidina 1%, fotodocumentados e descritos. A presença de espermatozoides na luz do túbulo seminífero no indivíduo 7767 indica um período de máxima gametogênese, enquanto o lúmen dos túbulos seminíferos pouco evidentes, sem a presença de espermatozoides e de células gaméticas em divisão, caracteriza o indivíduo 11752 em período quiescente. Mediante os achados histológicos descritos no presente estudo, concluiu-se que Boa c. constrictor apresenta sazonalidade em relação à gametogênese, sendo esse padrão de sazonalidade associado ao período de cópulas relatado em literatura característico de serpentes com padrão pré-nupcial.(AU)

These snakes belong to the second largest group within the reptiles, being able to present seasonality regarding spermatogenesis, with discontinuous or continuous production. The present study aims to characterize Boa constrictor constrictor reproductive biology aspects from histological findings in testicles during periods of maximum activity (period of gametogenesis) and quiescence. The testicles of two specimens of Boa c. constrictor (7767 and 11752) were cut to a thickness of 3µm in microtome, stained with 1% toluidine blue, photodocumented and described. The spermatozoa presence in the seminiferous tubule lumen in individual 7767 indicates a period of maximum gametogenesis, whereas the seminiferous tubules lumen is not very evident without spermatozoa and the absence of dividing gametic cells characterizes individual 11752 in the quiescent period. Through the histological findings we concluded that Boa c. constrictor presents seasonality in relation to gametogenesis, and the pattern of reproductive seasonality observed along with the period of copulas reported in the literature resembles the pre-nuptial pattern.(AU)

Antioxidant action of alpha lipoic acid on the testis and epididymis of diabetic rats: morphological, sperm and immunohistochemical evaluation

ABSTRACT Introduction Chronic hyperglycemia is caused by diabetes mellitus-committed genital morphophysiology, and oxidative stress is one of the main factors involved in this process. Alpha lipoic acid (ALA) can prevent metabolic and morphological changes in diabetic individuals. Objectives In present study, we evaluated the effects of regular ALA consumption on the spermatogenesis and histoarchitecture in the male genital system of diabetic rats. Materials and Methods Thirty-two Wistar rats were divided into groups: Control (CG); Diabetic Control (DCG), receiving commercial diet: ALA Group (ALAG) and Diabetic ALA Group (DALAG), fed diets with added ALA (300 mg/Kg bw). The diabetic groups received a single injection of streptozotocin (60 mg/kg). After sixty days of the diet, the animals were euthanized, and semen, testis and epididymis samples were collected. A histomorphometric analysis was performed to determine the epithelial height, tubular and luminal diameter, tubular and luminal area of seminiferous tubules and each epididymal region. Sertoli cells were evidenced using the antivimenti antibody and were quantified. The results were statistically analyzed by the ANOVA test. Results At the end of the experiment, the DALAG glycemia was significantly lower than DCG. The histomorphometric parameters of the seminiferous and epididymal tubules did not show improvement in the DALAG. However, there was an improvement in the DALAG in terms of the concentration, motility and percentage of spermatic pathologies, as well as in the number of Sertoli cells (p<0.001). Conclusions The results demonstrated that supplementation with the ALA antioxidant retards testicular lesions and preserve the process of spermatogenesis in diabetes.

In vitro transplantation of spermatogonial stem cells isolated from human frozen-thawed testis tissue can induce spermatogenesis under 3-dimensional tissue culture conditions

BACKGROUND: Sperm production is one of the most complex biological processes in the body. In vitro production of sperm is one of the most important goals of researches in the field of male infertility treatment, which is very important in male cancer patients treated with gonadotoxic methods and drugs. In this study, we examine the progression of spermatogenesis after transplantation of spermatogonial stem cells under conditions of testicular tissue culture. RESULTS: Testicular tissue samples from azoospermic patients were obtained and then these were freeze-thawed. Spermatogonial stem cells were isolated by two enzymatic digestion steps and the identification of these cells was confirmed by detecting the PLZF protein. These cells, after being labeled with DiI, were transplanted in azoospermia adult mice model. The host testes were placed on agarose gel as tissue culture system. After 8 weeks, histomorphometric, immunohistochemical and molecular studies were performed. The results of histomorphometric studies showed that the mean number of spermatogonial cells, spermatocytes and spermatids in the experimental group was significantly more than the control group (without transplantation) (P < 0.05) and most of the cells responded positively to the detection of DiI. Immunohistochemical studies in host testes fragments in the experimental group express the PLZF, SCP3 and ACRBP proteins in spermatogonial cells, spermatocyte and spermatozoa, respectively, which confirmed the human nature of these cells. Also, in molecular studies of PLZF, Tekt1 and TP1, the results indicated that the genes were positive in the test group, while not in the control group. CONCLUSION: These results suggest that the slow freezing of SSCs can support the induction of spermatogenesis to produce haploid cells under the 3-dimensional testicular tissue culture.

