Influence of stage of cycle, corpus luteum location, follicle size, and number of large follicles on estradiol-17 beta concentrations in bovine follicles.

Concentrations of estradiol-17 beta in follicular fluid were correlated to follicular size, stage of estrous cycle, location of corpus luteum, and presence of large follicles. Paired ovaries were obtained from 481 nonpregnant cows at slaughter and follicles were classified as ipsilateral or contralateral to the corpus luteum. Follicular fluid estradiol-17 beta concentrations from 2494 small, 1485 medium, and 396 large follicles were quantified by radioimmunoassay. Stage of estrous cycle was estimated by visual examination of the corpus luteum. Follicles in stage 1 of the estrous cycle (d 1 to 4) had the highest estradiol-17 beta concentration and the smallest mean follicular diameter. Location of follicles relative to the corpus luteum had no influence on estradiol-17 beta concentrations. As follicular size increased, concentration of estradiol-17 beta also increased. The presence of a single large follicle did not affect the concentration of estradiol-17 beta in medium or small follicles. In contrast, if multiple large follicles occurred in the same cow, concentrations of estradiol-17 beta were significantly lower in medium but not small follicles.

Removal of glycosaminoglycans from bovine granulosa cells contributes to increased binding of hydrogen-3 heparin.

Granulosa cells from small or large bovine follicles were pretreated with enzymes that hydrolyze various glycosaminoglycans, and binding of [3H]-heparin to the granulosa was measured. Binding of [3H] heparin increased significantly after enzymatic pretreatments with chondroitinase ABC and fungal hyaluronidase, and similar results were obtained with granulosa from small and large follicles. No changes in binding of [3H] heparin were detected after hydrolyses with chondroitinase AC and heparinase in either follicle size. Heparitinase, which hydrolyzes heparan sulfate, led to a significant 50% increase in binding of [3H] heparin to granulosa from large follicles but was without effect in small follicles. These results suggest that the lower binding of [3H] heparin, which has been reported with follicular enlargement, may be due to heparan sulfate occupying or obstructing binding sites for heparin on granulosa from large follicles.

Relationships between bovine follicular fluid glycosaminoglycans and steroids.

The glycosaminoglycans chondroitin sulfate (CS) and heparan sulfate (HS) are present in follicular fluid. The present study evaluated estradiol (E), progesterone (P), CS, and HS concentrations from pools of 2633 small (less than 6 mm), 1702 medium (6-10 mm), and 491 large (greater than 10 mm) bovine follicles subdivided by relative E concentrations. E and P concentrations increased with follicle enlargement (P less than 0.05), but were inversely related within a follicle size. CS levels were reduced (P less than 0.05) in large (0.84 mg/ml) compared to small (1.18 mg/ml) and medium (1.36 mg/ml) follicles, while HS levels were decreased (P less than 0.05) in medium (0.34 mg/ml) and large (0.19 mg/ml) compared to small (1.10 mg/ml) follicles. Within a follicle size, CS and HS levels decreased significantly with increasing E. CS to HS ratios increased with follicle size and E concentration. The ratio of CS to HS was 4.4- and 1.6-fold higher, respectively, in medium and large follicular fluid samples classified healthy compared to atretic by their E content. These results support the idea that concentrations of glycosaminoglycans decrease with follicular maturation, and atretic follicles contain elevated levels of CS and HS.

Specific binding of the glycosaminoglycan 3H-heparin to bull, monkey, and rabbit spermatozoa in vitro.

In Vitro binding and some binding parameters of the glycosaminoglycan heparin to viable epididymal or ejaculated bull spermatozoa, ejaculated rabbit spermatozoa, and frozen-thawed rhesus monkey spermatozoa were investigated. Nonspecific binding was affected only by the concentration of 3H-heparin, whereas specific binding was saturable, reversible, and dependent on the pH, temperature, and calcium concentration of the incubation medium. Magnesium concentration dependence was observed in the presence of calcium but could not be detected in the absence of calcium. Bound 3H-heparin was displaced by several orders of magnitude greater concentrations of chondroitin sulfate. Scatchard plot analysis suggested multiple binding affinities for 3H-heparin to spermatozoa. 3H-heparin was shown to bind to sperm heads and flagella. Fluorescein-labeled heparin bound to acrosomal, postacrosomal, and flagellar membranes. It was concluded that the specific binding of heparin involved a proteinaceous component on, or intercalated with, spermatozoal membranes. Thus, glycosaminoglycans present in the female reproductive tract may contribute to sperm capacitation and enhance the likelihood of successful fertilization in mammals.

Binding of the glycosaminoglycan [3H]heparin to bovine granulosa cells varies with size and estrogen content of ovarian follicles.

Bovine ovarian follicles were individually aspirated to obtain granulosa cells and follicular fluid, and estrogen concentrations in fluid were measured with a radioimmunoassay. Granulosa membranes were harvested by centrifugation and were pooled from follicles containing statistically high or low estrogen. Granulosa cell concentrations were estimated by comparing their protein content to cell standards. Results showed that cell numbers increased with increasing follicle size, and follicles with high estrogen concentrations possessed more granulosa than follicles with low estrogen. Specific binding of [3H]heparin decreased with advancing follicle size and was higher to granulosa harvested from follicles containing low estrogen.