Developmental programming: impact of prenatal testosterone excess on pre- and postnatal gonadotropin regulation in sheep

The goal of this study was to explore mechanisms that mediate hypersecretion of LH and progressive loss of cyclicity in female sheep exposed during fetal life to excess testosterone. Our working hypothesis was that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH (but not FSH) secretion and, thus, hypersecretion of LH in adulthood, and that this results from altered developmental gene expression of GnRH and estradiol (E2) receptors, gonadotropin subunits, and paracrine factors that differentially regulate LH and FSH synthesis. We observed that, relative to controls, females exposed during fetal life to excess testosterone, as well as the nor-aromatizable androgen dihydrotestosterone, exhibited enhanced LH but not FSH responses to intermittent delivery of GnRH boluses under conditions in which endogenous LH (GnRH) pulses were suppressed. Luteinizing hormone hypersecretion was more evident in adults than in prepubertal females, and it was associated with development of acyclicity. Measurement of pituitary mRNA concentrations revealed that prenatal testosterone excess induced developmental changes in gene expression of pituitary GnRH and E2 receptors and paracrine modulators of LH and FSH synthesis in a manner consistent with subsequent amplification of LH release. Together, this series of studies suggests that prenatal testosterone excess, by its androgenic action, amplifies GnRH-induced LH response, leading to LH hypersecretion and acyclicity in adulthood, and that this programming involves developmental changes in expression of pituitary genes involved in LH and FSH release.     

Hypertension caused by prenatal testosterone excess in female sheep

Polycystic ovary syndrome (PCOS), a leading cause of infertility, affects approximately 10% of women of reproductive age. The etiology and pathophysiology of PCOS are poorly understood. PCOS is multifaceted and includes reproductive abnormalities and components of the metabolic syndrome such as insulin resistance, obesity, dyslipidemia, and hypertension. Exposure to excess testosterone (T) during the prenatal period may predispose individuals to PCOS phenotype. The goal of this study was to determine whether hypertension and dyslipidemia occur in a well-characterized model of PCOS produced by prenatal treatment of sheep with T. Radiotelemetry was used to measure blood pressure over a 24-h period in conscious, undisturbed animals. To normalize circulating estradiol levels across treatment, control (n = 4) and prenatal T-treated (100 mg T propionate im twice weekly from days 30 to 90 of fetal life, n = 4) 2-yr-old females were ovariectomized, instrumented with a radiotelemetry transmitter, and clamped with early follicular phase levels of estrogen using an implant. Six days later, a 24-h recording period commenced. Prenatal T-treated sheep were hypertensive compared with control sheep, and heart rate tended to be higher. T-treated sheep had hyperglycemia, insulin resistance, hypernatremia, and hyperchloremia, and both total and LDL cholesterol tended to be higher. Plasma aldosterone and epinephrine were significantly lower in T-treated sheep, whereas norepinephrine was unchanged. This first-ever use of radiotelemetric blood pressure recordings in sheep demonstrates that mild hypertension, a risk factor reported in some women with PCOS, is also a feature of the sheep model of PCOS produced by prenatal T treatment.     

Developmental programming: deficits in reproductive hormone dynamics and ovulatory outcomes in prenatal, testosterone-treated sheep

Prenatal testosterone excess leads to neuroendocrine, ovarian, and metabolic disruptions, culminating in reproductive phenotypes mimicking that of women with polycystic ovary syndrome (PCOS). The objective of this study was to determine the consequences of prenatal testosterone treatment on periovulatory hormonal dynamics and ovulatory outcomes. To generate prenatal testosterone-treated females, pregnant sheep were injected intramuscularly (days 30-90 of gestation, term=147 days) with 100 mg of testosterone-propionate in cottonseed oil semi-weekly. Female offspring born to untreated control females and prenatal testosterone-treated females were then studied during their first two breeding seasons. Sheep were given two injections of prostaglandin F2alpha 11 days apart, and blood samples were collected at 2-h intervals for 120 h, 10-min intervals for 8 h during the luteal phase (first breeding season only), and daily for an additional 15 days to characterize changes in reproductive hormonal dynamics. During the first breeding season, prenatal testosterone-treated females manifested disruptions in the timing and magnitude of primary gonadotropin surges, luteal defects, and reduced responsiveness to progesterone negative feedback. Disruptions in the periovulatory sequence of events during the second breeding season included: 1) delayed but increased preovulatory estradiol rise, 2) delayed and severely reduced primary gonadotropin surge in prenatal testosterone-treated females having an LH surge, 3) tendency for an amplified secondary FSH surge and a shift in the relative balance of FSH regulatory proteins, and 4) luteal responses that ranged from normal to anovulatory. These outcomes are likely to be of relevance to developmental origin of infertility disorders and suggest that differences in fetal exposure or fetal susceptibility to testosterone may account for the variability in reproductive phenotypes.     

