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Endocrine disrupting chemicals (EDCs), such as bisphenol A (BPA) and 17α-ethinylestradiol (EE2), can have far reaching health effects, including transgenerational abnormalities in offspring that never directly contacted either chemical. We previously reported reduced fertilization rates and embryo survival at F2 and F3 generations caused by 7-day embryonic exposure (F0) to 100 μg/L BPA or 0.05 μg/L EE2 in medaka. Crossbreeding of fish in F2 generation indicated subfertility in males. To further understand the mechanisms underlying BPA or EE2-induced adult onset and transgenerational reproductive defects in males, the present study examined the expression of genes regulating the brain-pituitary-testis (BPT) axis in the same F0 and F2 generation male medaka. Embryonic exposure to BPA or EE2 led to hyperactivation of brain and pituitary genes, which are actively involved in reproduction in adulthood of the F0 generation male fish, and some of these F0 effects continued to the F2 generation (transgenerational effects). Particularly, the F2 generation inherited the hyperactivated state of expression for kisspeptin (kiss1 and kiss2) and their receptors (kiss1r and kiss2r), and gnrh and gnrh receptors. At F2 generation, expression of DNA methyltransferase 1 (dnmt1) decreased in brain of the BPA treatment lineage, while EE2 treatment lineage showed increased dnmt3bb expression. Global hypomethylation pattern was observed in the testis of both F0 and F2 generation fish. Taken together, these results demonstrated that BPA or EE2-induced transgenerational reproductive impairment in the F2 generation was associated with alterations of reproductive gene expression in brain and testis and global DNA methylation in testis., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

F1 hybrid New Zealand Black (NZB) x New Zealand White (NZW) (NZB/W) mice are hormone-sensitive models of the human disease systemic lupus erythematosus. In this study, NZB/W fetuses produced by pregnant NZB mice were compared with F1 C57BL/6 x DBA/2 (C57/DBA2) hybrid fetuses produced by nonautoimmune C57BL/6 females. Dams of both strains were treated with testosterone or the androgen blocker flutamide to alter the hormonal environment in late gestation. Hormonal changes in male fetuses carried by treated dams were of interest because hormonal manipulation using either testosterone or flutamide has been shown to increase longevity in male NZB/W offspring. Testosterone-implanted NZB dams developed the expected elevations in circulating maternal testosterone, whereas C57BL/6 dams treated with either testosterone or flutamide had elevated maternal serum testosterone concentrations. The treatment-induced changes in circulating testosterone in NZB dams and C57BL/6 dams were not reflected in serum from 18-day NZB/W or C57/DBA2 fetuses. Male NZB/W offspring from untreated control NZB dams had unexpectedly high levels of serum estradiol and alpha fetoprotein and relatively low extractable testicular testosterone, compared with nonautoimmune male control fetuses. Maternal testosterone treatments produced a significant decrease in serum estradiol in NZB/W male fetuses, and placental testosterone content was also reduced. Our findings suggest that placental androgen control is regulated differently in the autoimmune NZB-NZB/W vs. the nonautoimmune C57BL/6-C57/DBA2 maternal-placental-fetal unit.

Circulating estradiol is assumed not to contribute to sexual differentiation of the brain or other estrogen target tissues. The only estradiol available for binding to estrogen receptors is thought to be produced within brain cells by the aromatization of testosterone to estradiol as part of the action of androgen in the brain. However, we report that the concentration of free, biologically active serum estradiol (the concentration not bound to plasma proteins) was 0.54-2.17 pg/ml during the fetal and early neonatal period of sexual differentiation. These values were within the same concentration range for free estradiol observed in adult female rats throughout the estrous cycle (diestrus = 0.53 pg/ml; proestrus = 2.26 pg/ml), and estradiol clearly has physiological effects during diestrus as well as proestrus in adult females. When a stable, physiological blood concentration of [3H]estradiol of 49 pg/ml total (0.61 pg/ml free) was achieved with Silastic capsules in 2-day-old female pups, [3H]estradiol was recovered specifically bound to brain cell nuclei at approximately 2.7 fmol per pup brain or 12.4 fmol/mg DNA. The finding of brain uptake of circulating estradiol is contrary to current hypotheses. These findings suggest that estradiol in the fetal and neonatal circulation may be able to interact with testosterone and its metabolites to regulate sexual differentiation of the brain and other estrogen target tissues.

Mice differ in their adult reproductive characteristics as a function of whether they developed in utero between two male fetuses (2M males), which have higher testosterone levels, or between two female fetuses (0M males), which have higher estradiol levels. The present study was designed to further characterize biochemical parameters of 2M and 0M adult male mice. Activities of testicular steroidogenic enzymes, namely delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase, 17 alpha-hydroxylase, and C17,20-lyase (C21SCC P450), were measured by means of radiometric assays and HPLC fractionation of substrate and products. Activity of 5 alpha-reductase in both seminal vesicle and prostate was measured by similar techniques. Estrogen and androgen receptor concentrations, which indicate capacity to respond to steroid hormones, were also examined in the accessory sex organs. For both seminal vesicle and prostate, 5 alpha-reductase activities were approximately 60% greater in 2M males than in 0M males, indicating greater capacity to form dihydrotestosterone from testosterone in organs from 2M mice. No significant differences were found in testicular steroidogenic enzymes between 2M and 0M animals, whereas the trend for all three activities was higher for 2M males than for 0M males. While no differences were found in estrogen receptor concentrations, 0M prostates had three times the concentration of androgen receptors (occupied receptors) compared to 2M prostates. Our findings suggest that intrauterine fetal position exerts a significant influence on subsequent adult androgen metabolism and androgen responsiveness in reproductive organs of adult male mice.

