Morfologia do funículo espermático em caprinos da raça Bhuj Brasileira

Estudamos em 40 caprinos adultos da raça Bhuj Brasileira os aspectos histológicos do funículo espermático. Observamos que este se acha envolvido por uma cápsula de tecido conjuntivo fibroelástico denso, de espessura variável, pregueada em alguns pontos, e revestida por mesotélio que circunda todo o conjunto vásculo-nervoso, e projeta-se para formar o mesoducto deferente. Em posição subcapsular, verifica-se uma camada de tecido conjuntivo fibroelástico frouxo, de espessura variável, que circunda parcialmente o funículo espermático, isolando nas regiões deferencial e abdeferencial, conjuntos vásculo-nervosos, responsáveis pela nutrição do epidídimo. Na região do mesoducto deferente, o tecido subcapsular acompanhado de tecido adiposo constitui a camada interna deste meso, formando a sua adventícia e abrigando vasos e nervos deferenciais. Na região abdeferencial, pequenos acúmulos de tecido adiposo são vistos de permeio aos vasos e nervos desta região. Entre as artérias, veias e nervos testiculares, bem como entre os vasos das regiões deferencial e abdeferencial, observa-se o tecido conjuntivo denso, intervascular, rico em fibras elásticas, que constitui as adventícias contínuas destes vasos. O arranjo vascular mostra que o segmento da artéria testicular, contido no funículo espermático, apresenta trajeto sinuoso. Estando envolvido pelo plexo venoso pampiniforme, formado por veias testiculares desprovidas de válvulas de calibres variados, apresentando amplas comunicações entre si. As veias responsáveis pela drenagem do epidídimo e ducto deferente estão localizadas em posição subcapsular deferencial e abdeferencial e mostram-se providas de válvulas. O trato das artérias testiculares no funículo espermático apresenta como média e desvio padrão 134,6±38,1cm à direita, e 137,0±33,9cm à esquerda, não existindo diferenças estatisticamente significantes ao nível de 5%, quando comparamos a média do segmento da artéria testicular contida no funículo espermático direito em relação ao esquerdo.

Estudo morfológico dos órgãos genitais masculinos do guaxinim (Procyon cancrivorus)

Foram utilizados três animais, sendo um proveniente da Universidade de São Paulo e dois encaminhados pelo IBAMA (Proc. 02027.000.286/2004-92), excedente populacional de zoológico. Os aparelhos reprodutores masculinos "ex situ" foram fixados por imersão e dissecados. Foram retirados fragmentos de cada órgão e glândula anexa do aparelho reprodutor, os quais foram processados e incluídos pelas técnicas de inclusão em paraplast. Cada bloco foi cortado e os cortes foram corados por HE, picrosírius, reação de PAS com fundo de hematoxilina e tricromo de masson para a observação das estruturas ao microscópio óptico. Macroscopicamente, a região inguinal era composta pela uretra, músculo isquiocavernoso, músculos bulboesponjoso e bulbo cavernoso, músculo retrator do pênis, um par de testículos e o osso peniano ou báculo e ânus. A glande do pênis apresentou uma dilatação proximal (bulbo da glande) constituída pela parte dilatada do báculo. A posição dos testículos, dentro do escroto, era horizontal. A próstata apresentou-se com formato globoso, circundando a uretra. Microscopicamente, os testículos eram envoltos por uma cápsula de tecido conjuntivo denso, a túnica albugínea testicular. O ducto epididimário era provido de um epitélio pseudoestratificado com estereocílios. A uretra peniana apresentou-se circundada pelo corpo esponjoso e no restante do pênis apresentou o corpo cavernoso (tecido erétil). Os resultados macro e microscópicos encontrados até o momento são semelhantes aos achados no Canis familiaris (cão doméstico).

Epididimite ovina por Actinobacillus seminis no Estado de Pernambuco

Relata-se a ocorrência de orquite e epididimite ovina associada ao isolamento de Actinobacillus seminis no Estado de Pernambuco. Clinicamente observou-se aumento de volume nos testículos e epidídimos, dor e aumento de temperatura local à palpação, e atrofia testicular bilateral. Após o abate observou-se a presença de conteúdo purulento no epidídimo. À microscopia dos testículos observou-se espessamento da túnica albugínea, necrose de coagulação e calcificação de túbulos seminíferos, infiltrado inflamatório com predominância de linfócitos entre túbulos seminíferos, além de mineralização incipiente de túbulos. No epidídimo observou-se intensa proliferação de tecido conjuntivo ao redor dos ductos epididimários. O diagnóstico de orquite e epididimite por Actinobacillus seminis foi confirmado pela associação dos achados clínico-patológicos, isolamento e identificação da bactéria.

