Castration fails to prevent prenatally programmed hypertension in male rats.

Male offspring of rats that were modestly protein restricted during pregnancy become hypertensive as adults, whereas their female littermates remain normotensive. The purpose of this study was to determine the role of testosterone in promoting this sexual dimorphism of prenatally programmed hypertension. Rats were fed either a normal (19% protein, NP) or modestly protein-restricted (8.5% protein, LP) diet throughout pregnancy. Male offspring either remained intact or were castrated (CAS) at 30 days of age. Female offspring remained intact. At approximately 22 wk of age, the offspring were chronically instrumented for measurement of mean arterial pressure and renal function. Intact male LP offspring were hypertensive compared with male NP offspring (138 +/- 2 vs. 130 +/- 2 mmHg, P < 0.007), whereas female LP offspring were normotensive (123 +/- 1 vs. 122 +/- 2 mmHg in NP females). In CAS males, blood pressure in both diet groups was not different from that in intact males of the same group (138 +/- 3 mmHg in LP CAS males, and 131 +/- 2 mmHg in NP CAS males). Glomerular filtration rate and effective renal plasma flow were also not significantly affected by castration. However, castration significantly reduced protein excretion in LP males to levels not different from those in NP CAS and intact males. Renal histopathology scores showed a similar pattern. Thus removal of androgens by castration failed to provide any protective effect against the hypertension programmed by maternal protein restriction. Castration also failed to abolish the sex difference in blood pressure in both diet groups. These findings suggest that the lifelong presence of normal levels of testicular hormones does not play a major role either in maintaining baseline blood pressure higher in males than in females, or in promoting further elevations in blood pressure in males due to prenatal undernutrition. However, androgens such as testosterone may promote renal injury in LP males.

Expression of steroid hormone receptors in the fetal sheep brain during the critical period for sexual differentiation.

The objective of this study was to examine the expression of receptors for androgen, estrogen, and progesterone in the fetal sheep brain during the critical period for sexual differentiation. We isolated mRNA from the hypothalamus-preoptic area (HPOA), amygdala (AMYG), medulla (MD), frontal cortex (FCTX) and olfactory bulbs (OB) of fetal sheep that were delivered on day 64 of gestation. Using a ribonuclease protection assay and species-specific cRNA probes, we measured mRNA expression levels of androgen receptor (AR), estrogen receptor alpha (ERalpha) and progesterone receptor (PR). ERalpha and AR mRNA were expressed in all of the tissues tested and highest in the HPOA. PR mRNA was measured in HPOA and AMYG only and was significantly higher in male than in female fetuses. We conclude that the fetal brain is a target site for circulating steroid hormones. These data have implications for the steroid dependent development of sexually dimorphic brain functions in sheep.

The volume of a sexually dimorphic nucleus in the ovine medial preoptic area/anterior hypothalamus varies with sexual partner preference.

Sheep are one of the few animal models in which natural variations in male sexual preferences have been studied experimentally. Approximately 8% of rams exhibit sexual preferences for male partners (male-oriented rams) in contrast to most rams, which prefer female partners (female-oriented rams). We identified a cell group within the medial preoptic area/anterior hypothalamus of age-matched adult sheep that was significantly larger in adult rams than in ewes. This cell group was labeled the ovine sexually dimorphic nucleus (oSDN). In addition to a sex difference, we found that the volume of the oSDN was two times greater in female-oriented rams than in male-oriented rams. The dense cluster of neurons that comprise the oSDN express cytochrome P450 aromatase. Aromatase mRNA levels in the oSDN were significantly greater in female-oriented rams than in ewes, whereas male-oriented rams exhibited intermediate levels of expression. Because the medial preoptic area/anterior hypothalamus is known to control the expression of male sexual behaviors, these results suggest that naturally occurring variations in sexual partner preferences may be related to differences in brain anatomy and capacity for estrogen synthesis.

Estrogen synthesis in fetal sheep brain: effect of maternal treatment with an aromatase inhibitor.

