Epidermal growth factor replaces estrogen in the stimulation of female genital-tract growth and differentiation.

The in vivo studies presented here demonstrate that epidermal growth factor (EGF) is an important autocrine and/or paracrine mediator of estrogen-induced growth and differentiation in mouse uterus and vagina. An antibody specific for EGF significantly inhibited estrogen-induced uterine and vaginal growth, thereby implicating EGF involvement in estrogen action. Furthermore, EGF administered via slow-release pellets in ovariectomized mice acted as a potent uterine and vaginal mitogen as well as an inducer of vaginal keratinization. Experiments with ovariectomized, adrenalectomized, hypophysectomized mice indicated that EGF mitogenesis does not require pituitary or adrenal hormones. Treatment with EGF also mimicked estrogen in the induction of uterine lactoferrin (a major estrogen-inducible secretory protein) mRNA and protein. These data suggest that EGF has estrogen-like effects in the promotion of cell growth in the reproductive tract and that EGF may serve as an important mediator of estrogen action in vivo.     

Decreased expression of Wnt7a mRNA is inversely associated with the expression of estrogen receptor-alpha in human uterine leiomyoma

Wnt-7a gene not only guides the development of the anterior-posterior axis in the female reproductive tract, but also plays a critical role in uterine smooth muscle pattering and maintenance of adult uterine function. This gene is also responsive to changes in the levels of sex steroid hormone in the female reproductive tract. To explore the molecular mechanisms underlying the pathogenesis of uterine leiomyoma, the expression of Wnt7a mRNA in the leiomyoma has been assessed. RT-PCR was performed on uterine leiomyomas and the adjacent myometria. Of 30 cases of leiomyomas studied, 67% showed a decreased mRNA level as compared to the paired myometria. On the other hand, estrogen receptor-alpha (ER-alpha) mRNA is hyper-expressed in 67% of the leiomyomas as compared to their paired myometrium. An inverse association at mRNA expression was found between Wnt7a and ER-alpha. Miller et alhas shown that fetal exposure of DES results in de-regulation of Wnt7a during uterine morphogenesis. Referring to their results, we have postulated that hypersensitivity of leiomyoma cells to estrogen may deregulate the Wnt7a expression. Decreased expression of Wnt7a may lead to loss of control in patterning of the myometrium and result in development of leiomyoma     

Coupling of dual signaling pathways: epidermal growth factor action involves the estrogen receptor.

Epidermal growth factor (EGF) reproduces many of the effects of estrogen on the murine female reproductive tract and may partially mediate estrogen-induced growth and differentiation. This study was performed to investigate the mechanism by which EGF elicits estrogen-like actions in the whole animal. EGF was administered to adult ovariectomized mice by slow release pellets implanted under the kidney capsule. The induction of uterine DNA synthesis and phosphatidylinositol lipid turnover by EGF or administration of diethylstilbestrol (5 micrograms/kg), a potent estrogen, was attenuated by the estrogen receptor antagonist ICI 164,384. Furthermore, EGF mimicked the effects of estrogen on enhanced nuclear localization of the estrogen receptor and the formation of a unique form of the estrogen receptor found exclusively in the nucleus. These results suggest that EGF may induce effects similar to those of estrogen in the mouse uterus by an interaction between the EGF signaling pathway and the classical estrogen receptor. The demonstration of cross-talk between polypeptide growth factors and steroid hormone receptors may be of importance to our understanding of the regulation of normal growth and differentiation as well as the mechanisms of transmission of extracellular mitogen signals to the nucleus.     

Mouse calbindin-D(9k) gene expression in the uterus during late pregnancy and lactation

