Sex-related differences in blood and gonad levels of testosterone in silver fox fetuses

The mass of silver fox fetuses of both sexes, their gonads, and adrenals, and the levels of testosterone in blood serum and in gonads and adrenals were determined from day 31 of gestation and every five days thereafter until its termination. Marked sex-related differences were revealed: the blood and gonad levels of testosterone in male fetuses were much higher than those in female fetuses. The fetal adrenals contained significantly less testosterone than the gonads. No sex-related differences in the content of testosterone in the fetal adrenals were found. No differences were found in the body and adrenal mass in female and male fetuses at all the developmental stages studied, while the mass of ovaries exceeded that of testes from day 45 of gestation. The data obtained suggest sex dimorphism in the production of testosterone by gonads in silver foxes appears after day 35 and appears to correspond to the period of morphological differentiation of gonads.     

Radioimmunoassay of testosterone in late fetal and newborn rabbit plasma, gonads and adrenal glands]

A radioimmunoassay was used for measuring testosterone in the plasma, gonads and adrenals of 28, 29, 30 and 31-day-old rabbit fetuses of both sexes and newborns. A marked sex difference was shown in the concentrations of testosterone in plasma and in gonads whereas in adrenals the levels of testosterone were low in both sexes (34 to 147 pg/10 mg). In male fetuses, plasma testosterone levels increased from the 28th (133 +/- 20 pg/ml) to the 31st day (361 +/- 119 pg/ml) of intrauterine life, reaching then the values observed in the newborns (387 +/- 73 pg/ml). Plasma from males, on the other hand contained, at all stages studied, significantly more testosterone than plasma from female fetuses (21 +/- 6 to 41 +/- 11 pg/ml) and female newborns (42 +/- 6 pg/ml). In the same way, fetal testicular testosterone concentrations varying from 1 382 +/- 218 to 2 317 +/- 333 pg/10 mg were similar to those measured in the newborns (1 940 +/- 304 pg/10 mg) and significantly higher than fetal (13 to 34 pg/10 mg) or neonatal (44 pg/10 mg) ovarian concentrations. These results showed at evidence the endocrine activity of the fetal testis during this period.     

Fetal fluid steroids and their relationship to gonadal steroid secretion in single and twin bovine fetuses

Steroid concentrations in the fetal fluids of 153 single and 69 twin bovine pregnancies, ranging in age from 35 to 125 d of gestation, were studied to compare gonadal steroid secretions in vitro with the concentrations found in amniotic and allantoic fluids during the early stages of sex differentiation. Among the steroids measured in fetal fluids, only the testosterone level showed a correlation with the amount secreted by the gonads. Significantly higher concentrations of testosterone were associated with male fetuses than with female fetuses. The concentrations of androstenedione, estradiol and estrone in both fetal fluid compartments were generally correlated with age, reflecting the extra-gonadal source of steroids in these fluids. Androstenedione levels in fetal fluids were significantly higher in twins than in singletons, suggesting that this parameter may be useful for the diagnosis of fetal sex and/or type of pregnancy.     

Androgen receptors in fetal rabbit lung and the effect of fetal sex on the levels of circulating hormones and pulmonary hormone receptors

High affinity (KD = 0.2 nM), low capacity (3.6-5.0 fmol/mg protein), androgen-specific binding proteins with characteristics typical of androgen receptors were identified in the lungs of rabbit fetuses between the 26 and 29th day of gestation and in the lungs of adult rabbits. While androgen receptor concentrations increased significantly from late gestation to adulthood (P less than 0.01), no sex-related differences were observed in either the binding affinities or concentrations of the receptors at any age tested. Similarly, no sex-related differences were found in the levels of progesterone, cortisol and cortisone in the fetal circulation, or in the levels of progesterone receptors, glucocorticoid receptors and beta-adrenergic receptors in the fetal lung at 26 days of gestation. It is concluded that the fetal lung interacts directly with circulating androgens via specific androgen receptors and that the suggested male disadvantage with respect to lung maturation in the perinatal period does not appear to be associated with sex-related differences in the levels of pulmonary androgen, glucocorticoid, progesterone or beta-adrenergic receptors.     

