Proliferation Of Uterine Stromal Cells Research Articles

Early pregnancy exposure to beta-cypermethrin compromises endometrial decidualisation in mice via downregulation of cyclin D3, CDK4/6, and p21.

Beta-cypermethrin (β-CYP) is a highly effective broad-spectrum insecticide that can potentially affect female reproduction. However, little is known about the effect of β-CYP on uterine decidualisation, which is a vital process by which the uterus provides a suitable microenvironment for pregnancy maintenance. Therefore, we focused on the effect and mechanism of β-CYP on endometrial decidualisation during early pregnancy in mice. The results indicated that the expression levels of HOXA10, BMP2, and IGFBP1 was significantly downregulated in the decidual tissue and primary endometrial stromal cells of pregnant and pseudopregnant mice following β-CYP treatment. Serum E2 concentration was significantly increased, whereas P4 concentration and oestrogen receptor (ERα) and progesterone receptor (PRA) expression were significantly downregulated following β-CYP exposure. The number of polyploid decidual cells was lower in the β-CYP-treated group. Furthermore, β-CYP significantly downregulated the protein expression levels of CDK4 and CDK6, and the mRNA expression levels of cyclin D3 and p21. The number of foetuses per female in the first litter was markedly reduced following exposure to β-CYP. In summary, early pregnancy exposure to β-CYP may result in defective endometrial decidualisation via compromised proliferation of uterine stromal cells and reduced expressions of cyclin D3, CDK4/6, and p21 in mice.

CGMP-dependent protein kinase II determines β-catenin accumulation that is essential for uterine decidualization in mice.

In the early phase of pregnancy, decidualization is an indispensable event after mammal embryo implantation, accompanied by proliferation and differentiation of uterine stromal cells. Type II cGMP-dependent protein kinase (Prkg2) belongs to the family of serine/threonine kinase, which plays multiple roles in cellular signaling pathways to control proliferation and differentiation. However, the regulatory function and molecular mechanism of Prkg2 in decidualization are still unknown. In this study, we show that Prkg2 has a gradually increased expression pattern during peri-implantation and artificial decidualization, and the expression of Prkg2 is induced by estrogen and progesterone in the ovariectomized mouse uteri and primary cultured uterine stromal cells, the process of which is blocked by treating with estrogen receptor (ER) antagonist (ICI-182,780) and progesterone receptor (PR) antagonist (RU-486). Inhibition of Prkg2 activity by HA-100 promotes uterine stromal cell proliferation but compromises decidualization with decreased expression of prolactin family 8, subfamily a, member 2. In addition, the functional regulation of decidualization by Prkg2 is accomplished by its induced phosphorylation of glycogen synthase kinase-3β (GSK-3β) at serine-9, which results in accumulation of β-catenin in the decidual cells. Taken together, our findings demonstrate that estrogen and progesterone upregulate the expression of Prkg2 in uterine stromal cells depending on ER and PR; Prkg2 promotes phosphorylation of GSK-3β at serine-9 and inactivates it, leading to the accumulation of β-catenin and promoting the process of decidualization. In addition to revealing the regulatory mechanism of Prkg2 that ensures the success of uterine decidualization, our findings will contribute to the understanding in the maintenance of early pregnancy.

Hmgb3 Induces the Differentiation of Uterine Stromal Cells Through Targeting Ptn.

Uterine decidualization is crucial for placenta formation and pregnancy maintenance. Although previous studies have reported that high mobility group box 3 (Hmgb3) is involved in the regulation of cellular proliferation and differentiation, little is known regarding its physiological role in uterine decidualization. Here, in situ hybridization result exhibited a dynamic expression pattern of Hmgb3 messenger RNA (mRNA) during early gestation, and it was mainly localized to the decidua on days 6 to 8 of gestation. Consistently, elevated Hmgb3 expression was noted in the decidualizing stromal cells after intraluminal oil infusion. In uterine luminal epithelium of ovariectomized mice, estrogen induced the accumulation of Hmgb3 mRNA, which was dependent on the existence of implanting blastocyst. Simultaneously, Hmgb3 could stimulate the proliferation of uterine stromal cells and promote the expression of Prl8a2, a reliable marker for stromal cell differentiation. Further analysis evidenced that Hmgb3 might modulate the expression of pleiotropin (Ptn) in uterine stromal cells. Moreover, silencing of Ptn could impede the upregulation of Prl8a2 elicited by Hmgb3 overexpression, while overexpression of Ptn reversed the repressive effects of Hmgb3 siRNA on Prl8a2 expression. Collectively, Hmgb3 may direct uterine decidualization through targeting Ptn.