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium binding protein mainly expressed in duodenum, placenta and uterus. In order to understand the expression pattern and regulation of uterine CaBP-9k gene, the expression of CaBP-9k mRNA and its regulation by estrogen (E2) and progesterone (P4) were investigated in the mouse uterus during late pregnancy (from day 12 to 18) and lactation. The expression levels of uterine CaBP-9k, estrogen receptor alpha (ERalpha) and progesterone receptor (PR) mRNAs were measured by Northern blot analysis. The expression levels of mouse uterine CaBP-9k mRNA gradually increased from pregnancy day 16 (P16), peaked at P18 (6.0-fold vs. P12) and declined at birth and during lactation. The expression levels of ERalpha and PR mRNAs indicated a similar fluctuation as CaBP-9k mRNA, suggesting the role of sex steroids/receptors in the regulation of CaBP-9k gene. To investigate effect of steroid hormone on CaBP-9k mRNA expression, three groups of animals were injected (s.c) with steroid hormone antagonists (RU486, tamoxifen, and ICI182780), respectively. RU486, a P4 antagonist, induced a significant decrease in CaBP-9k mRNA expression at 48 (3.2-fold) and 72 h (3.8-fold). However, tamoxifen and ICI182780, E2 antagonists, had no effect on CaBP-9k mRNA expression. Combined treatment with RU486 and ICI182780 did not further decrease the expression level of CaBP-9k mRNA when compared with RU486 treatment at 48 and 72 h. In addition, the treatment with RU40555, a glucocorticoid/progesterone antagonist, resulted in a decrease at 48 and 72 h following treatment. These results indicate that E2 is not likely involved in the regulation of CaBP-9k gene in the mouse uterus during late pregnancy and lactation. In conclusion, the present results suggest that P4, not E2 is a key regulator of CaBP-9k mRNA expression during late pregnancy and lactation.     

Lipin1 regulation by estrogen in uterus and liver: implications for diabetes and fertility

Estrogens are essential for fertility and also have important effects on regulation of adiposity and the euglycemic state. We report here that lipin1, a candidate gene for lipodystrophy and obesity that is a phosphatidic acid phosphatase critical in regulation of cellular levels of diacylglycerol and triacylglycerol and a key regulator of lipid utilization, is rapidly and robustly down-regulated in the uterus by estradiol via the estrogen receptor. Lipin1 is expressed predominantly in the uterine luminal and glandular epithelium, and during the estrous cycle, lipin1 is lowest when blood levels of estrogen are highest. Lipin1 is expressed throughout all cells in the liver of ovariectomized female mice, and a sustained down-regulation is observed at the mRNA, protein and immunohistochemical levels after estrogen administration. Because the coupling of proper energy use and availability is central to reproduction, we also investigated expression of lipin1 in the uterus and liver of several mouse models of diabetes. Nonobese diabetic (NOD) mice, which have high blood levels of estrogen and impaired fertility, were severely deficient in lipin1 in the uterus and liver, which, interestingly, could be restored by insulin treatment. By contrast, nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice, which do not develop diabetes, showed normal levels of lipin1. Our findings of lipin1 regulation by estrogen in two key target organs suggest a new role for this lipid-regulating phosphatase not only in central metabolic regulation but also in uterine function and reproductive biology. Estrogen regulation of lipin1 may provide a mechanistic link between estrogens, lipid metabolism, and lipid signaling.     

Estrogen induces CCN5 expression in the rat uterus in vivo

Estrogen plays an important role in the normal physiology as well as various pathologies of the uterus. Given the nature of uterine remodeling during the reproductive cycle and pregnancy, we sought to determine whether CCN5, a gene that we have shown to be important in smooth muscle cell proliferation and migration, is an estrogen-induced gene in the uterus. In the present study, we demonstrate that levels of CCN5 mRNA and protein expression were 5-fold higher in uteri from proestrous females relative to metestrous females, a finding consistent with estrogen induction of the CCN5 gene. Ovariectomized rats treated with exogenous estrogen or estrogen and progesterone exhibited 4- to 8-fold higher levels of CCN5 mRNA and protein than animals treated with either progesterone or vehicle alone. Analysis of rat uterine sections using immunohistochemistry demonstrates CCN5 localization throughout the uterus, including the endometrium and endometrial glands as well as the myometrium. Thus, our data indicate that CCN5 is positively regulated by estrogen in the rat uterus and suggests that this gene may play an important role in maintaining normal uterine physiology.     

Neonatal estrogen exposure disrupts uterine development in the postnatal sheep

Postnatal development of the ovine uterus between birth and postnatal day (PND) 56 involves budding differentiation of the endometrial glandular epithelium from the luminal epithelium (LE) followed by extensive coiling and branching morphogenesis of the tubular glands. To determine the short- and long-term effects of estrogen on neonatal ovine uterine development after PND 14, neonatal sheep were randomly assigned at birth (PND 0) to be treated daily with estradiol-17beta benzoate (EB; 0, 0.01, 0.1, 1, or 10 microg/kg body weight.d) during one of two developmental periods (PND 14-27 or 42-55). All ewes were hemiovariohysterectomized at the end of EB treatment on either PND 28 or 56, and the remaining uterine horn and ovary removed on PND 112. Immediate responses to EB treatment included dose- and age-dependent increases in uterine wet weight, thickness of the endometrium, myometrium, and LE, but decreases in endometrial glands on PND 28 and 56. Transient exposure to EB decreased gland number and thickness of the endometrium and LE on PND 112 but did not affect extrauterine reproductive tract structures. The mechanism of estrogen inhibition of uterine development did not involve effects on cell proliferation. Real-time PCR analyses found that EB exposure disrupted normal patterns of growth factor (IGF-I, IGF-II, fibroblast growth factor-7, fibroblast growth factor-10, and hepatocyte growth factor) and receptor mRNA expression in the uterus. Transient exposure of the neonatal ewe to estrogens during critical periods specifically alters growth factor networks that perturb normal development of the uterus, leading to permanent alterations in uterine structure and function.     