The aim of the present study was to determine whether the fetal lamb brain has the capacity to aromatize androgens to estrogens during the critical period for sexual differentiation. We also determined whether administration of the aromatase-inhibitor 1,4,6-androstatriene-3,17-dione (ATD) could cross the placenta and inhibit aromatase activity (AA) in fetal brain. Eight pregnant ewes were utilized. On Day 50 of pregnancy, four ewes were given ATD-filled Silastic implants, and the other four ewes received sham surgeries. The fetuses were surgically delivered 2 wk later (Day 64 of gestation). High levels of AA (0.8-1.4 pmol/h/mg protein) were present in the hypothalamus and amygdala. Lower levels (0.02-0.1 pmol/h/mg protein) were measured in brain stem regions, cortex, and olfactory bulbs. The Michaelis-Menten dissociation constant (K(m)) for aromatase in the fetal sheep brain was 3-4 nM. No significant sex differences in AA were observed in brain. Treatment with ATD produced significant inhibition of AA in most brain areas but did not significantly alter serum profiles of the major sex steroids in maternal and fetal serum. Concentrations of testosterone in serum from the umbilical artery and vein were significantly greater in male than in female fetuses. No other sex differences in serum steroids were observed. These data demonstrate that high levels of AA are found in the fetal sheep hypothalamus and amygdala during the critical period for sexual differentiation. They also demonstrate that AA can be inhibited in the fetal lamb brain by treating the mother with ATD, without harming fetal development.

Relationship of serum testosterone concentrations to mate preferences in rams.

This study examined systemic testosterone concentrations in rams that were classified according to their sexual behavior and partner preference as either female-oriented (FOR), male-oriented (MOR), or asexual (NOR). For this purpose, we measured testosterone concentrations under three separate conditions: in conscious rams during the nonbreeding season (June) and breeding season (November), and in anesthetized rams during the breeding season. Basal testosterone concentrations in conscious rams were not different among the three groups (P > 0.05) in either season. However, when rams were anesthetized, mean systemic concentrations of testosterone in FORs (mean +/- SEM, 13.9 +/- 7.4 ng/ml serum) were greater (P < 0.05) than in NORs (0.9 +/- 0.1 ng/ml), but not in MORs (2.2 +/- 6.2 ng/ml), whereas testosterone concentrations were not different between MORs and NORs (P > 0.05). Concentrations of testosterone in the spermatic vein of FORs (127 +/- 66 ng/ml) were greater (P < 0.05) than in MORs (41 +/- 10 ng/ml) and NORs (19 +/- 7 ng/ml). Serum LH concentrations were not different. Cortisol was higher (P < 0.05) in anesthetized MORs (25.1 +/- 4.2 ng/ml) and NORs (27.2 +/- 4.4 ng/ml) than in FORs (10.9 +/- 1.8 ng/ml). These results demonstrate that circulating testosterone concentrations are related to sexual behavior only when rams are bled under anesthesia. Thus, differences in basal androgen concentrations in adulthood cannot be responsible for expression of male-oriented preferences or low libido in sheep. Instead, functional differences must exist between the brains of rams that differ in sexual preference expression.

Hormonal influences on sexual partner preference in rams.

Domestic rams display a naturally occurring variation in sexual partner preference, such that 6-10% of range-bred populations prefer male sexual partners (male-oriented) in contrast to the majority of rams that prefer female sexual partners (female-oriented). Male-oriented rams exhibit hormone profiles and stress responses distinctly different from their heterosexual counterparts. These differences include reduced circulating levels of testosterone that arise after anesthetization. Lower levels of aromatase activity in the medial preoptic area and estrogen receptor in the amygdala were also measured in male-oriented versus female-oriented rams and may represent an important link to sexual behavior that should be investigated. It is anticipated that the male-oriented ram model will be useful for studies aimed at identifying both the activational and organizational components and the neuronal substrates of male sexual partner preferences.