A. Carranco

Glycosamineglycan sulfate as acrosomal reaction-inducing factor of follicular fluid.

Follicular fluid from different mammalian species possesses two factors responsible for the induction of capacitation: a sperm-stimulating factor and an acrosomal reaction-inducing factor. The glycosamineglycan-sulfate (GAGs) extracted from pig follicular fluid induce acrosome reaction in pig spermatozoa which exhibit no morphological difference between the GAGs-induced reaction and the natural one. Acrosomal reaction commenced 30 min after the addition of GAGs and depended on GAGs concentration reaching 80% of acrosomal reacted spermatozoa after 6 hr of incubation with 7 mg of GAGs/ml. Chemical composition differs with the chemical data that characterize them as proteoglycans since those we obtained were practically protein free (2%). Another difference resides in the uronic acid content, which is almost twofold higher (59%). Electron microscope observations of the acrosomal reacted spermatozoa revealed that the addition of 10 mg/ml of trypsin soybean inhibitor did not interfere with any of the acrosomal reaction steps. The active capacitating factors may also originate from the follicular fluid released into the genital tract during ovulation.

Plasma gonadotropins and gonadal steroids in children treated with cyclophosphamide.

Twelve boys (4.7 to 15.4 years) and five girls (4.3 to 14.1 years) treated with cyclophosphamide, 50 to 100 mg/day for periods of two to 12 months were studied. Plasma follicle-stimulating hormone, luteinizing hormone, Δ 4 -androstenedione, testosterone, 17β-estradiol, 17-hydroxyprogesterone, and progesterone were determined by radioimmunoassay in four to six samples from each patient. At time of the study, cyclophosphamide had not been given for 1.0 to 5.3 years. Results were compared to age-matched healthy boys and girls. Four boys treated when prepubertal had a normal hormonal profile. Three out of seven boys treated during stage II of puberty had elevated FSH and LH levels, low Δ 4 -androstenedione levels, but normal plasma testosterone (0.9 to 2.7 years after therapy). Follow-up studies in two of these three boys (4.0 and 6.1 years, respectively, after therapy) disclosed azzospermia in one and severe oligospermia in the other; each had elevated plasma FSH concentrations. In the remaining four boys of this group with previously normal hormonal profiles, follow-up studies in three of them (4.0 to 7.9 years after cyclophosphamide therapy) disclosed normal sperm counts and plasma FSH in one boy and mild oligospermia with normal plasma concentration in two. No evidence of primary ovarian failure was detected in any of the girls. It is suggested that caution should be maintained not only in the total dose and duration of cyclophosphamide therapy, but also in the degree of pubertal development at time of initial therapy.

Sleep Deprivation Reduces Circulating Androgens in Healthy Men

The acute effect of sleep deprivation on the pituitary-testis axis was evaluated in 13 healthy men. To study such association, the circulating levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), Androstenedione (A), Testosterone (T), Dihydro-testosterone (DHT) and Estradiol (E2) were measured along with Cortisol (C) before and after sleep deprivation. Morning (8:00 AM) venous blood samples were obtained prior and after a continuous restless period of 24 hr and the values were analyzed by the paired Students t test. There was a significant and parallel decrease of each androgen and E2 but not of FSH, L.H. PRL, or C, associated with the acute sleep deprivation.

Heparin-Induced Nuclei Decondensation of Mammalian Epididymal Spermatozoa

Decondensation of mammalian epididymal spermatozoa nuclei has been induced by exposure of intact spermatozoa to heparin, including those species in which ejaculated sperm were not susceptible to this treatment. This process occurred in the absence of any disulfide bond cleaving reactant. Swelling of caput epididymal spermatozoa nuclei commenced about 30 min after the addition of heparin, reaching 88% in rat, 33% in rabbit, 26% in pig, and 62% in bull of swelled nuclei after 6 hr of incubation at 37 degrees C with 5000 USP of heparin per ml. Corpus epididymal spermatozoa nuclei of rat and rabbit underwent decondensation at 50 degrees C reaching 24% and 22% of swelled nuclei, respectively, after 6 hr of incubation. The nuclei of the sperm cells of pig and bull from this epididymal region remained highly condensed as well as the nuclei of the cauda epididymal spermatozoa of all the species assayed. Electron microscope observations of the caput epididymal spermatozoa nuclei treated with heparin revealed that the chromatin is organized into nuclear bodies joined by a network of cross-linked and branched chromatin fibers in the species studied.

A Simultaneous Assay to Quantitate Plasma and Endometrial Hormone Concentrations

A method for radioimmunoassay determination of hormones in both plasma and endometrium is presented. Total estrogen (TE) and progesterone (P) concentrations were measured simultaneously in plasma and endometrium in 59 women throughout the menstrual cycle. TE values in endometrium showed an increase of 0.45 ng/gm wet tissue weight on days 7 to 9, reaching a peak of 4.89 ng/gm wet weight at midcycle; values of 2.2 ng/gm wet weight were constant during the secretory phase. The endometrial P concentrations were 5.31 and 44.93 ng/gm wet weight during the proliferative and luteal phases, respectively. Plasma P levels during the proliferative phase were below 1 ng/ml, in comparison with values above 5.71 ng/ml during the luteal phase. The quadratic coefficients of correlation between plasma and endometrial concentrations of TE and P were 0.8 and 0.9, respectively, indicating that under such conditions modifications in the amount of circulating hormones are reflected in the target tissue. These simultaneous studies may permit further investigation of the role of circulationg hormones in local biologic phenomena.

Male pronuclei formation release of phosphorylation of histone H-3 during decondensation of human sperm nuclei activated in vitro by heparin.

