Expression of rat renin in mammalian cells and its purification.

Rat renin cDNA was transfected into COS-7 and Chinese hamster ovary (CHO) cells and expressed under the control of the Simian Virus 40 early promoter. Conditioned media of the transfected cells showed renin activity only after trypsin treatment, suggesting prorenin was secreted into the medium. From the trypsinized serum-free culture of the transfected CHO cells active renin was purified to homogeneity by a simple three-step procedure. The active renin had similar specific activity, molecular weight, Km, pH optimum, and isoelectric point compared to native renin. The amino-terminal sequence was the same as that deduced from the renin cDNA. This suggests that the recombinant rat renin is similar to kidney renin in many respects, and is easily obtained by the present procedures.

Quantitative determination of follicle size distribution in the guinea pig ovary after hemicastration and PMSG treatment.

In order to investigate the action point of intraphysiological or supraphysiological elevation of FSH during the preovulatory period on follicular development, adult guinea pigs underwent unilateral ovariectomy on days 10, 12 and 14 of the estrous cycle (N = 6 each group). Thereafter, guinea pigs were injected twice daily with either vehicle or pregnant mare's serum gonadotropin (PMS). After 2 days, the remaining ovaries were removed. The resected ovaries were fixed, embedded in paraffin, serially sectioned (7 microns) and stained with Azan. All follicles greater than 70 microns were classified by size and atretic stage. The follicular size distribution was not affected by hemicastration at day 10, although the ratio of atretic follicles (greater than 400 microns) decreased from 51% to 32% (P less than 0.01). Hemicastration at day 12 increased the largest nonatretic population (70-99 microns group) from 17% to 26%, and the ratio of atretic follicles (greater than 400 microns) decreased from 35% to 23%. The peak size distribution of follicles was shifted from 70-99 microns to 200-299 microns by PMS, and follicles 600-899 microns in size contained an increased percentage of atresia, which resulted in the bimodal distribution of viable follicles greater than 400 microns. These data suggest that 2 day hemicastration promotes an influx of primordial follicles into growing follicles and suppresses the atretic process by a different mechanism depending on the date of hemicastration in the estrous cycle. Conversely, hemicastration + PMS accelerated viable follicle growth to increase the percentage of atresia.

Flow cytometric quantification of rat spermatogenic cells after hypophysectomy and gonadotropin treatment.

DNA flow cytometry was evaluated as a tool to analyze stage-specific changes that occur in absolute cell numbers in the testes. Hypophysectomy was selected as a model system for perturbing testicular cell types, since the cytological sequelae of this treatment post-hypophysectomy in the rat are well documented in the literature. Rat spermatogenic cells in stages II-V, VII, and IX-XIII of the seminiferous epithelial cycle (as defined by Leblond and Clermont, 1952) were quantified in numbers per standard length of seminiferous tubule by DNA flow cytometry after hypophysectomy and subsequent gonadotropin treatment. In agreement with previous histological studies, we found that acrosome- and maturation-phase spermatids disappeared from the seminiferous epithelium after 17 days post-hypophysectomy, whereas meiosis and early spermiogenesis continued at least 164 days. The number of meiotic cells and round spermatids gradually decreased after hypophysectomy. Changes were observed as early as Day 6 post-hypophysectomy. Treatment with human chorionic gonadotropin (hCG) alone maintained most cell numbers within normal limits, and follicle-stimulating hormone (FSH) was needed in addition to hCG to maintain the normal number of cells with the amount of DNA contained in primary spermatocytes and spermatogonia in G2/M-phase (4C) in stages IX-XIII and elongated spermatids (1C') in stages II-V of the epithelial cycle. The absolute numbers of spermatogenic cells at different phases of maturation provide a useful reference for quantitative studies of spermatogenesis. Pathological changes in the seminiferous epithelium can be detected and quantified by DNA flow cytometry.

Plasminogen activator secretion in the rat seminiferous epithelium after hypophysectomy and gonadotropin treatment.

