Immunolocalization of steroidogenic enzymes in the vaginal mucous of Galea spixii during the estrous cycle.

BackgroundThe synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase and CPR expression in vaginal mucosa of Galea spixii (Spix cavy) by immuno-histochemical and western immunoblot analyses.MethodsStages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus. The ovaries and testis were used as positive control tissues for immunohistochemistry.ResultsData from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues.ConclusionCurrent results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression. Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens

Steroidogenesis during Dry and Wet Season of Free-Living Male Scorpion Mud Turtle (<em>Kinosternon scorpioides</em>)

This study aimed to elucidate seasonal reproductive patterns by associating the immunolocalization of the steroidogenic enzymes responsible for estrogen and androgen synthesis, cytochromes P450 aromatases (P450 arom), 17-hydroxylase/17,20-lyase (P450c17) respectively, as well as the redox partner NADPH cytochrome P450 oxido-reductase (CPR) in the male mud turtle (Kinosternon scorpioides). The animals were collected at São Bento, Maranhão, Brazil. Testicles and epidydimes from 38 adult animals were characterized by immunohistochemistry (IHC) and western blotting. Data were analyzed in two seasons: dry and wet season. In addition, the gonadosomatic index, morphometry, concentrations of testosterone, corticosterone, and estradiol were analyzed. The changes in biometrics, cell morphometry, and testicular steroidogenesis between the wet and dry seasons support that estrogen produced by the Leydig cells might influence germ cell production during spermatogenesis, and the expression of the enzymes P450 arom and P450c17 in the Leydig cells that play a role in testicular quiescence. We found that the P450arom, P450c17, and CPR are present in the testes and epididymides at both seasons. These findings support a state of male reproductive quiescence during the dry season and active spermatogenesis in the wet season

Origin and distribution of the ischiatic nerve in mixed-breed sheep

O presente trabalho estudou, por meio de dissecações, a origem e distribuição do nervo isquiático em 30 fetos de ovinos (Ovis aries) sem raça definida, machos ou fêmeas, obtidos de abortos, natimortos e mortes naturais de fêmeas gestantes de núcleos criatórios na região do Triângulo Mineiro, estado de Minas Gerais, Brasil. Injetou-se formaldeído a 10% por meio de canulação da aorta descendente torácica e, posteriormente, o material foi mantido submerso na referida solução por um período mínimo de 48 horas antes do início das dissecações. O nervo isquiático originou-se do ramo ventral do último nervo espinhal lombar, ora do sexto nervo espinhal lombar (L6), ora do sétimo (L7), quando presente, e dos ramos ventrais dos primeiro e segundo nervos espinhais sacrais (S1 e S2, respectivamente), podendo apresentar a contribuição do ramo ventral do terceiro nervo espinhal sacral (S3). O nervo isquiático cedeu ramos aos músculos: glúteo superficial, glúteo médio, glúteo acessório, glúteo profundo, gêmeo, quadrado femoral, adutor, bíceps femoral, semitendinoso e semimembranoso. Os ramos terminais do nervo isquiático foram os nervos tibial e fibular comum, tendo suas origens distalmente ao trocânter maior do fêmur. Estatisticamente, através da aplicação do teste de Wilcoxon (0,05), não houve diferenças significativas entre as frequências dos ramos musculares do nervo isquiático e os antímeros, independentemente do número de ramos musculares.Current research studied, by dissection, the origin and distribution of the ischiatic nerve in 30 fetuses of mixed-breed male or female sheep (Ovis aries), obtained from abortions, stillborns and natural deaths of pregnant females on farms in the Triângulo Mineiro region, state of Minas Gerais, Brazil. Formaldehyde 10% was injected through the cannulation of descending thoracic aorta and the material was kept immersed in the solution for at least 48 hours before dissection. The ischiatic nerve originated from the ventral branch of the last lumbar spinal nerve, either from the sixth lumbar spinal nerve (L6) or from the seventh (L7), when present, and from the ventral branches of first and second sacral spinalnerves (S1 and S2, respectively), and possibly from the ventral branch of third sacral spinal nerve (S3). The ischiatic nerve provided branches to the superficial gluteal, middle gluteal, accessory gluteal, deep gluteal, gemelli, quadratus femoris, adductor, biceps femoris, semitendinosus and semimembranosus muscles. The tibial and common peroneal nerves were the terminal branches of the ischiatic nerve, originating distally towards the greater trochanter of the femur bone. Wilcoxon’s test (0.05) showed that statistically there were no significant differences between the frequencies of the muscular branches of the ischiatic nerve and the antimeres, regardless of the number of muscular branches

