RESUMO
In vitro preservation of the male gamete is a challenge in the development of artificial insemination techniques for domestic animals. Specific strategies and diluents have been developed for the preservation of the fertilizing ability of the semen for each species. However, the epididymal medium has been demonstrated to be the best sperm environment to maintain sperm viability over several days and weeks for mammals. The aims of this study were to evaluate the motility and in vivo fertility of ram epididymal spermatozoa when the semen was stored for up to 4 days at 4°C undiluted in epididymal plasma. The study was undertaken with two ovine breeds (Ile de France and Corriedale). The motility of epididymal spermatozoa was better preserved in the undiluted epididymal fluid than when epididymal spermatozoa were diluted in classic ovine extender such as skim milk. During storage, the decrease in the percentage of motile sperm was lower if the epididymal spermatozoa were collected immediately after epididymal sampling than 24 h after castration or animal death. The fertility obtained after cryopreservation of the stored sperm and subsequent intrauterine insemination ranged from 55% to 24% following 24 to 96-h sperm storage. There was a linear regression relationship between fertility and the number of motile sperm inseminated for both breeds. These results show that it is possible to keep epididymal sperm motile and fertile for several days without dilution. Such a method of sperm preservation could be a final possibility for animals of high genetic value or for endangered species when the collection of semen before death of the animal is not possible.
Assuntos
Criopreservação/veterinária , Fertilidade , Preservação do Sêmen/veterinária , Ovinos/fisiologia , Espermatozoides/fisiologia , Animais , Criopreservação/métodos , Epididimo/fisiologia , Fertilização in vitro/veterinária , Inseminação Artificial , Modelos Lineares , Masculino , Sêmen/fisiologia , Análise do Sêmen/veterinária , Preservação do Sêmen/métodos , Carneiro DomésticoRESUMO
Seminal plasma is composed of secretions from the epididymis and the accessory sex glands and plays a critical role in the fertilising ability of spermatozoa. In rams, analysis of seminal plasma by GeLC-MS/MS has allowed the identification of more than 700 proteins, including a high abundance of Binder of Sperm family proteins (BSP1, BSP5, SPADH1, SPADH2), the spermadhesin family (bodhesin2), lactoferrin and newly identified proteins like UPF0762 (C6orf58 gene). When spermatogenesis was stopped by scrotal insulation, changes in the proteome profile revealed the sperm origin of 40 seminal proteins, such as glycolysis pathway enzymes, the chaperonin containing TCP1 (CCT) complex and the 26S proteasome complex. Sperm mobility after liquid preservation (24h in milk at 15°C) is male dependent and can be correlated to differences in the seminal plasma proteome, detected by spectral counting. The negative association of zinc alpha-2 glycoprotein (ZAG) with semen preservation was confirmed by the use of recombinant human ZAG, which induced an increase in mobility of fresh sperm, but then decreased sperm mobility after 24h of incubation. Several sperm membrane proteins interacting with the cytoskeleton, glycolysis enzymes and sperm-associated proteins involved in capacitation correlated with better liquid storage and can be considered as seminal biomarkers of sperm preservation. BIOLOGICAL SIGNIFICANCE: Extensive analysis of the ram seminal plasma proteome reveals a complex and diverse protein composition. This composition varies between males with different sperm preservation abilities. Several proteins were shown to originate from the spermatozoa and positively correlate with sperm liquid preservation, indicating that these proteins can be traced as sperm biomarkers within the seminal plasma. The zinc alpha-2 glycoprotein (ZAG) was found to have a biphasic effect on sperm mobility, with a short-term stimulation followed by a long-term exhaustion of sperm mobility after a 24h preservation period.
Assuntos
Preservação do Sêmen , Sêmen/metabolismo , Proteínas de Plasma Seminal/metabolismo , Ovinos/metabolismo , Espermatozoides/metabolismo , Animais , Humanos , Masculino , Espermatozoides/citologiaRESUMO
Sperm transit in the female tract is one of the key factors in the success of fertilization after artificial insemination in sheep species. However, its study is limited by the absence of in vivo imaging methods. The imaging of ram sperm in the female genital tract was made possible using the confocal fibered microscopy and fluorescent stains adapted to spermatozoa. Our results show the active role of the uterotubal junction in the selection of sperm during their transit.