Utility of maternal serum total testosterone analysis for fetal gender determination in Asian elephants (Elephas maximus).

It has been shown in some species that fetal testes produce testosterone early in gestation. This study investigated the possibility that fetal testosterone may be reflected in maternal serum levels in the Asian elephant (Elephas maximus). Weekly serum samples were collected from seventeen pregnant captive Asian elephants and analyzed via radioimmunoassay (RIA) for total testosterone levels. Nine of the cows carried male fetuses and eight carried female fetuses. A non-random pattern over time (P<0.01) was observed in cows carrying either a male or female fetus. Mean maternal serum total testosterone was significantly higher in cows carrying male versus female fetuses (P<0.01). Mean trimester values indicate that first trimester values are not significantly different among male versus female groups. The second and third trimester values of cows carrying male fetuses were higher than cows carrying female fetuses, (P<0.01 and <0.05, respectively). The results of this study show that it is possible via RIA of maternal serum for total testosterone to determine the gender of calves during gestation.     

Serum testosterone concentrations in advanced pregnancy in water buffaloes (Bubalus bubalis)

Peripheral testosterone levels were measured by radioimmunoassay of 65 serum samples taken at various intervals from 11 buffaloes in advanced pregnancy. Average testosterone levels measured at day 210 of gestation (248 ± 39 pg/ml) remained similar up to the last week of gestation. No difference in testosterone levels was recorded between buffaloes carrying male and female fetuses. Pregnant buffaloes had significantly higher testosterone levels than non-pregnant buffaloes.     

Sex-specific divergence of antioxidant pathways in fetal brain,liver, and skeletal muscles

The sex-specific divergence of antioxidant pathways in fetal organs of opposite-sex twin is unknown and remains urgently in need of investigation. Such study faces many challenges, mainly the ethical impossibility of obtaining human fetal organs. Opposite-sex sheep twins represent a unique model for studying a sex dimorphism for antioxidant systems. The activity of total superoxide dismutase (SOD), SOD1, SOD2, glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT), the content of total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were measured in brain, lung, liver, kidney, and skeletal muscles of female and male fetuses collected from sheep twin pregnancies at day 65 of gestation. Lipid peroxidation was assessed by measuring melondialdehyde (MDA) tissue content. Male brain has greater total SOD and SOD1 activities than female brain. Female liver has greater SOD2 activity than male liver. Male liver has greater GR activity than female liver. Male liver has higher total GSH and GSSG content than female liver. Male skeletal muscles have higher total GSH, GSH, and GSSG content than female skeletal muscles. Female brain and liver have higher MDA content than male brain and liver. This is the first report of a sex dimorphism for fetal organ antioxidative pathways. Brain, liver, and skeletal muscles of male and female fetuses display distinct antioxidant pathways. Such sexually dimorphic responses to early life oxidative stress might be involved in the sex-related difference in fetal development that may have a long-term effect on offspring. Our study urges researchers to take into consideration the importance of sex as a biologic variable in their investigations.     

Androgens in the bovine fetus and dam (38567).

Umbilical arterial and venous blood, and fetal testes were taken from 38 bovine fetuses at 90, 180 or 260 days of gestation. Concurrently blood also was taken from the jugular, and from the uterine artery and vein of the dams. Testosterone and androstenedione were determined by radioimmunoassays. Fetal testicular homogenates had 0.96 and 0.35 mug/g of testosterone and 0.39 and 0.50 mug/g of androstenedione at 180 and 260 days of gestation, respectively. Males had five to tenfold more serum testosterone and about twofold more androstenedione than female fetuses at each trimester of gestation. Male fetal blood testosterone decreased (P less than 0.01) from 2.7 to 0.3 ng/ml between 90 and 260 days of gestation. But, maternal testosterone and androstenedione increased (P less than 0.05) during gestation in cows with males, but not in cows with female fetuses. Testosterone was higher (P less 0.05) in cows carrying males than in cows with female fetuses. Androstenedione was higher in blood leaving the placenta on both the maternal and on the vetal sides suggesting placental synthesis of androstenedione.     