Stereological and morphological analysis of the effects of aging on spermatogenesis in rat testis

Abstract Purpose: To quantify, through stereological and morphometric analysis, spermatogenesis in rats undergoing the natural aging process. Methods: Seventy-two male Wistar rats were divided into 6 equal groups according to age at the time of killing: 3, 6, 9, 12, 18, and 24 months. All the rats were subjected orchiectomy and collection of testicular parenchymal fragments for histological and morphometric analysis. The numerical density of spermatids was calculated using a stereological study, and morphometric analysis was conducted to measure the height of the germinal epithelium and the area of the seminiferous tubules. Results: We found that the 18 and 24 months groups showed a significant reduction in the number of round spermatids. However, the height of the germinal epithelium was not significantly different between the groups. The area of seminiferous tubules was also significantly reduced in the elderly rats compared to that in the young ones. Conclusion: Aging of rats showed a significant reduction in the number of round spermatids and the area of the seminiferous tubules, more pronounced in the rats at 18 and 24 months of life.

Impact of testicular sperm extraction and testicular sperm aspiration on gonadal function in an experimental rat model

ABSTRACT Purpose To assess the impact of sperm retrieval on the gonadal function of rats with impaired spermatogenesis by comparing testicular sperm extraction (TESE) to aspiration (TESA). The efficacy of these procedures to sperm obtainment was also compared. Materials and Methods A pilot study showed impaired spermatogenesis, but normal testosterone (T) production after a bilateral orchidopexy applied to 26 rats, which were randomly assigned into four groups: TESE (n=7), TESA (n=7), SHAM (n=6) and Control (n=6). The T levels were measured through comparative analysis after the orchidopexy. Results There was no statistical difference in the animal's baseline T levels after orchidopexy in comparison to the controls: the TESE and TESA groups, 6.66±4.67ng/mL; the SHAM group (orchidopexy only), 4.99±1.96ng/mL; and the Control, 4.75±1.45ng/mL, p=0.27. Accordingly, no difference was found in the postoperative T levels: TESE, 5.35±4.65ng/mL; TESA, 3.96±0.80ng/mL; SHAM, 3.70±1.27ng/mL; p=0.4. The number of sperm cells found through TESE (41.0±7.0) was significantly larger than that found through TESA (21.3±8.1, p=0.001). Moreover, higher tissue weight was found through TESE (0.09±0.02g versus 0.04±0.04g, p=0.04). Conclusions The testicular sperm capture performed in rats through extraction or aspiration, after orchidopexy, did not significantly decrease the T levels. The amount of sperm found through testicular sperm extraction was higher than that through testicular sperm aspiration.

In vitro chemokine (C-C motif) receptor 6-dependent non-inflammatory chemotaxis during spermatogenesis

BACKGROUND: Chemokine (C-C motif) receptor 6 (CCR6) is present in sperm and plays a significant role in sperm motility and chemotaxis acting in the reproductive tracts. However, the expression and functional significance of CCR6 in testis are still poorly understood, especially in the process of spermatogenesis. METHODS AND RESULTS: CCR6 was expressed in spermatogenic cell lines and its expression was shown in an age-dependent upregulation manner from puberty to adulthood in mouse testis. Immunostaining results confirmed the localization of CCR 6 in testis. Further chemotaxis assays demonstrated that spermatogenic cells GC-1 and -2 exhibited a directional movement toward CCR6-specific ligand such as CCL20 or Sertoli cells in vitro. CONCLUSIONS: The present findings indicate that CCR6 is involved in the chemotaxis of spermatogenic cells in vitro and promotes chemotaxis under non-inflammatory conditions during normal spermatogenesis.

Permanent germinal epithelium and reproductive cycle of Atractosteus tropicus (Lepisosteiformes: Lepisosteidae) males, Tabasco, Mexico / Epitelio germinal permanente y ciclo reproductivo en machos de Atractosteus tropicus (Lepisosteiformes: Lepisosteidae) Tabasco, México

Abstract:The tropical gar A. tropicus plays an important ecological role as it regulates other fish stocks in different water bodies in Southeastern México. Nevertheless, wild populations are declining, and one conservation alternative is the aquaculture production and basic knowledge of reproductive biology; for males, this requires the study of germ and somatic structures of testes, to characterize the reproductive cycle, and to provide basic knowledge for exploitation and conservation models and strategies. With this aim, a total of 24 males with an average sL = 47.2 cm were collected from wild populations from the Laguna Pomposú, municipality of Jalpa de Mendez (18°19' - 93°01'12" W), Tabasco, Mexico. Fish were collected with a trawl net and were transported live to the Tropical Aquaculture Laboratory, División Académica de Ciencias Biológicas (DACBiol), Universidad Juárez Autónoma de Tabasco (UJAT). Males were killed by prolonged immersion in MS222. Testes samples were collected from each specimen and were processed using the standard histological procedures, that consisted of dehydration in an ascending ethanol series, xylol, embedding in paraffin, sectioning at 7 µm, and staining with hematoxylin-eosin (HE). The diameter of 20 seminiferous tubules (Dst), height of germinal epithelium (Hge), gonadosomatic index (GSI) and gonad volume (gV) were determined monthly. Based on morphometric and morpho-physiological characteristics, the testes consisted of a network of anastomosed tubules with non-restricted cystic spermatogenesis, and a permanent germinal epithelium. This is the first report of a permanent germinal epithelium in A. tropicus. Five reproductive classes were histologically identified: Class I Regressed; Class II Early Maturation; Class III Mid Maturation; Class IV Late Maturation; Class V Regression. Monthly GSI, gV and Dst values were lower in January and February, the testis showed spermatozoa remains and a regenerating discontinuous germinal epithelium. In March spermiogenesis increased and proliferation of spermatogonia decreased. Male tropical gar followed a seasonal reproductive cycle, indicated by the monthly variation of the reproductive classes and the reproductive season processes observed, and for which temperature and rainfall seem to stimulate reproductive activity and spermiation. Rev. Biol. Trop. 64 (4): 1597-1609. Epub 2016 December 01.

