<p>The vervet monkey, Chlorocebus aethiops, an Old World monkey, has been often used in biomedical research programs (toxicological studies and fertility) because of the inaccessibility of relevant human tissues. Data from nonhuman primates have been a vital component of advances in areas such as infertility, contraception, and other reproductive processes because of the phylogenetic closeness of the primates to humans. The aims and objectives of the study were 1) to describe the gross morphology, histology and ultrastructure of the male reproductive system, 2) to describe and compare the processes of spermatogenesis and spermiogenesis of the vervet monkey to humans and other nonhuman primates, and 3) to evaluate the vervet monkey as a possible experimental model for future human reproductive studies.</p> <p>Twenty-nine adult male vervet monkeys, aged between 5 and 11 years, were used. Gross morphological features of different organs of the reproductive tract were recorded. Light and electron microscopic techniques, and methacrylate sections were used on selected tissues of the reproductive tract. The results showed that the vervet monkey has a male&nbsp / reproductive system similar to many non-human primates studied and man. The epididymis was distinctively subdivided into the caput, corpus, and the caudal regions. No significant differences were observed on the epithelial height of these three regions. Four cell types, apical, principal, and basal cells, and the intraepithelial lymphocytes were observed. The basal cell distribution showed significant differences among three regions of the epididymis (P &le / 0.01). There were numerous phagocytic vesicles found in three regions of the epididymis. The Sertoli cells showed perforated sleeve-like processes which encased elongated and mature spermatids ready for spermiation. The nuclei of the Sertoli cells were found to be multilobed (4 to 5) compared to the less lobular nuclei of the human Sertoli cells (2 to 3). The Leydig cells showed typical features of steroidogenic cells with abundant smooth endoplasmic reticulum, numerous large mitochondria, and few rough endoplasmic reticulum.</p> <p>It was concluded that the gross morphology and structure of the reproductive tract of the vervet monkey has many similarities to humans and other mammals. Secondly, the organization of spermatogenesis is similar to that found in humans, and is commonly known as a helical arrangement. The results further suggest that the vervet monkey could be regarded as suitable model for human male reproductive studies.</p> <p>&nbsp / </p>

<p>The vervet monkey, Chlorocebus aethiops, an Old World monkey, has been often used in biomedical research programs (toxicological studies and fertility) because of the inaccessibility of relevant human tissues. Data from nonhuman primates have been a vital component of advances in areas such as infertility, contraception, and other reproductive processes because of the phylogenetic closeness of the primates to humans. The aims and objectives of the study were 1) to describe the gross morphology, histology and ultrastructure of the male reproductive system, 2) to describe and compare the processes of spermatogenesis and spermiogenesis of the vervet monkey to humans and other nonhuman primates, and 3) to evaluate the vervet monkey as a possible experimental model for future human reproductive studies.</p> <p>Twenty-nine adult male vervet monkeys, aged between 5 and 11 years, were used. Gross morphological features of different organs of the reproductive tract were recorded. Light and electron microscopic techniques, and methacrylate sections were used on selected tissues of the reproductive tract. The results showed that the vervet monkey has a male&nbsp / reproductive system similar to many non-human primates studied and man. The epididymis was distinctively subdivided into the caput, corpus, and the caudal regions. No significant differences were observed on the epithelial height of these three regions. Four cell types, apical, principal, and basal cells, and the intraepithelial lymphocytes were observed. The basal cell distribution showed significant differences among three regions of the epididymis (P &le / 0.01). There were numerous phagocytic vesicles found in three regions of the epididymis. The Sertoli cells showed perforated sleeve-like processes which encased elongated and mature spermatids ready for spermiation. The nuclei of the Sertoli cells were found to be multilobed (4 to 5) compared to the less lobular nuclei of the human Sertoli cells (2 to 3). The Leydig cells showed typical features of steroidogenic cells with abundant smooth endoplasmic reticulum, numerous large mitochondria, and few rough endoplasmic reticulum.</p> <p>It was concluded that the gross morphology and structure of the reproductive tract of the vervet monkey has many similarities to humans and other mammals. Secondly, the organization of spermatogenesis is similar to that found in humans, and is commonly known as a helical arrangement. The results further suggest that the vervet monkey could be regarded as suitable model for human male reproductive studies.</p> <p>&nbsp / </p>

Este estudo teve como objetivos caracterizar modificações no tamanho testicular e concentrações sanguíneas de testosterona relacionadas à idade, avaliar a presença de espematozóides epididimários, mensurar o diâmetro dos túbulos seminíferos e determinar o grau de maturação testicular, através da presença das células germinativas mais avançadas no epitélio seminífero de machos Crioulos no período da peri-puberdade. Os animais foram castrados cirurgicamente. Foi avaliada a circunferência torácica para estimativa do peso corporal. Trinta e quatro equinos foram agrupados em quatro categorias. O grupo I (GI) com quatro potros com idade de até 14 meses. O grupo II (GII) composto por sete animais com mais de 14 meses e menos de 17 meses. No terceiro grupo (GIII) foram incluídos 14 animais com mais de 17 meses e menos de 19 meses de idade e o último grupo (GIV) composto por nove equinos com mais de 19 meses e menos de 34 meses de idade. Após a castração os testículos foram pesados e medidos. Um fragmento foi coletado para posterior avaliação histológica e verificação do diâmetro dos túbulos seminíferos, além da presença de células germinativas do epitélio seminífero em diferentes fases de desenvolvimento. Uma amostra de sangue foi coletada para posterior determinação da concentração plasmática de testosterona. O peso médio dos animais no GI aumentou de 232 kg para 321 kg nos garanhões do GIV. Não houve diferença significativa entre medidas dos testículos direito e esquerdo. O peso e volume