Developmental programming: exogenous gonadotropin treatment rescues ovulatory function but does not completely normalize ovarian function in sheep treated prenatally with testosterone

Prenatal testosterone treatment leads to LH excess as well as ovarian follicular and ovulatory defects in the adult. These disruptions may stem from LH excess, abnormal FSH input, compromised ovarian sensitivity to gonadotropins, or intrinsic ovarian defects. To determine if exogenous gonadotropins rescue ovarian and ovulatory function of testosterone-treated sheep, the release of endogenous LH and biopotent FSH in control and prenatal testosterone-treated sheep was blocked with a GnRH antagonist during the first two breeding seasons and with LH/FSH coadministered in a manner approximating natural follicular phase. An acidic mix of FSH was administered the first 36 h at 2-h intervals and a less acidic mix for the next 12 h at 1-h intervals (different FSH preparations were used each year), and ovulation was induced with hCG. Circulating FSH and estradiol responses to gonadotropins measured in 2-h samples differed between treatment groups in Year 1 but not in Year 2. Ovarian follicular distribution and number of corpora lutea (in ewes that ovulated) tracked by ultrasonography and luteal progesterone responses were similar between control and prenatal testosterone-treated females but differed between years. Furthermore, hCG administration induced large cystic and luteinized follicles in both groups of females in Year 2, although the growth rate differed between control and prenatal testosterone-treated females. Our findings provide evidence that 1) ovulatory response in prenatal testosterone-treated females can be rescued with exogenous gonadotropins, 2) resultant follicular response is dependent on the nature of gonadotropic input, and 3) an abnormal follicular milieu may underlie differences in developmental trajectory of cystic follicles in prenatal testosterone-treated females.     

Developmental programming in sheep: administration of testosterone during 60-90 days of pregnancy reduces breeding success and pregnancy outcome

Evidence suggests that exposure to excess steroids during critical periods of fetal development leads to reproductive disorders. Exposure of female lambs to excess testosterone (T) from Days 60 to 90 of gestation (T60-90; term, 147 days) delayed onset of the LH surge and resulted in to male-typical reproductive behavior. The objectives of this study were to test the ability of T60-90 ewes to mate, conceive and lamb during the first three breeding seasons (Years 1, 2 and 3). Pregnant Suffolk ewes were injected with T propionate in cottonseed oil (100mg, im twice weekly) or vehicle (control; C) from Days 60 to 90 of gestation. In Year 1, ewes (C=12, T60-90=12) were kept with a vasectomized ram for 3 months and markings/visual observation of copulations were recorded. Rams had paint applied to their chest to facilitate detection of estrus and mating. All C but only three T60-90 ewes were marked (P<0.001). All ewes were then estrus-synchronized with two injections of prostaglandin F2alpha (20mg, im) given 11 days apart and allowed to mate with a painted, fertile ram. Nine of 12 C and 4 of 12 T60-90 ewes (P=0.1) were mated. Based on estrus and long-term monitoring of progesterone, more C than T60-90 became pregnant (82 and 18%, respectively; P<0.01). In Year 2, to maximize ram exposure, two C and two T60-90 estrus-synchronized ewes were placed with a painted, fertile ram at a time and mated ewes were removed to a nearby pen to force mating with others. Twenty-four hour video monitoring revealed the rams mated more C than T60-90 ewes (83 and 25%, respectively; P=0.01). In both Years 1 and 2, the rams preferred C over T60-90 ewes; therefore in Year 3 rams were given access only to T60-90 ewes. Only four T60-90 estrus-synchronized ewes were placed with a painted ram at a time. Not given an option, 91% of the T60-90 ewes were marked resulting in 4 of 11 (36%; first-service pregnancy rate in the breeding herd was 91%) ewes becoming pregnant to the synchronized estrus. Collectively these studies showed that fertility in T60-90 females was severely compromised, even after overcoming ram preference for controls.     

