The quantification of testicular cells during the postnatal development in two Caviomorph rodents: the guinea pig (Cavia porcellus) and the cutia (Dasyprocta agouti)

ABSTRACT The germinative, Sertoli and Leydig cells of two caviomorph rodents (Cavia porcellus and Dasyprocta agouti) were counted as well as the estimation of the total volume of the testis and the total volume of seminiferous tubules and interstitium in prepubertal, pubertal and adult animals. The number of spermatogonia, spermatocytes and spermatids cells increased during the pubertal phase in both rodents, notably the spermatid cells. The spermatocyte and spermatid slightly decreased in the adult of both rodents, but the increment in spermatogonia cells number was seen, mainly in cutias. The number of Sertoli cells increased in pubertal rodents, but in the adult the number reduced. Substantial number of Leydig cells was counted in pubertal and adult guinea pigs. In cutias, the number of Leydig cells increased in pubertal phase and decline in adults. The design-based stereological method has proven to be unbiased and reliable to be applied in reproduction studies.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu) / Avaliação morfológica e morfométrica de espermatozoides de catetos (Pecari tajacu)

The aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries (Pecari tajacu). Semen from 10 males was obtained by electroejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic alterations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through morphometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

O objetivo deste estudo foi comparar diferentes métodos de coloração para avaliação da morfologia espermática por microscopia de luz e também descrever a morfometria completa de espermatozoides de catetos (Pecari tajacu). Sêmen de 10 machos foi obtido por eletroejaculação e avaliado quanto à motilidade espermática, vigor e concentração. Foram preparados por três diferentes métodos de coloração: Rosa de Bengala, Azul de Bromofenol e Eosina-Nigrosina. Os esfregaços foram avaliados por microscopia de luz, e determinado o percentual das alterações morfológicas. Ainda, a análise da morfometria espermática foi realizada por microscópio de luz acoplado a um softwere de análise de imagens. Os esfregaços corados com Rosa de Bengala apresentaram melhores resultados de visualização da cauda, peça intermediária e cabeça dos espermatozoides. O uso do corante Eosina-Nigrosina não permitiu uma adequada impregnação e alguns dos espermatozoides apresentaram pouco contraste com o fundo da lâmina. Uma maior incidência de cauda fortemente enrolada foi verificada com o uso do corante Azul de Bromofenol (P<0.05). Através da avaliação morfométrica foi observada que a cauda ocupa a maior proporção (89%) do espermatozoide, e a cabeça apresenta-se discretamente alongada. De acordo com os resultados, o uso do corante Rosa de Bengala é recomendado para a avaliação morfológica de espermatozoides de catetos.

Período de permanência de espermatozoides em glândulas hospedeiras de espermatozoides e glândulas infundibulares em codorna de corte / Stay period of sperm in sperm-storage glands and in infundibular glands in quails

Para determinar o tempo de permanência de espermatozoides nas glândulas hospedeiras de espermatozoides (GHEs) e nas glândulas infundibulares (GIs) de codorna de corte (Coturnix coturnix coturnix), foram utilizados 12 machos e 66 fêmeas, totalizando 78 codornas em fase reprodutiva. As fêmeas foram distribuídas em 11 grupos e acasaladas por 24 horas em gaiolas individuais. Os machos, utilizados de modo intercalado, foram separados do contato com as fêmeas e colocados em descanso. As aves do grupo-controle (G0 - seis fêmeas) foram abatidas no início do experimento, enquanto as 60 fêmeas acasaladas foram distribuídas em 10 grupos (G1 a G10, com seis fêmeas cada) e abatidas a cada período de 24 horas, de forma sequencial. Fragmentos foram obtidos da região uterovaginal e do infundíbulo e submetidos às análises histológica, histoquímica e histométrica com técnicas de rotina. Os resultados morfométricos mostraram que 46% das GHEs continham espermatozoides em seu lume no primeiro dia após o acasalamento, diminuindo gradativamente nos dias posteriores chegando a 3% no quinto dia. Nesse período, os espermatozoides ascendem em direção às GIs, onde permanecem viáveis e férteis por, pelo menos, 96 horas após deixarem as GHEs, possibilitando a postura de ovos férteis por 10 dias, em média, após o acasalamento.

