Omar Hernández-Pérez

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.

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.

MALE REPRODUCTIVE CYCLE OF MEXICAN BIG-EARED BATS, CORYNORHINUS MEXICANUS (CHIROPTERA: VESPERTILIONIDAE)

Abstract Morphological observations of male genital tracts obtained from wild, adult Mexican big-eared bats (Corynorhinus mexicanus) revealed only one but long annual reproductive cycle showing the existence of temporal asynchrony of its reproductive functions, as is characteristic of temperate-zone vespertilionid and rhinolophid chiropterans. Testes were largest in August, whereas maximum development of epididymides and accessory sex glands complex was observed 1 and 3 months later. High value of relative body condition of individuals was observed from May to June, when testicular enlargement commenced, suggesting that recrudescence of spermatogenesis in the adult bats is dependent on a good body condition.

Re-Evaluation of the Role of Spermatozoa as Inducers of Protein Synthesis by the Rabbit Endometrium

The effect of spermatozoa as inducers of protein synthesis by the rabbit endometrium was studied. The presence of spermatozoa increased the leucine incorporation into proteins from 15.6 +/- 1.8 to 44.8 +/- 4.3 dpm X 10(-3)/mg DNA after 2 hr of incubation. There was no difference in the amount of incorporated leucine induced by live or by dead spermatozoa. Latex particles also induced an increase on protein synthesis (24 +/- 3.1 dpm X 10(-3)/mg DNA). These results seem to indicate that this increase in protein synthesis was nonspecific. The number of contaminant cells was always significantly greater in the recovered incubation medium after incubation with highly metabolically active sperm than with dead cells (230 +/- 85 and 86 +/- 33 X 10(-3) cells respectively). This leukocytic effect was smaller than that found in In vivo systems and may play a significant role on the proposed activity of sperm cells on the endometrium.

Effect of intrauterine copper on the nucleic acids, polysome pattern, and glycoprotein composition of the human endometrium

The modifications induced by the intrauterine release of copper in the macromolecular and glycoprotein composition of the proliferative and secretory human endometrium were studied. In addition, the endometrial changes produced in women users of copper-T intrauterine devices in the polysome pattern and in the content of ribonucleoprotein particles were determined during the secretory phase. A group of ten untreated normal women (control group) and 15 users of 200 mm.-2 copper-T intrauterine device were selected for this study from the outpatient clinic of the Hospital de Ginecologia y Obstetricia No. 2 del Instituto Mexicano del Seguro Social. The main changes observed in the copper-T users were: a significant decrease in the endometrial content of RNA in both phases of the menstrual cycle, a significant decrease in protein in the secretory phase, and drastic changes in the fucose-sialic acid ratios, which decreased during the proliferative and increased during the secretory phases. Normal human secretory endometrium contained 4.89 plus os minus 0.28 (mean plus or minus standard error (S. E.) mg. ribonucleoprotein particle per gram wet weight, while the endometrium of the Cu-T users showed a significant decrease to 2.52 plus or minus 0.17 (mean plus or minus S. E.). In addition, the Cu-T induced a decrease in the heavy components of the polysome pattern with a concomitant increase in the lighter components.

Proteolytic and antiproteolytic activities in goat antral follicles

Abstract Goat antral follicles were dissected and classified initially by size into three groups: 6 mm. Follicles less than 3 mm diameter were studied without any further classification. All others were classified according to their gross stereoscopic microscope appearance into four sub-groups. Histological observation showed that sub-group 1 consisted mainly of non-atretic follicles, sub-group 2 of follicles in the early stages of atresia, while sub-groups 3a and 3b were formed by secondary and tertiary atretic follicles. Tryptic and anti-trypic activites were measured in follicular fluid, homogenized follicular cells and empty follicular sacs, using a fluorometric procedure. Antitryptic activity in follicular fluid was found to be directly correlated with progress of atresia and inversely with follicular growth, being smallest in big (>6 mm), non-atretic follicles and consistently higher in small ( 6 mm non-atretic follicles. High trypsin-like activity was found in the cell walls of non-atretic or early atretic follicles. This activity was significantly lower in sub-group 3a follicles and was almost absent in tertiary atretic follicles (sub-group 3b). In contrast, trypsin-like activity in granulosa cells recovered by centrifugation of follicular fluid was twice as high in sub-group 3a as in sub-groups 1 and 2 and almost five times higher than in sub-group 3b.

