Pituitary ERα Research Articles

Perinatal DEHP exposure modulates pituitary estrogen receptor α and β expression altering lactotroph and somatotroph cell growth in prepuberal and adult male rats

Phthalates are synthetic chemicals widely used to make polyvinylchloride (PVC) soft and flexible. Of these, Di-(2-ethylhexyl) phthalate (DEHP) is the most commonly used, with high human exposure occurring as early as the fetal developmental stage and affecting the endocrine system. We focused on the perinatal DEHP effects on pituitary estrogen receptor (ER) expression in male rats, explored their impact on lactotroph and somatotroph cell growth, and evaluated the direct effects of this phthalate on pituitary cell cultures. Our results showed that DEHP perinatal exposure was unable to modify the ERα+ pituitary cell number from prepuberal rats, but increased ERβ+ cells. In adulthood, the pituitary ERα+ cells underwent a slight decrease with ERβ showing the greatest changes, and with a significant increase observed in somatotroph cells. Also, in vitro, DEHP reduced the ERα+ cells, increased the percentage of ERβ+ pituitary cells and modified the Ki67 index, as well as decreasing the lactotrophs and increasing the somatotroph cells. In conclusion, the present study showed that DEHP induced ER expression changes in normal pituitary glands from male rats in in vivo and in vitro conditions, suggesting that DEHP could differentially modulate lactotroph and somatotroph cell growth, possibly as a consequence of ER imbalance.

The phthalate DEHP modulates the estrogen receptors α and β increasing lactotroph cell population in female pituitary glands

Humans are exposed to numerous endocrine disruptors on a daily basis, which may interfere with endogenous estrogens, with Di-(2-ethylhexyl) phthalate (DEHP) being one of the most employed. The anterior pituitary gland is a target of 17β-estradiol (E2) through the specific estrogen receptors (ERs) α and β, whose expression levels fluctuate in the gland under different contexts, and the ERα/β index is responsible for the final E2 effect. The aim of the present study was to evaluate in vivo and in vitro the DEHP effects on ERα and β expression in the pituitary cell population, and also its impact on lactotroph and somatotroph cell growth.Our results revealed that perinatal exposure to DEHP altered the ERα and β expression pattern in pituitary glands from prepubertal and adult female rats and increased the percentage of lactotroph cells in adulthood. In the in vitro system, DEHP down-regulated ERα and β expression, and as a result increased the ERα/β ratio and decreased the percentages of lactotrophs and somatotrophs expressing ERα and β. In addition, DEHP increased the S + G2M phases, Ki67 index and cyclin D1 in vitro, leading to a rise in the lactotroph and somatotroph cell populations. These results showed that DEHP modified the pituitary ERα and β expression in lactotrophs and somatotrophs from female rats and had an impact on the pituitary cell growth. These changes in ER expression may be a mechanism underlying DEHP exposure in the pituitary gland, leading to cell growth deregulation.

Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Polycystic ovary syndrome (PCOS) is a hypothalamic-pituitary-gonadal (HPG) axis disorder. PCOS symptoms most likely result from a disturbance in the complex feedback regulation system of the HPG axis, which involves gonadotrophic hormones and ovarian steroid hormones. However, the nature of this complex and interconnecting feedback regulation makes it difficult to dissect the molecular mechanisms responsible for PCOS phenotypes. Global estrogen receptor α (ERα) knockout (KO) mice exhibit a disruption of the HPG axis, resulting in hormonal dysregulation in which female ERα KO mice have elevated levels of serum estradiol (E2), testosterone, and LH. The ERα KO females are anovulatory and develop cystic hemorrhagic ovaries that are thought to be due to persistently high circulating levels of LH from the pituitary. However, the role of ERα in the pituitary is still controversial because of the varied phenotypes reported in pituitary-specific ERα KO mouse models. Therefore, we developed a mouse model where ERα is reintroduced to be exclusively expressed in the pituitary on the background of a global ERα-null (PitERtgKO) mouse. Serum E2 and LH levels were normalized in PitERtgKO females and were comparable to wild-type serum levels. However, the ovaries of PitERtgKO adult mice displayed a more overt cystic and hemorrhagic phenotype when compared with ERα KO littermates. We determined that anomalous sporadic LH secretion caused the severe ovarian phenotype of PitERtgKO females. Our observations suggest that pituitary ERα is involved in the estrogen negative feedback regulation, whereas hypothalamic ERα is necessary for the precise control of LH secretion. Uncontrolled, irregular LH secretion may be the root cause of the cystic ovarian phenotype with similarities to PCOS.-Arao, Y., Hamilton, K. J., Wu, S.-P., Tsai, M.-J., DeMayo, F. J., Korach, K. S. Dysregulation of hypothalamic-pituitary estrogen receptor α-mediated signaling causes episodic LH secretion and cystic ovary.

