RESUMO
Acrosome reaction (AR) induced by low temperature has been used to evaluate sperm function; it correlates adequately with the fertilization percentages in vitro. In this study, the technique of AR induction by low temperature was used to evaluate the effect in the protection of the acrosome by cryopreservatives normally used in human semen cryopreservation. Donor sperm selected by use of the migration sedimentation technique was incubated in human tubal fluid medium, added to dimethyl sulphoxide 1 m, ethylene glycol 0.75 m, glycerol 1 m, incubated at 4 degrees C and 20 degrees C (as a control) for 18 h, and then for 3 h at 37 degrees C in a cell incubator. The AR was evaluated by triple stain in 100 viable spermatozoa. The effect of cryopreservatives on acrosome preservation in samples incubated for 18 h at 4 degrees C was as follows: 78% intact acrosome for glycerol, 77.8% intact acrosome for dimethyl sulphoxide and 96.2% intact acrosome for ethylene glycol (P < 0.0025 compared with glycerol and dimethyl-sulphoxide). The sperm samples incubated with cryopreservatives for 18 h at 20 degrees C did not show an increase in the percentage of AR in samples incubated with glycerol and ethylene glycol, while a significant variation was observed in the sample incubated with dimethyl sulphoxide (P < 0.001). Additional incubation for 3 h at 37 degrees C significantly increased the AR only in the sample incubated with glycerol (P < 0.001). Acrosome preservation is essential in the fertilization process and the evaluation of acrosome reaction induction by low temperature test was satisfactory. This test proves that ethylene glycol presents a greater protective effect on the acrosome preservation of human spermatozoa.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Preservação do Sêmen/métodos , Espermatozoides/efeitos dos fármacos , Reação Acrossômica/fisiologia , Dimetil Sulfóxido/farmacologia , Etilenoglicol/farmacologia , Fertilização/fisiologia , Glicerol/farmacologia , Humanos , Masculino , Espermatozoides/fisiologia , TemperaturaRESUMO
Alpha-glucosidase activity (EC.3.2.1.20) is present in human seminal plasma, and the neutral form of the enzyme originates almost exclusively from the epididymis. In this study, the specific immunocytochemical location of alpha-glucosidase in the human epididymis was evaluated using a polyclonal antibody. Furthermore, a spectrophotometric assay was employed to assess epididymal obstruction in infertile patients. The enzymatic activity of alpha-glucosidase free of prostate isoform (AGFPI) was determined spectrophotometrically at 405 nm. According to AGFPI activity, patients with leucocytospermia, oligozoospermia and azoospermia were recorded as having normal values or low values indicating epididymal obstruction. Specific immunochemistry staining was demonstrated in the cytoplasmic cells at the epithelial level, in the transition area and in the efferent ducts. The values of the three groups and the control were as follows (mean +/- SEM): normozoospermia (control): 20.2 +/- 1.4 mU ml(-1); azoospermia: normal value: 17.6 +/- 2.2 mU ml(-1), low value: 7.4 +/- 1.8 mU ml(-1); oligozoospermia: normal value: 22.3 +/- 2.5 mU ml(-1), low value: 7.3 +/- 0.7 mU ml(-1); leucocytospermia: increase value: 38.9 +/- 3.7 mU ml(-1), low value: 11.1 +/-1.3 mU ml(-1). This study suggests that determination of alpha-glucosidase might be helpful to evaluate functions of the epididymis and particularly to exclude epididymal obstruction.