Assuntos
Genitália Feminina/fisiologia , Microscopia Confocal/métodos , Ovinos/fisiologia , Transporte Espermático , Espermatozoides/citologia , Animais , Feminino , Masculino , Microscopia Confocal/instrumentaçãoRESUMO
The major challenge of follicular fluid proteomic analysis is the presence of high-abundance proteins that originate from plasma. These proteins can prevent the detection of lower abundant ones, produced locally by follicle cells and that may have important roles in follicular activity. In this study, the novel technology called hexapeptide ligand library was evaluated to enrich the low-abundance proteins in follicular fluid of human (HFF), porcine (PFF) and equine (EFF) prior 2D-PAGE. Our results showed that the new strategy enabled detection of many new protein spots, increased resolution and highly improved the intensity of low-abundance proteins by 2D-PAGE.
Assuntos
Eletroforese em Gel Bidimensional/veterinária , Líquido Folicular/química , Cavalos , Biblioteca de Peptídeos , Proteínas/análise , Suínos , Animais , Feminino , Humanos , Proteômica/métodosRESUMO
The proteins that are neosynthesized and secreted in the different regions of the human epididymis were determined by in vitro biosynthesis of epididymal tubules, and the luminal proteins were collected by microperfusion of each tubule. The preparations were analyzed by two-dimensional gel electrophoresis and the proteins were identified by mass spectrometry. Some of the major proteins identified corresponded to serum compounds such as albumin, transferrin and alpha-1-antitrypsin. The other proteins identified included lactotransferrin, clusterin, PEBP, NCP2/CTP/HE1, HE3, Crisp, actin, calmodulin, E12, PGDS, l-lactate dehydrogenase, malate dehydrogenase, carbonic anhydrase, triose phosphate isomerase, glutamyltransferase, glutathione S-transferase P, thioredoxin peroxidase, superoxide dismutase, cathepsin D and cystatin. Epididymal activity is highly regionalized in most species. However, in this study in humans, there were only minor changes in the major proteins secreted. It is suggested that this specificity might be related to the difference between species in the location of the epididymis where sperm become fertile.
Assuntos
Epididimo/metabolismo , Proteoma/análise , Proteoma/metabolismo , Adulto , Eletroforese em Gel de Poliacrilamida , Epitélio/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas/análise , Proteínas/metabolismoAssuntos
Doenças dos Cavalos/diagnóstico , Infertilidade Masculina/veterinária , Sêmen/química , Proteínas de Plasma Seminal/análise , Animais , Proteínas de Transporte/análise , Glicoproteínas/análise , Cavalos/fisiologia , Infertilidade Masculina/diagnóstico , Oxirredutases Intramoleculares/análise , Lipocalinas , Masculino , Peptidil Dipeptidase A/análise , Proteínas de Transporte VesicularRESUMO
Boar sperm from the proximal caput epididymis were co-incubated with 1, 4, 7, 10 and 14-day old caput, corpus and cauda epididymal cultures for 24, 48 and 72 h. Boar kidney epithelial cells (LLC-PK1) and ECM alone were used as negative controls. Sperm motility, morphology and membrane integrity were studied to evaluate boar sperm maturation in vitro. Our results showed that epithelial cell monolayers (10, 14-day old) create a suitable microenvironment for the survival of proximal caput sperm and the maintenance of sperm motility over a 72 h period. Moreover, corpus epididymal tubule fragments in culture (1, 4-day old) are capable of promoting the migration of the cytoplasmic droplet along the sperm tail after 24h of co-incubation.
