Effects of feed additives and veterinary drugs on aldosterone production and release by porcine adrenal cells in vitro.

The preparation of suspensions of porcine adrenocortical cells is described. Within the conditions adopted, the cell suspension responded to various agents as expected. It was possible to screen drugs (standard range 0.3-100 microM, incubation period 1 h) for actions on the production/release of aldosterone by the cortical cells using 1 microM deoxycorticosterone as substrate. Progesterone, pregnenolone or corticosterone were also used as substrates. Feed additives of the quinoxaline type induced a slowly developing inhibition of aldosterone production/release by the cell suspension, which was virtually irreversible. During the standard 1 h incubation period inhibitions of up to 22 +/- 2% of control were observed, which increased upon prolongation of the incubation by 2 h. With 100 microM cyadox the inhibition increased from 19 +/- 2% to 35 +/- 2% with prolonged incubation. Ten nitrofuran compounds exerted a more rapidly developing inhibition (by up to 79 +/- 1% of control) of aldosterone production/release, which was reversible. A submaximal inhibition with 10 microM furazolidone of 21 +/- 5% increased to 40 +/- 1% with longer incubation. The concentrations at which these compounds exerted this effect in vitro were well below the peak blood plasma concentrations encountered after administration of the drugs in therapeutic doses. Polyether-antibacterials/ionophores rapidly inhibited aldosterone production/release (to 26 +/- 1% of control) and this effect was completely reversible. The nitroimidazole compounds tested did not affect aldosterone production/release when deoxycorticosterone or progesterone were used as substrates. With use of corticosterone and to a lesser extent with pregnenolone as substrates a clear inhibition (to 73 +/- 3% of control) of aldosterone production was obtained. Amprolium in concentrations up to 100 microM, with deoxycorticosterone as substrate, did not induce a significant change in aldosterone production/release by the suspension of adrenocortical cells. In the same dose range tylosin and roxarsone induced a small but significant inhibition (by up to 10 +/- 3% of control) of aldosterone production/release, which was not dose-dependent. It is concluded that a wide range of growth-promoting drugs may be able to change aldosterone production/release in the animal.

Testicular weight, tubular diameter and number of Sertoli cells in rats are decreased after early prepubertal administration of an LHRH-antagonist; the quality of spermatozoa is not impaired.

To suppress gonadotropin secretion during the sensitive period in development of the testes, immature male rats were treated with an antagonist of luteinizing hormone-releasing hormone (LHRH; ORG. 30276) from postnatal days 6-15. Previously, it has been demonstrated that this treatment results in delayed pubertal development, decreased testicular weight, impaired fertility and adult sexual behavior. In the present experiments it was investigated whether the decreased testicular weight was correlated with morphological changes in the testis. Also, by using an artificial insemination technique, the biological activity of spermatozoa of adult male rats, treated during early prepuberty with the LHRH antagonist (LHRH-A), was tested. The present results demonstrated a decrease in the diameter of the testicular tubuli of LHRH-A-treated rats. The number of Sertoli cells per tubular cross-section was also smaller. But qualitatively no differences could be observed in the testis. All stages of maturation of the seminiferous epithelium were equally frequently represented in LHRH-A-treated males compared with controls. Artificial insemination using spermatozoa obtained from the epididymis of LHRH-A-treated rats, resulted in a pregnancy rate of 100%, similar to the control rate. From the present data, we conclude that the infertility in adult male rats, treated with an antagonist to LHRH during prepubertal life, does not result from malfunction in the maturational processes in the germinal cells and the testes as a whole, despite the observation of changes in the testicular morphology. The infertility of LHRH-A-treated male rats can be explained by the observed impairment of sexual behavior. We suggest, that a central action of the antagonist of LHRH when administered to immature male rats may lead to permanent changes in the development of sexual behavior.

Effects of LHRH antagonist administration to immature male rats on sexual development.

Gonadotropin secretion in immature male rats was inhibited by administration of a potent LHRH antagonist (LHRH-A): from 6 to 15 days of age (early onset/short-term treatment), from 6 to 48 days of age (early onset/long-term treatment) or from 22 to 31 days of age (late onset/short-term treatment). Balano-preputial separation was retarded by 9 or 13 days (short-term treatments) or by about 40 days (long-term treatment). Adult testicular weight was lowered and plasma FSH was increased after early, but not after late onset of LHRH-A treatment. Plasma LH and testosterone levels were not affected by any of the LHRH-A treatments. Fertility was diminished after early onset LHRH-A administration only. Adult precopulatory and copulatory behavior were severely affected after early onset of LHRH-A treatment. Intensity of precopulatory anogenital inspection was increased. The copulatory pattern was incomplete with absence of ejaculatory behavior during sexual behavior tests. Sexual behavior was not affected after late onset of LHRH-A treatment. Thus, administration of LHRH-A to immature male rats delays balano-preputial separation irrespective of the age of onset of LHRH-A treatment. In contrast, effects on adult FSH levels, testicular weight, fertility and sexual behavior depend on age and duration of LHRH-A administration.