In vitro regulation of sheep ovarian surface epithelium (OSE) proliferation by local ovarian factors

The ovarian surface epithelium (OSE) forms a lining around the entire ovary and actively participates in the ovulatory cycle. To investigate how specific growth factors and hormones affect OSE proliferation, the present study used sheep as a model to examine the effects of follicular and luteal products on the proliferation of sheep OSE cells in culture, and to analyse the influences of large antral follicles and corpora lutea (CL) on the expression of gonadotrophin receptors (FSHR and LHR) in the OSE. In the present study, follicular fluids from medium and large follicles, and extracts of corpora lutea stimulated the growth of OSE cells. The results of the present study showed that factors in the follicular fluid can induce OSE proliferative activity, and this stimulation effect could not be attributed to steroids in the follicular fluid since oestrogen and progesterone treatments failed to stimulate OSE cells. The expression of LH and FSH receptors over large follicles (5 mm or larger) was two and four times higher than those over stroma and CL, respectively. In conclusion, OSE proliferation in cycling sheep is associated with underlying developing follicles and CL, mediated by, at least in part, the up-regulation of gonadotrophin receptors, and facilitated by the action of mitogenic glycopeptides and growth factors, but not steroids.     

Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O(2)) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy.     

The role of Eag and HERG channels in cell proliferation and apoptotic cell death in SK-OV-3 ovarian cancer cell line

Background  

The voltage gated potassium (K⁺) channels Eag and HERG have been implicated in the pathogenesis of various cancers, through association with cell cycle changes and programmed cell death. The role of these channels in the onset and progression of ovarian cancer is unknown. An understanding of mechanism by which Eag and HERG channels affect cell proliferation in ovarian cancer cells is required and therefore we investigated their role in cell proliferation and their effect on the cell cycle and apoptosis of ovarian cancer cells.     

Adiponectin receptor agonist AdipoRon induces apoptotic cell death and suppresses proliferation in human ovarian cancer cells

Molecular and Cellular Biochemistry - The aim of our study is to explore the regulation of C1QTNF1-AS1 on its target miR-221-3p/SOCS3 in human hepatocellular carcinoma (HCC). To explore the...     

Developmental changes of hypothalamic, pituitary and striatal tachykinins in response to testosterone: influence of prenatal melatonin.

Substance P (SP) and neurokinin A (NKA), members of the family of mammalian tachykinins, are involved in the regulation of many physiological functions and are widely distributed in mammalian tissues. In this report, the effects of prenatal melatonin on the postnatal developmental pattern of NKA, and SP, and on testosterone secretion were investigated. Also, tachykinin response to the administration of testosterone propionate (TP) was studied. The brain areas studied were medio-basal-hypothalamus, pituitary gland and striatum. Male rat offspring of control or melatonin treated mother rats were studied at different ages of the sexual development: infantile, juvenile or prepubertal periods, and pubertal period. Both groups received exogenous TP (control-offspring+TP and MEL-offspring+TP), or the vehicle (control-offspring+placebo and MEL-offspring+placebo). Hypothalamic concentrations of all peptides studied in control-offspring+placebo remained at low levels until the juvenile period, days 30-31 of age. After this age, increasing concentrations of these peptides were found, with peak values at puberty, 40-41 days of age, then declining until adulthood. In the MEL-offspring+placebo a different pattern of development was observed; hypothalamic concentrations of NKA and SP from the infantile period until the end of juvenile period were significantly higher than in control-offspring+placebo. TP administration exerted a more marked influence on MEL-offspring than on control-offspring and prevented the elevation in tachykinin concentrations associated with prenatal melatonin treatment. TP administration to control-offspring resulted in significantly reduced (P < 0.05) tachykinin concentration only at 40-41 days of age, and increased (P < 0.01) during infantile period as compared to control-offspring+placebo. Pituitary NKA concentrations were lower than in the hypothalamus. In control-offspring+placebo pituitary NKA levels did not show significant changes throughout sexual development. A different developmental pattern was observed in MEL-offspring+placebo, with significantly increased (P < 0.05) pituitary NKA concentrations at 35-36 days of age than in control-offspring+placebo. TP administration to control-offspring influenced pituitary NKA levels at the end of the infantile and pubertal periods, showing at both stages significantly higher (P < 0.05) NKA levels as compared to control-offspring+placebo. NKA levels in MEL-offspring+TP were only affected at 21-22 days of age, showing significantly increased (P < 0.01) values as compared to MEL-offspring+placebo. Striatal tachykinin concentrations in control-offspring did not undergo important modifications throughout sexual development, but during the prepubertal period they started to increase. Maternal melatonin and TP injections produced short-lived alterations during the infantile period. The results showed that prenatal melatonin delayed the postnatal testosterone secretion pattern until the end of the pubertal period and postnatal peptide secretion in brain structures. Consequently, all functions depending of the affected areas will in turn, be affected.     