Developmental Programming: Insulin Sensitizer Prevents the GnRH-Stimulated LH Hypersecretion in a Sheep Model of PCOS

Prenatal testosterone (T) treatment recapitulates the reproductive and metabolic phenotypes of polycystic ovary syndrome in female sheep. At the neuroendocrine level, prenatal T treatment results in disrupted steroid feedback on gonadotropin release, increased pituitary sensitivity to GnRH, and subsequent LH hypersecretion. Because prenatal T-treated sheep manifest functional hyperandrogenism and hyperinsulinemia, gonadal steroids and/or insulin may play a role in programming and/or maintaining these neuroendocrine defects. Here, we investigated the effects of prenatal and postnatal treatments with an androgen antagonist (flutamide [F]) or an insulin sensitizer (rosiglitazone [R]) on GnRH-stimulated LH secretion in prenatal T-treated sheep. As expected, prenatal T treatment increased the pituitary responsiveness to GnRH leading to LH hypersecretion. Neither prenatal interventions nor postnatal F treatment normalized the GnRH-stimulated LH secretion. Conversely, postnatal R treatment completely normalized the GnRH-stimulated LH secretion. At the tissue level, gestational T increased pituitary LHβ, androgen receptor, and insulin receptor-β, whereas it reduced estrogen receptor (ER)α protein levels. Although postnatal F normalized pituitary androgen receptor and insulin receptor-β, it failed to prevent an increase in LHβ expression. Contrarily, postnatal R treatment restored ERα and partially normalized LHβ pituitary levels. Immunohistochemical findings confirmed changes in pituitary ERα expression to be specific to gonadotropes. In conclusion, these findings indicate that increased pituitary responsiveness to GnRH in prenatal T-treated sheep is likely a function of reduced peripheral insulin sensitivity. Moreover, results suggest that restoration of ERα levels in the pituitary may be one mechanism by which R prevents GnRH-stimulated LH hypersecretion in this sheep model of polycystic ovary syndrome-like phenotype.

Initiation of active immunization against testosterone during early puberty alters negative feedback regulation of the hypothalamic-pituitary-testicular axis in rabbits

To investigate the effects of antitestosterone immunization, initiated during early puberty, on hypothalamic-pituitary-testicular feedback in rabbits, 16 early pubertal male rabbits were randomly allocated into 2 groups (n = 8), control or immunized against testosterone-3(O-carboxymethyl)oxime-BSA in Freund adjuvant at 4 mo of age (with a booster immunization 4 wk later). Blood samples (for antibody titers and hormone concentrations) were collected at 2- or 4-wk intervals after immunization. Compared with controls, antitestosterone immunization triggered: a substantial and sustained antibody response (P < 0.01); increases in serum concentrations of luteinizing hormone (LH) and testosterone and testis weight and volume (P < 0.05); hyperplasia of testicular interstitial tissue with clustered and hypertrophic Leydig cells; and greater (P < 0.05) enzyme protein and messenger RNA (mRNA) expression levels for testicular cholesterol side-chain cleavage cytochrome P-450, 17α-hydroxylase cytochrome P-450, and 3β-dydroxysteroid dehydrogenase. Furthermore, immunoneutralization of testosterone upregulated mRNA expressions for genes in sex steroid negative feedback loops, including androgen receptor (AR), estrogen receptor alpha (ER-α), kisspeptin encoded gene (kiss-1) and kisspeptin receptor (G-coupled receptor 54) and gonadotropin-releasing hormone (GnRH) in the hypothalamic arcuate nucleus, GnRH receptor and LH-β in pituitary, and AR, inhibin-α and βA subunits in testes (P < 0.05). However, immunization did not affect mRNA expressions for follicle-stimulating hormone β, AR, and ER-α in pituitary, or ER-α in testes. We concluded that antitestosterone immunization in male rabbits, initiated during early puberty, increased GnRH mRNA expression, and in turn LH synthesis by reducing testicular feedback signaling. Reduction of direct steroidal effects on the testis may also have increased testosterone secretion. Consequently, there was an accelerated testicular development during puberty and enhanced testicular function after puberty, which likely conferred prolonged reproductive advantages.