Lactoferrin expression in the mouse reproductive tract during the natural estrous cycle: correlation with circulating estradiol and progesterone.

Lactoferrin (LTF), an iron-binding glycoprotein present in most exocrine secretions and in the secondary granules of polymorphonuclear leucocytes (PMN), is regulated by estrogen in the mouse reproductive tract. We investigated the expression of LTF mRNA and protein during the natural estrous cycle to increase our understanding of how this uterine secretory protein is regulated under physiological conditions. There was a positive correlation between LTF mRNA expression in the genital tract and serum estradiol (E2) concentrations. When E2 peaked in proestrus, LTF mRNA and protein were expressed in the uterus; however, during metestrus, when both E2 and progesterone levels were high, LTF mRNA was expressed, while LTF protein was decreasing. LTF protein expression may be hindered by progesterone or some other local factor in the endometrial epithelium after ovulation. Immunohistochemistry demonstrated two distinct staining patterns for LTF in the vaginal and endometrial epithelium. In one staining pattern, the colorimetric reaction was noted over the cytoplasm, and in the other, the nuclear region stained more intensely. This suggests the possibility that in addition to its known role as a secretory protein, LTF may be transported to the nucleus, serving an autocrine role. Our results also indicated that LTF protein is a useful marker for tracking PMN. Nonproliferating epithelial cells in the vagina and endometrium may synthesize chemotactic and/or adhesion molecules for PMN.     

Estrogen inhibits calbindin-D28k expression in mouse uterus.

The cellular localization and hormonal controls of calbindin-D9k expression in the rodent reproductive tract have suggested new functions for this protein. The present studies were undertaken to extend the earlier studies of calbindin-D9k to the related protein, calbindin-D28k. Immunohistochemical studies revealed that calbindin-D28k was absent from female rat reproductive tissues, but was abundantly expressed in immature mouse uterus and oviduct. Immunoreactivity was restricted to the endometrial and glandular epithelium of the uterus and the oviductal epithelium. Neither 1,25-dihydroxyvitamin D- nor strontium-containing diets (to blunt 1,25-dihydroxyvitamin D production) affected expression of calbindin-D28k. Uterine, but not oviductal, calbindin-D28k decreased markedly at sexual maturity; this pattern persisted in pregnant mice and was reproduced in immature mice by the administration of estradiol (3 micrograms/day for 3 days). RNA extraction and Northern analyses demonstrated that estrogen markedly decreased calbindin-D28k mRNA abundance in the uterus, but not in the oviduct. These findings suggest that estrogen affects mammalian calbindin-D28k expression and represent a rare example of estrogen-induced down-regulation of gene expression.     

Temporal expression and location of colony-stimulating factor 1 (CSF-1) and its receptor in the female reproductive tract are consistent with CSF-1-regulated placental development.

During pregnancy the mouse uterine epithelial synthesis of the mononuclear phagocyte growth factor designated colony-stimulating factor 1 (CSF-1) is regulated by female sex steroids. To study the role of CSF-1 in the pregnant female reproductive tract, the temporal expression and cellular sites of synthesis of CSF-1 and CSF-1 receptor (CSF-1R) mRNA were determined. CSF-1 mRNA, predominantly the 2.3-kilobase (kb) form, was first detected by in situ hybridization in uterine epithelium prior to implantation on day 3 and subsequently increased, reaching a peak at days 14-15. Its expression was restricted to the uterine epithelium at all stages of gestation and was not localized to areas of implantation. CSF-1R mRNA was first detected in maternal decidua at day 6. It was expressed in the decidua basalis during placentation, after which its expression declined. At day 7.5, trophectodermal cells also expressed CSF-1R mRNA; during placentation, it was found also in the diploid trophoblasts. The high level of CSF-1R mRNA expression by trophoblast giant cells was independent of their location around the conceptus. There was a differential distribution of CSF-1R mRNA expression in the mature placenta, with expression in the giant trophoblastic layer greater than spongiotrophoblastic layer greater than labyrinthine layer until term. Yolk sac cells also expressed low levels of CSF-1R mRNA. The coincidence of uterine CSF-1 mRNA expression and CSF-1 synthesis with both placental growth and CSF-1R mRNA expression in decidual cells and trophoblasts strongly implicates CSF-1 in the regulation of placental growth and differentiation.     

