John P. Lydon

Defective mammary gland morphogenesis in mice lacking the progesterone receptor B isoform

Progesterone (P) regulates female reproduction via two nuclear receptors, PR-A and PR-B. Although both receptors display overlapping and distinct transcription regulatory properties, their individual physiological roles are unclear. To address the physiological role of PR-A, we generated a mouse model in which expression of PR-B was specifically ablated (PRBKO–/–). We show that selective activation of PR-A in PRBKO–/– mice is sufficient to elicit normal ovarian and uterine responses to P but results in reduced mammary gland morphogenesis. In the absence of PR-B, pregnancy-associated ductal sidebranching and lobuloalveolar development are markedly reduced due to decreased ductal and alveolar epithelial cell proliferation and decreased survival of alveolar epithelium. In an effort to elucidate the molecular genetic signaling pathways that are differentially regulated by PRs in the mammary gland, we have identified receptor activator of nuclear factor κB ligand (RANKL) as a paracrine mediator of P-dependent alveologenesis. Further, we demonstrate that the defects in PRBKO–/– mice are associated with an inability of PR-A to activate the RANKL signaling pathway in response to P. Our data indicate that functional interaction between PR-A and PR-B is not required for reproductive activity and that selective modulation of PR-A activity by progestin agonists may have a protective effect against both uterine and mammary gland hyperplasias.

Reproductive phenotypes of the progesterone receptor null mutant mouse

Although progesterone has been traditionally associated with the establishment and maintenance of mammalian pregnancy, a number of studies have implicated physiological roles of this steroid hormone in other reproductive events. At present most of the downstream molecular and cellular mechanisms by which progesterone exerts its effects are unclear; however, the progesterone signal is known to be mediated initially by the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors. In most tissues studied, the PR is induced by ovarian estrogen via the estrogen receptor (ER), thereby implying that many of the observed reproductive physiological responses attributed to PR could conceivably be due to the combined effects of progesterone and estrogen. Therefore, to define clearly the distinct roles of progesterone and estrogen in vivo and to understand better progesterone function in a physiological context, we recently have generated a novel mouse strain in which both forms of the PR were ablated using gene targeting/embryonic stem cell techniques. Surprisingly, both male and female embryos, homozygous for the PR null mutation, developed to adulthood at the normal Mendelian frequency with no deviation in the sex ratio. Although developmental defects have yet to be detected in the adult male PR homozygote, extensive reproductive abnormalities were observed in the female. The reproductive phenotypes consisted of an inability to ovulate, uterine hyperplasia and inflammation, severely limited mammary gland development and an impairment in the induction of a sexual behavioral response. Collectively, these results provide direct in vivo evidence for progesterones role as a pleiotropic coordinator of diverse reproductive events that together ensure female fertility. Finally, we believe that this animal model will be an invaluable tool in exploring the effects of progesterone in physiological systems other than reproduction and may, in the future, help to redefine progesterone not just as a sex steroid hormone but also as a key regulator of diverse physiological processes.

Progesterone-dependent regulation of female reproductive activity by two distinct progesterone receptor isoforms

The steroid hormone, progesterone, is a central coordinator of all aspects of female reproductive activity. The physiological effects of progesterone are mediated by interaction of the hormone with specific intracellular progesterone receptors (PRs) that are expressed from a single gene as two protein isoforms and that are members of the nuclear receptor superfamily of transcription factors. Analysis of the structural and functional relationships of each isoform using in vitro systems has demonstrated that the PR-A and PR-B proteins have different transcription activation properties when liganded to progesterone. More recently, selective ablation of the PR-A and PR-B proteins in mice had facilitated examination of the contribution of the individual PR isoforms to the pleiotropic reproductive activities of progesterone. Analysis of the phenotypic consequences of these mutations on female reproductive function has provided proof of concept that the distinct transcriptional responses to PR-A and PR-B observed in cell-based transactivation assays are reflected in a distinct tissue-selective contribution of the individual isoforms to the reproductive activities of progesterone. In PR-A knock-out mice, in which the expression of the PR-A isoform is selectively ablated (PRAKO), the PR-B isoform functions in a tissue-specific manner to mediate a subset of the reproductive functions of PRs. Ablation of PR-A does not affect response of the mammary gland or thymus to progesterone but results in severe abnormalities in ovarian and uterine function leading to female infertility. More recent studies using PR-B knock-out (PRBKO) mice have shown that ablation of PR-B does not affect either ovarian, uterine or thymic responses to progesterone but results in reduced mammary ductal morphogenesis and alveologenesis during pregnancy. Thus, PR-A is both necessary and sufficient to elicit the progesterone-dependent reproductive responses necessary for female fertility, while the PR-B isoform is required to elicit normal proliferative and differentiative responses of the mammary gland to progesterone. This review will summarize our current understanding of the selective contribution of the two PR isoforms to progesterone action.

