Placental steroidogenesis in the rat: progesterone production by tissue of the basal zone.

Previous reports suggested that the rat placenta had a rather limited capacity for steroidogenesis, particularly with respect to progesterone production. We found that the basal zone in the rat placenta, which contains the steroidogenic giant cells, can be isolated by simple surgical separation, and using them for in vitro incubations, significant conversion of pregnenolone-7 alpha-3H to progesterone-1H could be demonstrated. The 5alpha-reduced metabolites of progesterone, as well as compounds of the delta4 pathway, including 17alpha-hydroxyprogesterone, androstenedione and testosterone were also isolated and characterized. When progesterone-7alpha-3H was added as precursor, most of it remained unmetabolized, but qualitatively identical products were isolated as were found when pregnenolong-7alpha-3H was used as precursor. Incubations of the whole placenta and of labyrinth tissue yielded relatively much lower progesterone-3H from pregnenolone-7alpha-3H. Our results have thus established that the rat placenta is a steroidogenic organ, just as in many other species. Past difficulties in isolating progesterone from in vitro studies may possibly be due to the intense activity of the enzyme 5alpha-reductase when the whole placenta was used.

Placental steriodogenesis in the rat: comparison of normal and giant placentae.

Placental hypertrophy was induced in pregnant rats by daily treatment with estrone (0.5 microng) and progesterone (4 mg) from day 3 through 19 combined with ovariectomy on day 12 of pregnancy. The ratio of basal zone to whole placenta was 47% by weight in these enlarged placentae on day 20 of pregnancy but only 34% in normal placentae. In each case, the basal zone tissue was homogenized and the 10,000 x g supernatant fraction was used for in vitro incubation with [7alpha-3H]pregnenolone as the added substrate. The normal placental tissue synthesized more progesterone and testosterone but less androstenedione and 5alpha-pregnane-3,20-dione than the giant placentae. Utilization of the pregnenolone substrate by the giant placentae was subnormal. Addition of 200 IU of human chorionic gonadotropin (hCG) in vitro to normal basal zone placentae sharply increased the production of progesterone, 17alpha-hydroxyprogesterone, androstenedione and 5alpha-pregnane-3,20-dione. The giant placentage failed to respond to hCG. Thus, steroid-induced hypertrophied placentae in rats do not participate in a compensatory mechanism for steroid hormone production toward pregnancy maintenance.

Age-related changes in the metabolism of [3H] testosterone in vitro by the epididymis of the immature rat.

We examined the production in vitro of 5 alpha-reduced metabolites from testosterone by the rat epididymis during pubertal maturation. Minced caput and cauda epididymides from 30-,45-, and 55-day-old rats were incubated with [3H] testosterone for 2h. Analysis of the radioactive metabolites revealed both similarities and differences in the metabolic patterns compared to those reported for adult rats. As in adults, 5 alpha-dihydrotestosterone was the most abundant metabolite produced by both epididymal segments at all three ages, and it was formed in larger quantities in the caput epididymidis than in the cauda. However, [3H] testosterone metabolism by the epididymis of the immature rat was characterized by a lower formation of 5 alpha-androstane-3 alpha,17 beta-diol and higher production of 5 alpha-androstane-3,17-dione than in adults. Production of these two metabolites by the caput region increased and decreased respectively, toward adult levels, with increasing age. In addition, the amount of [3H] testosterone metabolized was higher with tissues from prepubertal rats (30 days of age) than with those from rats 55 days of age. These data suggest that testosterone metabolism in the caput begins to change to that of the adult during the period of pubertal maturation but apparently not until later in the cauda epididymidis.

Effects of short-term bilateral and unilateral castration and androgen replacement on the metabolism of [3H] testosterone in vitro by the epididymis of the immature rat.

The in-vitro metabolism of [3H] testosterone by the epididymis of the pubertal rat (55 days of age) has been examined after short-term bilateral and unilateral castration and androgen replacement. Bilateral castration did not decrease the metabolism of [3H] testosterone, but did result in a decline in the proportion of 5 alpha-dihydrotestosterone produced and an increase in that of 5 alpha-androstane-3 alpha, 17 beta-diol, androsterone and 5 alpha-androstane-3,17-dione. Changes in metabolism occurred in the caput within 2 days after surgery, but not until 5 days after surgery in the cauda epididymidis. Daily testosterone treatment, which maintained prostatic growth in bilaterally castrated animals, did not restore normal androgen metabolism and increased further the production of androsterone and 5 alpha-androstanedione by the cauda. In unilaterally castrated animals, androgen metabolism in epididymal tissue from the operated side was normal in the cauda but was indistinguishable in the caput from that of bilaterally castrated rats. These results indicate that (a) androgen metabolism by the caput, compared to that by the cauda, responds more quickly to androgen withdrawal and (b) that in the short term, normal androgen metabolism by the caput, but not the cauda, is dependent upon the presence of the ipsilateral testis. Furthermore, testosterone alone proved an inadequate replacement for bilateral castration which implies that the pubertal rat testis secretes additional compounds which are essential for normal function of the epididymis.

Development of hypothalamic-pituitary-adrenal response to stress in rats made hypothyroid by exposure to thiouracil from conception.

The functional maturation of the hypothalamic-pituitary-adrenal (HPA) axis has been studied in rats of 20-35 days of age made hypothyroid by the administration of thiouracil from conception. Basal concentrations of corticosterone in serum were normal in hypothyroid animals. Ether stress led to an increase corticosterone content of the adrenal glands of hypothyroid and normal rats but not to a rise in serum corticosterone of hypothyroid rats until 30 days of age. Corticosterone secretion in response to ACTH administration was subnormal in hypothyroid rats. The hypothyroid state delays the development of the hypothalamic-pituitary-portion of the HPA axis until 30 days of age and causes a diminution in adrenal response to ACTH beyond this time.