testicular apresentaram variações mais acentuadas nos animais no GIV e diferiram significativamente em relação aos animais mais jovens. Valores plasmáticos de testosterona não diferiram entre grupos e peso corporal. O diâmetro dos túbulos seminíferos apresentou variação mais marcante nos garanhões do GIV, aumentando de 89,13 μm nos potros do GI para 168,24 μm nos garanhões do GIV. Em média, 17,5% dos túbulos seminíferos não apresentaram células germinativas no GI, decrescendo para 5,4% no GII, 5,2% no GIII e 0,8% no GIV. O número de túbulos contendo espemátides maduras e espermatozóides aumentou conforme o avanço da idade dos animais. As variações mais acentuadas no incremento do volume testicular e aumento do diâmetro dos túbulos seminíferos coincidiram com a uma elevada presença de espermatozoides nos túbulos. Em conclusão, todos os animais com 20 meses de idade ou mais alcançaram a puberdade e os primeiros espermatozóides epididimários foram encontrados em potros com 16 meses. Ao testículo atingir um volume e peso testicular de 16cm3 e 23g, respectivamente, todos os animais apresentaram espermatozoides epididimários. / The objectives of this study were to characterize age-associated changes in testicular size and blood concentrations of testosterone, as well as evaluate the presence of epididymal sperm. The testicular maturity was evaluated by measuring seminiferous tubule diameter and the presence of the most advanced germ cells in the seminiferous epithelium of colts in the peri–puberty period. The animals were surgically castrated and thorax circumference was taken to estimate the body weight. Thirty four male horses were grouped into four categories: Group I (GI) with four foals aged up to 14 months; Group II (GII) with seven animals over 14 months and less than 17 months; The third group (GIII) included 14 animals over 17 months and less than 19 months; Group (GIV) was composed of nine horses over 19 months and under 34 months of age. After castration the testes were weighed and measured. A segment was collected for subsequent histological evaluation, including measurement of the diameter of the seminiferous tubules and the presence of germ cells of the seminiferous epithelium at different stages of development. A blood sample was collected for determination of plasma testosterone. The average body weight of the animals increased significantly from 232 kg in GI to 321 kg for horses in GIV. There was no difference between measures taken on right and left testes. The testicular weight and volume was greater in animals of GIV and differed significantly compared to the younger animals. Plasma levels of testosterone did not differ between age groups. The diameter of the seminiferous tubules increased from 89.13 μm in foals of GI to 168.24 μm in horses of GIV. On average, 17.5% of the seminiferous tubules had no germ cells in animals of GI, decreasing to 5.4% in GII, 5.2% in GIII and 0.8% in GIV. The number of tubules containing mature spermatids and spermatozoa increased with age. Most significant variations in the increase of testicular volume and diameter of seminiferous tubules were associated with a high presence of spermatozoa in the tubules. In conclusion all animals with 20 months of age or more had reached puberty in the present study and the first spermatozoa appeared in 16 month old colts. All animals presented epididymal sperm when testicular volume and weight were over 16cm3 and 23g, respectively.

Este estudo teve como objetivos caracterizar modificações no tamanho testicular e concentrações sanguíneas de testosterona relacionadas à idade, avaliar a presença de espematozóides epididimários, mensurar o diâmetro dos túbulos seminíferos e determinar o grau de maturação testicular, através da presença das células germinativas mais avançadas no epitélio seminífero de machos Crioulos no período da peri-puberdade. Os animais foram castrados cirurgicamente. Foi avaliada a circunferência torácica para estimativa do peso corporal. Trinta e quatro equinos foram agrupados em quatro categorias. O grupo I (GI) com quatro potros com idade de até 14 meses. O grupo II (GII) composto por sete animais com mais de 14 meses e menos de 17 meses. No terceiro grupo (GIII) foram incluídos 14 animais com mais de 17 meses e menos de 19 meses de idade e o último grupo (GIV) composto por nove equinos com mais de 19 meses e menos de 34 meses de idade. Após a castração os testículos foram pesados e medidos. Um fragmento foi coletado para posterior avaliação histológica e verificação do diâmetro dos túbulos seminíferos, além da presença de células germinativas do epitélio seminífero em diferentes fases de desenvolvimento. Uma amostra de sangue foi coletada para posterior determinação da concentração plasmática de testosterona. O peso médio dos animais no GI aumentou de 232 kg para 321 kg nos garanhões do GIV. Não houve diferença significativa entre medidas dos testículos direito e esquerdo. O peso e volume testicular apresentaram variações mais acentuadas nos animais no GIV e diferiram significativamente em relação aos animais mais jovens. Valores plasmáticos de testosterona não diferiram entre grupos e peso corporal. O diâmetro dos túbulos seminíferos apresentou variação mais marcante nos garanhões do GIV, aumentando de 89,13 μm nos potros do GI para 168,24 μm nos garanhões do GIV. Em média, 17,5% dos túbulos seminíferos não apresentaram células germinativas no GI, decrescendo para 5,4% no GII, 5,2% no GIII e 0,8% no GIV. O número de túbulos contendo espemátides maduras e espermatozóides aumentou conforme o avanço da idade dos animais. As variações mais acentuadas no incremento do volume testicular e aumento do diâmetro dos túbulos seminíferos coincidiram com a uma elevada presença de espermatozoides nos túbulos. Em conclusão, todos os animais com 20 meses de idade ou mais alcançaram a puberdade e os primeiros espermatozóides epididimários foram encontrados em potros com 16 meses. Ao testículo atingir um volume e peso testicular de 16cm3 e 23g, respectivamente, todos os animais apresentaram espermatozoides epididimários. / The objectives of this study were to characterize age-associated changes in testicular size and blood concentrations of testosterone, as well as evaluate the presence of epididymal sperm. The testicular maturity was evaluated by measuring seminiferous tubule diameter and the presence of the most advanced germ cells in the seminiferous epithelium of colts in the peri–puberty period. The animals were surgically castrated and thorax circumference was taken to estimate the body weight. Thirty four male horses were grouped into four categories: Group I (GI) with four foals aged up to 14 months; Group