Reproductive functions of the progesterone receptor isoforms: lessons from knock-out mice.

Progesterone plays a central coordinate role in diverse reproductive events associated with establishment and maintenance of pregnancy. In humans and other vertebrates, the biological activities of progesterone are mediated by two proteins, A (PR-A) and B (PR-B) that arise from the same gene and function as progesterone activated transcription factors that exhibit different transcription regulatory activities in vitro. Mice lacking both PR isoforms (PRKO mice) exhibit pleiotropic reproductive abnormalities. To address the physiological role of the individual isoforms, we have selectively ablated PR-A expression in mice (PRAKO). We have demonstrated that PR-B mediates a subset of the reproductive functions of P. Ablation of PR-A does not affect responses of the mammary gland or thymus to P but results in severe abnormalities in ovarian and uterine function. Analysis of urine function of PRAKP mice reveals an unexpected P-dependent proliferative activity of PR-B in the epithelium and provides evidence that the tissue-specific reproductive effects of this isoform are due to specificity of target gene transactivation rather than differences in tissue-specific expression relative to PR-A. Taken together, our data indicate that PR-A and PR-B act in vivo as two functionally distinct transcription factors.

Indian hedgehog is a major mediator of progesterone signaling in the mouse uterus

The hedgehog family of morphogens are regulators of cell proliferation, differentiation and cell-cell communication. These morphogens have been shown to have important roles in organogenesis, spermatogenesis, stem cell maintenance and oncogenesis. Indian hedgehog (encoded by Ihh) has been shown to be expressed in the uterine epithelium under the control of the steroid hormone, progesterone. Although in vivo and in vitro studies have shown that progesterone achieves its effects on uterine function through epithelial-stromal cross-talk, molecular mediator(s) for this cellular communication pathway have not been elucidated. Using new experimental approaches that ablate Ihh specifically in Pgr-positive uterine cells of the mouse, we demonstrate that Ihh is an essential mediator of Pgr action in the uterus, and expression of this factor is critical in mediating the communication between the uterine epithelium and stroma required for embryo implantation.

Progesterone receptors in reproduction: functional impact of the A and B isoforms.

Progesterone (P) is a key regulator of female reproductive activity. The effects of P are mediated by two progesterone receptor (PR) proteins, termed A and B, that arise from a single gene and act as ligand-activated transcription factors to regulate the expression of reproductive target genes. Null mutation of the PR gene in mice (PRKO) leads to pleiotropic reproductive abnormalities. This paper will review the reproductive functions of PRs delineated using the PRKO mouse. Further, we will summarize the structure and functional properties of PRs and discuss how functional differences between the PR-A and PR-B isoforms are likely to impact on the overall physiological role of the receptor in reproductive systems.