Gross and microscopic morphology of the coati tongue (Nasua nasua)

O quati (Nasua nasua) é um animal que pertence à Família Procyonidae. Foram utilizados três animais ortotanasiados, de ambos os sexos, provenientes do Criatório Científico de Animais Silvestres, Centro Universitário Fundação de Ensino Octávio Bastos (Cecrimpas, Unifeob) autorizado pelo IBAMA (Proc.02027.003731/04-76). Para a análise macroscópica, as línguas foram retiradas, analisadas e foto-documentadas. Para análise microscópica, as línguas foram processadas rotineiramente pela técnica de microscopia eletrônica de varredura e inclusão em Paraplast; pela técnica de microscopia de luz os fragmentos foram cortados em micrótomo, com espessura média de 5mm e corados em HE e Picrosírius com fundo de hematoxilina. Os resultados macroscópicos e microscópicos mostram que a língua do quati apresenta papilas filiformes, fungiformes, valadas e cônicas sendo estas distribuídas nas regiões rostralis, medialis e caudalis. Histologicamente, a língua do quati é revestida por um epitélio pavimentoso estratificado queratinizado apresentando camada basal, espinhosa, granulosa e córnea com fibras de músculos estriados esqueléticos longitudinais e transversais e diversas glândulas. De acordo com os resultados pode-se concluir que a língua do quati possui características macroscópicas e microscópicas semelhantes aos canídeos, tendo como diferença o número de papilas valadas e o grau de queratinização.The coati (Nasua nasua) is an animal that belongs to the Procyonidae family. For this study we used three orthotanasized animals of both sexes, obtained from the Scientific Center for Wild Animal Breeding, University Center Octavio Bastos Educational Foundation (Cecrimpas, Unifeob), authorized by IBAMA (Proc.02027.003731/04-76). For microscopic analysis, tongues were processed routinely through electron microscopy scanning and inclusion in Paraplast; for light microscopy, the fragments were cut by microtome with an average thickness of 5mm and stained with H&E and Picrosirius counterstained with hematoxylin. The macroscopic and microscopic results show that the tongue of coati has filiform, fungiform, vallate and conical papillae distributed in the rostralis, medialis and caudalis regions. Histologically, the tongue is lined by a keratinized stratified squamous epithelium with spinous, granulous and keratin basal layer, with striated longitudinal and transverse skeletal muscles fibers, and several glands. We can conclude that coati's tongue has macroscopic and microscopic features similar to the ones of Canidae, with differences in the number of vallate papillae and degree of keratinization

Immunolocalization of steroidogenic enzymes in the vaginal mucous of Galea spixii during the estrous cycle

Abstract\ud \ud Background\ud The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase and CPR expression in vaginal mucosa of Galea spixii (Spix cavy) by immuno-histochemical and western immunoblot analyses.\ud \ud \ud Methods\ud Stages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus. The ovaries and testis were used as positive control tissues for immunohistochemistry.\ud \ud \ud Results\ud Data from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues.\ud \ud \ud Conclusion\ud Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression. Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens.This work was supported by grants from São Paulo Research Foundation/\ud FAPESP, Sao Paulo, Brazil (Process Number: 2011/03655-2) and the National\ud Council of Scientific Researches/CNPq (Process Number: 402220/2010-2)