II (GII) with seven animals over 14 months and less than 17 months; The third group (GIII) included 14 animals over 17 months and less than 19 months; Group (GIV) was composed of nine horses over 19 months and under 34 months of age. After castration the testes were weighed and measured. A segment was collected for subsequent histological evaluation, including measurement of the diameter of the seminiferous tubules and the presence of germ cells of the seminiferous epithelium at different stages of development. A blood sample was collected for determination of plasma testosterone. The average body weight of the animals increased significantly from 232 kg in GI to 321 kg for horses in GIV. There was no difference between measures taken on right and left testes. The testicular weight and volume was greater in animals of GIV and differed significantly compared to the younger animals. Plasma levels of testosterone did not differ between age groups. The diameter of the seminiferous tubules increased from 89.13 μm in foals of GI to 168.24 μm in horses of GIV. On average, 17.5% of the seminiferous tubules had no germ cells in animals of GI, decreasing to 5.4% in GII, 5.2% in GIII and 0.8% in GIV. The number of tubules containing mature spermatids and spermatozoa increased with age. Most significant variations in the increase of testicular volume and diameter of seminiferous tubules were associated with a high presence of spermatozoa in the tubules. In conclusion all animals with 20 months of age or more had reached puberty in the present study and the first spermatozoa appeared in 16 month old colts. All animals presented epididymal sperm when testicular volume and weight were over 16cm3 and 23g, respectively.

Este estudo teve como objetivos caracterizar modificações no tamanho testicular e concentrações sanguíneas de testosterona relacionadas à idade, avaliar a presença de espematozóides epididimários, mensurar o diâmetro dos túbulos seminíferos e determinar o grau de maturação testicular, através da presença das células germinativas mais avançadas no epitélio seminífero de machos Crioulos no período da peri-puberdade. Os animais foram castrados cirurgicamente. Foi avaliada a circunferência torácica para estimativa do peso corporal. Trinta e quatro equinos foram agrupados em quatro categorias. O grupo I (GI) com quatro potros com idade de até 14 meses. O grupo II (GII) composto por sete animais com mais de 14 meses e menos de 17 meses. No terceiro grupo (GIII) foram incluídos 14 animais com mais de 17 meses e menos de 19 meses de idade e o último grupo (GIV) composto por nove equinos com mais de 19 meses e menos de 34 meses de idade. Após a castração os testículos foram pesados e medidos. Um fragmento foi coletado para posterior avaliação histológica e verificação do diâmetro dos túbulos seminíferos, além da presença de células germinativas do epitélio seminífero em diferentes fases de desenvolvimento. Uma amostra de sangue foi coletada para posterior determinação da concentração plasmática de testosterona. O peso médio dos animais no GI aumentou de 232 kg para 321 kg nos garanhões do GIV. Não houve diferença significativa entre medidas dos testículos direito e esquerdo. O peso e volume testicular apresentaram variações mais acentuadas nos animais no GIV e diferiram significativamente em relação aos animais mais jovens. Valores plasmáticos de testosterona não diferiram entre grupos e peso corporal. O diâmetro dos túbulos seminíferos apresentou variação mais marcante nos garanhões do GIV, aumentando de 89,13 μm nos potros do GI para 168,24 μm nos garanhões do GIV. Em média, 17,5% dos túbulos seminíferos não apresentaram células germinativas no GI, decrescendo para 5,4% no GII, 5,2% no GIII e 0,8% no GIV. O número de túbulos contendo espemátides maduras e espermatozóides aumentou conforme o avanço da idade dos animais. As variações mais acentuadas no incremento do volume testicular e aumento do diâmetro dos túbulos seminíferos coincidiram com a uma elevada presença de espermatozoides nos túbulos. Em conclusão, todos os animais com 20 meses de idade ou mais alcançaram a puberdade e os primeiros espermatozóides epididimários foram encontrados em potros com 16 meses. Ao testículo atingir um volume e peso testicular de 16cm3 e 23g, respectivamente, todos os animais apresentaram espermatozoides epididimários. / The objectives of this study were to characterize age-associated changes in testicular size and blood concentrations of testosterone, as well as evaluate the presence of epididymal sperm. The testicular maturity was evaluated by measuring seminiferous tubule diameter and the presence of the most advanced germ cells in the seminiferous epithelium of colts in the peri–puberty period. The animals were surgically castrated and thorax circumference was taken to estimate the body weight. Thirty four male horses were grouped into four categories: Group I (GI) with four foals aged up to 14 months; Group II (GII) with seven animals over 14 months and less than 17 months; The third group (GIII) included 14 animals over 17 months and less than 19 months; Group (GIV) was composed of nine horses over 19 months and under 34 months of age. After castration the testes were weighed and measured. A segment was collected for subsequent histological evaluation, including measurement of the diameter of the seminiferous tubules and the presence of germ cells of the seminiferous epithelium at different stages of development. A blood sample was collected for determination of plasma testosterone. The average body weight of the animals increased significantly from 232 kg in GI to 321 kg for horses in GIV. There was no difference between measures taken on right and left testes. The testicular weight and volume was greater in animals of GIV and differed significantly compared to the younger animals. Plasma levels of testosterone did not differ between age groups. The diameter of the seminiferous tubules increased from 89.13 μm in foals of GI to 168.24 μm in horses of GIV. On average, 17.5% of the seminiferous tubules had no germ cells in animals of GI, decreasing to 5.4% in GII, 5.2% in GIII and 0.8% in GIV. The number of tubules containing mature spermatids and spermatozoa increased with age. Most significant variations in the increase of testicular volume and diameter of seminiferous tubules were associated with a high presence of spermatozoa in the tubules. In conclusion all animals with 20 months of age or more had reached puberty in the present study and the first spermatozoa appeared in 16 month old colts. All animals presented epididymal sperm when testicular volume and weight were over 16cm3 and 23g, respectively.

