Samuel A. Sholl

Androgen receptors are differentially distributed between right and left cerebral hemispheres of the fetal male rhesus monkey

In humans there are apparent sex differences in verbal and spatial abilities as well as several cortical pathologies. These differences may arise as the result of prenatal androgen exposure and its effect on the development of the cerebral cortex. With this in mind, we have examined androgen receptor (AR), aromatase (AROM) and 5 alpha-reductase (5 alpha R) levels in the cerebral cortex of Day 70 male and female fetal rhesus monkeys (Macaca mulatta). Receptor and enzyme levels were evaluated in both right (Rt) and left (Lft) temporal (TMP) and frontal (FR) lobes of the cerebral cortex. AR levels in FR-Rt of male subjects were higher than levels in FR-Lft (for each and every subject, P less than 0.05), while in females, there was no consistent pattern in the distribution of the receptor between the two sides of FR. In contrast, AR values in TMP-Lft of male subjects were consistently higher than in TMP-Rt (P less than 0.05). As with the FR, females exhibited no consistent pattern in the distribution of AR between the two TMP sides. AROM and 5 alpha R levels were similar, regardless of sex, between both sides of the two cortical lobes indicating that the AR distribution pattern is not a general biochemical phenomenon. The differential cortical distribution of AR in fetal males versus females lends support to the hypothesis that prenatal androgens from the fetal testes may effect the differentiation of sexually dimorphic, side-specific cortical activity.

Estrogen receptors in the rhesus monkey brain during fetal development.

Estrogen receptor (ER) levels were measured in brain tissue cytosol from fetal male and female rhesus monkeys at Days 70, 100 and 160 postconception. The brain regions which were examined included medial basal hypothalamus (MBH), amygdala (AMG), cerebral cortex (CTX) and cerebellum (CB). For comparison, brain tissues were also obtained from an adult female, and muscle (MUS) and genital tract (GEN, ovaries + uterus) ER values were measured in several Day 70 fetuses. Tissues were dissected and homogenized as previously described. Cytosol was passed through a microcolumn of Lipidex 1000 to remove interfering lipids and incubated with [3H]Moxestrol (4 nM) in the presence or absence of 500 nM Moxestrol. Incubations were carried out for 24 h at 4 degrees C, and free and bound ligand separated by Sephadex LH-20 gel filtration. In one case (Day 160 male fetus), saturation analysis yielded an estimate of apparent Kd of 0.46 x 10(-9) M and indicated that maximal specific binding was achieved at a ligand concentration of 1-2 nM. There was no sex difference at any stage of development (ANOVA). A significant age effect (P less than 0.002) was noted for the MBH and CB but not for any of the other tissues examined. In the MBH the significance of this effect was due to a progressive increase in ER levels with fetal age and into adulthood. In contrast, CB levels exhibited a progressive decline with age. These studies revealed that the ER is present during brain development. Thus any estrogens derived from the aromatization of circulating fetal androgens could potentially exert an influence upon brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen synthesis and metabolism in the hamster blastocyst, uterus and liver near the time of implantation

The steroidogenic potential of hamster tissues, just prior to implantation of the blastocyst in the uterus, was characterized by incubating blastocysts (14) and pieces of endometrium with [1, 2-3H]-androstenedione for 24 h. [3H]-2-Methoxyestradiol was synthesized, but intermediate estrogens were not found. To obtain a more quantitative assessment and comparison of steroidogenic activity, especially aromatase activity, in these tissues as well as in the uterine myometrium and liver and to increase the possibility of recovering estradiol, microsomes were isolated from 244 blastocysts and portions of the other tissues. Microsomes were incubated with [1 alpha, 2 alpha-3H]-testosterone plus [1 beta,2 beta-3H]-testosterone for 6 h. During this time [3H]-metabolites were synthesized by all tissues as indicated by HPLC. [3H]-Androstenedione was noted and values were higher than control levels (medium alone or microsomes from uterine flush fluid) in all samples but liver. [3H]-Estradiol was detected at an elevated level only in the blastocyst sample; however, addition of unlabeled estradiol during the subsequent incubation of endometrial, myometrial and liver microsomes increased the recovery of [3H]-estradiol. Identities of [3H]-2-methoxyestradiol from the first experiment and [3H]-androstenedione and [3H]-estradiol from the second experiment were confirmed by recrystallization. The formation of 3H2O from [beta-3H]-testosterone was used as an index of aromatase activity. After subtracting control medium values, blastocysts were 24-fold more active (dpm/microgram protein) than the endometrium and myometrium in synthesizing 3H2O. While there was no difference in synthetic potential between endometrium and myometrium, aromatase activity in these tissues was greater than that of the liver. Microsomes from uterine flush fluid displayed no capacity for synthesizing 3H2O indicating that the elevated blastocyst levels were not caused by contaminating endometrial cells. These results indicate that all of the tissues examined have the capacity to metabolize C19-steroids to a variety of hormones, including estrogens, and further, that estrogen metabolism occurs rapidly in these tissues. This capacity may be important for providing a suitable hormonal milieu at the time of implantation.

