Endoplasmic reticulum stress response and the regulation of endometrial interferon-beta production.

Objective: To gain an understanding of the potential role of endoplasmic reticulum (ER) stress in the endometrial compartment during early pregnancy, a highly understudied area.

Design: This study examined the regulation of interferon-β (IFNβ) in response to ER stress in human decidualized and nondecidualized endometrial cells (human endometrial stromal cells [HESCs]) in vitro. In vivo, we examined ER stress and the IFNβ levels locally in the mouse endometrium before and after implantation at embryonic day (E)1, E3, and E6.

Preimplantation apoptotic endometrial caspase-3-mediated phospholipase A2 activation: a potential component in programming uterine receptivity.

Objective: To examine the activation and consequence of uterine apoptotic caspase-3 action on 1 day after coitus (dpc) in the pregnant mouse. We have previously demonstrated that in a pregnant uterus, caspase-3 activation from mid to late gestation isolated to the myometrial compartment is largely nonapoptotic and controls uterine quiescence. Additionally, we had demonstrated that apoptotic caspase-3 activation isolated to the endometrial compartment at term regulated endometrial prostaglandin synthesis.

Steroidogenic Factor 1 (Nr5a1) is Required for Sertoli Cell Survival Post Sex Determination.

The elevated level of Steroidogenic Factor 1 (Nr5a1, Sf-1) expression in the male gonadal development pathway, post sex determination, implies a vital role in testis gonadal differentiation. In this study we generated Sertoli cell-specific Nr5a1 KO mice (SC-SF-1) at E14.5, which coincides with testis development post sex determination, using the Amh-Cre mouse model.

ARGLU1 is a transcriptional coactivator and splicing regulator important for stress hormone signaling and development.

Stress hormones bind and activate the glucocorticoid receptor (GR) in many tissues including the brain. We identified arginine and glutamate rich 1 (ARGLU1) in a screen for new modulators of glucocorticoid signaling in the CNS. Biochemical studies show that the glutamate rich C-terminus of ARGLU1 coactivates multiple nuclear receptors including the glucocorticoid receptor (GR) and the arginine rich N-terminus interacts with splicing factors and binds to RNA.

Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy.

Background: Circulating estrogen (E2) levels are high throughout pregnancy and increase towards term, however its local tissue specific actions vary across gestation. For example, myometrial E2 regulated uterotonic action is disabled until term, whereas it's proliferative function is maintained in the breast. We have identified gestationally regulated splicing events, mediated by hnRNPG and modulated by E2 that generate alternatively spliced estrogen receptor alpha (ERα) variants (ERΔ7 and ERα46) in the myometrium.

Preconditioning the uterine unfolded protein response maintains non-apoptotic Caspase 3-dependent quiescence during pregnancy.

The prevention of apoptotic caspase 3 activation through biological preconditioning, mediated through the modulation of the unfolded protein response has been demonstrated to ameliorate multiple pathophysiologies. The maintenance of non-apoptotic caspase 3 activity by the unfolded protein response within the pregnant uterus has previously been proven to be critical in inhibiting uterine myocyte contractility during pregnancy. Here we report that the pregnant uterus utilizes an unfolded protein response-preconditioning paradigm to conserve myometrial caspase 3 in a non-apoptotic state in order to effectively inhibit uterine contractility thereby preventing the onset of preterm labor.

Thinking Outside the Box.

A broad definition of preconditioning is "the preparation for a subsequent action." Mounting evidence demonstrates that novel remote preconditioning paradigms, in which protective stimuli experienced locally can capacitate systemic tolerance and enhanced cell viability upon exposure to ensuing cellular insults, have been largely successful in the field of cardiovascular ischemia/reperfusion injury. To ensure successful protective preconditioning, some models (including the uterus) have been demonstrated to activate the unfolded protein response (UPR), which is a cellular stress response controlled at the level of the endoplasmic reticulum.

The Unfolded Protein Response Regulates Uterine Myocyte Antioxidant Responsiveness During Pregnancy.

There is considerable evidence that implicates oxidative stress in the pathophysiology of human pregnancy complications. However, the role and the mechanism of maintaining an antioxidant prosurvival uterine environment during normal pregnancy is largely unresolved. Herein we report that the highly active uterine unfolded protein response plays a key role in promoting antioxidant activity in the uterine myocyte across gestation.

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique - usually untranslated - first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1.

Restoration of spermatogenesis and male fertility using an androgen receptor transgene.

Androgens signal through the androgen receptor (AR) to regulate male secondary sexual characteristics, reproductive tract development, prostate function, sperm production, bone and muscle mass as well as body hair growth among other functions. We developed a transgenic mouse model in which endogenous AR expression was replaced by a functionally modified AR transgene. A bacterial artificial chromosome (BAC) was constructed containing all AR exons and introns plus 40 kb each of 5' and 3' regulatory sequence.

Bcrp1 transcription in mouse testis is controlled by a promoter upstream of a novel first exon (E1U) regulated by steroidogenic factor-1.

Alternative promoter usage is typically associated with mRNAs with differing first exons that contain or consist entirely of a 5' untranslated region. The murine Bcrp1 (Abcg2) transporter has three alternative promoters associated with mRNAs containing alternative untranslated first exons designated as E1A, E1B, and E1C. The E1B promoter regulates Bcrp1 transcription in mouse intestine.

