Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) proton ATPases, on the capacitation of rabbit spermatozoa.

Mammalian spermatozoa maintain precisely regulated ionic gradients that must be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membranes of three metabolically different types of ATPases. The modifications induced during in vitro capacitation of rabbit spermatozoa by the specific inhibition of V-type H+-ATPases with bafilomycin A were studied. We used chlortetracycline binding to rabbit spermatozoa to monitor capacitation, and the coomassie brilliant blue method to identify acrosome-reacted sperm cells. There was a significant difference between the percentage of epididymal (66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, after a 6-h incubation period in the presence of Ca2+ without ATPase inhibitor. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16+/- 8%, epididymal and ejaculated spermatozoa, respectively) and eliminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reactions induced by the addition of A23187 (12 +/- 5, 23+/- 6 and 31 +/- 5 after 15, 30 and 45 min). The presence of 0.2 micromol l-1 bafilomycin during the capacitation incubation induced a significant decrease in the acrosome reaction percentages (4 +/- 2, 8 +/- 3 and 14 +/- 4 after 15, 30 and 45 min). The addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate that vacuolar ATPases play an important role during rabbit sperm capacitation. However, once the spermatozoa have been capacitated, V-type ATPases do not have a significant participation during the acrosome reaction.

[The male factor. II. Spermatozoa. Structure and function]. / El factor masculino. II. El espermatozoide. Estructura y funcionamiento.

In the "male factor" entity, the structural and functional correlation determining the sperm fertilizing capacity is constituted by a group of cellular factors that must be evaluated in the semen of the infertile men. Structurally the spermatozoa of the head, the middle piece and the flagellum. The head has a highly condensed haploid nucleus, surrounded by a thin layer of cytoplasmic material, which is covered in a cap-like fashion by the membrane limiting the acrosome. This last organelle, which has characteristics similar to those of a secretory granule, secretes in a programmed way the hydrolytic enzymes that facilitate the fertilization process. The middle piece contains the mitocondrail sheet, responsible for the energy metabolism of the sperm cell. The flagellum has the same basic structure of other cilia or flagella, but also has particular characteristics due to the presence of the outer dense fibers and the fibrous sheath. In the semen analysis from infertile men the abnormalities most frequently observed belong to the number, morphology, variability, motility of capacitation-acrosome reaction of the sperm cells. However, due to the apparent multifactorial etiology of the male factor, now a days we only have few options for medical or pharmacological treatment. In this paper we review the morphology and ultrastructure considered as "normal" in the human spermatozoa, as well as we describe the most frequent alterations in these parameters. At the same time, we discuss the impact of sperm motility and of the capacitation-acrosome reaction process in male fertility.

Enzyme-linked immunosorbent assay (ELISA) with mycobacterial crude antigens for the sero-epidemiological diagnosis of active tuberculosis.

In search for reliable, nonexpensive procedures for tuberculosis diagnosis suitable for seroepidemiological studies in leprosy-endemic areas, enzyme-linked immunosorbent assays (ELISAs) with whole intact bacilli, whole lipid-free bacilli and protein-enriched soluble extracts from the H37Rv Mycobacterium tuberculosis strain were evaluated. Sera tested came from 47 active, pulmonary tuberculosis adult cases, 60 household contacts of active tuberculosis cases, 20 lepromatous leprosy adult patients, and 67 healthy adult controls obtained from low and high leprosy and tuberculosis endemicity areas. There was no influence of such endemicity levels in the number of positive results in control sera. Antibody levels obtained with each of the antigens in ELISAs were significantly different in tuberculosis patients and the control groups. Ten percent of tuberculosis contacts were positive with some of the antigens and three of them showed suggestive chest radiographs. The best combination for a high number of positive results with tuberculosis sera and low positive results with leprosy sera was the BCG soluble extract (91% and 15%, respectively). This preparation also yielded excellent sensitivity and specificity values for tuberculosis (91.5% and 92.5%, respectively). These data suggest that BCG soluble extract ELISAs could provide helpful information to estimate tuberculosis prevalence only in leprosy-free areas, under a situation of unavailability of purified antigens. In pulmonary cases, sputum microscopic examination and culture have higher sensibility than serodiagnosis; therefore, the utilization of BCG soluble extract ELISAs as a diagnostic aid in individual patients with suspected active tuberculosis only can be useful in extrapulmonary cases.

Alterations in pulsatile luteinizing hormone and follicle-stimulating hormone secretion in idiopathic oligoasthenospermic men: assessment by deconvolution analysis--a clinical research center study.