Dopamine receptor activation inhibits estrogen-stimulated transforming growth factor-alpha gene expression and growth in anterior pituitary, but not in uterus.

Transforming growth factor-alpha (TGF alpha) has been localized to the anterior pituitary, specifically to the lactotroph and somatotroph cell populations, by our previous studies. Since pituitary lactotrophs are known to undergo growth in response to estrogens, we have used an estradiol-induced pituitary hyperplasia/adenoma model. Estradiol treatment resulted in induction of TGF alpha mRNA in anterior pituitary, evident by 48 h, preceding actual macroscopic growth, which attained a maximum greater than 500% by 12 weeks. This rapid effect of estradiol also enhanced PRL mRNA, but did not affect other species of mRNA encoding for proenkephalin, D2 receptor mRNA, or hexosaminidase-A. TGF alpha mRNA remained elevated for the duration of rapid pituitary growth. D2 receptor activation by its agonist bromocriptine resulted in marked attenuation of TGF alpha mRNA preceding regression of growth. Coadministration of bromocriptine with estradiol resulted in an involution of pituitary size, indicating the overriding influence of dopamine in spite of a continued estrogenic stimulus. Epidermal growth factor receptor mRNA was not affected by any of these manipulations, suggesting that the receptor was not coregulated in this tissue similarly to TGF alpha. Estradiol also induced uterine TGF alpha mRNA and marked growth of the organ, but TGF alpha in this location was not regulated by dopamine. These results indicate that TGF alpha in the anterior pituitary is rapidly induced by estrogen in a time course preceding the growth of the gland. Estrogen-induced TGF alpha is rapidly attenuated by D2 dopamine receptor activation and is accompanied by a regression of pituitary growth. Interaction between these opposing hormonal/transmitter responses will determine the growth potential of the anterior pituitary.     

Developmental changes of the oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of the rat--an analysis by in situ hybridization

This study employed an in situ hybridization technique to compare the cellular expression of oestrogen receptor (ER) subtypes, ER alpha and beta, in the female reproductive organ of the rat during prenatal and postnatal periods. Diffuse signals of ER alpha and beta mRNAs were co-expressed in the foetal ovary; they were weak and inconsistent before onset of gonadal differentiation, but increased in intensity with age. ER beta mRNA signals in the ovary sharply increased in intensity to adult levels by postnatal days 6-7, whereas those of ER alpha mRNA remained unchanged after birth. ER alpha was the sole subtype expressed during the prenatal period from the oviduct to the vagina, being localized mainly to the sub-epithelial stromal cells, and remained predominant thereafter. Signals for ER alpha mRNA in the epithelia were confined to the oviduct during prenatal and early postnatal periods; those in uterine and vaginal epithelia first appeared by postnatal days 4-5 and 6 respectively. Expressions of ER beta mRNA in the reproductive tract were absent during the prenatal period, and were weakly expressed during the postnatal period. Thus, oestrogen action in the developing ovary may be co-mediated by both ER alpha and beta, whereas ER alpha may be the primary mediator in the differentiation and growth of the female reproductive tract.     

Influence of estrogens on mouse uterine epidermal growth factor precursor protein and messenger ribonucleic acid