Resumen:A. tropicus tiene un papel ecológico importante, como regulador de otras poblaciones de peces, en los cuerpos de agua de México, pero sus poblaciones silvestres se reducen. Una alternativa de conservación es el cultivo, el cual requiere caracterizar el ciclo reproductivo por medio del estudio de estructuras germinales y somáticas de los testículos, conocimientos que son básicos para formar modelos de aprovechamiento y conservación. Se capturaron mensualmente tres machos sexualmente maduros (N = 24), con un promedio de sL = 47.2 cm en Laguna de Pomposú, Jalpa de Méndez (18°19´59" N - 93°01´12" W), Tabasco, México, de octubre 2009 a septiembre 2010. La técnica de captura fue red de arrastre, se transportaron vivos al laboratorio de acuicultura tropical, DACBiol, UJAT. Los machos recolectados se sacrificaron con baños de inmersión en sobredosis de MS222, los testículos se procesaron para análisis histológico. Se determinó mensualmente el diámetro de 20 túbulos seminíferos (Dst), altura de epitelio germinal (Hge), índice gonadosomático (GSI) y volumen de gónada (gV). Características morfo-fisiológicas del testículo muestran que está constituido de una red de túbulos anastomosados con espermatogénesis quística no restringida, y un epitelio germinal permanente, de nuestro conocimiento es la primera vez que se reporta este tipo de epitelio en Holostei (Lepisosteiformes: Lepisosteidae). Se identificaron cinco clases reproductivas: Clase I Recrudescencia, Clase II Madurez temprana, Clase III Madurez intermedia, Clase IV Maduración tardía, Clase V Regresión, que al contrastarlo con el valor mensual de los indicadores sexuales "GSI, gV, Dst" muestra un patrón de variación; durante enero-febrero se presentan valores bajos, se observa un epitelio germinal discontinuo en regeneración; durante marzo se incrementa la proliferación de espermatogonias disminuyendo la espermatogénesis. Los machos de A. tropicus muestran una actividad reproductora estacional anual, explicado por las variaciones mensuales de los indicadores reproductores, donde la temperatura y la precipitación parecen tener un papel importante como factores que estimulan la actividad reproductora y por tanto la espermiación.

Aspects of spermatogenesis and microscopic testicular morphology in Greater Rhea, Rhea americana (Linnaeus, 1758) / Aspectos da espermatogênese e morfologia microscópica testicular em Emas, Rhea americana (Linnaeus, 1758)

The purpose of this study was to study the microscopic morphology of the testicular parenchyma of Rhea americana birds. Fifty-four 2.5±0.5 year-old male adults bred in captivity. were used. During commercial slaughter, samples of testis were collected in November/2005, December/2006 and May/2007, in order to compare possible differences. The samples underwent optical microscopy analysis and measurements of seminiferous tubule (ST) total diameters, lumen, epithelium thickness and the relative volume of parenchyma. The ST had circular form in transverse cross sections. November/2005 and December/2006 samples had many types of germinative cells and spermatozoa in lumen, but in May/2007 the samples of epithelium were poor regarding meiotic and mitotic pictures, and it was difficult to find any spermatozoon; in many tubules the lumen was inexistent or diminished. In December/2006 and May/2007 the averages were: tubule diameter 110.3 and 5.3mµ, lumen 52.4 and 4.5mµ, epithelium thickness 57.8 and 0.7mµ respectively. The volumetric proportions were: seminiferous epithelium 75.6 and 75.9, cysts in epithelium 2.1 and 1.0, ST 93.3 and 84.0, interstitium 6.2 and 15.6 respectively. The sperm reserves were: 19.7±2 and 0±0 x109 sperm cells in December 2006 and May 2007 respectively. Microscopic measures of seminiferous tubules, spermatic cells and diameter of the nuclei were presented. These data confirm reproductive seasonality, with breeding season in spring-summer with sperm production. A great variation n parenchyma, when compared breeding was noticeable.(AU)