Assuntos
Epididimo/citologia , Epididimo/fisiologia , Espermatozoides/crescimento & desenvolvimento , Suínos , Animais , Membrana Celular/ultraestrutura , Técnicas de Cocultura , Células Epiteliais , Masculino , Motilidade dos Espermatozoides , Espermatozoides/anormalidades , Espermatozoides/ultraestruturaRESUMO
PHTF1 is an 84-86-kDa membrane protein found in the endoplasmic reticulum of male germ cells in rodents. There are no evident signs of PHTF1 in the spermatozoa released into the lumen of the seminiferous tubules but PHTF1 is present in the epididymal epithelium. Characterization of the epididymal Phtf1 messenger by Northern blot and reverse transcription-PCR identified a 3-kilobase transcript in the epididymis, similar to that previously reported in the testis. The transcript is present in the proximal part of the epididymis and it appears when the rats reach 4 wk of age. Through immunofluorescence analysis, PHTF1 was localized in the principal cells of the initial segment and the caput epididymis. Colocalization with different markers indicated PHTF1 is in the endoplasmic reticulum saccules applied to the trans face of the Golgi system. Western blot analyses revealed a shorter form of the protein--about 56-kDa versus the 84-kDa form found in the testis. Using the canine epididymal cell line CIM 20, transfected by N- and C-terminal myc-tagged PHTF1, we demonstrated that the 56-kDa epididymal form could result from proteolytical processing.
Assuntos
Epididimo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Células Cultivadas , Cães , Epididimo/citologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E Subtipo EP2 , Testículo/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
When mammalian spermatozoa exit the testis, they show a highly specialized morphology; however, they are not yet able to carry out their task: to fertilize an oocyte. This property, that includes the acquisition of motility and the ability to recognize and to fuse with the oocyte investments, is gained only after a transit through the epididymis during which the spermatozoa from the testis travel to the vas deferens. The exact molecular mechanisms that turn these cells into fertile gametes still remain mysterious, but surface-modifying events occurring in response to the external media are key steps in this process. Our laboratory has established cartographies of secreted (secretomes) and present proteins (proteomes) in the epididymal fluid of different mammals and have shown the regionalized variations in these fluid proteins along the epididymis. We have found that the main secreted proteins are common in different species and that enzymatic activities, capable of controlling the sperm surface changes, are present in the fluid. Our studies also indicate that the epididymal fluid is more complex than previously thought; it contains both soluble and particulate compartments such as exosome-like vesicles (epididymosomes) and certainly specific glycolipid-protein micelles. Understanding how these different compartments interplay to modify sperm components during their transit will be a necessary step if one wants to control and to ameliorate sperm quality and to obtain valuable fertility markers helpful to establish a male fertility based genetic selection.
Assuntos
Epididimo/citologia , Fertilidade , Maturação do Esperma/fisiologia , Espermatozoides/fisiologia , Testículo/citologia , Animais , Líquidos Corporais/química , Membrana Celular/fisiologia , Epididimo/química , Epididimo/fisiologia , Fertilidade/fisiologia , Masculino , Proteínas/fisiologia , Proteoma/fisiologia , Espermatozoides/ultraestrutura , Ducto Deferente/citologiaRESUMO
Northern blot analysis suggested that the boar epididymis produces closely related counterparts to human epididymal proteins HE1, HE3, HE4, HE5 and HE12. 'Full-length' cloning by nucleic acid and amino acid sequence similarity was achieved by RT-PCR methods in the case of the porcine counterparts of HE3 and HE4, while the homologues of HE5 and HE12, despite their cross-hybridization during Northern blot analysis, have not yet been cloned. The two novel porcine cDNAs were derived from moderately abundant epididymal mRNAs that were 75 and 83% identical to HE3 and HE4 cDNAs, respectively. To emphasize their relationship to the corresponding HEs, they were named Se3 and Se4 cDNAs. Their open reading frames predicted small secretory proteins with 55% (Se3) and 76% (Se4) conserved amino acids. Monospecific antipeptide antibodies to HE secretory proteins identified He3- and HE12-related proteins on Western blots of porcine epididymal fluid and semen. Both Northern and Western analyses indicated that the Se proteins were produced in a regionalized pattern and accumulated in the cauda fluid.