Cortisol production in fetal adrenals of the silver fox

The present study was designed to examine cortisol production by the silver fox fetal adrenals and their response to ACTH at different periods of prenatal life. Serum levels of cortisol were determined on days 35, 40, 45 and 50 of gestation (term on day 52) in embryos of both sexes. Cortisol content in adrenal tissue homogenates and its in vitro adrenal production were also investigated at the same time points. Hormones were measured by RIA. The levels of cortisol changed slightly during embryonic life. The adrenal content and the in vitro production of cortisol increased sharply and progressively (by 4-10 times from days 35 to 50 of gestation). The rises in cortisol content and in vitro production were associated with a rapid growth of the fetal adrenals. There were no sex differences in cortisol level and its adrenal content. ACTH (50 mIU per sample) increased the in vitro cortisol production by the adrenals in the two sexes on all the studied days (by 3-4 times on day 35 and by 1.4-1.7 times on day 50). These results indicate that 1) silver fox fetal adrenals are capable of synthesising cortisol; 2) ACTH is a potent activator of the in vitro cortisol production during embryonic life in this species.     

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.     

Activation of phospholipases during masculine differentiation of embryonic genitalia

We have investigated whether the androgen-induced masculine differentiation of the sex organs involves an induction of phospholipases. We have measured phosphatidylinositol-specific phospholipase C and phosphatidylcholine-specific phospholipase A2 in the reproductive tract of male and female mouse (CD-I) fetuses at the 18th day of gestation. We report here that (1) the activity of these two enzymes is higher in the male genitalia than in the female genitalia; (2) exogenous testosterone at the 13th to 17th day of pregnancy induces both phospholipase A2 and phospholipase C in the female fetal genitalia; and (3) prenatal administration of cyproterone acetate, an antiandrogen, known to produce feminized males, completely prevents the stimulation of phospholipase A2 and C by testosterone in the female fetuses. In the male fetuses, however, cyproterone acetate inhibits the PLC activity but is unable to alter phospholipase A2 activity. These findings provide evidence that the mechanism by which testosterone organizes the genitalia may involve a modification of phospholipases A2 and C.     

Ontogeny of secretory patterns of LH release and effects of gonadectomy in the chronically catheterized pig fetus and neonate

Two experiments were conducted to determine the ontogeny of secretory patterns of luteinizing hormone (LH) release and effects of gonadectomy on the characteristics of LH secretion in the chronically catheterized pig fetus and neonate. To study secretory patterns in intact animals, blood samples were collected from 44 pig fetuses and their mothers (Days 81, 99, 109 and 113 of gestation) as well as from 25 neonates (Days 4 and 8) every 15 min for 3 h (2 h on Day 81). The results indicate that the fetal adenohypophysis secretes occasional pulses of LH as early as Day 81 of fetal life. Fetal and maternal mean LH levels are low (0.25-0.50 ng/ml) at all gestational ages, with lowest values just before birth (Day 113 post coitum). Four-day-old neonates show a significant increase in pulse frequency (male and female) as well as pulse amplitude (female), relative to fetal values, leading to significant augmentations in mean LH levels. This is associated with reductions in both 17 beta-estradiol and progesterone. By 8 days of age significant sex differences in mean LH levels (males greater than females) appear. Testosterone/5 alpha-dihydrotestosterone levels (males) are low prenatally but are significantly increased after birth, possibly due to the stimulating effects of increasing LH levels. To study the gonadal control of LH secretion, forty-one 105-day-old fetuses and thirty-eight 4-day-old neonates were chronically catheterized and were either gonadectomized or remained as sham or control animals. Forty-eight and 96 h after surgery, blood samples were taken every 15 min for 3 h. No significant changes are detectable at 96 h in mean LH, pulse frequency and amplitude in female or male fetuses or in neonates. While significant reductions in testosterone levels are observed at 96 h in the male fetus and neonate, progesterone concentration is reduced only in the neonate. In the castrated female, on the other hand, neither fetus nor neonate display significant changes in circulating levels of progesterone and 17 beta-estradiol at 96 h. It is concluded that the pituitary of the pig is able to discharge LH with occasional pulses as early as Day 81 of fetal life; however, the pituitary remains suppressed until after birth, probably due to high circulating nongonadal steroids in the fetal compartment.     