Sperm-Storage Tubules (SSPs) and Infundibular Tubules (ITs) are the structures responsible for sperm storage in the oviduct of birds, snakes, alligators and turtles after mating. Aiming to determine length of stay of sperm-storage tubules (SSPs) and infundibular tubules (ITs) cutting quail, Coturnix coturnix coturnix, we used 12 males and 66 females, totaling 79 quails in the reproductive phase. The females were allocated into 11 groups and mated for 24 hours in individual cages. The males used were merged and separated from contact with females and placed at rest. The poultry of the control-group (G0 six females) was slaughtered at the beginning of the experiment, the 60 previously mated females were allocated into 10 groups (G1 to G10, with six females each) and were slaughtered sequentially. On the 10th day, the last group (G10) was shot. The fragments obtained from the utero-vaginal region and the infundibulum of each female underwent histological techniques, immunohistochemistry and morphometry routine. The morphometric results showed that GHEs had 46% of the sperm in his heat on day 1 after mating, decreasing gradually in the after days reaching 3% on day 5. At this time they increase toward the infundibular tubules, where they remain viable and fertile for at least another 96 hours (4 days) after leaving the SSPs, allowing these birds to lay fertile eggs for 10 days on average after mating.

Bull spermatozoa express receptors for platelet-activating factor

Platelet-activating factor (PAF; 1-O-Alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) is a ubiquitous phospholipid that is implicated in the mediation of a wide variety of reproductive processes. To better understand the role of PAF in bovine reproduction, it was designed experiments to: (a) determine whether bull spermatozoa express receptors for PAF and (b) study the effect of exogenous PAF on in vitro sperm physiology (i.e., capacitation, acrosome reaction, motility, and fertilizing ability). Bull sperm express PAF receptor as determined by two approaches: RT-PCR and immunofluorescence. However, exposure of spermatozoa to different concentrations of exogenous PAF (10-11-10-6 M) did not affect capacitation, acrosome reaction or motility. Consistent with these findings, coculture of gametes in medium containing increasing concentrations of PAF (1 x 10-8-8 x 10-6 M) did not improve in vitro fertilization outcome as measured by percentage of inseminated oocytes reaching 2-cell stage 48 h after fertilization. In contrast, PAF at 8 x 10-6 M concentration significantly inhibited IVF. In conclusion, although bull sperm have PAF receptors, exposure of bull spermatozoa to exogenous PAF failed to enhance the sperm function parameters measured in this study. Additional studies are warranted to elucidate the biological role of PAF on bull spermatozoa.

El factor activador de plaquetas (PAF; del inglés Platelete Activating Factor; 1-O-Alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) es un fosfolípido ampliamente distribuido que participa como mensajero mediador en diferentes procesos reproductivos. Para comprender mejor la participación del PAF en la fisiología espermática bovina se diseñaron experimentos para: (a) determinar si los espermatozoides de toro expresan receptores para PAF y (b) estudiar el efecto del PAF sobre el comportamiento de los espermatozoides bovinos in vitro (capacitación, reacción acrosomal y capacidad fertilizante). De acuerdo a los resultados obtenidos por RT-PCR e inmunofluorescencia, los espermatozoides de toro expresan receptores para PAF. Sin embargo, la exposición de los espermatozoides a concentraciones crecientes de PAF exógeno (10-11-10-6 M) no afectó la capacitación, reacción de acrosoma ni la motilidad. En concordancia con estos hallazgos, el cocultivo de gametas (ovocitos y espermatozoides) en medio al cual se le había adicionado PAF (1 x 10-8-8 x 10-6 M) no mejoró la tasa de fertilización medida como el porcentaje de ovocitos inseminados que alcanzaron el estadio de 2 células 48 hs después de la inseminación. Por el contrario, PAF a una concentración de 8 x 10-6 M inhibió significativamente la tasa de fertilización. En conclusión, a pesar de que los espermatozoides bovinos poseen receptores para PAF, el agregado de PAF al medio de cultivo no mejora las funciones espermáticas examinadas en el presente trabajo. Otros estudios serán necesarios para dilucidar la participación del PAF en la fisiología espermática del toro.