O objetivo do estudo foi estudar a morfologia microscópica do parênquima testicular de emas (Rhea americana). Foram utilizados 54 machos adultos criados em cativeiro de 2,5±0,5 anos de idade. Durante o abate comercial foram coletadas amostras de testículos em novembro/2005, dezembro/2006 e maio/2007, para efeitos de comparação. As amostras foram processadas e para microscopia ótica de rotina para análise. Foram medidas diâmetro total de túbulos seminíferos (ST), lúmen, espessura do epitélio e a proporção volumétrica dos componentes do parênquima. O ST apresentou forma circular nas seções transversais. Em novembro/2005 e dezembro/2006, se observaram os tipos de células germinativas e espermatozoides no lúmen. Em maio/2007, as amostras de epitélio se observaram escassas meioses e imagens de mitose e era difícil de ver qualquer espermatozoide, em muitos dos túbulos o lúmen era inexistentes ou diminuído de tamanho. Em dezembro/2006 e maio/2007, as médias das características estudadas foram: diâmetro dos túbulos 110,3 e 5,3 mµ, lúmen 52,4 e 4,5mµ, espessura do epitélio 57,8 e 0,7mµ, respectivamente. As proporções volumétricas foram: epitélio seminífero 75,6 e 75,9, cistos no epitélio 2,1 e 1,0, túbulos seminíferos 93,3 e 84,0, interstício 6,2 e 15,6, respectivamente. Foram apresentadas medidas microscópicas de túbulos seminíferos, diâmetro dos núcleos das espermátides. Estes dados confirmam a sazonalidade reprodutiva, com época de reprodução na primavera - verão, com a produção de esperma. Foi perceptível uma grande variação nas medidas do parênquima testicular, quando se comparou a estação reprodutiva.(AU)

Cellular development of the germinal epithelium during the gametogenic cycle of the golden mussel Limnoperna fortunei (Bivalvia: Mytilidae)

AbstractThe golden mussel Limnoperna fortunei is an invasive species that has quickly dispersed and colonized several potential different habitats distributed all over the world, causing environmental and economic impacts. Thus, in order to contribute to a better understanding of the reproductive aspects of L. fortunei, we described the cellular dynamic of the male and female germinal epithelium during the annual reproductive life history of this species, with the use of high resolution histology. An approximate of 1 200 specimens of L. fortunei were sampled periodically on the upper Paraná River floodplain (Brazil), from March 2010 to December 2012. Based on the activity of the germinal epithelium and consequent germ cell development, this study has resulted in the recognition of the following reproductive phases: Developing, Spawning Capable, Regressing and Regenerating. In the characterization of these phases, the following germ cells were described for males: spermatogonia, primary and secondary spermatocytes, spermatids and spermatozoa. Cell nests, oogonia, early prophase oocytes, previtellogenic oocytes and vitellogenic oocytes (early vitellogenic oocytes, middle vitellogenic oocytes and full-grown oocytes) were described for females. The morphological data and reproductive parameters obtained, showed the value of the cellular dynamics of the germinal epithelium, for the understanding of the cyclic gonadal events during the adult reproductive life of the mollusk in general. These results on the gametogenesis of this invasive species may be a fundamental tool for the development of control strategies and programs implementation, to reduce their proliferation and impacts in natural local environments. Rev. Biol. Trop. 64 (2): 521-536. Epub 2016 June 01.

ResumenEl mejillón dorado Limnoperna fortunei (Dunker, 1857) es una especie invasora que se ha dispersado rápidamente y ha colonizado diferentes hábitats potenciales distribuidos por todo el mundo, causando impactos ambientales y económicos. El control de esta especie depende del conocimiento de los aspectos reproductivos. Entre marzo 2010 y diciembre 2012 fueron muestreados periodicamente alrededor de 1 200 especímenes de L. fortunei en la planicie de inundación del Alto Rio Paraná, Brasil. Con el fin de contribuir con una mejor comprensión de los aspectos reproductivos de L. fortunei, se describió la dinámica celular del epitelio germinativo masculino y femenino durante la vida reproductiva anual de esta especie, mediante histología de alta resolución basada en la actividad del epitelio germinal y el consecuente desarrollo de las células germinales. En este estudio se reconocieron las siguientes fases reproductivas: desarrollo, capacidad de desove, regresión y regeneración. En los machos se describieron las siguientes células germinales: espermatogonias, espermatocitos primarios y secundarios, espermátidas y espermatozoides. Mientras que en las hembras se estudiaron: nidos celulares, ovogonias, ovocitos profásicos tempranos, ovocitos pre-vitelogénicos y ovocitos vitelogénicos (ovocitos vitelogénicos tempranos, ovocitos vitelogénicos y ovocitos completamente desarrollados).Los datos morfológicos y parámetros reproductivos que se muestran aquí confirman cómo la dinámica celular del epitelio germinal permite la comprensión de los acontecimientos cíclicos gonadales durante la vida reproductiva de adultos del molusco, en general. El conocimiento de la gametogénesis de esta especie invasora puede convertirse en una herramienta fundamental para el desarrollo de estrategias de control y aplicación de programas para disminuir su proliferación en ambientes naturales.

Influence of scrotal bipartition on spermatogenesis yield and sertoli cell efficiency in sheep / Influência da bipartição escrotal sobre o rendimento da espermatogênese e eficiência das células de Sertoli em ovinos

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.