Assuntos
Clonagem Molecular , Proteínas Secretadas pelo Epidídimo/química , Suínos/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , DNA Complementar/química , DNA Complementar/genética , Proteínas Secretadas pelo Epidídimo/genética , Proteínas Secretadas pelo Epidídimo/metabolismo , Epididimo/metabolismo , Regulação da Expressão Gênica , Humanos , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência , Suínos/genéticaRESUMO
The 94-kDa ram epididymal fluid form of the sperm membrane-derived germinal angiotensin I-converting enzyme (ACE) was purified by chromatography, and some of its enzymatic properties were studied. For the artificial substrate furanacryloyl-L-phenylalanylglycylglycine (FAPGG), the enzyme exhibited a Michaelis constant (K(m)) of 0.18 mM and a V(max) of 34 micromoles/(min x mg) and for hippuryl-L-histidyl-L-leucine a K(m) of 2.65 mM and a V(max) of 163 micromoles/(min x mg) under the defined standard conditions (300 mM NaCl and 50 mM Tris; pH 7.5 and 8.3, respectively). The FAPGG hydrolysis was decreased by 82.5% and 67.5% by EDTA and dithioerythritol, respectively, and was totally inhibited by specific ACE inhibitors such as captopril, P-Glu-Trp-Pro-Arg-Pro-Glu-Ile-Pro-Pro, and lisinopril. Optimum activity for FAPGG was with pH 6.0, 50 mM chloride, and 500 microM zinc. Under the various conditions tested, bradykinin, angiotensin (Ang) I, Ang II, and LHRH were competitors for FAPGG. Bradykinin and angiotensin I were the best competitors. The enzyme cleaved Ang I into Ang II, and the optimal conditions were with pH 7.5 and 300 mM chloride. The relationship between the carboxypeptidase activity in seminal plasma and the prediction of fertility of young rams was also studied. These results indicated a correlation between sperm concentration and ACE activity in semen but showed no statistically significant correlation between such activity and fertility of the animal. Finally, we tested the role of ACE in fertilization; no difference in the in vitro fertilization rate was observed in the presence of 10(-4) M captopril.
Assuntos
Epididimo/enzimologia , Peptidil Dipeptidase A/metabolismo , Ovinos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Captopril/farmacologia , Membrana Celular/enzimologia , Ditioeritritol/farmacologia , Ácido Edético/farmacologia , Fertilização in vitro/veterinária , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Masculino , Oligopeptídeos/metabolismo , Peptidil Dipeptidase A/fisiologia , Sêmen/enzimologia , Solubilidade , Contagem de Espermatozoides , Espermatozoides/ultraestruturaRESUMO
The nucleus of mammalian spermatozoa is surrounded by a rigid layer, the perinuclear theca, which is divided into a subacrosomal layer and a postacrosomal calyx. Among the proteins characterized in the perinuclear theca, calicin is one of the main components of the calyx. Its sequence contains three kelch repeats and a BTB/POZ domain. We have studied the association of boar calicin with F-actin and the distribution of boar and human calicin during spermiogenesis compared with the distribution of actin. Calicin was purified from boar sperm heads under nondenaturating conditions. The molecule bound actin with high affinity (K(d) = approximately 5 nM), and a stoichiometry of approximately one calicin per 12 actin monomers was observed. Gel filtration studies showed that calicin forms homomultimers (tetramers and higher polymers). According to immunocytochemical results, calicin is present (together with actin) in the acrosomal region of round spermatids and is mainly localized in the postacrosomal region of late spermatids and spermatozoa. Taken together, the results suggest that the affinity of calicin to F-actin allows targeting of calicin at the subacrosomal space of round spermatids, and that its ability to form homomultimers contributes to the formation of a rigid calyx.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Separação Celular , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Proteínas do Citoesqueleto/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas In Vitro , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Cabeça do Espermatozoide/química , Cabeça do Espermatozoide/fisiologia , Espermatozoides/química , Suínos , Testículo/citologiaRESUMO