Egr2 mediates the differentiation of mouse uterine stromal cells responsiveness to HB-EGF during decidualization.

Although Egr2 is involved in regulating the folliculogenesis and ovulation, there is almost no data describing its physiological function in embryo implantation and decidualization. Here, we showed that Egr2 mRNA was distinctly accumulated in subluminal stromal cells around implanting blastocyst on day 5 of pregnancy as well as in estrogen-activated implantation uterus. Estrogen induced the expression of Egr2 in uterine epithelia. Elevated expression of Egr2 mRNA was also observed in the decidual cells. Silencing of Egr2 by specific siRNA weakened the proliferation of uterine stromal cells and reduced the expression of Ccnd1, Ccnd3, Cdk4, and Cdk6. Furthermore, Egr2 advanced the expression of Prl8a2, Prl3c1, and Pgr, the well-established differentiation markers for decidualization. Administration of exogenous recombinant heparin-binding EGF-like growth factor (rHB-EGF) to uterine stromal cells resulted in an increase in the level of Egr2 mRNA. Moreover, siRNA-mediated attenuation of Egr2 impeded the stimulation of HB-EGF on stromal cell differentiation. Knockdown of Egr2 led to a reduction in the expression of Cox-2, mPGES-1, Vegf, Trp53, and Mmp2. Further analysis found that Egr2 may serve as an intermediate to mediate the regulation of HB-EGF on Cox-2, mPGES-1, Vegf, Trp53, Mmp2, and Ccnd3. Collectively, Egr2 may play an important role during embryo implantation and decidualization.

High Mobility Group Box 1 Regulates Uterine Decidualization through Bone Morphogenetic Protein 2 and Plays a Role in Kruppel-Like Factor 5-Induced Stromal Differentiation

Background/Aims: High mobility group box 1 (Hmgb1) is associated with a variety of physiological processes including embryonic development, cell proliferation and differentiation, but little information is available regarding its biological role in decidualization. Methods: In situ hybridization, real-time PCR, RNA interference, gene overexpression and MTS assay were used to analyze the spatiotemporal expression of Hmgb1 in mouse uterus during the pre-implantation period, and explore its function and regulatory mechanisms during uterine decidualization. Results: Hmgb1 mRNA was obviously observed in uterine epithelium on day 2 and 3 of pregnancy, but its expression was scarcely detected on day 4 of pregnancy. With the onset of embryo implantation, abundant Hmgb1 expression was noted in the subluminal stromal cells around the implanting blastocyst at implantation sites. Meanwhile, the accumulation of Hmgb1 mRNA was visualized in the decidual cells. Hmgb1 advanced the proliferation of uterine stromal cells and induced the expression of prolactin family 8, subfamily a, member 2 (Prl8a2), a reliable differentiation marker for decidualization. In uterine stromal cells, cAMP analogue 8-Br-cAMP up-regulated the expression of Hmgb1, but the up-regulation was abrogated by protein kinase A (PKA) inhibitor H89. Silencing of Hmgb1 by specific siRNA impeded the induction of 8-Br-cAMP on Prl8a2. Further analysis evidenced that Hmgb1 was a critical mediator of Kruppel-like factor 5 (Klf5) function in stromal differentiation. Knockdown of bone morphogenetic protein 2 (Bmp2) prevented the up-regulation of Prl8a2 elicited by Hmgb1 overexpression, whereas addition of exogenous recombinant Bmp2 protein (rBmp2) reversed the repression of Hmgb1 siRNA on Prl8a2 expression. Conclusion: Hmgb1 may play an important role during mouse uterine decidualization.

Ptn functions downstream of C/EBPβ to mediate the effects of cAMP on uterine stromal cell differentiation through targeting Hand2 in response to progesterone.