Com o objetivo de avaliar o efeito da bipartição escrotal sobre a espermatogênese em ovinos, foram utilizados os testículos de 12 ovinos sem raça definida oriundos de criadouros do município de Patos-PB, Brasil, distribuídos em dois grupos, GI de seis animais com bipartição escrotal e o GII de seis animais sem bipartição escrotal. Realizou-se a aferição da biometria testicular, em seguida, os testículos foram coletados, fixados em Bouin e fragmentos foram processados para obtenção de lâminas histológicas. Foi estimado o rendimento da espermatogênese e eficiência das células de Sertoli contando-se as células da linhagem espermatogênica no estádio I do Ciclo do Epitélio Seminífero, bem como as células de Sertoli. Os resultados foram submetidos à análise de variância pelo programa ASSISTAT v.7.6 e os valores médios foram comparados pelo teste Student-Newman-Keuls (SNK) a 5% de significância. Os parâmetros de biometria testicular não apresentaram diferença estatística (p>0,05) entre os grupos. Os rendimentos meiótico, espermatogênico e a eficiência das células de Sertoli mostraram-se superiores em animais bipartidos (p<0,05), enquanto o rendimento mitótico não diferiu (p>0,05) entre GI e GII. Os resultados indicaram existir superioridade nos parâmetros espermatogênicos de ovinos bipartidos, sugerindo que estes animais apresentam, assim como constatado em caprinos, indicativo de melhores índices reprodutivos.

El síndrome de Pasqualini: hipoandrogenismo con espermatogénesis conservada

Pasqualini y Bur publican el primer caso de eunucoidismo con espermatogénesis conservada en 1950 en la Revista de la Asociación Médica Argentina. El síndrome de hipoandrogenismo con espermatogénesis incluye: (a) eunucoidismo bien definido, (b) testículos de volumen normal con espermatogénesis completa, llegando a espermatozoides maduros en una elevada proporción de tubos seminíferos, con células de Leydig indiferenciadas e inmaduras, (c) compensación funcional completa mediante la administración de gonadotrofina coriónica, mientras ésta se aplique (d) gonadotrofinas urinarias totales dentro de límites normales, y (e) esta definición fue ampliada con la actividad normal de las otras hormonas adenohipofisarias y la ausencia de malformaciones congénitas en la mayoría de los casos. En la fisiopatogenia del síndrome de Pasqualini, conocido también como síndrome del "eunuco fértil", se demostró primero la ausencia de hormona luteinizante (LH) en el plasma y orina de estos pacientes. El segundo gran avance fueron los estudios funcionales y genéticos que validaron la hipótesis de un déficit funcional de LH en estos hombres, extendido luego a las mujeres. Varios grupos, incluyendo el nuestro, demostrarían en estos casos una LH con diferentes grados de actividad inmunológica pero biológicamente inactiva, a partir de una o más mutaciones invalidantes en el gen LHB. Por último, la comprensión acabada del síndrome de Pasqualini permitiría revertir el fenotipo y la infertilidad de estos pacientes a partir de la utilización de gonadotrofina coriónica y las modernas técnicas de fertilidad in vitro. Este artículo es una revisión histórica y un homenaje a la memoria de Rodolfo Q. Pasqualini.

Pasqualini and Bur published the first case of eunuchoidism with preserved spermatogenesis in 1950 in Revista de la Asociación Médica Argentina. The hypoandrogenism with spermatogenesis syndrome included: (a) eunuchoidism, (b) testis with normal spermatogenesis and full volume, with mature spermatozoa in a high proportion of seminiferous tubes and undifferentiated and immature Leydig cells (c) full functional compensation through the administration of chorionic gonadotropin hormone, while hCG is administered (d) total urinary gonadotrophins within normal limits (e) this definition supposes the normal activity of the pituitary and the absence of congenital malformations in general. A first step in the understanding of the physiopathogeny of Pasqualini syndrome or the so called "fertile eunuch" syndrome was the absence of LH in plasma and urine of patients. The second breakthrough was the functional and genetic studies that validated the hypothesis of a functional deficit of LH in these men: it will then also be described in some women. Different groups including ours demonstrated in these cases a LH with varying degrees of immunological activity but biologically inactive in most of the patients, due to one or more inactivating mutations in the LHB gene. Finally, the full comprehension of Pasqualini syndrome allowed to reverse the hypoandrogenic phenotype and to restore fertility in these patients through the use of chorionic gonadotropin and the modern in-vitro fertility techniques. This article is an historical review and a tribute to the memory of Rodolfo Q. Pasqualini.

Differential expression and localization of ADAM10 and ADAM17 during rat spermatogenesis suggest a role in germ cell differentiation

BACKGROUND: Extracellular metolloproteases have been implied in different process such as cell death, differentiation and migration. Membrane-bound metalloproteases of the ADAM family shed the extracellular domain of many cytokines and receptor controlling auto and para/juxtacrine cell signaling in different tissues. ADAM17 and ADAM10 are two members of this family surface metalloproteases involved in germ cell apoptosis during the first wave of spermatogenesis in the rat, but they have other signaling functions in somatic tissues. RESULTS: In an attempt to further study these two enzymes, we describe the presence and localization in adult male rats. Results showed that both enzymes are detected in germ and Sertoli cells during all the stages of spermatogenesis. Interestingly their protein levels and cell surface localization in adult rats were stage-specific, suggesting activation of these enzymes at particular events of rat spermatogenesis. CONCLUSIONS: Therefore, these results show that ADAM10 and ADAM17 protein levels and subcellular (cell surface) localization are regulated during rat spermatogenesis.