Enzymes expression: of the cytochrome P450 aromatase, NADPHcytochrome P450 redutase and cytochrome P450c17 (17-&alpha;-hidroxilase/17, 20-lyase) in the vagina of female cavies (Galea spixii, Wagler, 1831)

Para a metabolização de hormônios esteroides sexuais é essencial a participação de enzimas esteroidogênicas. A enzima citocromo P450c17 é responsável pela produção de andrógenos e a enzima citocromo P450 aromatase é responsável pela produção de estrógenos, sendo que, ambas as enzimas necessitam formar um complexo com uma enzima parceira, denominada NADPH citocromo P450 redutase, para realizar a metabolização destes hormônios essenciais para a diferenciação sexual. Objetivou-se imunolocalisar as três enzimas acima citadas no tecido vaginal de fêmeas de roedores Galea spixii. Para tanto, o experimento foi desenvolvido utilizando técnicas de citologia esfoliativa vaginal para definição das fases do ciclo estral, técnicas de microscopia de Luz, Eletrônica de Varredura e testes de imunohistoquímica para detecção das enzimas: citocromo P450c17, citocromo P450 aromatase, e NADPH citocromo P450 redutase. Constatou-se no presente estudo que o ciclo estral das fêmeas de preás silvestres do semiárido apresenta ciclo estral com duração de 15,85 ± 1,4 dias, com a formação e ruptura da membrana de oclusão vaginal durante cada ciclo estral. A vagina apresenta um clitóris hipertrofiado com o óstio uretral que se abre no topo do mesmo, e ausência de um vestíbulo vaginal. O epitélio da mucosa vaginal sofre modificações proliferativas, se espessando e se adelgando de acordo com a respectiva fase do ciclo estral. As enzimas citocromo P450 aromatase e NADPH redutase estão imunolocalizadas durante todo o ciclo estral no epitélio e tecido conjuntivo da vagina de Galea spixii. Por outro lado, a enzima P450c17 está imunolocalizada no epitélio vaginal durante todas as fases do ciclo estral, porém não está presente no estro. Com estes dados pode-se sugerir que ocorre uma metabolização local de hormônios estrógenos e andrógenos no tecido vaginal o que podem estar relacionado com a proliferação celular, variação na vascularização e inervação e ruptura da membrana de oclusão vaginal.To the metabolism of sex steroid hormones is essential the participation of esteroidogenic enzymes. The enzyme cytochrome P450c17 is responsible for androgen production; and the enzyme aromatase cytochrome P450 is responsible for estrogen production, however, both enzymes require forming a complex with an enzyme partner called NADPH cytochrome P450 reductase to perform the metabolism of these hormones, which are essential for sexual differentiation. This study aimed to immunolocalization of the three enzymes above mentioned in the vaginal tissue from female rodents Galea spixii. Therefore, the experiment was carried out using vaginal cytology techniques for definition of the estrous cycle phases, techniques for light microscopy, scanning electron and immunohistochemical tests for the detection of enzymes: cytochrome P450c17, cytochrome P450 aromatase, and NADPH cytochrome P450 reductase. It was found that the estrus cycle of Galea spixii female lasts 15.85 ± 1.4 days, with the formation and rupture of vaginal closure membrane during each oestrous cycle. The vagina has a hypertrophied clitoris with the urethral orifice that opens on top of it, and it is observed the absence of a vaginal vestibule. The vaginal mucosal epithelium undergoes proliferative changes, with thickening and thinning according to the respective estrous cycle phase. The enzymes cytochrome P450 aromatase and NADPH reductase are immunolocated throughout the oestrous cycle in the epithelium and connective tissue of the vagina from Galea spixii. On the other hand, the enzyme cytochrome P450c17 is immune located in the vaginal epithelium during all stages of the estrous cycle, except at estrous. With these data may be suggested that there is a local estrogens and androgens metabolism in vaginal tissue which may be associated with cell proliferation, vascularization and innervation variation besides the formation and rupture of the vaginal closure membrane