Abdominal and pelvic ultrasound study of the maned wolf (Chrysocyon brachyurus)

The aim of the present study was the ultrasound characterization of the abdominal and pelvic regions of five maned wolves kept in captivity at the Triage Center of Wild Animals of the Federal University of Viçosa (Centro de Triagem de Animais Silvestres, Universidade Federal de Viçosa). This characterization included descriptions of ultrasonographic aspects and measurements of various structures using B-mode ultrasound. Biometric data were collected to assess the existence of significant linear correlations between these measurements and the measurements obtained by ultrasound. Additionally, hematological and serum biochemistry evaluations of the animals were performed. The ultrasound findings were similar to those available in the literature on domestic dogs, which were used for comparison as a result of the lack of published data regarding maned wolves. The latter species showed characteristics closely resembling those of the former, differing in the spleen and left renal cortex echogenicities, in the appearance of the prostatic and testicular regions and in the hepatic portal vein morphology. In the current study, the biometric values were similar to those previously published; however, no data regarding thoracic perimeter, modified crown-rump length or thoracic depth were found in the literature for this Canidae species. Statistical analysis showed the existence of a strong negative correlation between the modified crown-rump length and left renal length, between the modified crown-rump length and the right renal volume, between the thoracic perimeter and the height at the cranial pole of the left adrenal gland and between the thoracic perimeter and the height at the caudal pole of the left adrenal gland. Laboratory findings, including segmented neutrophil, eosinophil, monocyte and lymphocyte counts and the serum levels of glucose, ALT, alkaline phosphatase, urea, total protein, globulin, creatine phosphokinase, triglyceride, sodium, phosphate, potassium and chloride, were inconsistent with values found by other authors. The ultrasound is a diagnostic imaging method that must be further explored in the medicine of wild animals; therefore, additional research in this area is required.

Análise morfológica e morfométrica das gônadas de preguiça (Bradypus variegatus Schinz, 1825)

A preguiça pode ser designada, zoologicamente, como um mamífero da classe Eutheria, ordem Edentata, subordem Xenarthra e família Bradypodidae. O gênero Bradypus é constituído de três espécies distintas: a preguiça-de-bentinho (B. tridactylus), restrita à região amazônica; a preguiça-comum (B. variegatus), de ampla distribuição, ocorrendo nas Américas Central e do Sul e a preguiça-de-coleira (B. torquatus), única seriamente ameaçada de extinção. Percebe-se a necessidade de uma investigação mais aprofundada sobre a espécie B. variegatus, a fim de contribuir de forma efetiva com a elaboração de tratados de anatomia da fauna silvestre, além de buscar mais informações sobre a anatomia do sistema reprodutor do bicho preguiça (Bradypus variegatus Schinz, 1825) e, desta forma, aplicar os novos conhecimentos na sua preservação. Utilizamos quatro indivíduos machos e três fêmeas, pertencentes ao banco de espécies da Área de Anatomia do Departamento de Morfologia e Fisiologia Animal da Universidade Federal Rural de Pernambuco (DMFA/UFRPE), as quais foram dissecados e evidenciamos a vascularização das gônadas, bem como suas localizações e aspectos morfológicos e morfométricos dos testículos e ovários. Como resultados, observou-se que o macho possui testículos localizados no interior do espaço intraabdominal, ligados às glândulas adrenais por um ligamento de peritônio. A vascularização não é feita por um plexo pampiniforme, mas por uma artéria e uma veia testicular. Os testículos possuem, em média, volume igual a 1,42cm³ e espessura, largura e comprimento, respectivamente iguais a 0,98, 1,23 e 1,45cm. Os ovários também estão no interior do espaço intra-abdominal, no fundo do útero, protegidos por uma bolsa ovariana, composta por mesovário e mesossalpinge. A vascularização é realizada por um plexo arteriovenoso que irriga e drena o útero, e suas ramificações na parede uterina atingem os ovários. Os ovários possuem, em média, espessura, largura e comprimento, respectivamente iguais a 0,37, 0,73 e 0,62 cm.