Ptn is a pleiotropic growth factor involving in the regulation of cellular proliferation and differentiation, but its biological function in uterine decidualization remains unknown. Here, we showed that Ptn was highly expressed in the decidual cells, and could induce the proliferation of uterine stromal cells and expression of Prl8a2 and Prl3c1 which were two well-established differentiation markers for decidualization, suggesting an important role of Ptn in decidualization. In the uterine stromal cells, progesterone stimulated the expression of Ptn accompanied with an accumulation of intracellular cAMP level. Silencing of Ptn impeded the induction of progesterone and cAMP on the differentiation of uterine stromal cells. Administration of PKA inhibitor H89 resulted in a blockage of progesterone on Ptn expression. Further analysis evidenced that regulation of progesterone and cAMP on Ptn was mediated by C/EBPβ. During in vitro decidualization, knockdown of Ptn could weaken the up-regulation of Prl8a2 and Prl3c1 elicited by C/EBPβ overexpression, while constitutive activation of Ptn reversed the repressive effects of C/EBPβ siRNA on the expression of Prl8a2 and Prl3c1. Meanwhile, Ptn might mediate the regulation of C/EBPβ on Hand2 which was a downstream target of Ptn in the differentiation of uterine stromal cells. Attenuation of Ptn or C/EBPβ by specific siRNA blocked the stimulation of Hand2 by progesterone and cAMP. Collectively, Ptn may play a vital role in the progesterone-induced decidualization pathway.

Gja1 acts downstream of Acvr1 to regulate uterine decidualization via Hand2 in mice.

Although Gja1 has been proved to play an important role in uterine decidualization, its regulatory mechanism remains largely unknown. Here, we showed that Gja1 was highly expressed in the decidual cells and promoted the proliferation of uterine stromal cells and expression of Prl8a2 and Prl3c1, which were two well-known differentiation markers for decidualization. Further analysis revealed that Gja1 might act downstream of Acvr1 and cAMP to regulate the differentiation of uterine stromal cells. Administration of cAMP analog 8-Br-cAMP to Acvr1 siRNA-transfected stromal cells resulted in an obvious increase of Gja1 expression, whereas PKA inhibitor H89 impeded the induction of Gja1 elicited by Acvr1 overexpression, indicating that cAMP-PKA signal mediates the regulation of Acvr1 on Gja1 expression. In uterine stromal cells, knockdown of Gja1 blocked the cAMP induction of Hand2 Moreover, siRNA-mediated downregulation of Hand2 impaired the stimulatory effects of Gja1 overexpression on the expression of Prl8a2 and Prl3c1, whereas constitutive expression of Hand2 reversed the inhibitory effects of Gja1 siRNA on stromal differentiation. Meanwhile, Gja1 might play a vital role in the crosstalk between Acvr1 and Hand2 Collectively, Gja1 may act downstream of cAMP-PKA signal to mediate the effects of Acvr1 on the differentiation of uterine stromal cells through targeting Hand2.

WINGLESS (WNT) signaling is a progesterone target for rat uterine stromal cell proliferation.

Preparation of mammalian uterus for embryo implantation requires a precise sequence of cell proliferation. In rodent uterus, estradiol stimulates proliferation of epithelial cells. Progesterone operates as a molecular switch and redirects proliferation to the stroma by down-regulating glycogen synthase kinase-3β (GSK-3β) and stimulating β-catenin accumulation in the periluminal stromal cells. In this study, the WNT signal involved in the progesterone-dependent proliferative switch was investigated. Transcripts of four candidate Wnt genes were measured in the uteri from ovariectomized (OVX) rats, progesterone-pretreated (3 days of progesterone, 2mg/daily) rats, and progesterone-pretreated rats given a single dose (0.2µg) of estradiol. The spatial distribution of the WNT proteins was determined in the uteri after the same treatments. Wnt5a increased in response to progesterone and the protein emerged in the periluminal stromal cells of progesterone-pretreated rat uteri. To investigate whether WNT5A was required for proliferation, uterine stromal cell lines were stimulated with progesterone (1µM) and fibroblast growth factor (FGF, 50ng/mL). Proliferating stromal cells expressed a two-fold increase in WNT5A protein at 12h post stimulation. Stimulated stromal cells were cultured with actinomycin D (25µg/mL) to inhibit new RNA synthesis. Relative Wnt5a expression increased at 4 and 6 h of culture, suggesting that progesterone plus FGF preferentially increased Wnt5a mRNA stability. Knockdown of Wnt5a in uterine stromal cell lines inhibited stromal cell proliferation and decreased Wnt5a mRNA. The results indicate that progesterone initiates and synchronizes uterine stromal cell proliferation by increasing WNT5A expression and signaling.

Differential expression and regulation of Ido2 in the mouse uterus during peri-implantation period.