Análise morfológica e funcional do processo espermatogênico em cobaios (Cavia porcellus) da pré-puberdade até a pós-puberdade / Morphological and functional analysis of spermatogenesis in guinea pigs (Cavia porcellus) from pre-puberty to post-puberty

Este estudo descreveu as análises morfológica e funcional do processo espermatogênico em cobaios (Cavia porcellus) de cinco (S5); seis (S6); nove (S9) e onze (S11) semanas de idade (N=5/grupo). Os aspectos analisados incluíram a contagem das populações celulares presentes no estádio 1 do ciclo do epitélio seminífero (CES), eficiência das mitoses espermatogoniais (RMi), produção meiótica (RMe), rendimento geral da espermatogênese (RGE), índice de células de Sertoli (ICS) e capacidade de suporte das células de Sertoli (CSCS). Os resultados mostraram que número médio de espermatogônias A, espermatócitos primários em pré-leptóteno/leptóteno, espermatócitos primários em paquíteno, células espermatogênicas totais e células de Sertoli mostraram variações numéricas em função da idade, entretanto, não detectadas estatisticamente, enquanto espermátides arredondadas aumentaram significativamente na puberdade e depois se estabilizaram. A produção espermatogênica de cobaios de 5 a 11 semanas não atingiu o ponto de estabilização e o RMi, RMe, RGE, ICS e CSCS mostraram variação numérica significativa em função da idade. Os resultados demonstraram que Cavia porcellus na pós-puberdade 2 são um modelo experimental vantajoso para estudos de processos de reconhecimento homólogos, alinhamento, e sinapses durante a prófase meiótica; o rendimento intrínseco da espermatogênese em cobaios é semelhante ao relatado para ratos Wistar, pacas e cutias (Dasyprocta sp.) e menor do que em preás, enquanto que a eficiência funcional das células de Sertoli é superior a de cutias e ratos Wistar e inferior à de pacas, rato espinhoso e catetos. Concluiu-se que em cobaios a espermatogênese está completamente estabelecida na semana 6 de idade, indicando a fase púbere do desenvolvimento sexual, e até a semana 11 eles não atingiram a produção espermática diária máxima e, portanto, a maturidade sexual.

This study describes the morphological and functional analysis of spermatogenesis in guinea pigs (Cavia porcellus) with five (W5), six (W6), nine (W9) and eleven (W11) weeks of age (n=5/group). The aspects analyzed include counts of cell populations present in stage 1 of seminiferous epithelium cycle (SEC), efficiency of spermatogonial mitosis (EMi), meiotic production (EMe), overall yield of spermatogenesis (EOS), Sertoli cell index (SCI) and carrying capacity of Sertoli cells (CCSC). The results showed that the average number of spermatogonia type A, primary spermatocytes in pré-leptóteno/leptóteno, primary spermatocytes in pachytene, total spermatogenic cells and Sertoli cells showed numerical variations according to age; however they were statistically not detected, while round spermatids increased significantly at puberty and then stabilized. The spermatogenic production of 5 to 11-week-old guinea pigs did not reach the stabilization point, and the RMi, RME, EOS, SCI and CCSC showed significant number variation as a function of age. The results demonstrate that Cavia porcellus in post-pubertal stage 2 are an advantageous experimental model to address studies on the processes of homologous recognition, alignment, and synapsis during meiotic prophase; intrinsic yield of spermatogenesis in guinea pigs is similar to Wistar rats, paca and agouti (Dasyprocta sp.) and lower than in cavies, whereas the functional efficiency of Sertoli cells is higher than in agouti and Wistar rats, and lower than in pacas, spiny rat and collared peccaries. We conclude that in guinea pigs the spermatogenesis is fully established at 6 weeks of age, indicating the pubertal stage of sexual development, and until week 11 they do not reach the maximum daily sperm production and therefore sexual maturity.

Seasonal variation of energy reserves and reproduction in neotropical free-tailed bats Molossus molossus (Chiroptera: Molossidae) / Variação sazonal das reservas energéticas e a reprodução do morcego neotropical de cauda livre Molossus molossus (Chiroptera: Molossidae)

Seasonal variation is a key factor regulating energy metabolism and reproduction in several mammals, including bats. This study aimed to track seasonal changes in the energy reserves of the insectivorous bat Molossus molossus associated with its reproductive cycle. Adult males were collected during the four neotropical annual seasons in Viçosa - MG, Brazil. Blood and tissues were collected for metabolic analysis and testes were removed for histology and morphometry. Our results show that liver and breast muscle glycogen concentrations were significantly lower in winter. The adiposity index was significantly higher in the fall compared to winter and spring. Seminiferous tubules were greater in diameter in animals captured in fall and winter, indicating a higher investment in spermatic production during these seasons. The percentage of Leydig cells was higher in summer compared to fall and winter. We suggest that M. molossus presents a type of seasonal reproduction with two peaks of testicular activity: one in fall, with higher sperm production (spermatogenesis), and another in summer, with higher hormone production (steroidogenesis). The metabolic pattern may be associated with reproductive events, especially due to the highest fat storage observed in the fall, which coincides with the further development of the seminiferous tubules.