To investigate the nature of neuroendocrine disturbances of the hypothalamo-pituitary-gonadal axis in idiopathic male infertility, we studied 14 infertile men with oligoasthenozoospermia (OLIGO) and 15 age-, body mass index-, and community-matched euspermic controls by blood withdrawal at 10-min intervals for 12 h to encompass basal (8-h) and exogenous GnRH-stimulated (4-h) pulsatile release of LH and FSH (by immunoradiometric assay) as well as testosterone (by RIA). Deconvolution analysis was used to estimate endogenous LH and FSH half-lives, secretory burst frequency, amplitude, duration, and mass. OLIGO men exhibited normal serum concentrations of total, free, and percent dialyzable testosterone and estradiol, but distinct dynamic alterations within the LH and FSH axes; namely (P < 0.05), 1) a prolonged half-life of LH (OLIGO, 95 +/- 19 min; control, 80 +/- 9.3 min) and a reduced half-life of FSH (OLIGO, 260 +/- 79 min; control, 320 +/- 93 min); 2) a low LH, but normal FSH, secretory burst frequency (OLIGO, 12 +/- 3.4; control, 15 +/- 3.0 LH pulses/day); 3) a decreased serum testosterone peak frequency (OLIGO, 16 +/- 4.3; control, 21 +/- 3.2 peaks/day); and 4) an amplified mass of LH (1.1- to 1.3-fold higher in OLIGO) and FSH (2.4- to 2.7-fold higher in OLIGO) secreted per burst basally as well as after GnRH injection. These disturbances were readily distinguishable from the neuroendocrine dysregulation described in other states of male hypogonadotropism (e.g. uremia, fasting, and aging).

[Comparative study of the morpho-functional features of the sperm of fertile and infertile men and their relation to fertilizing ability]. / Estudio comparativo de las características morfo-funcionales de los espermatozoides de varones fértiles e infértiles y su relación con la habilidad fertilizante.

The recent advances in assisted reproduction procedures have helped to the better understanding of the female reproductive physiology and pathology, however, male infertility remains as a poor explained medical problem, nevertheless it occurs in almost 50% of infertile couples. Oligoasthenozoospermia is one of the more common causes of male infertility, therefore we measured in 10 patients with this diagnosis and in 10 fertile euspermic men, besides the parameters included in the standard semen analysis, the quantitative motility (photography method), capacitation-acrosome reaction and the sperm ability to fertilize zona-free hamster oocytes, with the aim to correlate the morphofunctional characteristics of the male gametes with their fertilizing capacity. The results showed significant differences in every parameter studied, including the correlation analysis. In relation with the in vitro induction of the acrosome reaction in both groups, we found significant correlations of the sperm fertilizing ability and the progressive sperm motility with this parameter (fertile group: RS = 0.834, P less than 0.005 & RS = 0.612, P less than 0.05; infertile group: RS = 0.986, P less than 0.001 & RS = 0.536, P less than 0.05 respectively), nevertheless the sperm rate which completed this process was low in relation to the total sperm population even in the fertile men (9.4 +/- 2.0% & 4.4 +/- 2.5% acrosome reacted cells after 18 h of incubation, in the fertile and infertile males respectively). The results also showed the presence of fully capacitated spermatozoa in both groups, since they penetrated the zona-free hamster eggs and decondensed their chromatin (73.9 +/- 13.4% & 10.4 +/- 7.7% penetrated eggs in the euspermic and oligoasthenozoospermic individuals respectively), however, the spermatozoa from the oligoasthenozoospermic men showed low polyspermy indexes too (0.1 penetrated spermatozoa/inseminated oocyte). In this last group we found, in addition, that the mean sperm velocity and the abnormal sperm morphology rate showed significant correlations with the fertilizing ability of the male gametes too (RS = 0.986, P less than 0.005 & RS = -0.942, P less than 0.005. respectively). These data allow us to suggest that before an infertile man is involved in any assisted reproduction program, the presence of possible morphofunctional alterations in the spermatozoa be analyzed, with the aim to be able to make a better prognosis about the success with these patients.

[Inhibin, steroid hormone, and gonadotropin concentration in blood from fertile and infertile males and its relationship with testicular function]. / Concentración de inhibina, hormonas esteroides y gonadotrofinas en el suero sanguíneo de varones fértiles e infértiles y su relación con la función testicular.