A ocitocina (OT) é um neuropeptídio hipotalâmico, que dentre suas funções na fêmea destaca-se a contração uterina durante o parto e a ejeção do leite. No entanto, estudos vêm demonstrando importantes funções endócrinas e parácrinas no trato reprodutivo masculino. Evidenciando a possível ação conjunta entre OT e a Globulina ligadora de hormônios sexuais (SHBG). Entretanto, em cães não existem informações disponíveis quanto sua atuação. Assim, estudos direcionados aos receptores de ocitocina (OTR) e SHBG e suas funções no sistema reprodutor masculino, mais especificamente na fisiologia espermática, são de suma importância para os conhecimentos da fisiologia reprodutiva para posterior aplicação em biotecnologias reprodutivas em pequenos animais e humanos, fomentando também novas perspectivas para a utilização terapêutica da ocitocina em enfermidades reprodutivas. Portanto, o objetivo deste estudo é verificar a expressão gênica e proteica do OTR e SHBG no testículo e epidídimo de cães, correlacionando tais dados com a qualidade espermática e dosagem de testosterona. Para tal, foram coletados testículos e epidídimos de 26 cães em idade reprodutiva (1 a 5 anos). Após a orquiectomia, foi realizada a coleta dos espermatozoides provenientes da cauda do epidídimo e então, as amostras foram analisadas quanto à motilidade computadorizada do sêmen (CASA), integridade de membrana plasmática (Eosina/Nigrosina), integridade de membrana acrossomal (Fast Green / Rosa Bengala) e atividade mitocondrial (3´3 Diaminobenzidine). A imunolocalização do OTR e SHBG foi realizada através de imunoistoquímica e imunofluorescência. E a análise de expressão gênica, através da Reação em cadeia da polimerase em tempo real (qRT PCR). E da expressão proteica, através do Western Blotting. Foram encontradas correlações significantes e positivas entre as expressões gênicas do OTR e do SHBG, tanto no testículo como no epidídimo. Além disto, a expressão do OTR no testículo correlacionou-se positivamente com espermatozoides com membrana acrossomal íntegra e negativamente com a porcentagem de células com baixa atividade mitocondrial. Já o SHBG do testículo, correlacionou-se positivamente com a concentração de espermatozoides, porcentagens de células com membrana plasmática e acrossomal íntegras, motilidade, motilidade progressiva e velocidade rápida, e negativamente com a porcentagem de células com baixa atividade mitocondrial. Por outro lado, no epidídimo, a expressão gênica do SHBG apresentou correlação positiva com a porcentagem de células com membrana plasmática íntegra e expressão proteica de SHBG no testículo. Quanto a expressão proteica, o OTR no testículo obteve correlação positiva com testosterona e negativa com atividade mitocondrial nula, já no epidídimo, ocorreu correlação positiva com integridade de membrana acrossomal e negativa também com atividade mitocondrial nula. Em relação ao SHBG, houve correlação positiva com a expressão gênica do SHBG no epidídimo, células normais e padrões de velocidade. E na imunoistoquímica foi possível observar a imunomarcação do OTR e SHBG na musculatura lisa e células de Leydig do testículo e OTR na musculatura lisa do epidídimo. No entanto, não houve imunomarcação do SHBG no epidídimo, assim como expressão proteica. Nossos resultados demonstraram que o OTR e SHBG são expressos nos testículos e epidídimos de cães e que estão relacionados a funções espermáticas importantes, sendo essenciais para o sucesso reprodutivo / Oxytocin (OT) is a hypothalamic neuropeptide that plays important and well known roles in the female such as uterine contraction during childbirth and milk ejection. Notwithstanding, studies have shown important endocrine and paracrine functions also in the male reproductive tract, highlighted by the possible joint action between OT and sex hormone-binding globulin (SHBG). In dogs, however, there is no information available with regards to the role of these hormones in the reproductive function. Thus, studies directed to oxytocin (OTR) and SHBG receptors and their functions in the male reproductive system, specifically with regards to sperm physiology. Such knowledge is essential to understand the reproductive physiology for the subsequent use in reproductive biotechnologies in small animals and humans, especially by providing new perspectives for the therapeutic use of oxytocin in reproductive disorders. Therefore, the aim of this study is to assess the gene and protein expression of OTR and SHBG in the testis and epididymis of dogs, correlating these data with sperm quality and testosterone dosage. To this end, testis and epididymis were collected from 26 dogs in reproductive age (1 to 5 years). After orchiectomy, collection of sperm from the cauda epididymis was carried out and then the samples were analyzed for computerized motility of semen (CASA), plasma membrane integrity (eosin / nigrosine), acrosome membrane integrity (Fast Green / rose Bengal) and mitochondrial activity (3\'3 Diaminobenzidine). The immunolocalization of OTR and SHBG was performed by immunohistochemistry and immunofluorescence. Gene expression analysis was performed by real time polymerase chain reaction (qRT - PCR). The protein expression was further assessed by Western Blotting. Significant positive correlations were found between the gene expressions of OTR and SHBG in both the testis and epididymis. Furthermore, the OTR expression in testis was positively correlated to sperm with intact acrosome