Assuntos
Epididimo/enzimologia , Infertilidade Masculina/enzimologia , Infertilidade Masculina/etiologia , Doenças Testiculares/complicações , Doenças Testiculares/diagnóstico , alfa-Glucosidases/metabolismo , Adulto , Animais , Western Blotting , Constrição Patológica , Eletroforese em Gel de Poliacrilamida , Humanos , Soros Imunes , Imuno-Histoquímica , Masculino , Coelhos , Sêmen/enzimologia , Espectrofotometria , Distribuição TecidualRESUMO
BACKGROUND: The presence of leukocytes, detected by peroxidase test in semen, can be a good indicator of infections in the male genital tract. Peroxidase positive cells have been positively correlated with elevated values of elastase, one of the major proteases liberated by granulocytes at the inflammation place. However, seminal granulocytes may not be adequately detected by the peroxidase test in comparison with immunological methods. AIM: To correlate the determination of peroxidase positive cells with the elastase level in the seminal plasma. MATERIAL AND METHODS: Seminal plasma from 64 patients with a high number of round cells (> 106/ml) in semen, was studied. Correlation analysis was done using the Pearson correlation coefficient. RESULTS: No correlation between the level of granulocyte elastase and the number of peroxidase positive cells (r = 0.2237, p > 0.05), or even the number of round cells (r = 0.03934, p > 0.05) was observed. CONCLUSIONS: Our results suggest that the determination of peroxidase positive cells is not a reliable indicator of leukocytes in the seminal plasma and their absence do not discard a silent genital tract infection.
Assuntos
Humanos , Masculino , Ensaios Enzimáticos Clínicos , Doenças dos Genitais Masculinos/diagnóstico , Elastase de Leucócito/análise , Infecções/diagnóstico , Peroxidase/análise , Sêmen/enzimologia , Reprodutibilidade dos Testes , Granulócitos/enzimologia , Leucócitos/enzimologia , Biomarcadores/análise , Sêmen/citologiaRESUMO
Sperm culture media used for in vitro fertilization (IVF) procedures are important factors concerning the viability, motility and acrosomal integrity of spermatozoa. The aim of this study was to investigate the effects of three different sperm diluting media, tissue culture medium (TCM-199), sperm culture medium (Sp-TALP) and human tubular fluid (HTF) supplemented with varying concentrations of bovine serum albumin (1, 4 and 6%) or polyvinyl alcohol (0.8%) on the acrosomal integrity, motility and viability of canine spermatozoa. Ejaculates collected from four dogs were diluted in all media and spermatozoa were separated from seminal plasma by the swim-up technique. Sperm progressive motility was assessed using a phase contrast microscope. Viability and acrosomal integrity were evaluated using a dual stain technique (Giemsa-Trypan blue). The results demonstrated that the number of live canine spermatozoa was similar in culture media supplemented or not supplemented with macromolecules. A minimal concentration of albumin (1%) in the three media showed similar effects on vitality, motility and acrosomal integrity, as had higher concentrations (4 and 6%). The percentage of acrosome-intact spermatozoa was markedly higher after HTF (94.1%) than after TCM-199 (70.1%) or Sp-TALP (71.0%) without supplementation. It is concluded that serum bovine albumin, irrespective of the concentration, preserved sperm viability and function, and HTF is the most suitable medium for preserving the acrosome in canine spermatozoa prepared for in vitro manipulation through short incubation.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Meios de Cultura/farmacologia , Cães/fisiologia , Álcool de Polivinil/farmacologia , Albumina Sérica/farmacologia , Capacitação Espermática/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacos , Animais , Bovinos , Inseminação Artificial/veterinária , Masculino , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Albumina Sérica/administração & dosagemRESUMO