Secretion of plasminogen activators (PA) by seminiferous tubules of hypophysectomized and hypophysectomized gonadotropin-treated rats was studied. Adult male hypophysectomized rats were treated with daily subcutaneous injections of saline, FSH (5 i.u.), hCG (100 i.u.), or FSH + hCG. At six and 10 days posthypophysectomy, segments of seminiferous tubules at stages VII-VIII of the epithelial cycle were isolated for culture in a serum-free medium. PA activity was determined by an indirect solid-phase radiometric assay from conditioned media. PA secretion was significantly decreased in seminiferous tubules from rats treated with saline, FSH, or hCG, whereas normal PA secretion was found in seminiferous tubules of FSH + hCG-treated rats. It is concluded that both FSH and LH, via stimulation of androgen secretion, are needed for the maintenance of normal PA secretion by rat seminiferous tubules.

Reduction of fertility in the male rat by systemic treatment with follicle regulatory protein.

Forty-five-day-old rats received daily injections of follicle regulatory protein (FRP). After 15, 30, 45, and 70 days of therapy, serum was measured for testosterone, androstenedione, estradiol, and follicle-stimulating hormone levels. Testes were evaluated for sperm head counts, plasminogen activator activity, weight, and length of seminiferous epithelial stages. In no case was serum follicle-stimulating hormone concentration reduced in FRP-treated rats. After 75 days of treatment, there was a significant decrease in the number of the sperm head counts. After 60 days of treatment, the length of the dark zone of the tubule was longer than that of control. Pregnancy rates for FRP-treated rats were reduced after 45 and 60 days of treatment. In conclusion, systemic injection of FRP alters seminiferous epithelial function by reducing development of mature sperm.

Disruption of seminiferous epithelial function in the rat by ovarian protein.

The granulosa cell produces an inhibitor of aromatase activity, which recently was purified to homogeneity (follicle-regulatory protein: FRP). Since extracts of testicular homogenates also contain factor(s) with biological properties similar to FRP, including inhibition of granulosa cell aromatase, we examined the effects of ovarian FRP on testicular function. Forty-five-day-old rats received daily FRP injections (100 micrograms or 300 micrograms). After 15, 30, 45, and 70 days of therapy, (n = 5 each group), trunk serum was measured for testosterone, androstenedione, estradiol, and FSH levels by established radioimmunoassays (RIA). One testis from each rat was homogenized, centrifuged, and evaluated for sperm head counts; the other testis was dissected by transillumination, and the length of seminiferous epithelial stages determined. After 15 (control: 4.8 +/- 0.2 mm; 100 micrograms: 6.0 +/- 0.3 mm; 300 micrograms: 6.6 +/- 0.3 mm) and 30 days (control: 4.6 +/- 0.2 mm; 100 micrograms: 6.3 +/- 0.2 mm; 300 micrograms: 5.9 +/- 0.2 mm) of treatment the length of the "strong" seminiferous tubule segment in FRP-treated rats was greater than in control rats (p less than 0.05). Serum levels of steroids and FSH were similar in all groups. After 30, 45, and 70 days of treatment, the sperm head counts for the 100-micrograms and 300-micrograms dosages were 26%, 29%, 30% and 20%, 34%, and 24% of control values, respectively. By 70 days of treatment, cycle Stage VII was markedly reduced or absent in FRP-treated rats, and their round spermatids contained ring chromatin; both conditions indicate degeneration. FRP (50 micrograms/ml) was added to rat Sertoli cell cultures for 4 days after which transferrin and androgen-binding protein (ABP) were measured. FRP enhanced Sertoli cell secretion of ABP (58 vs. 138 +/- 7 microliters eq/culture) and transferrin (2.1 vs. 4.7 +/- 0.6 microgram/culture). In conclusion, systemic injection of FRP alters seminiferous epithelial function by reducing maturation of mature sperm forms. Adding FRP to Sertoli cells in culture enhances secretion of transferrin and ABP; this suggests that maturation of the germinal elements may be linked to the secretory function of seminiferous tubules.