Gastric estrogen increases pituitary estrogen receptor α and prolactin mRNAs during the different pathological conditions of the liver

Mammalian liver is an estrogen-responsive tissue mediated by hepatic estrogen receptors. Although Ueyama et al. (Endocrinology 143:3162-3170, 2002) have reported the presence of aromatase and active production of gastric 17β-estradiol in parietal cells, there are a few studies on gastric 17β-estradiol exploring the relationship between gastro-hepato function and the gastro-pituitary-gonadal axis. The alteration of gastric 17β-estradiol flow into the systemic circulation by portal vein ligation (PVL) or partial hepatectomy (PH), and the effect of gastric 17β-estradiol on the pituitary function was investigated. In the PVL rats, arterial 17β-estradiol increased 9.5 times that of controls on day 3, and gradually decreased near to control levels in the portal vein by 4 weeks, which was still 5 times higher than those in the arteries of the control rats. In the PH rats, arterial 17β-estradiol increased 2 times that of controls on day 3, and gradually decreased to the control levels. Regeneration and growth of the liver remnants were observed about 2 weeks after PH. In the PVL and PH animals, pituitary ERα and prolactin mRNAs levels increased, positively correlating with an increase of arterial 17β-estradiol levels. Both reduced LHβ mRNA. It is apparent that hepatic dysfunction causes changes in gastric 17β-estradiol levels in the systemic circulation; and that elevated gastric 17β-estradiol affects pituitary function(s). This data suggest that gastric 17β-estradiol has a pivotal role in the regulation of the gastro-hepato-pituitary axis.

Effects of Laser Irradiation of Acupuncture Points Shenshu on Ovariectomized Rats

The effect of laser acupuncture on obesity in postmenopausal women remains unknown. The aim of this study was to investigate the effects of this form of treatment using an animal model. Seventy-seven female Sprague-Dawley rats were assigned into seven groups: normal control (Normal), sham-operation control (Sham), ovariectomized (OVX) control rats (OVX), OVX rats treated with estrogen (OVX + E), and three groups of ovariectomized rats treated with laser acupuncture (OVX + L). Bilateral ovaries were removed to decrease estrogen levels. After 2 wk, semiconductor laser irradiation was administered to bilateral Shenshu (BL 23) acupoints of rats in the three OVX + L groups at 12, 30, and 60 J/cm(2), respectively. Changes in body weight and pituitary estrogen receptor (ER) mRNA expression were analyzed. Morphological differences in the uterus and pituitary glands were also observed. The OVX group exhibited marked weight gain and a significant decrease in pituitary ERα mRNA expression. Semiconductor laser irradiation at 30 J/cm(2) reduced body weight and increased ERα expression compared with the control, whereas irradiation at 12 or 60 J/cm(2) presented slightly weaker effects. Significant differences in pituitary ERβ mRNA were not observed due to lack of optical density data. The semiconductor laser irradiation of bilateral Shenshu (BL 23) acupoints can exert beneficial effects on OVX rats through reducing body weight and increasing pituitary ERα expression, and 30 J/cm(2) was the most effective dose among those used.

Expression of estrogen receptor α and β genes in the mediobasal hypothalamus, pituitary and ovary during the canine estrous cycle

Estrogen receptor α (ERα) and ERβ mRNA levels were measured in the mediobasal hypothalamus, the anterior pituitary and the ovary of beagle bitches at various stages of the estrous cycle. With polymerase chain reaction analysis we detected ERβ gene transcripts in all tissue samples. The levels of hypothalamic and pituitary ERα and β mRNAs increased from mid anestrus to proestrus and declined thereafter. In the ovary, ERα mRNA levels increased from proestrus to diestrus and were positively correlated with plasma progesterone levels ( r=0.62, P<0.01), whereas ERβ mRNA levels increased from mid anestrus to proestrus and were positively correlated with plasma estradiol-17β levels ( r=0.73, P<0.001). These results suggest that the rise in hypothalamic and pituitary ERα and β mRNAs is associated with termination of anestrus, and that increases in ovarian ERα and β mRNAs may be involved in initiating development of the follicle or corpora lutea.