Bmp2 Is Critical for the Murine Uterine Decidual Response

ABSTRACT The process of implantation, necessary for all viviparous birth, consists of tightly regulated events, including apposition of the blastocyst, attachment to the uterine lumen, and differentiation of the uterine stroma. In rodents and primates the uterine stroma undergoes a process called decidualization. Decidualization, the process by which the uterine endometrial stroma proliferates and differentiates into large epithelioid decidual cells, is critical to the establishment of fetal-maternal communication and the progression of implantation. The role of bone morphogenetic protein 2 (Bmp2) in regulating the transformation of the uterine stroma during embryo implantation in the mouse was investigated by the conditional ablation of Bmp2 in the uterus using the (PR-cre) mouse. Bmp2 gene ablation was confirmed by real-time PCR analysis in the PR-cre; Bmp2fl/fl (termed Bmp2d/d) uterus. While littermate controls average 0.9 litter of 6.2 ± 0.7 pups per month, Bmp2d/d females are completely infertile. Analysis of the infertility indicates that whereas embryo attachment is normal in the Bmp2d/d as in control mice, the uterine stroma is incapable of undergoing the decidual reaction to support further embryonic development. Recombinant human BMP2 can partially rescue the decidual response, suggesting that the observed phenotypes are not due to a developmental consequence of Bmp2 ablation. Microarray analysis demonstrates that ablation of Bmp2 leads to specific gene changes, including disruption of the Wnt signaling pathway, Progesterone receptor (PR) signaling, and the induction of prostaglandin synthase 2 (Ptgs2). Taken together, these data demonstrate that Bmp2 is a critical regulator of gene expression and function in the murine uterus.

The Antiproliferative Action of Progesterone in Uterine Epithelium Is Mediated by Hand2

A progesterone-regulated transcription factor regulates stromal-epithelial communication in early pregnancy. During pregnancy, progesterone inhibits the growth-promoting actions of estrogen in the uterus. However, the mechanism for this is not clear. The attenuation of estrogen-mediated proliferation of the uterine epithelium by progesterone is a prerequisite for successful implantation. Our study reveals that progesterone-induced expression of the basic helix-loop-helix transcription factor Hand2 in the uterine stroma suppresses the production of several fibroblast growth factors (FGFs) that act as paracrine mediators of mitogenic effects of estrogen on the epithelium. In mouse uteri lacking Hand2, continued induction of these FGFs in the stroma maintains epithelial proliferation and stimulates estrogen-induced pathways, resulting in impaired implantation. Thus, Hand2 is a critical regulator of the uterine stromal-epithelial communication that directs proper steroid regulation conducive for the establishment of pregnancy.

Stromal progesterone receptors mediate the inhibitory effects of progesterone on estrogen-induced uterine epithelial cell deoxyribonucleic acid synthesis.

The role of epithelial and stromal progesterone (P) receptors (PR) in the regulation of uterine epithelial DNA synthesis by P was investigated by analyzing the four types of tissue recombinants prepared with uterine stroma (S) and epithelium (E) from wild-type (wt) and PR knockout (PRKO) mice: wt-S + wt-E, PRKO-S + PRKO-E, wt-S + PRKO-E, and PRKO-S + wt-E. 17-Beta estradiol (E2) stimulated DNA synthesis in all four types of tissue recombinants. On the other hand, P inhibited E2-induced DNA synthesis only in tissue recombinants prepared with wild-type (PR-positive) stroma (wt-S + wt-E or wt-S + PRKO-E) but not knockout (PR-negative) stroma (PRKO-S + wt-E or PRKO-S + PRKO-E). These results clearly demonstrate that the inhibitory effect of P on uterine epithelial DNA synthesis is mediated by stromal PR. Epithelial PR is neither necessary nor sufficient for P inhibition of E2-induced epithelial DNA synthesis.

Dopamine activation of an orphan of the steroid receptor superfamily

The chicken ovalbumin upstream promoter transcription factor (COUP-TF) is a member of the steroid receptor superfamily and participates in the regulation of several genes. While a number of functions have been ascribed to COUP-TF, no ligand or activator molecule has been identified, and thus it is classified as one of a group of orphan receptors. Activation of COUP-TF by physiological concentrations of the neurotransmitter dopamine was observed in transient transfection assays. Treatment of transfected cells with the dopamine receptor agonist alpha-ergocryptine also activated COUP-dependent expression of a reporter gene. COUP-TF that contained a deletion in the COOH-terminal domain was not activated by these compounds. These observations suggest that dopamine may be a physiological activator of COUP-TF.