Virilizing effect of testosterone and its metabolites on the urovaginal septum of female fetal rats

A single injection of 50 microgram testosterone was given to fetal rats on day 17, 18, 19 or 20 of gestation. On day 21, the fetuses were removed from the mother under maternal ether anesthesia, and the length of the urovaginal septum was measured microscopically in female fetuses in order to assess the virilizing effect of testosterone. In fetuses treated with testosterone on day 17, the length of the urovaginal septum was comparable to that of oil-treated littermate controls. In fetuses treated on day 18, the length was significantly abridged compared with controls. In fetuses treated on day 19, the abridgment of the urovaginal septum was most marked. In fetuses treated on day 20, the length of the septum was again comparable to that of controls. The observations suggest that day 19 is the critical day for the virilizing effect of testosterone. Various amounts of testosterone and its metabolites including dihydrotestosterone, androstane-3 beta, 17beta-diol and androstane-3 alpha, 17beta-diol were injected into 19-day-old female fetuses, in order to test the dose relation to the virilizing effects of these steroids in terms of abridgment of the urovaginal septum. As a consequence, it was found that testosterone was the most effective for virilization.     

Endocrine antecedents of polycystic ovary syndrome in fetal and infant prenatally androgenized female rhesus monkeys

Experimentally induced fetal androgen excess induces polycystic ovary syndrome-like traits in adult female rhesus monkeys (Macaca mulatta). Developmental changes leading to this endocrinopathy are not known. We therefore studied 15 time-mated, gravid female rhesus monkeys with known female fetuses. Nine dams received daily s.c. injections of 15 mg of testosterone propionate (TP), and six received injections of oil vehicle (control) from 40 through 80 days of gestation (term, 165 days; range, +/-10 days). All fetuses were delivered by cesarean section using established methods at term. Ultrasound-guided fetal blood sample collection and peripheral venous sample collection of dams and subsequent infants enabled determination of circulating levels of steroid hormones, LH and FSH. The TP injections elevated serum testosterone and androstenedione levels in the dams and prenatally androgenized (PA) fetuses. After cessation of TP injections, testosterone levels returned to values within the reference range for animals in these age groups, whereas serum androstenedione levels in PA infants were elevated. The TP injections did not increase estrogen levels in the dams or the PA fetuses or infants, yet conjugated estrogen levels were elevated in the TP-injected dams. Serum levels of LH and FSH were elevated in late-gestation PA fetuses, and LH levels were elevated in PA infants. These studies suggest that experimentally induced fetal androgen excess increases gonadotropin secretion in PA female fetuses and infants and elevates endogenous androgen levels in PA infants. Thus, in this nonhuman primate model, differential programming of the fetal hypothalamo-pituitary unit with concomitant hyperandrogenism provides evidence to suggest developmental origins of LH and androgen excess in adulthood.     

