Adolfo Rosado

A kinetic study of the participation of zinc in human spermatozoa metabolism.

Abstract Incubation of washed human spermatozoa in the presence of 6 mM concentrations of EDTA, histidine and cysteine induces a release of about 75% of the zinc bound to the cells. No zinc is released by human spermatozoa when incubation is done in the absence of the mentioned reagents. No detectable amounts of calcium or magnesium were found to be released by the sperm cells under any of the experimental conditions tested. Zinc release induced by the presence of EDTA, histidine and cysteine is accompanied: 1) by a significant increase in oxygen uptake, both under basal conditions and in the presence of some substrates (glucose, pyrubate and succinate) and 2) by a significant increase in motility. This increase was greater with cysteine than with histidine, and greater with the latter than with EDTA. This behavior of human spermatozoa resembles that previously described for invertebrate spermatozoa and is related to the regulation of energy metabolism and probably to sperm capacitation.

Binding of 17-β-estradiol to the outer surface and nucleus of human spermatozoa

The binding of 3H-17-β-estradiol to human ejaculated spermatozoa and to its subcellular structures was studied. The binding kinetics of the labeled steroid to whole spermatozoa followed a parabolic pattern. Scatchard-type plots showed the presence of high-affinity binding sites (1.56 ± 0.23 × 104 per sperm cell) with an apparent Kd of 6.6 × 10−10 M. In competition experiments testosterone was partially effective in decreasing 17-β-estradiol binding, whereas progesterone and 17-α-estradiol were ineffective. Study of membrane fractions obtained from estradiol-labeled spermatozoa showed that under saturating conditions 75–84% of the bound steroid was bound to sperm membranes. Nuclear fractions obtained from estradiol-labeled spermatozoa showed only 10% of the total bound radioactivity. When isolated sperm nuclei were incubated in the presence of the purified receptor-17-β-estradiol complex obtained from the high-speed supernatant of human uterus almost no transfer of radioactivity to the nuclei was observed.

Amino Acid Pools in the Feto-Maternal System

The determinations of the amino acid pools, including tryptophan (by fluorescence spectrometry), in the arterial and venous blood of the mother, the arterial and venous blood of the fetus (cord blood), the amniotic fluid and the placenta was done in eight women at the moment of delivery. Only nine amino acids (asp, try, met, phe, ser, cys, lys, gly, thr) were significantly retained and four (arg, glu, pro and glu-NH2) were significantly released by the fetal tissues. In contrast with this behavior most amino acids were retained by the maternal tissue, try, phe and hist showing the highest retention. When the amino acids are grouped as essential and nonessentials, the maternal tissues retained both categories without apparent discrimination, while the fetal tissues retained essential amino acids preferentially. Our results emphasize the importance of the placenta as the regulating system of the fetal milieu under normal conditions. Thus human fetal blood levels of amino acids are patterned after the placental ones and not after the maternal values obtained at the same time. It is apparent that the placenta seems to function as a nonspecific retention filter for outgoing amino acids, but that its function is selective in respect to the release of amino acids into the fetal circulation.

Metalloproteinase activity during growth, maturation and atresia in the ovarian follicles of the goat

Metalloproteinases are an important group of hydrolytic enzymes which participate in interstitial matrix degradation during tissue remodelling processes and therefore may be required during follicular growth and maturation. The activity of metalloproteinases (collagenases, gelatinase, and Pz-peptidase), was measured during growth, maturation and atresia of goat antral follicles. These follicles (n = 67) were separated by size and also classified into four groups: non-atretic (Group I); early atretic (Stage I) (Group II); moderately atretic (Stage II) (Group IIIa); and, late atretic (Stage III) (Group IIIb). Pz-peptidase was greater in granulosa than in thecal cells, and almost absent in follicular fluid. In non-atretic follicles, activity in granulosa cells increased with increasing follicle size, whereas activity peaked in 3-6 mm follicles in thecal cells. Atresia was associated with declining activity in thecal cells from follicles in the 3-6 mm range and in granulosa cells from the > 6 mm range. Interstitial collagenase activity was significant and similar in granulosa and thecal cell extracts and low in follicular fluid from non-atretic follicles. Activity increased significantly in thecal cells, but decreased significantly in granulosa cells from large (> 6 mm) non-atretic follicles. Atresia was associated with declining activity in both types cells and increasing activity in follicular fluid. Gelatinase activity was some times associated with five regions corresponding to molecular weights of 22.1, 30.7, 39.6, 63.8 and 71.4 kDa, and rarely at 91.3 and 81.2 kDa. Overall activity declined with atresia in thecal cells from follicles in the 3-6 mm range, but not in those > 6 mm. In granulosa cells from follicles 3-6 mm, activity varied widely with stage of atresia, while in cells from follicles > 6 mm, activity was greatly increased in atretic follicles.