Analysis of sex and regional differences in androgen receptors in fetal rhesus monkey brain

Cytosolic androgen receptor levels were measured in different brain regions of fetal male and female rhesus monkeys (day 135 post-conception). The hypothalamus-preoptic area, amygdala and cerebellum exhibited higher receptor concentration in comparison to the cerebral cortex (parietal, temporal frontal, and occipital lobes), hippocampus and cingulate gyrus (P less than 0.001). No sexual difference in receptor concentrations was found except in the amygdala where fetal males exhibited slightly higher levels than females. The similarity in androgen receptor levels suggests that the developing brain of both sexes could be influenced by circulating androgens under normal or pathological conditions.

Aromatase, 5-Alpha-Reductase, and Androgen Receptor Levels in the Fetal Monkey Brain during Early Development

Aromatase, 5 alpha-reductase and cytosolic androgen receptor levels were measured in the medial basal hypothalamus (MHB), amygdala (AMG), cerebellum and cerebral cortex of male and female fetal rhesus monkeys on day 70 of gestation. Higher aromatase activities were noted in the MBH and AMG of male than female fetuses. In contrast, no sex differences were found for 5 alpha-reductase and androgen receptor levels. These data suggest that at this early stage of development, differentiation of the MBH and AMG of the male fetus may be more susceptible to androgen modification, by way of aromatization to estrogens, than corresponding areas in the female fetus. Moreover, based upon a comparison of the current data to that published previously for later stages of development, it is suggested that the sex differences in aromatase activity are not the result of androgen stimulation.

Serum progesterone and corpus luteum function in pregnant pigtailed monkeys (Macaca nemestrina)

Corpus luteum (CL) function and control during pregnancy and early lactation in the pigtailed macaque was investigated. Peripheral concentrations of progesterone (P) on day 10 of pregnancy were 12.98 +/- 2.21 ng/ml and decreased progressively to 7.96 +/- 1.27 ng/ml by day 21 of pregnancy. The concentration of P increased around day 27 of gestation and reached peak levels of 18.48 +/- 2.45 ng/ml on day 37, thereafter gradually decreasing to a nadir at about midgestation. Ten days before parturition P concentrations increased again (P < 0.05). Concentrations of P decreased from 6.62 +/- 1.48 ng/ml on the day of delivery to 2.16 +/- 0.43 ng/ml on day 2 of lactation and remained low thereafter. Ovariectomy on day 35 did not affect the normal course of gestation or the patterns of P secretion during pregnancy. However, in these ovariectomized animals, in spite of suckling, P was not detectable after parturition. In intact monkeys, serum concentrations of P in the utero-ovarian vein at days 80 and 159 of pregnancy were higher relative to the uterine vein. Incubation studies utilizing 3H-cholesterol as a substrate revealed that the CL were capable of synthesizing P on days 35 and 159 of gestation. Histologically, the CL contained active luteal cells at late pregnancy. Low serum concentrations of chorionic gonadotropin were detected on day 10 of gestation; concentrations of this hormone reached high levels between days 18 and 24 and the titers were nondetectable after day 40 of pregnancy. Luteinizing hormone was present in constant amounts in the circulation during pregnancy and lactation. These data suggest that the CL of pregnancy in the pigtailed monkey is functional or capable of functioning during various stages of pregnancy. However, the fetoplacental unit is the primary source of P during the latter 4.5 months of gestation. As in other primates, a functional CL is not required for maintenance of pregnancy after implantation nor for lactation. Thus, the physiological significance of CL function during pregnancy is unclear.

Neural uptake and metabolism of testosterone and dihydrotestosterone in the guinea pig

Neural tissues from adult, castrated male guinea pigs were examined for their capability to concentrate and metabolize [1,2-3H]testosterone (T) and [1,2-3H]dihydrotestosterone (DHT), both in vitro and in vivo. In vitro uptake of DHT and T was greater in the hypothalamus and anterior pituitary than in the cerebral cortex. With DHT as the substrate, the 800 times g particulate concentration of this compound was highest in the hypothalamus, although in this tissue, particulate concentration was less than that of the cytoplasm. In the cerebral cortex 5alpha-androstane-3,17-dione was the most abundant metabolite, whereas 5alpha-androstane-3,17-dione, 5alpha-androstane-3alpha,17beta-diol, and 5alpha-androstane-3beta,17beta-diol were all present in equivalent amounts in the hypothalamus and pituitary. Incubation with T resulted in the formation of DHT, 4-androstene-3,17-dione, and a compound with the mobility of 5alpha-(or 5beta-)androstane-3,17-dione. The radioactivity associated with DHT was the most prevalent in the pituitary (1.3%), and least prevalent in the cerebral cortex (0.6%), and in all cases cytoplasmic concentration of this compound exceeded the concentration in the particulate fraction. Recrystallization failed to confirm the presence of estradiol-17beta. Although there were no apparent tissue differences in the uptake of DHT or T 1 hour after their injection, intracellular distribution varied. In all tissues examined, that percentage of total radioactivity attributable to DHT was greatest in the 800 times g particulate preparations, particularly in the hypothalamus. Thus neural tissues in the guinea pig, as in other species, exhibit differential uptake and metabolism of androgen through which physiological and behavioral effects may be mediated.