LHRH Statin is a putative gonadal protein that increases the interval between two consecutive LHRH pulses. The present work was aimed at analyzing the immunological homology between LHRH Statin and the N-terminal region of the alphaC subunit of inhibin. Thus, rete testis fluid (RTF) proteins were purified by immunoaffinity chromatography using antibodies against residues 1-7 plus 7-30 (experiment 1, A-fractions) and 14-28 of the alphaC inhibin subunit (experiment 2, B-fractions), and the LHRH Statin activity of the fractions was examined by intracerebroventricular administration in castrated rams followed by RIA of plasma LH levels in 15-min blood samples. Fractions that bound to the immunoaffinity column with low affinity were eluted with 0.5 M NaCl, pH 7.4 (-F2); then highly bound fractions were eluted sequentially in acidic (pH 2.5, -F3) followed by basic conditions (pH 11.5, -F4). In experiment 1, RTF (40 microg, n = 4) and highly bound fractions (A-F3, 30 ng, n = 8, 150 ng, n = 3; A-F4, 120 ng, n = 5) decreased LH mean plasma levels between 4 and 6 h after injection by 39%, 29%, 43%, and 37%, respectively (P<0.001 to 0.01), while the weakly bound fractions (A-F2, 180 ng, n = 4) and albumin control (40 microg, n = 4) had no activity. In experiment 2, RTF (100 microg, n = 4) and B-F3 (100 ng, n = 3) decreased plasma LH levels by 48% and 38%, respectively (P<0.001 to 0.05), whereas B-F4 (100 ng, n = 4) and albumin control (100 microg, n = 4) had no effect. A fraction obtained from B-F3 by gel filtration had significant LHRH Statin activity (63%, n = 6, P<0.001). PAGE with colloidal gold staining revealed 3 high molecular weight bands and 5 low molecular weight bands in B-F3. The 3 high molecular weight bands were shown to belong to the clusterin family and did not appear to have LHRH Statin activity. The 5 low molecular weight bands were all labeled by anti-alphaC inhibin antibodies. Collectively, these results strongly suggest that LHRH Statin has some homology with the 14-28 alphaC inhibin sequence.
Assuntos
Líquidos Corporais/química , Epitopos/imunologia , Hormônio Liberador de Gonadotropina/imunologia , Inibinas , Peptídeos/imunologia , Rede do Testículo/metabolismo , Ovinos , Animais , Especificidade de Anticorpos , Cromatografia de Afinidade , Epitopos/análise , Epitopos/química , Hormônio Liberador de Gonadotropina/isolamento & purificação , Hormônio Liberador de Gonadotropina/farmacologia , Técnicas Imunológicas , Hormônio Luteinizante/sangue , Masculino , Peptídeos/análise , Peptídeos/isolamento & purificação , Homologia de SequênciaRESUMO
Proteins present in and secreted into the lumen of various regions of the stallion epididymis were characterized qualitatively and quantitatively by two-dimensional electrophoresis. Using this proteomic approach, 201 proteins were found in the lumen and 117 were found that were secreted by the epithelium in various parts of the organ. Eighteen proteins made up 92.6% of the total epididymal secretory activity, lactoferrin (41.2%) and clusterin (24.8%) being the most abundant. Procathepsin D, HE1/CTP (cholesterol transfer protein), GPX (glutathione peroxidase), beta-N-acetyl-hexosaminidase, and PGDS (prostaglandin D2 synthase) were the other major compounds secreted. The most abundant proteins found in the luminal fluid were albumin and the secreted proteins: lactoferrin, PGDS, GPX, HE1/CTP, and hexosaminidase. Three main secretory epididymal regions were identified from the protein pattern, i.e., regions E0-E2, E3-E5, and E6-E9. Region E0-E2 was characterized by the secretion of clusterin (53%), PGDS (44%), and GPX (6%). Region E3-E5 had the highest number of secreted proteins, the highest protein concentrations (60-80 mg/ml), and the highest spermatocrit value (85%). Lactoferrin (60% in E4), clusterin (29% in E3), hexosaminidase (10% in E3), and procathepsin D (6.9% in E4) were the most abundant proteins in this region. Region E6-E9, in which few region-specific secreted compounds were found, was characterized by a high quantity of lactoferrin in the luminal fluid (2-14 mg/ml). Comparison between the secretion of the major proteins and their concentrations in the lumen throughout the organ showed that the behavior of each protein is specific, in particular for the three isoforms of clusterin.