membrane and negatively to the percentage of cells with low mitochondrial activity. On the other hand, testicular SHBG was positively correlated with sperm concentration, percentage of sperm with intact plasma membrane and acrosome, motility, progressive motility and the percentage of RAPID sperm. Also, negative correlation was found between testicular SHBG and the percentage of cells with low mitochondrial activity. Furthermore, in the epididymis, SHBG gene expression was positively correlated to the percentage of cells with intact plasma membrane and protein expression of SHBG in the testis. In relation to the protein expression, the OTR in the testis correlated positively with blood plasma testosterone and negatively with sperm with no mitochondrial activity. In the epididymis, OTR protein expression correlated positively with sperm showing intact acrosome and negatively with cells with no mitochondrial activity. With regards to SHBG proteins expression, there was a positive correlation to SHBG gene expression in the epididymis, normal cells and some patterns of sperm velocity. In the immunohistochemistry, we observed the OTR and SHBG immunostainings in the smooth muscle and Leydig cells of the testis while, in the epididymis, the OTR immunostaining could be observed only in the smooth muscle. Interestingly, there was no immunostaining or protein expression of SHBG in the epididymis. Our results demonstrated that OTR and SHBG are expressed in the testis and epididymis of dogs and are related to important sperm functions, essential for reproductive success

Philosophiae Doctor - PhD / The vervet monkey, Chlorocebus aethiops, an Old World monkey, has been often used in biomedical research programs (toxicological studies and fertility) because of the inaccessibility of relevant human tissues. Data from nonhuman primates have been a vital component of advances in areas such as infertility, contraception, and other reproductive processes because of the phylogenetic closeness of the primates to humans. The aims and objectives of the study were 1) to describe the gross morphology, histology and ultrastructure of the male reproductive system, 2) to describe and compare the processes of spermatogenesis and spermiogenesis of the vervet monkey to humans and other nonhuman primates, and 3) to evaluate the vervet monkey as a possible experimental model for future human reproductive studies. Twenty-nine adult male vervet monkeys, aged between 5 and 11 years, were used. Gross morphological features of different organs of the reproductive tract were recorded. Light and electron microscopic techniques, and methacrylate sections were used on selected tissues of the reproductive tract. The results showed that the vervet monkey has a male reproductive system similar to many non-human primates studied and man. The epididymis was distinctively subdivided into the caput, corpus, and the caudal regions. No significant differences were observed on the epithelial height of these three regions. Four cell types, apical, principal, and basal cells, and the intraepithelial lymphocytes were observed. The basal cell distribution showed significant differences among three regions of the epididymis (P ≤ 0.01). There were numerous phagocytic vesicles found in three regions of the epididymis. The Sertoli cells showed perforated sleeve-like processes which encased elongated and mature spermatids ready for spermiation. The nuclei of the Sertoli cells were found to be multilobed (4 to 5) compared to the less lobular nuclei of the human Sertoli cells (2 to 3). The Leydig cells showed typical features of steroidogenic cells with abundant smooth endoplasmic reticulum, numerous large mitochondria, and few rough endoplasmic reticulum. It was concluded that the gross morphology and structure of the reproductive tract of the vervet monkey has many similarities to humans and other mammals. Secondly, the organization of spermatogenesis is similar to that found in humans, and is commonly known as a helical arrangement. The results further suggest that the vervet monkey could be regarded as suitable model for human male reproductive studies

1 AbstractAbstractAbstractAbstract Proteins secreted in the male reproductive tract play a key role in post-testicular development of sperm and in further steps needed for fertilization. Sperm maturation represents a key step in the reproduction process. Sperm, during the passage through the epididymis undergoes significant changes due to proteolytic and glycolytic activities in the epididymal fluid. Inhibitor of acrosin protects spermatozoa and reproductive epithelium against proteolytic degradation and also protects binding sites for ZP on sperm plasma membrane. In boar reproductive system acrosin inhibitor (AI) was found in seminal plasma and on sperm plasma membrane. Polyclonal antibody recognized AI in extracts of the cauda epididymidis, seminal vesicles, prostate, and Cowper's glands. Using immunofluorescence method has revealed the AI in the epithelium and lumen of these organs but also on the surface of epididymal and ejaculated spermatozoa. We registered the increasing signal of AI from caput to cauda epididymis. Gene expression of AI mRNA was detected in the epididymis, seminal vesicles, prostate, and Cowper's glands and increased gradually throughout the epididymal duct. In present study, we also monitored AI in boar epididymal fluid and spermatozoa along the organ. In the epididymis, AI may...