Ido2 is involved in tryptophan catabolism and immunity, but its physiological functions remain poorly understood. This study was undertaken to examine the expression and regulation of Ido2 gene in mouse uterus during the peri-implantation period. The results showed that Ido2 mRNA was highly expressed on day 4 of pregnancy and in the delayed implantation uterus. On days 5-8 of pregnancy, a low level of Ido2 expression was observed in the uteri. Simultaneously, Ido2 mRNA was also lowly expressed in the decidualized uterus. In the uterine stromal cells, 8-Br-cAMP could inhibit the expression of Ido2 mRNA. Moreover, Ido2 mRNA expression was gradually decreased after the stromal cells were treated with estrogen and progesterone and reached a nadir at 96 h. Further study found that overexpression of Ido2 could downregulate the expression of decidualization marker genes PRL, IGFBP1, and Dtprp under in vitro decidualization, while inhibition of Ido2 with devo-1-methyl-tryptophan (D-1-MT) could upregulate the expression of these marker genes. Under in vitro decidualization, overexpression of Ido2 could suppress the proliferation of uterine stromal cells and elevate the expression of Bax and MMP2 genes. On the contrary, Ido2 inhibitor D-1-MT could enhance the proliferation of stromal cells and expression of Bcl2 gene but decline the Bax/Bcl2 ratio. In the uterine stromal cells, estrogen and progesterone could induce the expression of Ido2 mRNA. These data indicate that Ido2 may be important for mouse embryo implantation and decidualization.

Dysregulation of Uterine Signaling Pathways in Progesterone Receptor-Cre Knockout of Dicer

Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNA (miRNA) have been implicated in several reproductive processes, the specific roles of Dicer and miRNA in uterine development are not known. To address the roles of miRNA in the regulation of key uterine pathways, we generated a conditional knockout of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor-Cre. These Dicer conditional knockout females are sterile with small uteri, which demonstrate significant defects, including absence of glandular epithelium and enhanced stromal apoptosis, beginning at approximately postnatal d 15, with coincident expression of Cre and deletion of Dicer. Specific miRNA (miR-181c, -200b, -101, let-7d) were down-regulated and corresponding predicted proapoptotic target genes (Bcl2l11, Aldh1a3) were up-regulated, reflecting the apoptotic phenomenon. Although these mice had normal serum hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/β-catenin canonical pathway, were dysregulated at the mRNA level. Importantly, uterine stromal cell proliferation in response to progesterone was absent, whereas uterine epithelial cell proliferation in response to estradiol was maintained in adult uteri. These data implicate Dicer and appropriate miRNA expression as essential players in the regulation of multiple uterine signaling pathways required for uterine development and appropriate function.

Cell cycle regulatory control for uterine stromal cell decidualization in implantation

Uterine stromal cell decidualization is integral to successful embryo implantation, which is a gateway to pregnancy establishment. This process is characterized by stromal cell proliferation and differentiation into decidual cells with polyploidy. The molecular mechanisms that are involved in these events remain poorly understood. The current concept is that locally induced factors with the onset of implantation influence uterine stromal cell proliferation and/or differentiation through modulation of core cell cycle regulators. This review will aim to address the currently available knowledge on interaction between growth factor/homeobox and cell cycle regulatory signaling in the progression of various aspects of decidualization.

Decidual Cells Produce a Heparin-binding Prolactin Family Cytokine with Putative Intrauterine Regulatory Actions

Pregnancy in mice and rats is associated with the production of a large family of hormones/cytokines related to prolactin (PRL). The hormones/cytokines are hypothesized to coordinate maternal and fetal adaptations to pregnancy. In this study, PRL-like protein-J (PLP-J, also known as PRL family 3, subfamily c, member 1 (Prl3c1)) is shown to be a product of the uterine decidua and a regulator of postimplantation intrauterine events. PLP-J-specific antibodies and a series of recombinant PLP-J proteins were generated and used to investigate PLP-J expression and as ligands for investigating biological targets. Decidual PLP-J migrates as a 29-kDa protein and localizes to a band of decidual cells surrounding the trophoblast cell layer on gestation day 8.5. PLP-J ligands specifically bound in situ to the surrounding uterine stromal cells and vasculature within the decidua of gestation day 8.5 implantation sites. We then investigated the in vitro actions of PLP-J on uterine stromal cells and endothelial cells. PLP-J specifically interacted with both cell populations. PLP-J promoted uterine stromal cell proliferation and inhibited endothelial cell proliferation. We determined that PLP-J does not interact with PRL receptors. Instead, PLP-J interacts with heparin-containing molecules, including syndecan-1, which is expressed in gestation day 8.5 pregnant uteri, as well as in uterine stromal cells and endothelial cells. The restricted expression of PLP-J and its specific interactions with uterine stromal cells and endothelial cells suggests that it acts locally and regulates decidual cell development and the endometrial vasculature.