Developmental programming of vascular dysfunction by prenatal and postnatal zinc deficiency in male and female rats

Micronutrient malnutrition during intrauterine and postnatal growth may program cardiovascular diseases in adulthood. We examined whether moderate zinc restriction in male and female rats throughout fetal life, lactation and/or postweaning growth induces alterations that can predispose to the onset of vascular dysfunction in adulthood. Female Wistar rats were fed low- or control zinc diets from pregnancy to offspring weaning. After weaning, offspring were fed either a low- or a control zinc diet until 81 days. We evaluated systolic blood pressure (SBP), thoracic aorta morphology, nitric oxide (NO) system and vascular reactivity in 6- and/or 81-day-old offspring. At day 6, zinc-deficient male and female offspring showed a decrease in aortic NO synthase (NOS) activity accompanied by an increase in oxidative stress. Zinc-deficient 81-day-old male rats exhibited an increase in collagen deposition in tunica media, as well as lower activity of endothelial NOS (eNOS) that could not be reversed with an adequate zinc diet during postweaning life. Zinc deficiency programmed a reduction in eNOS protein expression and higher SBP only in males. Adult zinc-deficient rats of both sexes showed reduced vasodilator response dependent on eNOS activity and impaired aortic vasoconstrictor response to angiotensin-II associated with alterations in intracellular calcium mobilization. Female rats were less sensitive to the effects of zinc deficiency and exhibited higher eNOS activity and/or expression than males, without alterations in SBP or aortic histology. This work strengthens the importance of a balanced intake of micronutrients during perinatal growth to ensure adequate vascular function in adult life.     

Differential effects of prenatal testosterone timing and duration on phenotypic and behavioral masculinization and defeminization of female sheep

The process of sexual differentiation leaves genetically female individuals at risk of being masculinized by exogenous androgens. Previous research with sheep indicates that exposure to excess testosterone from Gestational Day (GD) 30 to GD 90 of the 147-day gestation masculinizes and defeminizes behavior as well as genitalia. Lower doses and shorter durations produce animals with varying degrees of genital virilization and alterations of the hypothalamic-pituitary-gonadal axis, but to our knowledge, the effects on complex behavior and its prediction by the amount of external virilization have not been explored. Previous research in rodents has suggested that sexual differentiation of the central nervous system and the external genitalia can be dissociated. Therefore, we hypothesized that the extent of virilization of external genitalia would not be predictive of the lack of female-typical, or the presence of male-typical, mating behavior. To test this hypothesis, we compared control females, females exposed to exogenous testosterone from GD 30 to GD 90 (T60 females) that have virilized genitalia, and females exposed to testosterone from GD 60 to GD 90 (T30 females) that have female-typical genitalia. Both natural behavioral estrus in the flock and hormonally controlled behavioral tests were used to explore reproductive behavior. The T60 and T30 females exhibited more masculinized reproductive behavior than the controls; however, the T30 females also exhibited feminine behavior. Neither testosterone-treated group was receptive or was mounted at rates comparable to those of controls. These data illustrate that variation in the timing or duration of exposure to prenatal testosterone during a critical period for masculinization can have variable effects on defeminization and that the effects of testosterone on genitalia are not entirely predictive of behavior.     

Dual anticancer activity of 8-Cl-cAMP: inhibition of cell proliferation and induction of apoptotic cell death

8-Cl-cAMP induces apoptotic cell death in human cancer cells. To look at this more closely, we examined the changes in the levels of Bcl-2 family proteins during 8-Cl-cAMP-induced apoptosis of SH-SY5Y human neuroblastoma cells. Following the treatment with 8-Cl-cAMP, Bcl-2 was transiently down-regulated and Bad was increased continuously up to day 5. In addition, overexpression of Bcl-2 efficiently blocked the 8-Cl-cAMP-induced apoptosis, suggesting Bcl-2 family proteins may be involved in the 8-Cl-cAMP-induced apoptosis. The contribution of the apoptotic cell death and the inhibition of cell proliferation in the 8-Cl-cAMP-induced growth inhibition was closely monitored in the Bcl-2-overexpressing cells. Though the apoptosis was reduced significantly, no significant difference was observed in the inhibition of cell proliferation up to day 2 of 8-Cl-cAMP treatment. These results suggest that 8-Cl-cAMP exerts anticancer activity by two distinct mechanisms, i.e. , through the inhibition of cell proliferation as well as the induction of apoptosis. Supporting this notion was the observations that (1) suppression of apoptosis by zVAD did not abrogate 8-Cl-cAMP-induced inhibition of cell proliferation, and (2) 8-Cl-cAMP did not show additive inhibition of cell proliferation in RIIbeta-overexpressing cells.     