L’infertilité affecte environ 15% des couples en âge de se reproduire. Dans près de la moitié des cas, des facteurs masculins sont à la base de l’infertilité, quoique les causes exactes demeurent souvent inconnues. Les spermatozoïdes de mammifères subissent une série d’étapes de maturation avant d’acquérir la capacité de féconder un ovocyte. Les premiers changements ont lieu à l’intérieur de l’épididyme, où les spermatozoïdes gagnent la capacité de se mouvoir ainsi que de reconnaître et d’interagir avec l’ovocyte. Suite à l’éjaculation, ils doivent subir une seconde série de modifications à l’intérieur du tractus génital femelle, nommée capacitation. Nous avons préalablement démontré que chez le bovin, la famille de protéines BSP (Binder of SPerm) est essentielle à la capacitation. Des homologues des BSP ont aussi été isolés du fluide séminal de porc, de bouc, de bélier, de bison et d’étalon. Malgré la détection d’antigènes apparentés aux BSP dans le fluide séminal de souris et d’humain, les homologues des BSP n’ont jamais été caractérisés chez ces espèces. Nous avons émis l’hypothèse que des homologues des BSP seraient exprimés chez la souris et l’humain et joueraient un rôle dans la maturation des spermatozoïdes. Nous avons démontré que des séquences homologues aux BSP sont présentes dans les génomes murin et humain. Le génome murin contient trois séquences; Bsph1, Bsph2a et Bsph2b, tandis qu’une seule séquence (BSPH1) a été identifée chez l’humain. Les séquences d’ADNc de Bsph1, Bsph2a et BSPH1 ont été clonées, tandis que Bsph2b serait probablement un pseudogène. Les trois gènes sont exprimés uniquement dans l’épididyme et font partie d’une sous-famille distincte à l’intérieur de la famille des BSP. Chez les ongulés, les BSP sont exprimées par les vésicules séminales, sont ajoutées aux spermatozoïdes lors de l’éjaculation et représentent une proportion significative des protéines du plasma séminal. Au contraire, les BSP épididymaires ne sont retrouvées qu’en faibles quantités dans le fluide séminal. L’étude de leur rôle dans les fonctions spermatiques était donc plus difficile que chez les ongulés, où l’isolement des protéines natives du plasma séminal à l’aide de techniques de chromatographie était possible. Afin d’étudier sa fonction, nous avons exprimé BSPH1 recombinante dans E. coli. Les ponts disulfure des domaines de type-II caractéristiques de ces protéines ont fait en sorte que l’expression de BSPH1 fusionnée à une étiquette hexahistidine ou glutathion-S-transférase a donné lieu à des protéines insolubles dans les corps d’inclusion. La production de BSPH1 soluble a été possible grâce à l’ajout d’une étiquette thiorédoxine et l’expression dans une souche au cytoplasme oxidatif. BSPH1 a été purifiée par affinité et sa liaison aux partenaires connus des BSP, la phosphatidylcholine, les lipoprotéines de faible densité et la membrane des spermatozoïdes, suggérait que la protéine recombinante possédait sa conformation native et pouvait être utilisée pour des essais fonctionnels. La forme native de BSPH1 a été détectée dans le plasma séminal humain suite au fractionnement par gel filtration. La liaison de BSPH1 native à une colonne d’affinité à l’héparine a indiqué qu’elle partage aussi cette propriété de liaison avec la famille des BSP, et pourrait lier les GAGs semblables à l’héparine du tractus génital féminin. Une colonne d’immunoaffinité anti-BSPH1 a été préparée à l’aide d’anticorps générés contre des protéines recombinantes, et a permis d’isoler BSPH1 native à partir d’extraits de spermatozoïdes humains. Nos résultats montrent que BSPH1 native serait localisée dans les microdomaines « rafts » de la membrane. Sa masse moléculaire apparente était de 32 kDa, ce qui est supérieur à la masse prédite selon sa séquence en acides aminés, indiquant la présence probable de modifications post-traductionnelles, ou d’une migration anormale. L’effet de BSPH1 recombinante et des anticorps anti-BSPH1 sur la motilité, la viabilité et la capacitation a aussi été étudié. Les deux dernières variables ont été mesurées par un essai de cytométrie en flux, optimisé dans cette étude. Aucun effet des protéines recombinantes ou des anticorps sur la motilité et la viabilité des spermatozoïdes n’a été noté. Quoiqu’une stimulation modeste, quoique significative, de la capacitation ait été observée à la plus faible concentration de BSPH1, les concentrations plus élevées n’ont pas montré d’effet. De la même manière, les anticorps anti-BSPH1 n’ont pas eu d’effet significatif sur la capacitation. Ces résultats suggèrent que BSPH1 produite dans E. coli n’affecte pas la capacitation de façon marquée. Cependant, puisque BSPH1 native possède probablement des modifications post-traductionnelles, une protéine recombinante produite dans des cellules de mammifères pourrait affecter les fonctions spermatiques. De manière alternative, les BSP épididymaires remplissent peut-être un rôle différent dans les fonctions spermatiques que celles sécrétées par les vésicules séminales des ongulés. Les résultats décrits dans cette thèse pourraient contribuer à améliorer le diagnostic de l’infertilité masculine, ainsi que