Intrauterine contraception with the progesterone-T device

Abstract The possible mechanism of action of the progesterone-releasing intrauterine device has been studied by means of determination of the action that uterine washings have on the metabolism and capacitation of human and rabbit spermatozoa. Uterine washings from normal, untreated women, induce, in human spermatozoa, the same changes although of lesser magnitude, that have been previously reported as possibly participating in the uterine stage of capacitation. Uterine washings obtained from progesterone-T wearing women produce a significant decrease in oxygen uptake and glucose utilization, an inhibition of the BANA-hydrolytic activity, and changes in the tetracycline binding and release processes. All these changes support the hypothesis that at least part of the mechanism of action of the progesterone-releasing type of intrauterine devices is due to a direct capacitation inhibiting effect of the uterine secretion. This hypothesis is strengthened by the direct demonstration of inhibition of in vitro rabbit spermatozoa capacitation produced by these washings.

Inhibition of human sperm motility by calcium and zinc ions

Abstract Calcium and zinc chloride were effective in impairing the motility and viability of human spermatozoa at high molar concentrations. The effect of these ions on several enzymatic activities and on oxygen uptake was measured in order to explain the mechanism by which immobilization was produced. The possibility of using these salts in fertility regulation is suggested.

Exchange of Lipids Between Spermatozoa and Seminal Plasma in Normal and Pathological Human Semen

Normal and pathological semen were studied with regard to cholesterol and phospholipid content of sperm cells and seminal plasma. Spermatozoa from pathologic semen have similar concentrations of phospholipid-phosphorous and significantly higher cholesterol concentration than spermatozoa from normal semen. However, only oligoasthenospermic spermatozoa showed a significantly higher cholesterol/phospholipid ratio. Azoospermic seminal plasma showed the lowest values of both cholesterol and phospholipids, but the ratio of cholesterol to phospholipids was equal to that in normal spermatozoa. No significant difference was found in the cholesterol concentration of seminal plasma from oligoasthenospermic, asthenospermic, and normospermic subjects and only asthenospermic plasma showed a significantly lower concentration of this compound. Cholesterol and phospholipid exchange between sperm cells and seminal plasma was shown by the striking correlation between the lipid composition of seminal plasma with that of sperm cells.

Amino Acid and Protein Concentrations of Human Follicular Fluid

The amino acid and protein composition of human follicular fluid, obtained during surgery from women with polycystic ovaries, and of a simultaneously obtained sample of blood plasma were studied. In general, amino acid concentrations were higher in follicular fluid than in blood plasma: only the concentration of Cys was significantly lower in follicular fluid than in plasma, while Asp, Thr, Glu, Glu-NH2, Gly, Ala, and Met showed concentrations that were not significantly different in either biologic fluid. The concentration of basic amino acids, taken as group, was almost twice as high in follicular fluid as in plasma. The total protein concentration in follicular fluid was not significantly different from that in blood plasma. However, the follicular fluid albumin concentration was higher and globulin concentration lower than the respective concentrations in plasma. Polyacrylamide gel disc electrophoresis of follicular fluid showed some consistent differences, particularly in the alpha-globulin region, with the pattern observed in blood plasma. These findings are discussed in relation to the possible role of follicular fluid in capacitation and egg segmentation.

Modification of human sperm metabolism by the induced release of intracellular zinc

Incubation of washed human spermatozoa in the presence of 6 mM histidine induces a release of about 75% of the zinc bound to the cells. Zinc release induced by the presence of histidine was accompanied by: 1) a significant increase in the utilization of exogenous 14C-labelled glucose, which is reflected in an increase in the production of 14CO2, 2) a small but significant decrease (−14%) in the utilization of fructose when this sugar is added as exogenous substrate, and 3) a highly significant decrease in the endogenous sperm phospholipids (>30%), an effect which is not inhibited by the addition of exogenous substrates. This behavior of human spermatozoa resembles that previously described for invertebrate spermatozoa and seems to be related to the regulation of energy metabolism and probably to sperm capacitation.

Cyclic-amp receptors in the human spermatozoa membrane

Abstract The binding properties of cyclic-AMP to human spermatozoa were studied. Incubation of whole human spermatozoa and of head and tail fractions with 14 C-cyclic AMP induced the binding of 7.8 ± 0.86 pmoles of the nucleotide per 5 × 10 7 sperm cells showing an intrinsic association constant of 15 × 10 −8 M. No significant inhibition of cyclic-AMP binding was caused by the addition of one hundred fold excess of AMP. However, when the excess AMP reached a thousand fold a slight but significant reduction (10%) was observed. These values were not modified by using sperm homogenates instead of intact sperm cells, which suggested that cyclic-AMP binding is to surface membranes and not to protein released from broken or damaged sperm. Binding was found to be unrelated with pH, between 6 to 8, but depended on the temperature of the incubation medium, showing a maximum at 20°C. The blocking of membrane sulfhydryl groups significantly inhibited (48%) cyclic-AMP binding to sperm cells.