Steroid hormones, which affect development of reproductive traits, alter immune responses in rodents and appear to control severity of disease in F1 hybrid NZB/W mice, an animal model of systemic lupus erythematosus. We tested the hypothesis that exposure of NZB/W fetuses to altered hormonal environments would influence subsequent expression of autoimmune renal disease and affect longevity. NZB females, pregnant with NZB/W fetuses, were treated from Days 13-18 of gestation with testosterone or the antiandrogen, flutamide. Similar treatments were carried out in C57BL/6 dams mated to DBA/2 males to permit comparison with nonautoimmune hybrid mice. Serum concentrations of testosterone were greater in testosterone-implanted dams of both strains, but concentrations of estradiol were greater only in C57BL/6 dams treated with flutamide. Alpha fetoprotein (AFP), which binds estrogen and modulates immune responsiveness, was greater in serum from both groups of testosterone-treated dams, while flutamide treatment increased serum AFP only in NZB dams. We conclude that factors governing circulating estradiol and AFP differed in pregnant NZB and C57BL/6 females. Morphological analyses confirmed effects of hormonal manipulation on the developing fetuses. Testosterone implants resulted in female offspring with greater anogenital spaces, and treatment of dams with flutamide eliminated the expected difference between anogenital spaces in females and males. Effects of altered prenatal hormonal environments on immune-mediated disease in NZB/W offspring were examined in a longevity study. Early deaths were delayed in NZB/W females produced by flutamide-treated dams. An unexpected result was observed in NZB/W males. Male offspring from both testosterone- and flutamide-treated mothers lived longer than males from control dams. This paradox suggested that a characteristic shared by both groups of treated NZB dams had similar effects on the developing fetuses. It is proposed that elevated concentrations of AFP modulated the course of autoimmune disease and contributed to increased longevity in NZB/W offspring of treated dams.

We examined effects of maternal stress on prenatal serum concentrations of testosterone and estradiol and on postnatal reproductive traits in female mice from different intrauterine positions. On Day 18 of fetal life, control females positioned in utero between two male fetuses (2M females) had higher concentrations of testosterone and lower concentrations of estradiol in serum than control female fetuses located between two females (0M females). Control females positioned between a male and a female fetus (1M females) had intermediate levels of both hormones. Prior intrauterine position in control females accounted for differences in genital morphology (length of the anogenital separation) at birth and length of estrous cycles during adulthood. Maternal stress eliminated these postnatal differences due to prior intrauterine position: all 0M, 1M, and 2M female offspring of stressed mothers exhibited postnatal traits that were indistinguishable from those of control 2M females. Maternal stress resulted in an increase of over 1 ng/ml in serum testosterone in all female fetuses; the magnitude of the increase was similar for 0M, 1M, and 2M females. There was no effect of maternal stress on serum concentrations of estradiol in 0M and 2M female fetuses. Maternal stress resulted in a dramatic change in the postnatal traits of 0M females, whereas 2M females showed no change. Since the effect of maternal stress on sex steroids was similar among fetuses from different intrauterine positions but postnatal response to maternal stress varied by intrauterine position, other components of the endocrine system may mediate effects of maternal stress on these postnatal characteristics.

The production of live young during successive pregnancies was investigated in female CF-1 house mice (Mus musculus) identified at cesarean delivery as having developed in utero between 2 male fetuses (2M females) or not next to a male fetus (0M females). 2M female mice have previously been found to be exposed to higher concentrations of testosterone than 0M females during fetal life, presumably as a result of the transport of steroids between contiguous fetuses. 0M and 2M females were paired with stud males. The males were removed prior to delivery of a litter and replaced by other males when the litter was weaned. This process was repeated until: 1) a female did not become pregnant within 2.5 mo or 2) two successive litters were produced in which all of the pups were dead. In Experiment 1 females were first mated when 25 days old, and 2M females ceased producing litters containing live pups at a younger age and after fewer litters than did 0M females; however, many females were terminated from the study as a result of producing 2 successive litters of dead young rather than failing to become pregnant during a 2.5-mo period. There was a gradual decline in the number of live young produced by 0M females as a function of age and parity, but 2M females abruptly ceased producing any live young after producing a litter of normal size. For the last live litter, there were thus significantly fewer live young produced by 0M females than by 2M females. None of these differences were observed in Experiment 2, in which 0M and 2M females were mated for the first time beginning at 7 mo of age. The 2M females in this experiment ceased producing live young at a significantly older age than did the 2M females first mated at puberty. In contrast, there was no effect of age at initial mating on the age at which 0M females ceased producing live young. This finding suggests that exposure of 2M females to elevated titers of testosterone during fetal life results in a reduction in reproductive life span if they first become pregnant during the pubertal period.