les techniques de reproduction assistée et éventuellement, pourraient mener au développement de contraceptifs masculins. / Infertility affects approximately 15% of couples of reproductive age. In nearly half the cases, male factors are responsible, although causes underlying male infertility often remain unknown. Mammalian sperm undergo a series of maturational steps before acquiring the capacity to fertilize an oocyte. The first changes take place inside the epididymis, where sperm gain motility and the ability to recognize and interact with the oocyte. After ejaculation, sperm go through a second maturation event named capacitation, taking place inside the female reproductive tract. We previously showed that in the bovine species, proteins of the BSP (Binder of SPerm) family are essential for capacitation. Homologs of these proteins have also been isolated from boar, ram, goat, bison and stallion seminal fluid. Although BSP-related antigens have been detected in mouse and human seminal fluid, BSP homologs have never been characterized in these species. We hypothesized that BSPs would indeed be expressed in mice and humans and could be involved in sperm maturation. Our studies demonstrated that BSP-homologous sequences are present in the mouse and human genomes. The mouse genome contains three BSP-like sequences, Bsph1, Bsph2a and Bsph2b, whereas only one sequence (BSPH1) was identified in the human genome. The complete cDNA sequences of Bsph1, Bsph2a and BSPH1 were cloned, whereas Bsph2b is probably a pseudogene. The two murine and sole human genes are expressed uniquely in the epididymis, and are part of a distinct sub-family within the BSP superfamily. The BSPs of ungulates are expressed in the seminal vesicles, are added to sperm upon ejaculation and represent a significant proportion of seminal plasma proteins. In contrast, BSP proteins expressed in the mouse and human epididymides are found in very small quantities in seminal fluid. The study of their role in sperm functions was therefore less straightforward than for ungulate species, where direct isolation of the native proteins from seminal plasma was feasible using various chromatography techniques. In order to investigate the role of the human BSP protein, BSPH1, we expressed the recombinant protein in E. coli. Probably due to the multiple disulfide bonds within the fibronectin type-II domains characteristic of these proteins, expression of BSPH1 with a hexahistidine or glutathione-S-tranferase tag gave rise to insoluble protein trapped inside bacterial inclusion bodies. Successful expression of soluble BSPH1 was achieved when the protein was fused to a thioredoxin tag and expressed in a bacterial strain that possesses an oxidizing cytoplasm. This protein was purified using affinity chromatography techniques and tested for binding to known ligands of BSP proteins: phosphatidylcholine, low-density lipoproteins and the human sperm membrane. Since recombinant BSPH1 displayed all three binding properties, we concluded that it had assumed its native conformation and could be used in subsequent functional assays to determine its role in sperm functions. The native form of BSPH1 was detected in human seminal plasma after fractionation on a gel filtration column. Native BSPH1 also bound to a heparin-affinity column, indicating that it shares this binding property with the BSP family and may also bind heparin-like GAGs of the female reproductive tract. An anti-BSPH1 immunoaffinity column was prepared using antibodies generated with bacterially expressed recombinant proteins and was used to isolate native BSPH1 from human sperm extracts. In addition, our results show that BSPH1 probably localizes to detergent-resistant microdomains of the human sperm membrane. Its apparent molecular weight was 32 kDa, which is superior to that predicted by its amino acid sequence. Therefore, BSPH1 probably undergoes post-translational modifications or migrates abnormally during electrophoresis. The effect of recombinant BSPH1 protein and anti-BSPH1 antibodies on human sperm motility, viability and capacitation were also investigated. The latter two sperm functions were assayed using a flow cytometry technique optimized in this study. No effect of recombinant BSPH1 or antibodies on sperm motility or viability was noted. Although a modest yet significant stimulation of capacitation was observed at lower BSPH1 protein concentrations, higher concentrations showed no effect. In the same fashion, anti-BSPH1 antibodies showed no significant effect on capacitation. These results suggest that recombinant BSPH1 produced in E. coli does not appreciably affect capacitation. However, since native BSPH1 may be subject to post-translational modifications, it is possible that BSPH1 expressed in a mammalian system would affect sperm capacitation. Alternatively, epididymally expressed BSPs may play a somewhat different role in sperm functions than those secreted by the seminal vesicles of ungulates. The results described in this thesis could aid in better diagnosing male infertility, improving assisted reproduction and eventually, developing male contraceptives.