Estrogen Induces a Systemic Growth Factor Through an Estrogen Receptor-Alpha-Dependent Mechanism1

Estrogen induces proliferation of uterine epithelium through a paracrine action of estrogen receptor (ERalpha) in the underlying stroma. In ovariectomized mice primed with progesterone, estrogen stimulates proliferation in both the epithelium and the stroma. We set out to test whether a paracrine mode of action is involved in estrogen-induced proliferation of the uterine stroma. Epithelial and mesenchymal tissues derived from uteri of neonatal ERalpha null mice (ERalphaKO) or wild-type mice were separated and recombined in all four possible configurations (ERalpha+ or ERalpha- epithelium with ERalpha+ or ERalpha- mesenchyme) and grafted into female athymic mice. After 5 wk, hosts were ovariectomized and challenged with hormone treatment, and cellular proliferation was monitored by thymidine autoradiography. Results showed that, although the full response of the epithelium was dependent on an ERalpha-positive mesenchyme, stromal cell proliferation was independent of tissue ERalpha. This latter observation suggests that the response of the stroma was due to a systemic factor induced in the ERalpha-positive hosts. To test this possibility, pieces of whole uterus from neonatal wild-type or ERalphaKO mice were grafted into syngeneic wild-type or ERalphaKO hosts. In these whole-uterus grafts, estradiol stimulated ERalphaKO uterine stroma when they were grown in wild-type hosts but not when grown in ERalphaKO hosts. The epithelium of whole-uterus ERalphaKO grafts did not respond to estrogen, regardless of the host phenotype. These observations suggest that treatment of progesterone-primed mice with estradiol stimulates production of a systemic factor that is capable of inducing uterine stromal cell proliferation and that this systemic factor is produced by an ERalpha-dependent mechanism.

Gene expression profiling reveals progesterone-mediated cell cycle and immunoregulatory roles of Hoxa-10 in the preimplantation uterus.

Human infertility and recurrent pregnancy loss caused by implantation defects are poorly understood. Hoxa-10-deficient female mice have severe infertility and recurrent pregnancy loss due to defective uterine implantation. Gene expression profiling experiments reveal that Hoxa-10 is an important regulator of two critical events in implantation: stromal cell proliferation and local immunosuppression. At the time of implantation, Hoxa-10 mediates the progesterone-stimulated proliferation of uterine stromal cells. Hoxa-10 mutants express a stromal cell proliferation defect that is accompanied by quantitative or spatial alterations in the expression of two cyclin-dependent kinase inhibitor genes, p57 and p15. Hoxa-10 deficiency also leads to a severe local immunological disturbance, characterized by a polyclonal proliferation of T cells, that occurs in place of the normal progesterone-mediated immunosuppression in the periimplantation uterus.

Evaluation of a Tier I Screening Battery for Detecting Endocrine-Active Compounds (EACs) Using the Positive Controls Testosterone, Coumestrol, Progesterone, and RU486