Effects of Hyperthyroidism on Expression of Vascular Endothelial Growth Factor (VEGF) and Apoptosis in Fetal Adrenal Glands

This study investigated the expression of vascular endothelial growth factor (VEGF), vascular density, and apoptosis in fetal rat adrenal glands with hyperthyroidism in late gestation. Twelve mature female Wistar albino rats with the same biological and physiological features were used for this study. Rats were divided into two groups: control and hyperthyroidism. Hyperthyroidism was induced by daily subcutaneous injections of L-thyroxine (250 µg/kg) before pregnancy for 21 days and during pregnancy. Rats in the control and hyperthyroidism groups were caged according to the number of male rats. Zero day of pregnancy (Day 0) was indicated when the animals were observed to have microscopic sperm in vaginal smears. Pregnant rats were sacrificed on the 20th day of pregnancy; blood from each animal was collected to determine the concentrations of maternal adrenocorticotropic hormone and thyroxine. Rat fetuses were then quickly removed from the uterus, and the adrenal glands of the fetuses were dissected. VEGF expression, vascular density, and apoptosis were analyzed in fetal rat adrenal glands. Maternal serum levels of the ACTH and free thyroxine were significantly higher in the hyperthyroidism group than in the control group. Immunohistochemistry revealed that the number of VEGF positive cells and vessel density significantly increased in the hyperthyroidism rat fetal adrenal group compared with the control group. Hyperthyroidism did not change the fetal and placental weights and the number of fetuses. This study demonstrates that hyperthyroidism may have an effect on the development of rat adrenal glands mediated by VEGF expression, angiogenesis, and apoptosis.     

Genes, hormones and the risk factors in the development of the male phenotype]

The onset of the expression of Sry and other sex-determining genes such as SF-1, DAX-1, WT-1 and SOX family initiates the testis organogenesis from the bipotential primordium. The fetal testis produces anti-Mullerian hormone and testosterone. These two hormones play essential role in the further development of the male phenotype. The bases for the activity of the sexual function and behavior are created within frames of these processes. Interindividual differences in these characters may achieve high degrees. Alleles of the sex-determining genes and the genes of the other genetic systems which participate in regulation of reproduction may be responsible for this variability. For example, the inherited variations in testosterone levels in the blood are negatively correlated to the alpha2-adrenergic receptor densities in the hypothalamus in males of mouse strains. Testosterone level in the fetal blood during critical period of sexual differentiation is one of the key points through which genetic and ontogenetic factors affect male sexual development. We have found nearly twofold interstrain differences in testosterone levels in the blood of male rat fetuses of 2 strains. The rats with higher testosterone levels during intrauterine development have higher rates of sexual maturation and sexual activity in future life. Genetic differences were also found in sensitivity of fetal testosterone to disruptive influences. These differences may be the reason for the strain-specific effects of prenatal stress or glucocorticoid treatment on the male sexual development in rats and mice. Substances and treatments which are capable of changing testosterone levels and/or interaction of these hormones with their receptors: ionizing radiation, pesticides, xenoestrogenes, drugs, alcohol, various stressors are the risk factors of the male sexual development.     

Analysis of metopirone-induced hypertrophy of adrenals in fetal rats encephalectomized in utero

Pregnant rats were treated with 30 mg metopirone (M) each day for 2 days and autopsied on the third day in various gestational periods (Days 18-20, 19-21, and 20-22). Control rats were treated with saline alone (S). The adrenals of intact fetuses in M-treated dams were significantly heavier than those of intact fetuses in S-treated dams in every experimental period. In both M- and S-treated dams, the adrenals of encephalectomized (E) fetuses were lighter than those of intact littermates. However, in the experimental period of Days 18-20 and 19-21, the adrenals of E fetuses in M-treated dams were slightly but significantly heavier than those of similar E fetuses in S-treated dams. In contrast, in the experimental period Days 20-22, there was no significant difference in the weight of adrenals of E fetuses of M- and S-treated dams. These changes in fetal adrenal weight were reflected histologically in parallel changes in the size of adrenocortical cells. The observations suggest that the fetal adrenal hypertrophy following maternal treatment with metopirone can occur to some extent independent of the fetal brain, but that close to the end of gestation the hypertrophy occurs completely under the control of the fetal brain.