Androgen-receptor defect abolishes sex differences in nitric oxide and reactivity to vasopressin in rat aorta.

Contractions of rat thoracic aorta to vasopressin (VP) are threefold higher in females (F) than in males (M), primarily because nitric oxide (NO) attenuation of contraction is greater in M. To determine the role of the androgen receptor (AR) in this mechanism, vascular reactivity to VP was examined in thoracic aorta of the testicular-feminized male (Tfm) rat, which has an X-linked, recessive defect in AR function in affected M. Maximal contraction of normal aortas to VP was fourfold higher in F (4,128 +/- 291 mg/mg ring wt) than in M (971 +/- 133 mg); maximal response of Tfm (3,967 +/- 253 mg) was similar to that of normal F. N(G)-nitro-L-arginine methyl ester increased maximal response to VP threefold in M but had no effect in F or Tfm. In contrast, maximal contraction of normal aortas to phenylephrine was 43% higher in M (4,011 +/- 179 mg) than in F (2,809 +/- 78 mg); maximal response of Tfm (2,716 +/- 126 mg) was similar to that of normal F. N(G)-nitro-L-arginine methyl ester increased maximal response to phenylephrine by >50% in F and Tfm but had no effect in M. Maximal contractile response to 80 mM KCl did not differ among M, F, or Tfm. Thus androgens and normal vascular AR function are important in the greater NO-mediated attenuation of reactivity to VP in M than in F rat aorta, which may involve specific modulation of endothelial VP signal transduction pathways and NO release by androgens. These data also establish the importance of the Tfm rat as a model to study the effects of androgens on cardiovascular function.

The hypertensive Y chromosome elevates blood pressure in F11 normotensive rats.

Our laboratory has shown that the Y chromosome has a significant effect on blood pressure in the spontaneously hypertensive rat (SHR) model of hypertension and that the testes and androgen receptor contribute to the blood pressure rise. As an extension of our research, we have developed two new rat strains, SHR/a and SHR/y (F11) to study the Y chromosome. The objectives of the following research were 1) to study the blood pressure of rats with an SHR Y chromosome in a normotensive genetic background (SHR/y) or a normotensive Y chromosome in an SHR genetic background (SHR/a), 2) to determine the effect of male sex phenotype on the blood pressure of these rats, 3) to determine if testosterone replacement in castrated rats would restore blood pressure, and 4) to determine whether the Y chromosome from the SHR/y strain when crossed with a normotensive female can induce hypertension in androgen receptor-deficient male offspring. Blood pressure of male SHR/y rats was significantly higher than that of normotensive Wistar-Kyoto males (p < 0.01), and SHR/a males had significantly lower blood pressure compared with that of the parent SHR strain (p = 0.05). Testosterone replacement in castrated rats of both strains (SHR/a and SHR/y) restored blood pressure to control levels. Normotensive female King-Holtzman rats heterozygous for the testicular feminization gene were crossed with F11 SHR/a and SHR/y males.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotropin-releasing hormone-stimulated luteinizing hormone secretion by perifused pituitary cells from normal, gonadectomized, and testicular feminized rats.

To elucidate further the manner in which gonadal steroids influence the secretion of LH, we examined the effects of gonadectomy and the absence of functional androgen receptors on GnRH-induced LH release from dispersed rat anterior pituitary cells. Intact and gonadectomized (GNX) normal rats and androgen-resistant, testicular feminized (Tfm) animals from the King x Holtzman strain (a mutant strain that possesses defective androgen receptors) were used. Dispersed pituitary cells were perifused with Medium 199 during a 4-h equilibration period and then subjected to eight 2.5-min pulses of GnRH introduced at 30-min intervals at concentrations ranging from 0.03-100 nM. Basal LH secretion by cells from intact male and female rats was indistinguishable (P = 0.79) and was substantially lower (P less than 0.0001) than that by cells from GNX male and female animals. Basal LH secretion by cells from Tfm rats was significantly higher (P less than 0.01) than that by cells from intact animals, but lower (P less than 0.005) than that by cells from GNX animals. In response to GnRH, perifused pituitary cells from animals representing all experimental groups demonstrated concentration-dependent LH release. Pituitary cells from intact female rats showed an overall greater (P less than 0.05) response to GnRH than cells from intact male rats. Pituitary cells from Tfm rats demonstrated a greater GnRH-stimulated LH mean response than cells from intact male (P less than 0.0001) or intact female (P less than 0.0001) rats. Gonadectomy of male rats resulted in an overall GnRH-stimulated LH release similar to that exhibited by cells from gonadectomized female rats (P = 0.61). Cells from Tfm animals released more LH in response to GnRH than those from gonadectomized male and female rats (P less than 0.001). These data demonstrate that the release of LH in response to GnRH by pituitary cells from intact male rats (i.e. in the presence of androgen and functional androgen receptors) is less than that seen by cells from intact females rats. Since circulating levels of testosterone and estradiol are known to be elevated in the testicular feminized rat, the heightened GnRH-stimulated LH release by cells from such animals may reflect either the long term lack of androgenic influence and/or the combined effects of androgen resistance and elevated levels of circulating estrogens.

Genetic evidence for androgen-dependent and independent control of aromatase activity in the rat brain.