Administration of a GnRH-antagonist to immature rats affects subsequent female and male pubertal development differently.

Gonadotropin secretion was inhibited in immature male and female rats by sc administration of the GnRH-antagonist ORG30276 (GnRH-A) on days 6, 9, 12 and 15. In GnRH-A-treated females this resulted in suppression of the temporarily increased plasma LH and FSH levels, which normally occur in prepubertal female rats. Ovarian weight was decreased. Although vaginal opening in GnRH-A-treated rats occurred earlier, the age of 1st estrus and the number of ova shed at first ovulation were not affected. Fertility at 4 months of age was normal. After initial suppression of gonadotropin levels, the FSH levels in GnRH-A-treated males, however, sharply increased to about twice the control levels. Plasma FSH remained elevated at least until 4 months of age. The LH levels at adult age were not affected by antagonist treatment and neither were testosterone levels. Testicular weight was decreased by GnRH-A from about 50% on day 15 to about 30% at 4 months of age. Pubertal development as measured by balano-preputial separation was delayed by about 7-10 days. At 4 months of age fertility was decreased. Thus, suppression of early gonadotropin secretion by GnRH-A treatment had dramatic effects on subsequent pubertal development in the male, but not in the female rat.

Ovarian follicle dynamics in immature rats treated with a luteinizing hormone-releasing hormone antagonist (Org. 30276).

The high concentrations of gonadotropins present in immature female rats by the end of the second week of life were suppressed by treatment with an antagonist against luteinizing hormone-releasing hormone (LHRH-A; Org. 30276) on Days 6, 9, 12, and 15 of life. Differential ovarian follicle counts were made on Days 15, 22, 28, and on the day of first estrus of all growing follicles and follicles greater than or equal to 100 x 10(5) microns 3 (mostly antral). In LHRH-A-treated rats, a retardation of follicle growth was noted on Day 15, followed by a gradual loss of growing follicles that amounted to 20% on Day 22 and 40% on Day 28; at first estrus, the total population of growing follicles was only 50% of that present in control rats. Antral follicles, first present at 22 days of age, were lower in number at 28 days of age and at first estrus in LHRH-A-treated rats; this was true for both healthy and atretic follicles. Ovarian weights were significantly reduced in LHRH-A-treated rats at 15 and 28 days of age and on the day of first estrus. However, the numbers of corpora lutea following the first, and normally timed, ovulation were the same in both groups. It was concluded that for early recruitment of follicles to reach a full-sized pool of growing follicles at the age of puberty, high concentrations of gonadotropins early in life have a significant role.

Immunoreactive corticotropin releasing factor in adult and developing rat cerebellum: its presence in climbing and mossy fibres.

Corticotropin Releasing Factor (CRF) immunoreactivity was localized within the rat central nervous system with immunocytochemical methods, using a highly specific anti-serum to rat-CRF. In adult rats abundant CRF immunoreactivity (CRF-ir) was found in perikarya of the inferior olivary nucleus, in climbing fibres in the molecular layer, and mossy fibres in the granular layer of the cerebellum and in the cerebellar nuclei. CRF-ir climbing fibres were found in a longitudinal zonal pattern throughout the molecular layer. In developing cerebellum only a few CRF-positive fibres were seen on postnatal day 8. From day 8 onwards a gradual increase in CRF-ir was found in both the molecular and granular layer with formation of CRF-positive nests around Purkinje cellbodies starting at day 12. Subsequent outgrowth into the molecular layer could be followed, leading to an adult level of CRF-ir in the cerebellum at approximately day 22 postnatally. CRF-ir in mossy fibres has as adult appearance from approximately day 18. The temporal and morphological changes of CRF-ir in fibres in the cerebellum of immature rats followed closely the morphological development of climbing and mossy fibres. The present findings indicate that CRF (or related substances) are already present in both climbing and mossy fibres during their outgrowth into the cerebellum.

The distribution of vasotocin and isotocin in the brain of the rainbow trout.

The distribution of vasotocin and isotocin in the brain of the rainbow trout Salmo gairdneri was investigated by the unlabeled antibody enzyme method, by using purified antisera against arginine vasotocin and isotocin. In the preoptic nucleus no clear differences were observed in the distribution of vasotocin- and isotocin-containing cells. Vasotocin and isotocin innervation was found in most brain regions, though in general isotocin fibers were much more abundant. The area dorsalis pars medialis of the telencephalon, the saccus dorsalis, and the basal part of the nucleus recessus posterioris were found to be innervated by vasotocin and scarcely by isotocin fibers. In the nucleus habenularis, the nucleus recessus lobus lateralis, the nucleus preglomerulosus pars medialis, and the tectum mesencephali isotocin fibers prevailed. These findings in the trout brain are compared with the vasopressin and oxytocin innervation of the rat brain.