The role of inhibin in the testicular function and its relation with other hormone regulation spermatogenesis have not been elucidated in the human, therefore we studied by RIA the serum concentrations of this testicular secretion product, as well as those of LH, FSH, T and E2 and their possible correlation with some parameters evaluated by spermatobioscopy (cell number, motility, morphology and immature germinal cells) in fertile, oligozoospermic and azoospermic men. We did not find significant differences in inhibin concentration between fertile and oligozoospermic males; however in the azoospermic group the mean inhibin concentration was significantly higher (722.9 +/- 137.9 U/l in the fertile men; 658.5 +/- 147.1 and 963.1 +/- 300.9 U/l in the oligozoospermic and azoospermic groups respectively), in spite of data dispersion in the 3 study groups. Among the fertile males we found a negative significant correlation between the inhibin and the LH and FSH concentrations (p less than 0.05), while in the oligozoospermic patients this negative correlation was observed with T concentration. In the fertile males inhibin also showed correlation with the sperm percentage with normal morphology and with the immature germinal cells in semen; in the oligozoospermic group this glycoprotein showed correlation with the sperm count and the germinal cells concentration. We also found correlation between testosterone and/or LH concentrations and sperm count in both groups. These results indicate modifications in the hormonal concentrations regulating the testicular function and in their relationship, in men with testicular damage, which can render a change in the mechanisms controlling this function.(ABSTRACT TRUNCATED AT 250 WORDS)

[New concepts about the terminal mechanism of fertilization in mammals]. / Algunos conceptos nuevos sobre el mecanismo terminal de la fertilización en los mamíferos.

We briefly reviewed some ionic mechanisms participating in the fulfillment of the acrosome reaction processes, indicating the results obtained by the simultaneous and dynamic quantitation of sodium, potassium, calcium and hydrogen concentrations in the in incubation medium of human spermatozoa induced to achieve the acrosome reaction through the addition of cAMP and follicular fluid. At the same time, the participation of zinc and some enzymatic activities, like phospholipase A2, in the occurrence of this process, is indicated. A study is done about the sperm nuclear decondensation mechanisms, pointing at the importance of various participants in this event, like the disulfide groups reducing agents, some metal ions and the glycosaminoglycans, as well as a proposal of a mechanism which could be physiologically functional for the in vivo occurrence of this phenomenon. Finally we present some results explaining the DNA synthesis activation, which is repressed in the spermatozoa since the spermiogenesis final stages, and is indispensable for the chromosomic duplication required during egg segmentation.

[Several new concepts on the terminal mechanism of mammal fertilization]. / Algunos conceptos nuevos sobre el mecanismo terminal de la fertilización en los mamíferos.

We briefly reviewed some ionic mechanisms participating in the fulfillment of the acrosome reaction processes, indicating the results obtained by the simultaneous and dynamic quantitation of sodium, potassium, calcium and hydrogen concentrations in the in incubation medium of human spermatozoa induced to achieve the acrosome reaction through the addition of cAMP and follicular fluid. At the same time, the participation of zinc and some enzymatic activities, like phospholipase A2, in the occurrence of this process, is indicated. A study is done about the sperm nuclear decondensation mechanisms, pointing at the importance of various participants in this event, like the disulfide groups reducing agents, some metalic ions and the glycosaminoglycanes, as well as a proposal of a mechanism which could be physiologically functional for the in vivo occurrence of this phenomenon. Finally we present some results explaining the DNA synthesis activation, which is repressed in the spermatozoa since the spermiogenesis final stages, and is indispensable for the chromosomic duplication required during egg segmentation.

Demonstration of acid-fast bacilli in skin biopsies from indeterminate leprosy cases.

Because the correct diagnosis of indeterminate leprosy (IL) requires the finding of acid-fast bacilli in skin lesions from clinically and histopathologically suggestive cases, it is necessary to develop a reliable method for this purpose. This paper presents a simple procedure, available to every general laboratory, which consists in obtaining 2 suspensions: SI, by mincing and grinding the tissue in phosphate-buffered saline; and SII, after treating SI with NaOH solution and digesting with trypsin. In 22 IL skin biopsies, bacilli were directly observed in only 3 with the Ziehl-Neelsen (ZN) stain; and with the peroxidase-antiperoxidase method it was impossible to differentiate between nonspecific precipitate and true positive reactions. In contrast, 18 positive results from the same 22 samples were obtained when both SI and SII were evaluated with ZN stain. The logarithmic bacterial index was also increased in at least 7 cases.