A variação sazonal é um fator chave na regulação do metabolismo energético e da reprodução em vários mamíferos, incluindo os morcegos. O objetivo deste estudo foi avaliar as reservas energéticas do morcego insetívoro Molossus molossus ao longo das estações anuais associadas ao seu ciclo reprodutivo. Foram coletados machos adultos durante as quatro estações anuais na cidade de Viçosa - MG, Brasil. Para as análises metabólicas foram coletados tecidos e sangue, e os testículos foram removidos para análises histológicas e morfométricas. Os resultados mostram que as concentrações de glicogênio no fígado e músculo peitoral foram significativamente menores no inverno. O índice adiposo foi significativamente maior no outono em relação ao inverno e primavera. Os túbulos seminíferos apresentaram maiores diâmetros nos animais coletados no outono e inverno, indicando um maior investimento na produção espermática durante estas estações. A porcentagem de células de Leydig foi maior no verão em comparação com outono e inverno. Sugere-se que M. molossus apresenta um tipo de reprodução sazonal com dois picos de atividade testicular: uma no outono, com maior produção de espermatozóides (espermatogênese), e outra no verão, com maior produção de hormônio (esteroidogênese). O padrão metabólico pode estar associado a eventos reprodutivos, especialmente devido ao maior armazenamento de gordura observado no outono, que coincide com o desenvolvimento dos túbulos seminíferos.

Reproductive cycle of the neotropical cichlid yellow peacock bass cichla kelberi: a novel pattern of testicular development

The present study describes the testicular maturation phases (associating the germ cells development and the morphological changes suffered by the germinal epithelium along the whole year), and the testicular morphology in the yellow peacock bass Cichla kelberi, relating it to other species. For this purpose, 78 specimens were studied according conventional techniques of light microscope. The testes in C. kelberi were classified as unrestricted spermatogonial lobular, an apomorphic characteristic in the recent groups of Teleost. Furthermore, were defined five testicular maturation phases: Preparatory phase; Early Germinal Epithelium Development; Mid Germinal Epithelium Development; Late Germinal Epithelium Development and; Regression. Similar classifications were described to other species indicating that the testicular classifications based on this propose, can be applied to lots of fishes. However, besides it similarity, the testicular reproductive cycle of C. kelberi follows a different pattern in the Regression phase, on which the gonadal restructuration and the spermatogonial proliferation gathers at the same time. So, the testes in C. kelberi never return to the Preparatory phase to start a new reproductive cycle, being this one present only at the first reproductive cycle in this species. This fact also explains the absence of individuals totally spent after their first reproductive cycle.

O presente estudo descreve as fases de maturação testicular (associando o desenvolvimento das células germinativas e as alterações morfológicas sofridas pelo epitélio germinativo ao longo do ano), e a morfologia testicular do tucunaré amarelo Cichla kelberi, relacionando a outras espécies. Com este propósito, 78 indivíduos foram estudados de acordo com técnicas convencionais para microscopia de luz. Os testículos em C. kelberi foram classificados como lobular espermatogonial irrestrito, uma característica apomórfica encontrada nos grupos recentes dos teleósteos. Além disso, cinco fases de maturação testicular foram definidas para C. kelberi: Fase Preparatória; Desenvolvimento Inicial do epitélio germinativo; Desenvolvimento Intermediário do epitélio; Desenvolvimento Final do epitélio germinativo e Regressão. Classificações similares foram descritas para outras espécies, indicando que a classificação gonadal baseada nesta proposta, pode ser aplicada a muitas espécies de peixes. Entretanto, apesar desta similaridade, o ciclo reprodutivo testicular de C. kelberi segue um padrão diferente na fase de Regressão, na qual a reestruturação gonadal e a proliferação espermatogonial ocorrem ao mesmo tempo. Deste modo, os testículos em C. kelberi nunca retornam à fase Preparatória para começar um novo ciclo reprodutivo nesta espécie. Este fato também explica a ausência de indivíduos totalmente esgotados após seu primeiro ciclo reprodutivo.

Gonad maturation of Haemulon plumieri (Teleostei: Haemulidae) in Ceará state, Northeastern Brazil / Maturação gonadal de Haemulon plumieri (Teleostei: Haemulidae) no Nordeste do Brasil

To elucidate the reproductive cycle of any animal, it is first necessary to understand the development of the germ cells and how this process occurs throughout the reproductive tract. Thus, the aim of the present study was to understand the oogenesis and spermatogenesis, as well as the ovarian and testis maturation and type of spawning, of Haemulon plumieri. A total of 119 females and 136 males were analysed. Based on micro and macroscopic characteristics of gonads, the following stages of maturation for females were determined: immature, maturing, ripe, partially spawning, spawning and resting. For males, only the stages immature, maturing, ripe and spent were observed. We can conclude that the ovarian development occurs cyclically, in which the female can spawn more than two times during the reproductive season with the spawning “synchronous in more than two groups”. Since males do not present the “rest” stage after copulation, the maturation process is continuous.