Assuntos
Proteínas de Transporte , Epididimo/metabolismo , Cavalos/metabolismo , Chaperonas Moleculares , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Líquidos Corporais/química , Catepsina D/análise , Catepsina D/química , Catepsina D/metabolismo , Clusterina , Eletroforese em Gel Bidimensional , Epididimo/anatomia & histologia , Epitélio/metabolismo , Glutationa Peroxidase/análise , Glutationa Peroxidase/química , Glutationa Peroxidase/metabolismo , Glicoproteínas/análise , Glicoproteínas/química , Glicoproteínas/metabolismo , Cavalos/anatomia & histologia , Oxirredutases Intramoleculares/análise , Oxirredutases Intramoleculares/metabolismo , Lactoferrina/análise , Lactoferrina/metabolismo , Lipocalinas , Masculino , Dados de Sequência Molecular , Proteínas/análise , Proteoma , Análise de Sequência de Proteína , Contagem de Espermatozoides , Proteínas de Transporte Vesicular , beta-N-Acetil-Hexosaminidases/análise , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-beta-D-glucopy-ranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23-kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.
Assuntos
Epididimo/metabolismo , Glicoproteínas/metabolismo , Espermatozoides/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/imunologia , Imuno-Histoquímica , Masculino , Octoxinol , Fragmentos de Peptídeos/análise , Polietilenoglicóis , Ovinos , Solubilidade , Maturação do Esperma/efeitos dos fármacos , Testosterona/metabolismoRESUMO
The number of proteins secreted by the boar epididymis increased progressively from 1 mo of age to the adult period. The first specific secretory activity was revealed at 2 mo in the distal caput (hexosaminidase, clusterin, and lactoferrin) and in the corpus (train O/HE1). Train A and glutathione peroxidase specific to the proximal caput, and trains E and M specific to the corpus, appeared at 4 mo. At 5 mo, secretion of procathepsin L occurred in the middle caput and that of mannosidase and E-RABP in the distal caput. Approximately 48% of all the proteins secreted in the adult boar epididymis were dependent on the presence of androgens, either stimulated (33.6%) or repressed (14.4%); 47% were modulated by other factors, and 5% were unregulated. In the proximal caput, 50% of the specific secreted proteins were controlled essentially by factors emanating from the testis. In more distal regions, two proteins secreted in the corpus were regulated by factors from the anterior regions. The regionalization of the secretory activity of the epididymal epithelium resulted in a specific regulation for each protein, which was modulated according to the region of expression and influenced by either testicular or epididymal factors that remain to be identified.