The release and phosphorylation/dephosphorylation mechanisms of human spermatozoa histone during nuclei in vitro decondensation by heparin was studied. Washed sperm cells were incubated in the presence of 32P and in the absence or presence of heparin. The results showed an increase in the incorporation of 32P of 20 times greater in the presence of heparin than in the absence of heparin (the control sample). In some cases the incorporation of 32P into histones was confirmed by its isolation. To validate these results a phosphorylation kinetic of isolated sperm histone, used as a substrate, was performed. The amount of 32P was not a linear function of time, and maximal phosphorylation was reached in 60 min. A measurement of 32P incorporated as a function of the amount of histone, shows a linear relationship of up to 50 micrograms of protein, with a rapid saturation thereafter with the incorporation of 220 nm and with a KD = 442 x 10(-6) mol/L. 32P incorporation, independent of exogenous cAMP, was related to alkaline pH but was totally dependent on temperature--with a maximum of 37 degrees C. The only histone released was histone H-3. Phosphorylation/dephosphorylation is involved during male pronuclei formation.

Accumulation of Ethinylestradiol in Blood and Endometrium of Women Taking Oral Contraceptives: The Sequential Therapy *

A radioimmunoassay to quantitate ethinylestradiol (EE-2) in both plasma and endometrium is described. In 29 women under sequential oral contraceptive therapy (chlormadinone acetate, 2 mg, plus mestranol, 80 microgram) for 6 to 84 months, a single blood sample and a single endometrial sample were simultaneously obtained on different days of the pseudomenstrual cycle. In 24 women under 40 years of age the mean plasma EE-2 concentrations were similar during the first (989 +/- 94 pg/ml) and the second half of the cycle (1053 +/- 186 pg/ml) (P greater than 0.05). A similar finding was observed with regard to mean endometrial EE-2 concentrations (3.55 +/- 2.1 and 5.89 +/- 1.7 microgram/gm of wet tissue, respectively). On the other hand, five women over 40 years of age had plasma EE-2 concentrations similar to those of the previous group, but the mean endometrial EE-2 concentrations was 0.9 +/- 0.6 microgram/gm of wet tissue (P less than 0.05). Although plasma follicle-stimulating hormone and luteinizing hormone did not show midcycle peak values, complete suppression of both gonadotropins was not observed. These results show that endometrium has a great ability to concentrate EE-2, and this ability seems to be greater in women below age 40 than above. Whether or not this observation might be related to the known higher incidence of endometrial cancer in women less than 40 years old who have been under chronic sequential oral contraceptive therapy cannot be disclosed from this limited number of determinations.

Glycosaminoglycan-Sulfate as Plasma Membrane Component of Pig Spermatozoa

The effect of specific glycosaminoglycan-hydrolyzing enzymes on the ruthenium red staining of pig spermatozoa was studied. Washed spermatozoa were incubated at 35 degrees C in buffer or with neuraminidase 0.5 units/ml, heparinase 0.2 mg/ml, or chondroitinase ABC 2.0 units/ml. After incubation sperm cells were washed, stained with ruthenium red and studied under the electron microscope. Anionic sites in the surface of untreated spermatozoa follow regularly the plasma membrane, but present are numerous processes constituting what has been defined as the glycocalyx. Neuraminidase did not affect the distribution of ruthenium red on the surface of the spermatozoa, but eliminated almost completely the processes of the glycocalyx. Heparinase caused loss of the ruthenium red-stained sites on the membrane surface of pig spermatozoa with less influence on the dense processes of the glycocalyx. A similar loss of ruthenium red-stained sites was observed with nitrous acid treatment. A striking effect of treatment with chondroitinase ABC was the production of a typical acrosome reaction.

Ethynyl-estradiol (EE-2) content in blood and endometrium caused by oral contraceptives☆

Abstract A comparison of EE-2 concentrations in blood and endometrium of women under sequential therapy (Group I: chlormadinone acetate + mestranol; 2 mg + 80μg, n = 24) and under combined therapy (Group II: norgestrel + EE-2: 0.5 mg + 30μg, n = 23), was performed. By means of reliable radioimmunoassay techniques, simultaneous determinations were made of the synthetic estrogen in both plasma and endometrium covering several days of the pseudomenstrual cycle. The mean ± SD plasma EE-2 concentrations observed for Group I was 1021 ± 140 pg/ml and that for Group II was 83 ± 16pg/ml. while endometrial concentrations (wet tissue) were: 4.7 ± 1.9 μg/g and 89 ± 13 ng/g respectively. The results show: (1) Greater EE-2 concentrations in the endometrium than in plasma, in both groups. (2) The EE-2 concentrates 10 times more in blood and in the order of μg in endometrium of Group I than in Group II. Together with data presented in other papers of this series, these simultaneous studies may permit understanding the role of circulating hormones in local biologic phenomena.

Increased Acrosome-Reaction Inducing Activity of Glycosaminoglycans by Partial Hydrolysis

The possibility that partial hydrolysis of glycosaminoglycan-sulfates (GAGs) such as occurs during the last phases of follicular maturation could play some role in the activity of follicular fluid as an inducer of the acrosome reaction was explored. Hydrolysis of follicular fluid GAGs (ff-GAGs) for 30 min with low-pH HNO2 substantially increased (more than 3 times) its capacity to induce the acrosome reaction. This increase was significantly reduced when the time of hydrolysis was either shorter (10 min) or longer (60 min). Partial hydrolysis of spermatozoa GAGs by direct incubation of sperm cells with chondroitinase ABC was also capable of inducing the acrosome reaction.