Subcellular Distribution of Phospholipase A2 and Atpases During Capacitation and Acrosome Reaction in Guinea Pig Spermatozoa

The presence, distribution, and levels of phospholipase A2 and ATPases activities in those structures of the guinea pig spermatozoa that participate in the acrosome reaction were studied, both before and after capacitation, as well as during the acrosome reaction induced in vitro. Spermatozoa were collected from the cauda epididymis and incubated in the absence and presence of 1.15 mmol/L calcium, with and without the addition of 1 mumol/L A23187. Membrane fractions were recovered by vortexing and discontinuous sucrose density gradient centrifugation. Most of the Na+, K(+)-ATPase was recovered in the acrosome-free spermatozoa, but a clear, distinct presence of this enzyme was observed in the plasma membrane (25 against 101 nmoles Pi released per milligram of protein, respectively). The activity of this enzyme in the periacrosomal plasma and in the outer acrosomal membrane increased during calcium incubation. Ca2(+)-dependent ATPase was found in both membrane fractions, being higher in the periacrosomal plasma membrane. The addition of calcium induced a significant inhibition of this acrosomal ATPase, whereas the activity in the acrosome-free spermatozoa increased. The activity of phospholipase A2, under all experimental conditions, was found to be restricted to the soluble fraction.

Participation of DNA structure on sperm chromatin organization.

The in vitro interaction between purified bovine liver and sperm DNA with somatic histones, to form nucleosomes, and with bovine and salmon protamines were studied. DNAse or microccocal nuclease digestion of liver DNA-histone reassociated chromatin produced the expected polynucleosome type of fragments. Electrophoretic patterns of digested sperm-DNA nucleosomes were different. Micrococcal nuclease digestion produced mainly fragments smaller than 100 bp and some nucleosome-type particles. Under DNAse activity most of the products were smaller than 100 bp, indicating an increased susceptibility of the sperm DNA-histone complexes to the hydrolytic activity of both nucleases, particularly toward DNAse I. This differential susceptibility was confirmed by sucrose gradient spectrophotometric analysis. Acridine orange (AO) staining of histone-DNA reassociated nucleosomes showed significant differences in fluorescence intensity, sperm DNA-histone complexes being almost twice as fluorescent as liver DNA-histone complexes. On the contrary, liver DNA/protamine complexes stained with AO were consistently more fluorescent than sperm DNA-protamine complexes. Finally, no differences in either fluorescence intensity or spectra were observed when liver and sperm DNA were stained with AO after interaction with salmon protamines. The data suggest that sperm DNA has important structural characteristics that differentiates it from somatic DNA. These differences seem to be species specific and must surely play an important role on the determination of the dramatic sequence of that participates sperm chromatin organization.The in vitro interaction between purified bovine liver and sperm DNA with somatic histones, to form nucleosomes, and with bovine and salmon protamines were studied. DNAse or microccocal nuclease digestion of liver DNA-histone reassociated chromatin produced the expected polynucleosome type of fragments. Electrophoretic patterns of digested sperm-DNA nucleosomes were different. Micrococcal nuclease digestion produced mainly fragments smaller than 100 bp and some nucleosome-type particles. Under DNAse activity most of the products were smaller than 100 bp, indicating an increased susceptibility of the sperm DNA-histone complexes to the hydrolytic activity of both nucleases, particularly toward DNAse I. This differential susceptibility was confirmed by sucrose gradient spectrophotometric analysis. Acridine orange (AO) staining of histone-DNA reassociated nucleosomes showed significant differences in fluorescence intensity, sperm DNA-histone complexes being almost twice as fluorescent as liver DNA-histone complexes. On the contrary, liver DNA/protamine complexes stained with AO were consistently more fluorescent than sperm DNA-protamine complexes. Finally, no differences in either fluorescence intensity or spectra were observed when liver and sperm DNA were stained with AO after interaction with salmon protamines. The data suggest that sperm DNA has important structural characteristics that differentiates it from somatic DNA. These differences seem to be species specific and must surely play an important role on the determination of the dramatic sequence of that participates sperm chromatin organization.

Effect of cupric ions on the initiation protein synthesis rate in the human endometrium

The effect of cupric ions on the initiation protein synthesis rate of the human endometrium was studied. Addition of copper to the complete ribosomal system decreased the binding of [3H]Met-tRNA(i) to the isolated ribosomes with a plateau at about 70% inhibition with concentrations higher than 150 microM. The initiation activity was GTP-dependent with a maximum at 2 mM. This activity was very rapid, requiring 5 min to complete the reaction. Incubation of isolated initiation factors with copper (300 microM) inhibited the formation of the ternary complex. When the complete system was reconstituted with salt-washed ribosomes after ternary complex formation, no significant change on the inhibition pattern was observed. Addition of initiation factors to 5-min preincubated salt-washed ribosomes with 300 microM copper, after the elimination of excess copper, induced only a 12% decrease on Met-tRNA(i) binding. This effect was not modified by the presence of Sparsomycin, an elongation inhibitor. It was concluded that copper interferes with the initiation process, probably at the ternary complex formation level.