Assuntos
Epididimo/crescimento & desenvolvimento , Epididimo/metabolismo , Chaperonas Moleculares , Proteínas/metabolismo , Suínos/crescimento & desenvolvimento , Envelhecimento , Androgênios/farmacologia , Animais , Catepsinas/metabolismo , Clusterina , Glutationa Peroxidase/metabolismo , Glicoproteínas/metabolismo , Lactoferrina/metabolismo , Masculino , Orquiectomia , Precursores de Proteínas/metabolismo , Suínos/fisiologia , Testosterona/sangue , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
A lysosomal type alpha-D-mannosidase was successfully purified by DEAE-Sephacel, Red-Amicon and Superdex 200 column chromatographies from porcine cauda epididymal fluid. The purified enzyme consisted of 63 and 51 kDa subunits at equimolar amounts. It cleaved alpha1-2 linked mannosyl residues and less but significantly cleaved alpha1-3 and alpha1-6 linked mannosyl residues in the high-mannose oligosaccharides. The optimal pH to hydrolyze oligosaccharide was in the acidic pH range (pH 3.5 approximately 4.0). Total alpha-D-mannosidase activities in the porcine epididymal fluid increased from proximal to distal caput epididymis, which maintained to cauda epididymis. At least two kinds of alpha-D-mannosidase (lysosomal type enzyme and 135 kDa alpha-D-mannosidase (MAN2B2)) were contained in the porcine epididymal fluid. The activity of the lysosomal type enzyme is much higher than MAN2B2 at the physiological pH. These results suggest that the lysosomal type alpha-D-mannosidase is the predominantly active enzyme in the luminal fluid of porcine epididymis and that it participates in the glycoprotein modification on the sperm surface during epididymal transit.
Assuntos
Líquidos Corporais/enzimologia , Epididimo/enzimologia , Manosidases/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Carboidratos , Concentração de Íons de Hidrogênio , Lisossomos/enzimologia , Masculino , Manosidases/química , Dados de Sequência Molecular , Especificidade por Substrato , Suínos , alfa-ManosidaseRESUMO
A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.
Assuntos
Colesterol/metabolismo , Epididimo/metabolismo , Glicoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Líquidos Corporais/metabolismo , Proteínas de Transporte/isolamento & purificação , Clonagem Molecular , DNA Complementar/metabolismo , Epididimo/química , Biblioteca Gênica , Glicoproteínas/isolamento & purificação , Masculino , Dados de Sequência Molecular , Peso Molecular , Elastase Pancreática , Proteínas , RNA Mensageiro/análise , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Maturação do Esperma , Espermatozoides/metabolismo , Suínos , Proteínas de Transporte VesicularRESUMO
Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.
Assuntos
Feto/fisiologia , Transplante de Células-Tronco Hematopoéticas , Transplante Heterólogo/fisiologia , Envelhecimento/fisiologia , Animais , Feminino , Citometria de Fluxo , Cabras , Sobrevivência de Enxerto/fisiologia , Humanos , Cariotipagem , Ovinos/embriologiaRESUMO
SDS-PAGE analysis of luminal fluid from the ram testis and epididymis revealed a protein of about 105 kDa in the fluid in the caput epididymal region. The molecular mass of this fluid protein shifted from 105 kDa to 94 kDa in the distal caput epididymidis and remained at 94 kDa in the lower regions of the epididymis. The possible sperm origin of this protein was suggested by the decrease in intensity of a 105-kDa compound on the sperm plasma membrane extract and by its total disappearance from the fluid of animals with impaired sperm production caused by scrotal heating. The 94-kDa protein was purified from ram cauda epididymal fluid, and a rabbit polyclonal antiserum was obtained. This antiserum showed that membranes of testicular sperm and sperm from the initial caput were positive for the presence of an immunologically related antigen. The protein was immunolocalized mainly on the flagellar intermediate piece, whereas in some corpus and caudal sperm, only the apical ridge of the acrosomal vesicle was labeled. The purified protein was microsequenced: its N-terminal was not found in the sequence database, but its tryptic fragments matched the sequence of the angiotensin I-converting enzyme (ACE). Indeed, the purified 94-kDa protein exhibited a carboxypeptidase activity inhibited by specific blockers of ACE. All the soluble seminal plasma ACE activity in the ram was attributable to the 94-kDa epididymal fluid ACE. The polyclonal antiserum also showed that a soluble form of ACE appeared specifically in the caput epididymal fluid of the boar, stallion, and bull. This soluble form was responsible for all the ACE activity observed in the fluid from the distal caput to the cauda epididymidis in these species. Our results strongly suggest that the epididymal fluid ACE derives from the germinal form of ACE that is liberated from the testicular sperm in a specific epididymal area.