Morphologic and morphometric analysis of testis of Pseudis limellum (Cope, 1862) (Anura, Hylidae) during the reproductive cycle in the Pantanal, Brazil

The spermatogenesis of Pseudis limellum, from the Southern Pantanal, Brazil, was studied from July 1995 to May 1996, through histological sections of the testis. The cells could be differentiated as: primary spermatogonia, large cells, generally with bilobed nucleus; secondary spermatogonia, smaller cells, with darker cytoplasm, chromatin of radial form; primary and secondary spermatocytes, differentiated according to the different stages of the nucleus during the successive cells divisions. Furthermore, we observed cells in process of morphologic differentiation: rounded spermatids much smaller, with nucleus containing chromatin in compacting process and cytoplasm reduction; elongated spermatids, generally parallel organized in well defined bundles, with the anterior region directed toward the periphery of the seminiferous tubule and the tail directed toward the lumen. Spermatozoa are free in the lumen of the seminiferous tubule. All the cells are organized as cysts, which are supported by a large amount of Sertoli cells. The spermatogenesis in P. limellum is very similar to that of other anurans, but peculiarities were observed regarding the organization of the germ cells, the great amount of free Sertoli cells in the lumen of testis collected in May, and the long cytoplasmatic extensions of the cells bearing pigments and involving the seminiferous tubule. The diameter of the seminiferous tubule (SD) exhibited an annual mean of 251.79 +/- 37.57 microm. Spermatozoa number by seminiferous tubule (SN) exhibited an annual mean of 306.66 +/- 39.83, also with higher and lower values at each month. Variations in SD and SN were not significantly correlated with climatic variables. In this species, reproduction occurs throughout the year in ponds and flooded areas, despite the seasonal climate of the Pantanal. Although males varied in their annual reproductive activity, they were considered potentially reproductive in all months throughout the year.

Sobre el origen ontogénico del ser humano: La solución científica / On the ontogenetic origin of human beings: The scientific solution

Every living being is the result of a genome-environment interaction. Neither human oocytes nor spermatozoids have human functional genomes, but the zygote that they constitute may have a human functional genome and other functional genomes such as those of the hydatidiform mole, polyploids, and non-human living beings. When the zygotic human functional genome is integrated and activated, the biotic humanity is acquired. This may occur when the paternal chromatin decondenses; the nuclear environment and envelope of both nuclei are changed to constitute pronuclei; the replacement of sperm protamines by histones; genome imprinting modifications; centriole duplication; and more importantly, the fourfold genome replication. Other propositions on the origin of humans are: embryo implantation [6-7 days post fertilization, (dpf)]; the appearance of the antero-posterior axis; the limit for monozygote twining (13dpf) and the appearance of the neural tissue (16dpf). They are refuted because some mammals do not implant; embryo axes are present in the zygote; some animals regenerate complete individuals from each part in which they are divided; plants do not have neural system; a human whose brain was destroyed by cancer continues to be a human. Alternative propositions coming from philosophies, theologies, perceptive knowledge, beliefs and intuitions and based on conceptualizations like person, anima, soul, organization, socio-cultural relations are ideologically or religiously biased and based on irreducible beliefs such as faith. They lead to disagreement rather than to agreement.

Testicular maturation of Oligosarcus hepsetus (Cuvier) (Actinopterygii, Characidae) in a brazilian tropical reservoir

Seis classes de maturação dos testículos de Oligosarcus hepsetus (Cuvier, 1829), um Characiforme carnívoro de médio porte, foram descritas com base em técnicas macro e microscópicas. Um total de 175 indivíduos foi capturado mensalmente entre abril/2001 e junho/2002 no Reservatório de Lajes, Brasil, uma das maiores áreas represadas do Estado do Rio de Janeiro. As classes reprodutivas foram estabelecidas de acordo com mudanças na morfologia dos testículos e nos estádios das células germinativas: repouso, maturação inicial, maturação final, maduro, parcialmente esvaziado e totalmente esvaziado. Os testículos da classe em repouso apresentaram espermatogônias e espermatócitos ao longo da parede dos túbulos seminíferos, enquanto as espermátides foram encontradas no lúmen dos túbulos. Nos testículos em maturação inicial, a espermatogênese é intensa; em maturação avançada e maduros, os túbulos estão repletos de sêmen, que são característicos de peixes aptos a reprodução. Testículos esvaziados apresentaram sêmen residual nos túbulos seminíferos, coincidindo com a diminuição do IGS e com uma grande redução na espermatogênese. Em geral, a morfologia dos testículos e o desenvolvimento das classes de maturação de O. hepsetus no Reservatório de Lajes não diferiram de espécies similares em outros ambientes lênticos. Os menores valores do IGS para o reservatório oligotrófico de Lajes, quando comparados com lagos naturais eutróficos, sugerem que O. hepsetus pode estar alterando este aspecto de sua estratégia reprodutiva no ambiente artificial.