Cross-species withdrawal of MCL1 facilitates postpartum uterine involution in both the mouse and baboon.

A successful postpartum involution permits the postnatal uterus to rapidly regain its prepregnancy function and size to ultimately facilitate an ensuing blastocyst implantation. This study investigates the molecular mechanisms that govern the initiation of the involution process by examining the signaling events that occur as the uterus transitions from the pregnant to postnatal state. Using mouse and baboon uteri, we found a remarkable cross-species conservation at the signal transduction level as the pregnant uterus initiates and progresses through the involution process.

Antiapoptotic signaling via MCL1 confers resistance to caspase-3-mediated apoptotic cell death in the pregnant human uterine myocyte.

Our group has previously identified elevated levels of nonapoptotic active caspase 3 (CASP3) accompanied by increased prosurvival, antiapoptotic signaling in the pregnant mouse uterus during late gestation. We speculated that increased antiapoptotic signaling desensitized the pregnant uterine myocyte to the apoptotic action of uterine CASP3. This current study examines the mechanism by which the pregnant myocyte gains resistance to the apoptotic effects of increased uterine CASP3.

Elevated levels of uterine anti-apoptotic signaling may activate NFKB and potentially confer resistance to caspase 3-mediated apoptotic cell death during pregnancy in mice.

Preserving the uterus in a state of relative quiescence is vital to the maintenance of a successful pregnancy. Elevated cytoplasmic levels of uterine caspase 3 during pregnancy have been proposed as a potential regulator of uterine quiescence through direct targeting and disabling of the uterine contractile architecture. However, despite highly elevated levels of uterine caspase 3 during pregnancy, there is minimal evidence of apoptosis.

Hexose-6-phosphate dehydrogenase contributes to skeletal muscle homeostasis independent of 11β-hydroxysteroid dehydrogenase type 1.

Glucose-6-phosphate (G6P) metabolism by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) within the sarcoplasmic reticulum lumen generates nicotinamide adenine dinucleotide phosphate (reduced) to provide the redox potential for the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to activate glucocorticoid (GC). H6PDH knockout (KO) mice have a switch in 11β-HSD1 activity, resulting in GC inactivation and hypothalamic-pituitary-adrenal axis activation. Importantly, H6PDHKO mice develop a type II fiber myopathy with abnormalities in glucose metabolism and activation of the unfolded protein response (UPR).

Progesterone-regulated caspase 3 action in the mouse may play a role in uterine quiescence during pregnancy through fragmentation of uterine myocyte contractile proteins.

The appropriate timing of the onset of labor is critical to a successful pregnancy, with potentially devastating consequences resulting to both the mother and child with the onset of preterm labor. In this study, we tested the central hypothesis that progesterone maintains uterine quiescence through regulation of active uterine caspase 3. Using the mouse as our model system, we examined, by Western blot analysis, levels of active caspase 3 and its association with the degradation of uterine contractile proteins during pregnancy.

Complex role of the mitochondrial targeting signal in the function of steroidogenic acute regulatory protein revealed by bacterial artificial chromosome transgenesis in vivo.

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads.

Hexose-6-phosphate dehydrogenase knock-out mice lack 11 beta-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation.

The local generation of active glucocorticoid by NADPH-dependent, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) oxoreductase activity, has emerged as an important factor in regulating hepatic glucose output and visceral adiposity. We have proposed that this NADPH is generated within the endoplasmic reticulum by the enzyme hexose-6-phosphate dehydrogenase. To address this hypothesis, we generated mice with a targeted inactivation of the H6PD gene.

The transcription factors steroidogenic factor-1 and SOX9 regulate expression of Vanin-1 during mouse testis development.

We previously showed, using differential expression screening and in situ hybridization that Vanin-1, which encodes a glycosylphosphatidylinositol-linked membrane-associated pantetheinase, is expressed in a sex-specific manner during fetal gonad development in mice (Bowles, J., Bullejos, M., and Koopman, P.

Cell-specific knockout of steroidogenic factor 1 reveals its essential roles in gonadal function.

Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1, officially designated Nr5a1) have a compound endocrine phenotype that includes adrenal and gonadal agenesis, impaired expression of pituitary gonadotropins, and structural abnormalities of the ventromedial hypothalamic nucleus. To inactivate a conditional SF-1 allele in the gonads, we targeted the expression of Cre recombinase with a knock-in allele of the anti-Müllerian hormone type 2 receptor locus. In testes, Cre was expressed in Leydig cells.

Surfactant protein secreted by the maturing mouse fetal lung acts as a hormone that signals the initiation of parturition.

Parturition is timed to begin only after the developing embryo is sufficiently mature to survive outside the womb. It has been postulated that the signal for the initiation of parturition arises from the fetus although the nature and source of this signal remain obscure. Herein, we provide evidence that this signal originates from the maturing fetal lung.

Tissue-specific knockouts of steroidogenic factor 1.

Targeted gene disruption has produced knockout (KO) mice globally deficient in the orphan nuclear receptor steroidogenic factor 1 (SF-1). These SF-1 KO mice lacked adrenal glands and gonads, and also had impaired expression of gonadotropins in pituitary gonadotropes and marked structural abnormalities of the ventromedial hypothalamic nucleus (VMH). To define the roles of SF-1 within discrete sites of the hypothalamic-pituitary-steroidogenic organ axis, we have sought to make tissue-specific SF-1 KO mice (as reviewed here).