Effects of postnatal thyroxine administration on brain development, response to postnatal androgen and thyroid regulation in female rats.

Some developmental and functional manifestations of thyroxine (T4) administered on the first 2 days of postnatal life were studied in the female rat. Brain myelinogenesis estimated by brain esterified cholesterol concentration, and brain myelin age estimated by brain total cholesterol concentration, were subsequently determined. Thyroxine treatment resulted in a greater concentration of esterified cholesterol in the brain that saline treatment, but the latter appeared to delay the normal increase shown by non-injected controls. Thyroxine treatment resulted in total and free cholesterol levels similar to those of non-injected controls, these again being greater than those in saline-treated rats. Cholesterol concentrations in liver and serum were not affected by T4 or saline treatment. Administration of T4 to female rats before administration of 1-25 mg testosterone propionate on day 7 resulted in an ovarian and uterine weight response to human chorionic gonadotrophin (HCG, 1 i.u./day on days 23-26) on day 27 that was greater than that in litter-mates given saline at birth before testosterone propionate and HCG treatment. Postnatal T4 treatment alone in the female was also associated with a reduced thyroid and pituitary gland enlargement after 7 days of propylthiouracil feeding (0-015% in tap water, days 24-31 of life) when compared with either saline or non-injected controls.

Ovarian delta-5-3-beta-hydroxysteroid dehydrogenase in aging rats.

Ovaries of rats 1, 2, 4, and 18 months old revealed significant delta-5-3-beta-hydroxysteroid dehydrogenase (3beta-OHSD) activity when dehydroepiandrosterone (DHA), pregnenolone, and 16-dehydropregnenolone served as substrates. No enzyme activity was evident in ovaries 4 and 18 months of age when the substrates 17alpha-hydroxypregnenolone, pregnenolone sulfate, and dehydroepiandrosterone sulfate were used. A marked increase in enzyme activity occurred in the granulosae of mature vesicular follicles at 18 months of age. DHA, pregnenolone, and 16-dehydropregnenolone provided similar activities. On the other hand, interstitial enzyme activity exhibited an aging decline with DHA and 16-dehydropregnenolone but increased with pregnenolone. Corpora lutea were less evident in aging ovaries but did exhibit strong 3beta-OHSD activity when DHA was used. Pregnenolone use by corpora lutea was sharply increased in aging rats but no change occurred with 16-dehydropregnenolone. Thus aging was associated with changes in 3beta-OHSD distribution and use of steroid substrate; a change in function is suggested.

Effect of excess dietary methionine on rat pregnancy: influence on ovarian delta5-3beta-hydroxysteroid dehydrogenase activity.

The addition of 4% dl-methionine to a diet of 18% or 12% casein reduced pregnancy maintenance in the rat from 100% to 48% and 5%, respectively. Fetal weight was subnormal. The major influence of methionine was not due to reduced food intake as determined by pair-fed controls but was related to reduced ovarian function. Thus, administration of estrone (0.5 microng) and progesterone (4 mg) on each of days 3 to 20 restored pregnancy to 100% in methionine-fed rats, but fetal weight remained subnormal. Further ovarian delta5-3beta-hydroxysteroid dehydrogenase activity was subnormal in litter-resorbed, methionine-fed rats. Since adequate amounts of ovarian steroids are required for placenta formation, it is possible that placental development was impaired and served as the cause of fetal wastage in animals consuming excess methionine.

Simultaneous measurements of testosterone and three 5 alpha-reduced androgens in the venous effluent of immature rat testes in situ.

Simultaneous measurements were made by radioimmunoassay of testosterone, 17 beta-hydroxy-5 alpha-androstan-3-one (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol), and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-diol) in the testicular venous plasma (TVP) and peripheral plasma (PP) of 30, 45, and 55 day old rats. At 30 days of age, the preponderant androgen in both plasmas was 3 alpha-diol but testosterone predominated by day 55. Testosterone levels increased with age in both TVP (6.39, 15.08, and 54.93 ng/ml on days 30, 45, and 55 respectively) and PP (0.13, 0.56, and 1.02 ng/ml on days 30, 45 and 55 respectively) whereas 3 alpha-diol concentrations decreased in TVP (48.07 ng/ml, day 30; 24.85 ng/ml, day 55) though not peripherally (range: 0.41-0.52 ng/ml). DHT was low in both TVP and PP and appeared to rise only slightly although the increase was not statistically significant. Levels of 3 beta-diol remained low and unchanged. These observations suggest that the total androgen content of the venous effluent from the prepubertal rat testis is quite high and that significant changes in peripheral interconversions of androgens are occurring during sexual maturation.

Influence of dietary protein depletion and repletion on sex organ weight of male rats in relation to age.

Studies were made on the effect of protein-free diet (PFD) feeding and on recovery from PFD feeding on sex organ weight, serum testosterone, pituitary prolactin and pituitary growth hormone content of young (4 months old) and old (18 months old) male rats. After 20 days of protein depletion, the testis weight remained almost the same, but the ratio of testis weight to body weight was higher in the lower age group, demonstrating a greater resistibility to protein deficiency in young rats. The weight of the seminal vesicle and prostate decreased on ingestion of PFD in both age groups, but a more marked increase was shown in refed young rats. Feeding of PFD for 20 days significantly reduced pituitary prolactin, pituitary growth hormone and serum testosterone in both age groups. Refeeding of protein increased these hormones, but a more marked increase was also observed in the younger group. These results seem to support the hypothesis that one of the important characteristics of an aging organism is its reduced capacity to adapt to environmental changes.