L’infertilité affecte jusqu’à 15-20% des couples en âge de se reproduire. C’est pourquoi, mieux comprendre les mécanismes à la base de la fécondation est essentiel pour l’identification de nouvelles causes d’infertilité et l’optimisation des techniques de reproduction assistée. La capacitation est une étape de la maturation des spermatozoïdes qui se déroule dans le tractus génital femelle. Elle est requise pour la fécondation d’un ovocyte. Notre laboratoire a démontré que des protéines du plasma séminal bovin, appelées protéines Binder of SPerm (BSP), se lient aux phospholipides portant des groupements choline à la surface de la membrane des spermatozoïdes lors de l’éjaculation et promeuvent la capacitation. Ces protéines exprimées par les vésicules séminales sont ubiquitaires chez les mammifères et ont été étudiées chez plusieurs espèces dont l’étalon, le porc, le bouc et le bélier. Récemment, l’expression de gènes homologues aux BSP a été découverte dans les épididymes d’humains (BSPH1) et de souris (Bsph1 et Bsph2). Notre hypothèse est que les BSP chez ces deux espèces sont ajoutées aux spermatozoïdes lors de la maturation épididymaire et ont des rôles dans les fonctions spermatiques, similaires à ceux des protéines BSP bovines. Les protéines BSP humaines et murines représentent une faible fraction des protéines totales du plasma séminal. Pour cette raison, afin d’étudier leurs caractéristiques biochimiques et fonctionnelles, des protéines recombinantes ont été produites. Les protéines recombinantes ont été exprimées dans des cellules Escherichia coli origami B(DE3)pLysS en utilisant un vecteur d’expression pET32a. Suivant la lyse cellulaire, les protéines ont été dénaturées avec de l’urée et purifiées par chromatographie d’affinité sur ions métalliques immobilisés. Une fois liées à la colonne, les protéines ont été repliées à l’aide d’un gradient d’urée décroissant avant d’être éluées. Cette méthode a mené à la production de trois protéines recombinantes (rec-BSPH1 humaine, rec-BSPH1 murine et rec-BSPH2 murine) pures et fonctionnelles. Des expériences de chromatographie d’affinité et de co-sédimentation nous ont permis de démontrer que les trois protéines peuvent se lier à des ligands connus des protéines BSP comme la gélatine et l’héparine en plus de pouvoir se lier aux spermatozoïdes. Nos études ont également révélées que les deux protéines rec-BSPH1 peuvent se lier aux liposomes de phosphatidylcholine (PC) et sont capable de promouvoir la capacitation des spermatozoïdes. À l’opposé, rec-BSPH2 ne peut ni se lier aux liposomes de PC, ni stimuler la capacitation. Finalement, les protéines recombinantes n’ont aucun effet sur la réaction acrosomique ou sur la motilité des spermatozoïdes. Chez les bovins, les protéines BSP induisent la capacitation grâce des interactions avec les lipoprotéines de haute densité (HDL) et les glycosaminoglycanes. Puisque le HDL est également un joueur important de la capacitation chez la souris, le rôle de la protéine native BSPH1 murine au niveau de la capacitation induite par le HDL a été étudié. Les résultats obtenus suggèrent que, in vivo, la protéine BSPH1 de souris serait impliquée dans la capacitation via une interaction directe avec le HDL. Comme les protéines BSPH1 humaines et murines sont orthologues, ces résultats pourraient aussi s’appliquer à la fertilité humaine. Les résultats présentés dans cette thèse pourraient mener à une meilleure compréhension de la fertilité masculine et aider à améliorer les techniques de reproduction assistée. Ils pourraient également mener au développement de nouveaux tests diagnostiques ou de contraceptifs masculins. / Infertility can affect as much as 15-20% of couples of reproductive age. Therefore, elucidating mechanisms occurring during fertilization is needed to resolve cases of infertility and optimize assisted reproductive technology procedures. Sperm capacitation is a maturation step that takes place in the female genital tract and is deemed to be essential for sperm to fertilize an oocyte. Our laboratory has demonstrated that proteins from bovine seminal plasma called Binder of SPerm (BSP) proteins bind to choline phospholipids on the sperm membrane upon ejaculation and promote capacitation. These proteins expressed in seminal vesicles are ubiquitous amongst mammals and have been studied in many species including stallion, boar, ram and goat. More recently, the expression of BSP-homologous genes has been discovered in the epididymis of humans (BSPH1) and in mice (Bsph1 and Bsph2). We hypothesized that the BSP homologs in these two species are added to sperm during epididymal maturation and play similar roles in sperm functions as bovine BSP proteins. BSP proteins in humans and mice constitute only a minute percentage of the seminal plasma proteins. Thus, to study their biochemical and functional characteristics recombinant proteins were produced. Recombinant proteins were expressed in Escherichia coli origami B(DE3)pLysS cells using a pET32a expression vector. Following cell lysis, proteins were denatured using urea and purified by immobilized metal ion affinity chromatography. Once bound to the resin, proteins were refolded using a decreasing urea gradient after which they were eluted. This method led to the production of three pure, functional recombinant proteins (human rec-BSPH1, mouse rec-BSPH1 and mouse rec-BSPH2). Using affinity chromatography and co-sedimentation experiments, we were able to demonstrate that all three recombinant proteins bind known ligands of BSP proteins including gelatin, heparin and have the ability to bind to sperm. Studies also revealed that both rec-BSPH1 proteins bind to phosphatidylcholine (PC) liposomes and promote sperm capacitation. However, rec-BSPH2 neither binds to PC liposomes nor stimulates capacitation. Recombinant proteins had no effect on acrosome reaction or sperm motility. In bovine, BSP proteins promote sperm capacitation through interactions with high-density lipoproteins (HDL) and glycosaminoglycans. Since in mice HDL is also a major factor implicated in capacitation, the role of the native murine BSPH1 protein in HDL-induced capacitation was investigated. Results obtained suggest that, in vivo, murine BSPH1 protein could act in capacitation via a direct interaction with HDL. As human and murine BSPH1 are orthologs, these results could possibly also apply to human fertility. The results presented in this thesis could lead to a better understanding of male fertility and help improve assisted reproduction technology procedures. They could also lead to the development of diagnostic tests as well male contraceptives.