After previously examining 12 compounds with known endocrine activities, we have now evaluated 4 additional compounds in a Tier I screening battery for detecting endocrine-active compounds (EACs): a weak estrogen receptor (ER) agonist (coumestrol; COUM), an androgen receptor (AR) agonist (testosterone; TEST), a progesterone receptor (PR) agonist (progesterone; PROG), and a PR antagonist (mifepristone; RU486). The Tier I battery incorporates 2 short-term in vivo tests (5-day ovariectomized female battery; 15-day intact male battery) and an in vitro yeast transactivation system (YTS). The Tier I battery is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptors; steroid biosynthesis inhibitors (aromatase, 5alpha-reductase, and testosterone biosynthesis); or compounds that alter thyroid function. In addition to the Tier I battery, a 15-day dietary restriction experiment was performed using male rats to assess confounding due to treatment-related decreases in body weight. In the Tier I female battery, TEST administration increased uterine weight, uterine stromal cell proliferation, and altered hormonal concentrations (increased serum testosterone [T] and prolactin [PRL]; and decreased serum FSH and LH). In the male battery, TEST increased accessory sex gland weights, altered hormonal concentrations (increased serum T, dihydrotestosterone [DHT], estradiol [E2], and PRL; decreased serum FSH and LH), and produced microscopic changes of the testis (Leydig cell atrophy and spermatid retention). In the YTS, TEST activated gene transcription in the yeast containing the AR or PR. In the female battery, COUM administration increased uterine weight, uterine stromal cell proliferation, and uterine epithelial cell height, and increased serum PRL concentrations. In the male battery, COUM altered hormonal concentrations (decreased serum T, DHT, E2; increased serum PRL) and, in the YTS, COUM activated gene transcription in the yeast containing the ER. In the female battery, PROG administration increased uterine weight, uterine stromal cell proliferation, and uterine epithelial cell height and altered hormonal concentrations (increased serum progesterone and decreased serum FSH and LH). In the male battery, PROG decreased epididymis and accessory sex gland weights, altered hormonal concentrations (decreased serum T, PRL, FSH, and LH; increased serum progesterone and E2), and produced microscopic changes of the testis (Leydig cell atrophy). In the YTS, PROG activated gene transcription in the yeast containing the AR or PR. In the female battery, RU486 administration increased uterine weight and decreased uterine stromal cell proliferation. In the male battery, RU486 decreased epididymis and accessory sex gland weights and increased serum FSH and LH concentrations. In the YTS, RU486 activated gene transcription in the yeast containing the ER, AR, or PR. Dietary restriction data demonstrate that confounding due to decrements in body weight are not observed when body weight decrements are 10% or less in the Tier I male battery. In addition, minimal confounding is observed at body decrements of 15% (relative liver weight, T3, and T4). Hence, compounds can be evaluated in this Tier I at levels that produce a 10% decrease in body weight without confounding of the selected endpoints. Using the responses obtained for all the endpoints in the Tier I battery, a distinct "fingerprint" was produced for each type of endocrine activity against which compounds with unknown activity can be compared. These data demonstrate that the described Tier I battery is useful for identifying EACs and they extend the compounds evaluated to 16.

An ongoing validation of a Tier I screening battery for detecting endocrine-active compounds (EACs).

After previously examining an estrogen receptor agonist (17beta-estradiol), several additional compounds have been evaluated in a Tier I screening battery for detecting endocrine-active compounds (EACs): an estrogen receptor antagonist (ICI-182,780, ICI), an androgen receptor antagonist (flutamide, FLUT), a testosterone biosynthesis inhibitor (ketoconazole, KETO), a 5alpha-reductase inhibitor (finasteride, FIN), and an aromatase inhibitor (anastrozole, ANA). The Tier I battery incorporates two short-term in vivo tests (a 5-day ovariectomized female battery and a 15-day intact male battery) and an in vitro yeast transactivation system (YTS). The Tier I battery is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptors, steroid biosynthesis inhibitors (aromatase, 5alpha-reductase, and testosterone biosynthesis), or compounds that alter thyroid function. ICI administration decreased uterine estrogen and progesterone receptor number in the female battery, increased serum follicle-stimulating hormone (FSH) levels and caused spermatid retention in the male battery, and activated gene transcription in the YTS containing the estrogen receptor. FLUT administration increased uterine stromal cell proliferation in the female battery and decreased weights for all androgen-dependent tissues, induced Leydig cell hyperplasia, and caused hormonal alterations (increased testosterone (T), estradiol (E2), dihydrotestosterone (DHT), luteinizing hormone (LH), and FSH) in the male battery, and competed for binding to the androgen receptor in the YTS competition assay. In the male battery KETO decreased weights for all androgen-dependent tissues, caused hormonal alterations (decreased T and DHT and increased LH and FSH), and induced spermatid retention. FIN decreased seminal vesicle and accessory sex gland (ASG) unit weight and caused hormonal alterations (decreased DHT and increased LH, and PRL) in the male battery. KETO was judged not to affect any of the endpoints in the female battery. ANA decreased ASG unit weight and serum E2 levels in the male battery. Using the responses obtained for all the endpoints in the Tier I battery, a distinct "fingerprint" was produced for each type of endocrine activity against which compounds with unknown activity can be compared. These data demonstrate that the described Tier I battery is useful for identifying EACs.