Para se conhecer a biologia reprodutiva de qualquer animal, é fundamental o conhecimento prévio do desenvolvimento das células germinativas e como esse processo ocorre ao longo do trato reprodutivo. Desta forma, o objetivo do presente trabalho foi descrever a oogênese e a espermatogênese, bem como o desenvolvimento maturacional dos ovários e testículos, e o tipo de desova do Haemulon plumieri. Foram analisadas 119 fêmeas e 136 machos. A partir das características macro e microscópicas das gônadas, foram estabelecidos os seguintes estágios de maturação para fêmeas: imatura, em maturação, matura, parcialmente desovada, desovada e em repouso. Para os machos, foram identificados apenas os seguintes estágios: imaturo, em maturação, maturo e esvaziado. A partir das análises, pôde-se concluir que o desenvolvimento ovariano é cíclico, em que a fêmea pode desovar mais de duas vezes por estação, por apresentar a desova do tipo ‘sincrônica em mais de dois grupos'. Uma vez que os machos não apresentam o estágio ‘em repouso' após a cópula, o processo de maturação é contínuo.

Seasonal testicular changes in Dendropsophus minutus Peters, 1872 (Anura, Hylidae)

The reproductive cycle in anurans may be either continuous or discontinuous. These differences may be connected to seasonal climate changes and/or to anthropic activity. Forty adult male individuals of the Dendropsophus minutus species were collected during one year, in the municipality of Chapada dos Guimarães (Mato Grosso, Brazil). The testicles were studied under light and transmission electron microscopy. No variations were observed when the diameter of the seminiferous tubules and the thickness of the interstitial tissue were studied. However, changes in spermatogenesis were conspicuous and indicated that the reproductive cycle of D. minutus in Chapada dos Guimarães is discontinuous and seems related to variations in air temperature and rainfall.

Microscopia e morfometria de componentes tubulares de testículos de coelhos suplementados com geleia real / Microscopy and morphometry of tubular components of rabbit testis supplemented with royal jelly

Estudaram-se os efeitos da geleia real sobre a espermatogênese de coelhos tratados com diferentes concentrações de geleia real. Os tratamentos foram formados por três grupos: grupo-controle; grupo que recebeu 0,5mg/dia de geleia real; e grupo que recebeu 1,0mg/dia de geleia real. O estudo envolveu a morfometria testicular. Não houve diferença entre os tratamentos quanto aos pesos corporal (T1=3,20±0,19kg, T2=2,96±0,30kg e T3=3,21±0,37kg) e gonadal (T1=2,36±0,33g, T2=2,53±0,33g e T3=2,64±0,39g) e quanto aos índices gonadossomático (T1=0,15±0,02%, T2=0,17±0,03% e T3=0,16±0,02%) e tubulossomático (T1=0,06±0,01%; T2=0,07±0,01% e T3=0,06±0,01%). O diâmetro médio dos túbulos seminíferos (T1=225,95±13,27µm, T2=239,68±21,50µm e T3=231,57±15,94µm), a altura do epitélio seminífero (T1=66,05±5,37µm, T2= 73,47±9,11µm e T3=63,34±4,79µm) e o comprimento de túbulos seminíferos por testículo (T1=46,63±13,44m, T2=43,58±12,17m e T3=46,96±9,54m) e por grama de testículo (T1=19,50±2,68m, T2=17,12±3,91m e T3=17,78±1,98m) não diferiram entre tratamentos. Conclui-se que a suplementação com geleia real, nas doses utilizadas, não altera as características testiculares avaliadas.

This study aimed to investigate the effects of royal jelly on spermatogenesis in rabbits treated with different concentrations of RJ (Control; 0,5mg/day; and 1,0mg/day) using testicular morphometry. There was no significant difference between the body weight (T1= 3.20±0.19kg; T2= 2.96±0.30kg; T3=3.21±0.37kg) and gonadal weight (T1= 2.36±0.33g; T2= 2.53±0.33g; T3= 2.64±0.39g), gonadossomatic index (T1= 0.15±0.02%; T2= 0.17±0.03%. T3= 0.16±0.02%) and tubulossomatic index (T1= 0.06±0.01%; T2= 0.07±0.01%. T3= 0.06±0.01%) between treatments, showing that the percentage of body mass, and the percentage of seminiferous tubules allocated in testis were similar in the 3 experimental groups. Similarly, the mean diameter of the seminiferous tubules (T1= 225.95±13.27µm; T2=239.68±21.50µm; T3= 231.57±15,94µm), the height of the seminiferous epithelium (T1=66,05±5,37µm; T2=73.47±9.11µm; T3=63.34±4.79 µm) and length of seminiferous tubule for testis (T1=46.63±13.44m; T2=43.58±12.17m; T3=46.96±9.54m) and per gram of testis (T1=19.50±2.68m; T2=17.12±3.91m; T3=17.78±1.98m) did not differ statistically. It was concluded that supplementation with royal jelly, at the doses used, did not alter the testicular parameters evaluated here.