Effect of Long-Term Treatment with the Antiestrogen EM-652.HCl on Pituitary Estrogen Receptor Alpha and Prolactin mRNA Expression in Intact, Ovariectomized and Gonadotropin- Releasing Hormone-Treated Female Rats

Estrogen receptor α (ERα) is the predominant estrogen receptor subtype in the anterior pituitary gland. In order to assess the influence of the pure antiestrogen EM-652.HCl on ERα gene transcription, we have studied the effect of long-term administration of the antiestrogen in ovariectomized rats as well as in intact female rats treated or not with the GnRH-agonist D-trp⁶, des-Gly-NH₂¹⁰ GnRH ethylamide (GnRH-A), a treatment which induces pharmacological castration. To evaluate the degree of pituitary responsiveness to changes in estrogen exposure, prolactin (PRL) mRNA levels were also measured. ERα and PRL mRNA levels were evaluated by quantitative in situ hybridization. It was found that, 49 weeks after ovariectomy (OVX), pituitary ERα mRNA levels were decreased by 55%. Long-term administration (49 weeks) of EM-652.HCl to OVX animals resulted in a further 41% decrease in ERα mRNA. On the other hand, ovariectomy induced an 82% decrease in PRL mRNA levels while the administration of EM-652.HCl to OVX animals did not further decrease PRL mRNA. The administration of EM.652.HCl or GnRH-A alone to intact rats during 52 weeks did not significantly modify pituitary ERα mRNA levels. Concomitant administration of both GnRH-A and EM-652.HCl induced 41 and 47% decreases in ERα mRNA levels, when compared to the levels measured in vehicle-treated and GnRH-treated animals respectively. Combined administration of EM.652.HCl and GnRH-A induced 56 and 65% decreases in PRL mRNA, respectively. When EM-652.HCl was administered concomitantly with GnRH-A, the inhibitory effect on PRL mRNA levels was more marked than that observed in GnRH-A-treated animals. The present data demonstrate that when circulating estrogens are absent or maintained at very low levels by GnRH administration, EM-652.HCl can still depress ERα gene transcription. It is suggested that estrogens can positively regulate pituitary ERα gene transcription and that the antiestrogen EM-652.HCl can downregulate by itself pituitary ERα gene transcription.

Neonatal Imprinting and Regulation of Estrogen Receptor Alpha and Beta mRNA Expression by Estrogen in the Pituitary and Hypothalamus of the Male Rat

In male rodents, complete reproductive function is critically dependent on adequate estrogen action at different levels of the hypothalamic-pituitary-testicular axis. However, administration of high doses of estrogen during the critical period of neonatal differentiation results in multiple defects in the reproductive axis that disrupt male fertility, the molecular mechanism(s) behind such a phenotype remaining poorly characterized. The aim of this study was twofold: (1) to characterize the effects of neonatal estrogenization upon the pattern of estrogen receptors ERα and ERβ mRNA expression in the pituitary and hypothalamus of the male rat, and (2) to evaluate the ability of estrogen to acutely regulate pituitary and hypothalamic ERα and ERβ mRNA expression in the male rat. To achieve the first goal, groups of male rats were treated on the first day of life with estradiol benzoate (EB; 500 µg/rat), and pituitary and hypothalamic expression of ERα and ERβ mRNA levels were assayed by semiquantitative RT-PCR at different ages from the neonatal period until adulthood (days 1–75 of life). In addition, the mechanism(s) of altered pituitary expression of ERα and ERβ messages in neonatally estrogenized rats was explored by comparison of the effects of neonatal treatment with estrogen or a potent gonadotropin-releasing hormone (GnRH) antagonist. For the second goal, the acute effects of a single dose of EB (12.5–25 µg/rat) on hypothalamic and pituitary ERα and ERβ mRNA expression were assessed in prepubertal and adult male rats. Neonatal estrogenization permanently decreased pituitary ERα and ERβ mRNA expression levels respective to control values at all ages studied. This pattern of response was similar to the short-term effects of neonatal blockade of endogenous GnRH actions. In contrast, neonatal exposure to estrogen transiently increased the hypothalamic expression levels of ERα and ERβ messages. This effect was detected in neonatal (5-day-old), infantile (15-day-old) and prepubertal (30-day-old) rats, and it disappeared at puberty. In addition, estrogen was able to acutely regulate mRNA expression levels of its cognate receptor subtypes in the pituitary and hypothalamus of intact male rats. In adult (75-day-old) males, EB administration (25 µg/rat) induced a significant time-dependent decrease in pituitary ERα and ERβ mRNA levels. In contrast hypothalamic expression of ERα and ERβ messages was increased after acute exposure to EB (12.5 µg/rat) in prepubertal males (30 days old); yet differences in the time course of the response to estrogen were noticed between targets. In conclusion, our data indicate that estrogen is able to neonatally imprint and acutely regulate mRNA expression levels of ERα and ERβ in the pituitary and hypothalamus of the male rat. Regulation of pituitary and hypothalamic ERα and ERβ gene expression by the cognate ligand represents a novel mechanism whereby estrogen modulates its own biological actions upon different levels of the male reproductive axis throughout the life span.