Estudo morfológico e histomorfométrico sobre variações sazonais do ducto deferente de codorna da variedade italiana / Seasonal study quail’s vas deferens of italian variety: morphology and histomorphometry

No outono o ducto deferente de codorna da variedade Italiana foi observado como um ducto simples, delgado e retilíneo em toda a sua extensão. Assim sendo, secções histológicas transversais deste ducto mostraram-no com forma circular, sendo revestido por epitélio pseudoestratificado cilíndrico que forma pregas longitudinais. Essas pregas adentravam o lúmen tubular, que aparecia freqüentemente vazio de espermatozóides. Porém, no inverno, primavera e verão a aparência morfológica do ducto deferente era a de um túbulo grandemente enovelado. Logo, cada secção histológica transversal do ducto deferente, ao longo de toda a sua extensão, mostrava-se estruturada como secções tubulares dispostas paralelamente entre si. Estas secções tubulares apareciam irregulares quanto à forma e variáveis em número, estando interconectadas por tecido conjuntivo frouxo adventicial. Nestas observações no inverno, primavera e verão o lúmen tubular vaso-deferencial estava totalmente preenchido por espermatozóides e fluido seminal. Portanto, pôde-se concluir, inclusive com base em estudo prévio sobre a cinética testicular nesta variedade Italiana de codorna, que a produção de espermatozóides bem como a sua emissão, estocagem e ejaculação através do ducto deferente não cessam ao longo da maior parte do ano, exceto no outono a etapa quiescente do ciclo reprodutivo circum-anual desta ave doméstica.

In the autumn the vas deferens of the Italian variety of domestic quail appeared as a single, thin, and straight duct along its total extension. Thus, transversal histologic sections of this duct showed a circular tubular shape. The pseudoestratified columnar epithelium that lined the vas deferens presented longitudinal folds which invaded the lumen frequently empty of spermatozoa. Although in the winter, spring and summer the usual morphological appearance of the quail’s vas deferens was seen as a highly coiling duct. So, each transversal histologic section of the vas deferens, in all the segments, showed parallel cut sections of the duct being irregular in shape and variable in number and interconnected by the adventitial loose connective tissue. In these observations, the tubular lumen was totally performed by spermatozoa and seminal fluid. Consequently, with previous base on the testis kinetics of the Italian quail variety, it was concluded that the spermatozoa production, followed by emission, storage and ejaculation of spermatozoa through the vas deferens did not stop during the winter, spring and summer, but ceased in the autumn the quiescent phase of the circannual reproductive cycle in this bird.

Ultrastructure of spermatogenesis and sperm development in Saccocoelioides godoyi Kohn & Froes, 1986 (Digenea, Haploporidae)

Ultrastructural observations of spermatogenesis and sperm development of Saccocoelioides godoyi, an intestinal parasite of Leporinus friderici (Bloch, 1794) are described. The irregular-shaped spermatogonia form a peripheral layer, and show a prominent nucleus. Spermatocytes are larger than spermatogonia, and in the early stage present synaptonemal complex. Spermatids show nuclei smaller than the spermatocytes. Spermiogenesis is characterized by outgrowth of the zone of differentiation, presenting basal bodies, separated by an intercentriolar body. At the end of this process, the spermatozoa are released into the residual cytoplasmic mass. The spermatozoa of S. godoyi are elongate, similar to the pattern described for other Digenea, showing nuclei, mitochondria and two axonemes with the 9+1 configuration. The peripheral cortical microtubules on the dorsal and ventral faces are laterally interrupted

The use of periovulatory human cervical mucus in human sperm capacitation and in-vitro fertilization of foreign eggs.

The time requirement of a routine in-vitro fertilization experiment using human spermatozoa and zona-free hamster oocytes is more than one working day. We have used a simple process of simultaneous sperm washing and capacitation before challenging them against oocytes. Spermatozoa were recovered up to 90 minutes from the proximal end of a column of periovulatory human cervical mucus into Brinster, Whitten and Whittingham's (B.W.W.) medium. Without any further capacitation 50 microliters of these spermatozoal suspension, in the concentration of 2.5-3 X 10(6)/ml, fertilized 81% of a total of 100 eggs. A dramatic decrease in the fertilization rate (18%) was observed when these spermatozoa were further capacitated in-vitro for five hours before actual egg incubation for three hours. Both selection and accelerated capacitation may contribute to this result.