Sensitivity of a Tier I Screening Battery Compared to an in Utero Exposure for Detecting the Estrogen Receptor Agonist 17β-Estradiol

A Tier I screening battery for detecting endocrine active compounds (EACs) has been evaluated for its ability to identify 17β-estradiol, a pure estrogen receptor agonist. In addition, the responses obtained with the Tier I battery were compared to the responses obtained from F1 generation rats from a 90-day/one-generation reproduction study with 17β-estradiol in order to characterize the sensitivity of the Tier I battery against the sensitivity of an in utero exposure for detecting EACs. The Tier I battery incorporates two short-term in vivo tests (5-day ovariectomized female battery; 15-day Intact male battery) and an in vitro yeast transactivation system (YTS) for identifying compounds that alter endocrine homeostasis. The Tier I female battery consists of traditional uterotrophic endpoints coupled with biochemical and hormonal endpoints. It is designed to identify compounds that are estrogenic/antiestrogenic or modulate dopamine levels. The Tier I male battery consists of organ weights coupled with microscopic evaluations and a comprehensive hormonal assessment It is designed to identify compounds that have the potential to act as agonists or antagonists to the estrogen, androgen, progesterone, or dopamine receptor; steroid biosynthesis inhibitors (aromatase, 5 α-reductase, and testosterone biosynthesis); or compounds that alter thyroid function. The YTS is designed to identify compounds that bind to steroid hormone receptors (estrogen, androgen, and progesterone) and activate gene transcription. The profile generated for 17β-estradiol was characteristic of the responses expected with a pure estrogen receptor agonist. In the female battery, responses to 17β-estradiol included increases in uterine fluid imbibition, uterine weight, estrus conversion, uterine stromal cell proliferation, uterine epithelial cell height, uterine progesterone receptor content, serum prolactin and estradiol levels, and decreases in uterine estrogen receptor content and follicle stimulating hormone and luteinizing hormone levels. In the male battery, responses to 17β-estradiol included decreases in absolute testis and epididymi-des weights, decreases in relative weights for androgen-dependent tissues (prostate, seminal vesicles, and accessory sex gland unit), hormonal alterations (decreased serum testosterone, dihydrotest-osterone, and LH and increased serum prolactin levels), and microscopic alterations of the testis and epididymides. In the YTS for the estrogen receptor, 17β-estradiol had an EC50, value of 7.2 × 10−9 M, while DHT and progesterone had little cross-activation. The androgen and progesterone receptor systems were less selective in that 17β-estradiol activated these systems within 3 orders of magnitude of the primary ligand. In the 90-day/one-generation reproduction study, responses to dietary administration of 17β-estradiol included alterations in organ weights, developmental landmarks, and hormonal levels. Comparison of the responses obtained with our Tier I battery and an in utero exposure demonstrates that the Tier I screening battery is as sensitive as an in utero exposure for detecting 17β-estradiol-induced alterations in hormonal homeostasis.

Weak estrogenic activity from continuous-release pellets

In the process of evaluating a proprietary compound for weak estrogenic activity, two different types of dosing regimens were used, repeated daily dosing (three times/d) and continuous-release pellets. In studies using the proprietary compound, rats that were dosed via intraperitoneal injection showed no estrogenic responses, while those receiving the test compound via continuous-release pellets displayed several estrogenic responses. Because of the conflicting results, the control pellets were evaluated for estrogenic activity in the same battery of tests using the same number of pellets. In studies using only the control pellets, several estrogenic responses were observed including increased uterine weight, uterine stromal cell proliferation, estrous conversion, uterine progesterone receptor content, and decreased uterine estrogen receptor content. Animals receiving no pellet implant showed no estrogenic responses. In addition, a methylene chloride/DMSO extract of the control pellets promoted expression of a reporter gene controlled by the estrogen receptor and demonstrated competition with 17β-estradiol for binding to the human estrogen receptor. It is concluded that component(s) of the control pellets possess weak estrogenic activity.

Anin VivoBattery for Identifying Endocrine Modulators That Are Estrogenic or Dopamine Regulators