The effects of prenatal testosterone on wages: Evidence from Russia

Is in utero exposure to testosterone correlated with earnings? The question matters for understanding determinants of wage differences that have attracted so much attention among economists in the past decade. Evidence indicates that markers for early testosterone exposure are correlated with traits like risk-taking and aggressiveness. But it is not at all clear how such findings might map into labor market success. We combine unique data from the Russian Longitudinal Monitoring Survey with measured markers (2D:4D ratios) for testosterone exposure and find that lower digit ratios (higher T) correlate with higher wages for women and for men, when controlling for age, education and occupation. There is also some evidence of a potential non-linear, inverse U-effect of digit ratios on wages but this is sensitive to choice of specification. These findings are consistent with earlier work on prenatal T and success in careers (Coates et al., 2009) but inconsistent with the work of Gielen et al. (2016) who find differing effects for men and women.     

Effects of calmodulin antagonists on human ovarian cancer cell proliferation in vitro

Examined were effects of calmodulin antagonists (W-5 and W-7) on proliferation of two kinds of human cell lines, designated HR and KF, derived from serous cystadecarcinoma of the ovary. Although both W-5 and W-7 inhibited their cell proliferation in vitro, the degree of inhibition was more marked with W-5 rather than with W-7. HR cells had higher sensitivity to cisplatinum than KF cells, while KF cells had higher sensitivity to adriamycin. Combinations of calmodulin antagonists and anti-cancer drugs resulted in adjuvant effects with regard to the inhibition of their cell proliferation in vitro.     

Fetal programming: prenatal androgen disrupts positive feedback actions of estradiol but does not affect timing of puberty in female sheep

We studied the impact of prenatal androgen exposure on the timing of onset of puberty, maintenance of cyclicity in the first breeding season, and the LH surge mechanism in female sheep. Pregnant sheep were injected with testosterone propionate (100 mg i.m.) twice each week from Day 30 to Day 90 (D30-90) or from Day 60 to Day 90 (D60-90) of gestation (term = 147 days). Concentrations of plasma progesterone and gonadotropins were measured in blood samples collected twice each week from control (n = 10), D60-90 (n = 13), and D30-90 (n = 3) animals. Rate of weight gain and initiation of estrous behavior were also monitored. After the first breeding season, when the animals entered anestrus, competency of the gonadotropin surge system to respond to estradiol positive feedback was tested in the absence or presence of progesterone priming for 12 days. Prenatally androgenized females had similar body weight gain and achieved puberty (start of first progestogenic cycle) at the same time as controls. Duration of the breeding season and the number of cycles that occurred during the first breeding season were similar between control and prenatally androgenized sheep. In contrast, prenatal exposure to androgens compromised the positive feedback effects of estradiol. Onset of LH/FSH surges following the estradiol stimulus was delayed in both groups of androgenized ewes compared with the controls in both the absence and presence of progesterone priming. In addition, the magnitude of LH and FSH surges in the two animals that surged in the D30-90 group were only one third and one half, respectively, of the magnitudes observed in the control and D60-90 groups. The present findings indicate that disruption of the surge system can account for the fertility problems that occur during adulthood in prenatally androgenized sheep.     

Identification of MicroRNAs controlling human ovarian cell proliferation and apoptosis

Previous studies have shown that microRNAs (miRNAs) can control steroidogenesis in cultured granulosa cells. In this study we wanted to determine if miRNAs can also affect proliferation and apoptosis in human ovarian cells. The effect of transfection of cultured primary ovarian granulosa cells with 80 different constructs encoding human pre‐miRNAs on the expression of the proliferation marker, PCNA, and the apoptosis marker, Bax was evaluated by immunocytochemistry. Eleven out of 80 tested miRNA constructs resulted in stimulation, and 53 miRNAs inhibited expression of PCNA. Furthermore, 11 of the 80 miRNAs tested promoted accumulation of Bax, while 46 miRNAs caused a reduction in Bax in human ovarian cells. In addition, two selected antisense constructs that block the corresponding miRNAs mir‐15a and mir‐188 were evaluated for their effects on expression of PCNA. An antisense construct inhibiting mir‐15a (which precursor suppressed PCNA) increased PCNA, whereas an antisense construct for mir‐188 (which precursor did not change PCNA) did not affect PCNA expression. Verification of effects of selected pre‐mir‐10a, mir‐105, and mir‐182 by using other markers of proliferation (cyclin B1) and apoptosis (TdT and caspase 3) confirmed specificity of miRNAs effects on these processes. This is the first direct demonstration of the involvement of miRNAs in controlling both proliferation and apoptosis by ovarian granulose cells, as well as the identification of miRNAs promoting and suppressing these processes utilizing a genome‐wide miRNA screen. J. Cell. Physiol. 223: 49–56, 2010. © 2009 Wiley‐Liss, Inc.