Quantification of 20α- and 20β-dihydroprogesterone in plasma of the pregnant rhesus monkey

Abstract A competitive protein binding assay for 20α- and 20β-dihydroprogesterone is described which involves an initial chemical or enzymatic oxidation of these two isomers to progesterone. The assay can distinguish between 20α- and 20β-dihydroprogesterone and is sensitive to pg amounts of these two steroids. Venous steroid concentrations were measured in the pregnant rhesus monkey employing this assay. In this species the corpus luteum (CL) at days 22 and 157 of gestation is the primary ovarian source of 20α-dihydroprogesterone as indicated by a higher plasma concentration of this steroid in the ovarian vein draining the ovary containing the CL (+CL) than in the contralateral vein (−CL) (9.34 ng/ml versus 1.72 ng/ml, day 22; 7.52 ng/ml versus 1.96 ng/ml, day 157). By contrast the CL at day 50 appeared to secrete no 20α-dihydroprogesterone as evidenced by the essentially equal steroid levels in both ovarian veins. The CL synthesizes 20β-dihydroprogesterone only during early gestation (21–22 days) when the concentration of this steroid was 6.46 ng/ml and 0.87 ng/ml in the ovarian (+CL) and ovarian (−CL) veins, respectively. Synthesis of both 20α- and 20β-dihydroprogesterone occurs in the fetoplacental unit throughout pregnancy. This is indicated by higher steroid concentrations in the uterine vein than in the femoral vein. The results suggest both a qualitative and quantitative alteration in the luteal synthesis of 20α- and 20β-dihydroprogesterone with the advancement of gestation. The data also provide additional evidence that the steroidogenic activity of the CL is enhanced before parturition.

Patterns of 3β-hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal

3β-Hydroxysteroid dehydrogenaseΔ5−4isomerase activity (3Δ-HSDH) was examined in the rhesus monkey (Macaca mulatta) placenta and fetal adrenal at 135 and 155–162 days of gestation. Activity was evaluated in microsomes by the conversion of [3H]pregnenolone to [3H]progesterone. There was a 7-fold increase in enzyme activity in the whole adrenal (minus medulla) between the two stages of development. Combining data from both periods, enzyme activity was greater in the outer than in the inner region of the adrenal. No stage-dependent change in placental activity was evident. The temporal patterns in 3β-HSDH activity are consistent with corticoid and progesterone patterns in the circulation. Thus, the level of 3β-HSDH activity may be rate limiting in both the fetal adrenal and placenta. Enzyme activity was assessed in incubations which included unex-tracted, heat-treated, 100,000 g tissue supernatants. In both placental and adrenal incubations, competitive inhibition was noted. Ethyl ether extracts of 100,000 g tissue supernatants also inhibited 3β-HSDH in the respective tissues. GLC analysis of these extracts revealed the presence of putative dehydroepiandrosterone. Hormone levels and the nature of the inhibition that were observed are compatible with the conclusion that dehydroepiandrosterone can inhibit the conversion of pregnenolone to progesterone in vivo. The physiological importance of this remains to be determined.

Placental and luteal steroidogenesis in the pregnant rhesus monkey

Progesterone, 20alpha-dihydroprogesterone, estrone and estradiol-17beta concentrations were estimated by radioimmunoassay in blood plasma from uterine, uteroovarian and femoral veins of rhesus monkeys (Macaca mulatta) on days 22, 49, 128 and 160 of gestation. Steroids were consistently more concentrated in uterine and uteroovarian that in femoral venous plasma and in many cases levels in the uteroovarian vein were also higher than those in the uterine vein indicating luteal secretion of both progestins and estrogens thoughout gestation. In some animals, however, the corpus luteum appeared quiescent. As reflected in the decline in the uterine venous progesterone/estradiol-17beta concentration ratio, a shift in steroid contribution from the uterus and its contents occurred between days 22 and 49 of gestation with progesterone declining more rapidly than estradiol-17beta. Progesterone/20alpha-dihydroprogesterone was higher in both uterine and uteroovarian than in femoral venous plasma suggesting peripheral metabolism of progesterone to 20alpha-dihydroprogesterone.