We have combined several endpoints into a single 5-dayin vivoscreening procedure to identify estrogenic compounds and dopaminergic modulators, both of which play important roles in enhancing mammary tumorigenesis in rodents. The endpoints evaluated as markers of estrogenicity included increases in uterine fluid and vaginal cornification incidence, serum prolactin levels, uterine weight, uterine epithelial cell height, uterine stromal cell proliferation, and uterine progesterone receptor (PR) number and decreases in uterine estrogen receptor (ER) number. The endpoints evaluated for changes in dopamine regulation included increases in prolactin and decreases in growth hormone levels. The estrogen agonists estradiol (E2) and estriol (E3), the mixed estrogen agonist/antagonist tamoxifen (TAM), the full antiestrogen ICI-182,780 (ICI), and the dopamine modulators haloperidol (HAL) and reserpine (RES) were tested using a three-time/day (8-hr intervals) intraperitoneal dosing regimen in sexually mature ovariectomized female Crl:CD BR rats. All compounds were evaluated over a range of concentrations. Thisin vivobattery was used to evaluate the effects of different classes of endocrine modulators on the selected endpoints. For example, the estrogen receptor agonists E2 and E3 display a unique profile based on changes in the uterotrophic endpoints (estrus conversion, uterine fluid imbibition, increases in uterine weight, and uterine endometrial cell proliferation) where full and partial agonists can be distinguished by the magnitude of these responses. Both the estrogen receptor antagonist ICI and the dopamine modulators HAL and RES lack these uterotrophic responses. Dopamine modulators can be distinguished from estrogen receptor agonists by the profile of increased prolactin levels with no uterotrophic changes. Estrogen receptor antagonists can be distinguished from agonists by comparing their effects on ER, PR, and uterotrophic responses. For instance, the full estrogen receptor antagonist ICI decreased ER (to almost 0) and PR levels, but has no uterotrophic effects, while TAM decreases ER (to almost 0) and increases PR with uterotrophic effects. The most useful endpoints for distinguishing estrogen agonists and dopamine modulators were uterine fluid imbibition, uterine weight, uterine stromal cell proliferation, and serum prolactin levels. In order to distinguish an estrogen agonist from an antagonist, other endpoints, such as receptor levels, are necessary. The advantage of anin vivoscreen is that it utilizes a metabolically and physiologically defined system which is especially important with highly integrative systems such as the endocrine system. This battery can be used as a screening tool to identify potential endocrine modulators as well as to identify mode of action following adverse findings in toxicology studies. Last, additional endpoints may be added to identify other classes of endocrine modulators.

An in Vivo Battery for Identifying Endocrine Modulators That Are Estrogenic or Dopamine Regulators

We have combined several endpoints into a single 5-day in vivo screening procedure to identify estrogenic compounds and dopaminergic modulators, both of which play important roles in enhancing mammary tumorigenesis in rodents. The endpoints evaluated as markers of estrogenicity included increases in uterine fluid and vaginal cornification incidence, serum prolactin levels, uterine weight, uterine epithelial cell height, uterine stromal cell proliferation, and uterine progesterone receptor (PR) number and decreases in uterine estrogen receptor (ER) number. The endpoints evaluated for changes in dopamine regulation included increases in prolactin and decreases in growth hormone levels. The estrogen agonist estradiol (E2) and estriol (E3), the mixed estrogen agonist/ antagonist tamoxifen (TAM), the full antiestrogen ICI-182, 780 (ICI),and the dopamine modulators haloperidol (HAL) and reserpine (RES) were tested using a three-time/day (8-hr intervals) intraperitoneal dosing regimen in sexually mature ovariectomized female Crl:CD BR rats. All compounds were evaluated over a range of concentrations. This in vivo battery was used to evaluate the effects of different classes of endocrine modulators on the selected endpoints. For example, the estrogen receptor agonists E2 and E3 display a unique profile based on changes in the uterotrophic endpoints (estrus conversion, uterine fluid imbibition, increases in uterine weight, and uterine endometrial cell proliferation) where full and partial agonists can be distinguished by the magnitude of these responses. Both the estrogen receptor antagonist ICI and the dopamine modulators HAL and RES lack these uterotrophic responses. Dopamine modulators can be distinguished from estrogen receptor agonists by the profile of increased prolactin levels with no uterotrophic changes. Estrogen receptor antagonists can be distinguished from agonists by comparing their effects on ER, PR, and uterotrophic responses. For instance, the full estrogen receptor antagonist ICI decreased ER (to almost 0) and PR levels, but has no uterotrophic effects, while TAM decreases ER (to almost 0) and increases PR with uterotrophic effects. The most useful endpoints for distinguishing estrogen agonists and dopamine modulators were uterine fluid imbibition, uterine weight, uterine stromal cell proliferation, and serum prolactin levels. In order to distinguish an estrogen agonist from an antagonist, other endpoints, such as receptor levels, are necessary. The advantage of an in vivo screen is that it utilizes a metabolically and physiologically defined system which is especially important with highly integrative system such as the endocrine system. This battery can be used as a screening tool to identify potential endocrine modulators as well as to identify mode of action following adverse findings in toxicology studies. Last, additional endpoints may be added to identify other classes of endocrine modulators.