Estrogens stimulate the in vivo proliferation of epithelial cells of the mouse uterus. The cumulative evidence from several earlier studies suggests that the mitogenic effect of estrogens is mediated indirectly through a polypeptide growth factor. The primary focus of the present investigation was to determine whether an epidermal growth factor (EGF)-related polypeptide originates in the uterus of the immature or adult mouse under normal or altered estrogen status. Hybridization experiments revealed the presence of the 4.7-kilobase prepro-EGF mRNA in uteri of immature CD-1 mice. The level of this mRNA was augmented at least 2-fold in immature mice treated for 4 days with estrogen, but levels remained markedly low compared to those in submaxillary gland or kidney. Two preparations of pooled uterine luminal fluid from estrogen-treated immature mice contained EGF immunoreactivity (1.2 and 1.7 ng/ml) that was stable in response to acid (50 mM acetic acid) and heat. Negligible EGF (less than 20 pg/uterus) was detected in acid extracts of uteri from ovariectomized or cycling adult mice. After injection of 17 beta-estradiol (0.2 or 2.0 micrograms, ip), the levels of acid-extractable uterine EGF in ovariectomized adult mice up to 48 h after treatment were not different from those obtained with vehicle alone. Immunolocalization of EGF in the mouse uterus was demonstrated only after paraffin sections were first briefly treated with pronase. Staining was observed along the borders of luminal and glandular epithelial cells, especially at the apical region of the cells. Some staining was also observed in the myometrium; stromal cells were negative. Synthesis of the reactive material was apparently estrogen independent, since localization was retained in uteri of both ovariectomized and immature mice. Immunoblots of preparations of membranes from uterine homogenates or epithelial cells revealed a band at mol wt of about 130,000, which, along with other findings of the present study, suggests that EGF occurs predominantly as the membrane-bound precursor form in this organ, as has been previously shown for the kidney. Although the biological role of the precursor in the uterus is not known, we speculate that estrogens function in an autocrine circuit by stimulating processing of the membrane-bound EGF precursor. EGF elaborated by this mechanism might conceivably react with known complementary receptors on uterine epithelial cells to stimulate proliferation.     

Analysis of estrogen-regulated genes in mouse uterus using cDNA microarray and laser capture microdissection

The steroid hormone, estrogen, plays an important role in various physiological events which are mediated via its nuclear estrogen receptors, ERalpha and ERbeta. However, the molecular mechanisms that are regulated by estrogen in the uterus remain largely unknown. To identify genes that are regulated by estrogen, the ovariectomized mouse uterus was exposed to 17beta-estradiol (E2) for 6 h and 12 h, and the data were analyzed by cDNA microarray. The present study confirms previous findings and identifies several genes with expressions not previously known to be influenced by estrogen. These genes include small proline-rich protein 2A, receptor-activity-modifying protein 3, inhibitor of DNA binding-1, eukaryotic translation initiation factor 2, cystatin B, decorin, secreted frizzled-related protein 2, integral membrane protein 2B and chemokine ligand 12. The expression patterns of several selected genes identified by the microarray analysis were confirmed by RT-PCR. In addition, laser capture microdissection (LCM) was conducted to determine the expression of selected genes in specific uterine cell types. Analysis of early and late responsive genes using LCM and cDNA microarray not only suggests direct and indirect effects of E2 on uterine physiological events, but also demonstrates differential regulation of E2 in specific uterine cell types. These results provide a basic background on global gene alterations or genetic pathways in the uterus during the estrous cycle and the implantation period.     

Complex regulation of Calbindin-D(9k) in the mouse placenta and extra-embryonic membrane during mid- and late pregnancy

Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein mainly expressed in the duodenum, uterus and placenta, however, the role of CaBP-9k in the regulation of fetal growth remains to be elucidated. The present study was performed to investigate the expression pattern and regulation of CaBP-9k by antagonists of steroid hormones related with steroid hormone receptors during mid- and late pregnancy in mouse placenta and extra-embryonic membrane. The expression level of CaBP-9k increased in the placenta, while it decreased in the extra-embryonic membrane during pregnancy. The mRNA expression levels of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) appeared to increase in both placenta and extra-embryonic membrane during pregnancy, suggesting that the ER and PR mRNA and protein expressions of placental CaBP-9k are positively correlated, but expressions of extra-embryonic membrane CaBP-9k are reversely correlated with ERalpha and PR mRNA levels. In addition, the present study indicates that the expressions of CaBP-9k mRNA and protein are differentially up- or down-regulated by antagonists of estrogen (E2) and progesterone (P4) in mouse placenta and extra-embryonic membranes, which suggests that E2 and P4 may be dominant factors in the regulation of the CaBP-9k. In particular, RU486, an antagonist of P4, down-regulated the mRNA and protein levels of placental CaBP-9k, whereas it up-regulated the protein level of extra-embryonic membrane CaBP-9k. In conclusion, we demonstrated that the CaBP-9k is distinctly regulated in the mouse placenta and extra-embryonic membrane, probably via sex steroid hormones (E2 and P4) and their receptors through a complex pathway. Extended studies are needed to verify relevant factors to regulate CaBP-9k gene and to provide further insight into roles of CaBP-9k gene in these tissues for the control of reproductive functions.