Parâmetros anátomo-estruturais de orgãos reprodutivos de ovinos sem raça definida (SRD) nativos do Estado da Paraíba, com e sem bipartição escrotal: estudo do escroto e funículo espermático

Foram utilizados 42 ovinos sem raça definida, divididos segundo a configuração escrotal. Destes animais, 12 foram utilizados na investigação da biometria testicular e histologia da pele escrotal. Os demais foram destinados ao estudo do funículo espermático. Os animais foram agrupados em um grupo de 21 animais sem bipartição escrotal (GEI) e 21 com bipartição escrotal, (GEII), esta não atingindo 50% do comprimento do eixo longitudinal do escroto. Em cada grupo, em 6 animais foram coletados fragmentos da pele do escroto e em 5 do funículo espermáticos, e processados em rotina histológica e analisados em microscopia de luz; e em 10 foram injetados látex na artéria testicular para obtenção de moldes vasculares e obtenção do comprimento da artéria. Quando comparados os grupos GEI e GEII, não foram encontradas diferenças estatísticas significativas (p<0,05) entre a espessura do escroto (epiderme e derme), constituição histológica da pele escrotal, número de glândulas sudoríparas por área, comprimento do funículo espermático ou parâmetros biométricos testiculares. Entretanto, o comprimento total das artérias testiculares do GEI foi maior do que o GEII (p<0,05). Concluiu-se, com base nos parâmetros morfológicos analisados, que a bipartição escrotal em ovinos não influenciou na estrutura da pele, funículo ou biometria testicular quando comparado aos animais que não apresentavam esta característica. Outros estudos merecem atenção para desmistificar o porquê do aparecimento dessa característica em ovinos e se esta característica é ou não desejável para melhoria na produção desses animais em regiões de clima quente.

Triplex Doppler evaluation of the testes in dogs of different sizes

This study aimed to assess whether there are differences in Doppler velocimetry parameters between different sizes. Twenty dogs were equally divided into small and large groups used in this study. The dogs were evaluated using Triplex ultrasound. Testicular artery was located by Colour Doppler in the spermatic cord, marginal to the testes and intratesticular segments and then, spectral Doppler were used to calculate: peak systolic velocity (PSV), end diastolic velocity (EDV), resistance index (RI) and pulsatility index (PI). The mean testicular volume in the left side was significantly higher than the right side, in both groups. Doppler examination showed higher velocities (EDV) at spermatic cord in large dogs; marginal to the testes was observed higher velocities in small dogs; intratesticular region no differences were observed (P < 0.05) and within the groups differences between segments of the artery were also observed for each parameter. The results showed that there are differences in Doppler velocimetry parameters between different sizes.

Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.

Annual changes in serum calcium and inorganic phosphate levels and correlation with gonadal status of a freshwater murrel, Channa punctatus (Bloch)

Adult Channa punctatus murrels of both sexes (60-80 g) were collected locally from Ramgarh Lake during the second week of every month (10 individuals of each sex/month) throughout the year. Blood samples were collected and analyzed for serum calcium and phosphate levels by the methods of Trinder (1960) and Fiske and Subbarow (1925), respectively. Gonads were fixed to judge the state of maturation of the fish. Males exhibited no change in serum calcium levels throughout the year in correlation with testicular maturation. However, serum phosphate levels exhibited a rise in correlation with the increased gonadosomatic index. Females showed marked seasonal changes in serum calcium and phosphate levels which were associated with ovarian maturation (vitellogenesis).

Fertility of male adult rats submitted to forced swimming stress

We investigated whether stress interferes with fertility during adulthood. Male Wistar rats (weighing 220 g in the beginning of the experiment) were forced to swim for 3 min in water at 32ºC daily for 15 days. Stress was assessed by the hot-plate test after the last stressing session. To assess fertility, control and stressed males (N = 15 per group) were mated with sexually mature normal females. Males were sacrificed after copulation. Stress caused by forced swimming was demonstrated by a significant increase in the latency of the pain response in the hot-plate test (14.6 ± 1.25 s for control males vs 26.0 ± 1.53 s for stressed males, P = 0.0004). No changes were observed in body weight, testicular weight, seminal vesicle weight, ventral prostate weight or gross histological features of the testes of stressed males. Similarly, no changes were observed in fertility rate, measured by counting live fetuses in the uterus of normal females mated with control and stressed males; no dead or incompletely developed fetuses were observed in the uterus of either group. In contrast, there was a statistically significant decrease in spermatid production demonstrated by histometric evaluation (154.96 ± 5.41 vs 127.02 ± 3.95 spermatids per tubular section for control and stressed rats, respectively, P = 0.001). These data demonstrate that 15 days of forced swimming stress applied to adult male rats did not impair fertility, but significantly decreased spermatid production. This suggests that the effect of stress on fertility should not be assessed before at least the time required for one cycle of spermatogenesis.