Outer dense fibers (ODF) are structural elements in the mammalian sperm tail which surround the axoneme in the midpiece and principal piece and probably may help to maintain the elastic structures and elastic recoil of the sperm tail. In the present study, we have generated and characterized and describe a series of monoclonal antibodies (mAbs) against the 30 kDa major protein from boar ODF. For antibody screening an ELISA was developed using a newly developed method to fix the ODF proteins to the solid phase. A total of seven mAbs were selected and characterized by ELISA, Western blot and immunofluorescence. The mAbs recognize the major protein component of boar ODF on preparative Western blot and mark the mid- and principal piece of demembranated flagella. These mAbs also recognize the mid- and principal piece of demembranated human spermatozoa from normozoospermic patients, but not from those with asthenozoospermia. For the first time, we succeeded in obtaining hybridoma cell lines that secrete mAbs of class IgM, which react with the 30 kDa protein of boar ODF.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Choque Térmico , Proteínas/imunologia , Cauda do Espermatozoide/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , SuínosRESUMO
PURPOSE: Two types of glass wool were used to remove leukocytes in semen for evaluation of reactive oxygen species production by spermatozoa in oligozoospermic patients with leukocytospermia. METHODS: Semen samples were prepared using fine-structure glass wool (SpermFertil) and coarse-structure glass wool. In each treatment group, native semen was evaluated for sperm concentration, percentage motility, viability, leukocyte concentration, and production of reactive oxygen species. RESULTS: Electron microscopically, SpermFertil showed a higher number of leukocytes attached to the fibers compared to coarse-structure glass wool. Leukocytes in native semen and after glass wool filtration as determined by peroxidase cytochemistry confirmed this observation. Reactive oxygen species decreased from 45.303 counts/10(7) viable cells in native semen to 15.806 counts/10(7) cells in coarse structure wool and 7.465 counts/10(7) cells in Spermfertil, respectively. CONCLUSIONS: Removal of leukocytes from semen of oligozoospermic patients by means of glass wool filtration is a useful method to distinguish production of reactive oxygen species by leukocytes versus sperm cells.
Assuntos
Vidro , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/metabolismo , Humanos , Leucócitos/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Estresse Oxidativo , Peroxidase/análise , Peroxidase/metabolismo , Sêmen/química , Sêmen/metabolismo , Contagem de Espermatozoides , Motilidade dos EspermatozoidesRESUMO
PURPOSE: To improve the chances of successful in vitro fertilization, spermatozoa have to be separated from semen before insemination. Therefore, sperm preparation methods are of great importance. METHODS: To obtain sufficient numbers of spermatozoa from patients with cryptozoospermia or severe OAT syndrome, only Minipercoll centrifugation and migration-sedimentation (MS) are practicable methods. The present study was performed to compare these two methods with regard to sperm concentration, motility, vitality, morphology, and chromatin condensation. The number of spermatozoa obtained after minipercoll was higher than that after MS, but sperm quality in all parameters examined was clearly better after MS than after Minipercoll. In the second stage of this study, the MS method was used for preparation of the spermatozoa for intracytoplasmic sperm injection (ICSI). RESULTS: Over a period of 13 months, 159 cycles were treated by ICSI. Of 1045 aspirated oocytes, 790 were injected. The fertilization rate was 70.4% of injected oocytes (556 oocytes with clearly visible pronuclei). In 146 cases, embryonic transfer was achieved; 58 patients became pregnant (39.7% per transfer and 36.5% per cycle). CONCLUSIONS: Although the abortion rate was very high (18 women lost their embryos), the results demonstrate that the microinjection method can be successfully used in combination with a MS method for preparation of spermatozoa.
Assuntos
Fertilização in vitro , Espermatozoides/metabolismo , Aborto Espontâneo , Morte Celular , Movimento Celular , Separação Celular/métodos , Centrifugação , Cromatina/metabolismo , Transferência Embrionária , Feminino , Fertilização in vitro/métodos , Humanos , Infertilidade/metabolismo , Masculino , Microinjeções/métodos , Oócitos/metabolismo , Povidona/uso terapêutico , Gravidez , Resultado da Gravidez , Sêmen/metabolismo , Dióxido de Silício/uso terapêutico , UrologiaRESUMO
The state of the acrosomal membranes in human spermatozoa was studied by means of the hypo-osmotic swelling test and indirect immunofluorescence using anti-boar outer acrosomal membrane antibodies. The swelling phenomenon observed in the acrosomal region was characterized by expansion of the plasma membrane without modification of the outer acrosomal membrane.