To investigate the role of androgen receptors in the regulation of brain aromatase activity (AA) in adult rats, the levels of AA in discrete brain areas of androgen-insensitive testicular feminized (Tfm) rats were compared with those in their normal male littermates (NL). AA was measured in homogenates of brain tissue by using a radiometric assay that quantifies the production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. Initially, we assessed the capability of block-dissected tissues to aromatize androgens. We found that the AA in the amygdala and hypothalamus-preoptic area of Tfm rats was significantly lower (P less than 0.001) than the AA in NL despite the fact that circulating androgen concentrations in the Tfm were significantly higher. Kinetics studies demonstrated that the apparent Michaelis constant was equivalent for both groups (0.02-0.03 microM). Administration of testosterone propionate to castrated males produced 3 to 4-fold elevations of AA in NL, but did not affect brain AA in Tfm rats. To pinpoint specific sites where AA is affected in Tfm rats, we measured AA in 10 hypothalamic and limbic nuclei that were dissected from 300-micron frozen brain sections. Compared to NL, Tfm rats exhibited significantly lower levels of AA in all micro-dissected brain regions studied, except for the medial and cortical amygdala. These data provide genetic evidence for both androgen-dependent and independent regulation of AA in the rat brain.

Hemicastration causes and testosterone prevents enhanced uptake of [3H] thymidine by Sertoli cells in testes of immature rats.

Rat pups were hemicastrated and uptake of [3H] thymidine by Sertoli cells in the remaining testis was compared to that in testes of sham-operated pups at intervals of from 8 h to 21 days after surgery. Labeled thymidine was administered subcutaneously 2 h before sacrifice. Testes were processed for light microscope autoradiography and the percent of Sertoli cell nuclei that had incorporated [3H] thymidine was determined by scoring nuclei in tissue sections as labeled or unlabeled. The percentage of cells labeled was increased in hemicastrates over intact controls by 8 h after surgery and testicular hypertrophy became apparent in hemicastrates by the following day. Labeling of Sertoli cells in hemicastrates remained elevated for 4 days and then returned to normal. When plasma levels of gonadotropins were measured in both groups 4 days after surgery, follicle-stimulating hormone (FSH) was found to be more than twice normal in hemicastrates while luteinizing hormone (LH) was unchanged. The effect of testosterone on the response of Sertoli cells to hemicastration was also examined. In hemicastrates, 2 days of androgen therapy depressed, and an additional 2 days abolished, the proliferative response of the Sertoli cells. Our findings suggest that increased proliferation of Sertoli cells within the remaining testis is involved in the enlargement of the testis that follows hemicastration. They also imply that prevention of compensatory hypertrophy by testosterone involves interference with this response of Sertoli cells in some way. Finally, our data implicate FSH in control of Sertoli cell proliferation in vivo in immature rats.

Effect of gonadotrophin-releasing hormone on circulating levels of immunoreactive luteinizing hormone in fetal rats.

Concentrations of luteinizing hormone (LH) were measured in plasma samples obtained from rat fetuses after injecting them with synthetic gonadotrophin-releasing hormone (GnRH) in utero. On days 17 1/2, 18 1/2 or 19 1/2 post-conception (p.c.), fetuses in one uterine horn were injected subcutaneously with 100 ng GnRH in 2 microliters of saline. Fetuses in the other uterine horn were injected with saline (four litters at each gestational age). Plasma samples were collected beginning 30 min after injection of GnRH. The concentration of LH in 20 microliters of fetal plasma was measured by a microvolume radioimmunoassay. On day 17 1/2 p.c., LH was detectable only in plasma samples of GnRH-injected fetuses. In 18 1/2- and 19 1/2-day-old fetuses LH reached detectable levels (greater than 6 ng/ml) in most saline-injected fetuses. In 18 1/2- and 19 1/2-day-old fetuses of both sexes, plasma LH titers of GnRH-injected fetuses were significantly higher than in saline-injected littermates. On day 19 1/2 GnRH-treated females had higher levels of plasma LH than similarly treated males. Thus fetal rat pituitaries were shown to have the capacity to release LH in vivo in response to circulating GnRH as early as 17 1/2 days p.c.

A light and electron microscopic study of the pineal body of the nutria (Myocastor coypus).

Light and electron microscopic studies of the nutria (Myocastor coypus) pineal gland revealed pinealocytes interspersed among glial, vascular, and neuronal elements. Each pinealocyte possessed a single process that terminated within the parenchyma near the perivascular region. The eccentrically located nucleus in these cells contained euchromatic chromatin, a prominent nucleolus, and a highly infolded nuclear envelope. The cytoplasm was rich in mitochondria, Golgi complexes, and glycogen particles. The smooth endoplasmic reticulum (SER) was better developed thant he rough endoplasmic reticulum (RER) and polyribosomes were not abundant. Long profiles of subsurface cisterns constituted prominent cytoplasmic features, and these were most conspicuous in the regions of soma-somatic apposition. The bulbous endings of the pinealocyte processes were filled with clear, round, secretory vesicles. Dense-cored vesicles were rarely observed. Glia reminiscent of protoplasmic astrocytes displayed cytoplasmic processes that enveloped blood vessels, invested the pineal periphery, and intervened among the pinealocytes. They thus seemed to form a barrier between the meningeal capsule and vascular space on the one hand and the parenchyma on the other.