Reproductive ability of pubertal male and female rats

Ten Fisher rats 50 to 55 days of age made up the pubertal group, and ten rats 90 to 95 days of age served as the controls. The testicular and epididymal weights and volumes of the pubertal males were lower than those of the controls (P<0.001). There was also a difference in relative epididymal weight (P<0.001). The sperm of pubertal males was morphologically abnormal in 58.2% of cases, as opposed to only 3.8% in the controls (P<0.001). The mean number of spermatozoa in the control group was 11.9 × 10(6)/ml and their viability was 99.6%, while these values could not be determined for pubertal rats. Serum testosterone was higher in the pubertal animals than in the controls (2.52 ± 1.46 vs 0.92 ± 0.34 nM, P<0.01). The ovaries of control females were heavier than those of pubertal females (P<0.001) but there was no difference in their relative weights. Serum estradiol was similar in both groups (75.5 ± 12.8 vs 81.8 ± 14.7 nM, P>0.05). At the beginning of gestation, the pubertal dams weighed less than the controls (P<0.001) but following uterectomy the body weights were equal. Pubertal dams delivered fewer pups than the controls (8.1 ± 2.5 vs 10.4 ± 1.3, P<0.05). There was no difference in the body weights of their offspring or in the weights of their placentas. The results suggest that, in contrast to their female counterparts, pubertal male rats are not fully mature and have not reached complete reproductive capacity at 50-55 days of age.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20ºC until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37ºC for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 ± 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (± SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 ± 26.7, after: 213.7 ± 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Somatic cytogenetic and azoospermia factor gene microdeletion studies in infertile men

The objective of the present study was to determine the frequency of somatic chromosomal anomalies and Y chromosomal microdeletions (azoospermia factor genes, AZF) in infertile males who seek assisted reproduction. These studies are very important because the assisted reproduction techniques (mainly intracytoplasmic sperm injection) bypass the natural selection process and some classical chromosomal abnormalities, microdeletions of AZF genes or some deleterious genic mutations could pass through generations. These genetic abnormalities can cause in the offspring of these patients male infertility, ambiguous external genitalia, mental retardation, and other birth defects. We studied 165 infertile men whose infertility was attributable to testicular problems (60 were azoospermic, 100 were oligospermic and 5 were asthenospermic). We studied 100 metaphases per patient with GTG banding obtained from temporary lymphocyte culture for chromosomal abnormality detection and performed a genomic DNA analysis using 28 Y chromosome-specific sequence-tagged sites for Y AZF microdeletion detection. Karyotyping revealed somatic anomalies in 16 subjects (16/165 = 9.6%). Of these 16, 12 were in the azoospermic group (12/60 = 20%) and 4 were in the oligospermic group (4/100 = 4%). The most common chromosomal anomaly was Klinefelter syndrome (10/165 = 6%). Microdeletions of AZF genes were detected in 12 subjects (12/160 = 7.5%). The frequencies detected are similar to those described previously. These results show the importance of genetic evaluation of infertile males prior to assisted reproduction. Such evaluation can lead to genetic counseling and, consequently, to primary and secondary prevention of mental retardation and birth defects.

Estrogens and male reproduction: a new concept

The mammalian testis serves two main functions: production of spermatozoa and synthesis of steroids; among them estrogens are the end products obtained from the irreversible transformation of androgens by a microsomal enzymatic complex named aromatase. The aromatase is encoded by a single gene (cyp19) in humans which contains 18 exons, 9 of them being translated. In rats, the aromatase activity is mainly located in Sertoli cells of immature rats and then in Leydig cells of adult rats. We have demonstrated that germ cells represent an important source of estrogens: the amount of P450arom transcript is 3-fold higher in pachytene spermatocytes compared to gonocytes or round spermatids; conversely, aromatase activity is more intense in haploid cells. Male germ cells of mice, bank voles, bears, and monkeys express aromatase. In humans, we have shown the presence of a biologically active aromatase and of estrogen receptors (alpha and ß) in ejaculated spermatozoa and in immature germ cells in addition to Leydig cells. Moreover, we have demonstrated that the amount of P450arom transcripts is 30% lower in immotile than in motile spermatozoa. Alterations of spermatogenesis in terms of number and motility of spermatozoa have been described in men genetically deficient in aromatase. These last observations, together with our data showing a significant decrease of aromatase in immotile spermatozoa, suggest that aromatase could be involved in the acquisition of sperm motility. Thus, taking into account the widespread localization of aromatase and estrogen receptors in testicular cells, it is obvious that, besides gonadotrophins and androgens, estrogens produced locally should be considered to be physiologically relevant hormones involved in the regulation of spermatogenesis and spermiogenesis.