Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manne

Swine models of cystic fibrosis reveal male reproductive tract phenotype at birth

Nearly all male cystic fibrosis (CF) patients exhibit tissue abnormalities in the reproductive tract, a condition that renders them azoospermic and infertile. Two porcine CF models have been reported recently that include respiratory and digestive manifestations that are comparable to human CF. The goal of this study was to determine the phenotypic changes that may be present in the vas deferens of these porcine CF models. Tracts from CFTR[superscript -/-] and CFTR[superscript ΔF508/ΔF508] neonates revealed partial or total vas deferens and/or epididymis atresia at birth, while wild-type (WT) littermates were normal. Histopathological analysis revealed a range of tissue abnormalities and disruptions in tubular organization. Vas deferens epithelial cells were isolated and electrophysiological results support that CFTR[superscript -/-] monolayers can exhibit Na[superscript +] reabsorption but reveal no anion secretion following exposure to cAMP-generating compounds, suggesting that CFTR-dependent Clˉ and/or HCO[subscript 3]ˉ transport is completely impaired. SLC26A3 and SLC26A6 immunoreactivities were detected in all experimental groups, indicating that these two chloride-bicarbonate exchangers were present, but were either unable to function or their activity is electroneutral. In addition, no signs of increased mucus synthesis and/or secretion were present in the male excurrent ducts of these CF models. Results demonstrate a causal link between CFTR mutations and duct abnormalities that are manifested at birth

Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancyResearch in context

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. These variants allow for differential, gestationally regulated, modulation of the uterotonic action of E2. Methods: Human myometrium isolated from preterm and term non-laboring and laboring pregnant women were analyzed for ERα isoforms and splice factor levels. Lentiviral mediated shRNA knockdown of hnRNPG and overexpression of ERΔ7 were performed in human myometrial (hTERT-HM) cells. Functional 3D collagen contraction assays were executed. Findings: ERΔ7 acts as a dominant negative repressor of the uterotonic action of ERα66 and ERα46 isoforms through the regulation of the myometrial gap junction protein GJA1. Elimination of hnRNPG inhibits the generation of ERΔ7 while overexpression of ERΔ7 inhibited GJA1 expression. Moreover in vivo human myometrial hnRNPG levels decline at term in an E2 dependent manner resulting in a withdrawal of ERΔ7 levels and its tocolytic action at term. Interpretation: Our findings implicate the unique role of ERΔ7 as a modulator of myometrial quiescence and define the mechanism of ERΔ7 generation, through hormonally regulated splicing events. Fund: This study was supported by NIH OPRU U01 supplement (HD047905), University of Pittsburgh and Wayne State University Perinatal Research Initiative (USA). Keywords: Estrogen receptor alpha, Alternative splicing, hnRNPG, Myometrium, Parturition, GJA

Gestational regulation of ER stress signaling in the pregnant mouse uterus from E6–E19.

<p>A) Analysis of protein expression of DDIT3 and XBP1(S) in the nuclear fraction of a gestational series of mouse uteri from E6–19. NCOA3 was used as a loading control. Graphs of ROD of B) DDIT3 and C) XBP1(S) normalized to NCOA3. D) Representative western blots of cleaved (CL) and full length (FL) CASP12 in the cytoplasmic fraction across gestation. PDI was used as a loading control. ROD of E) CL CASP12 and F) FL CASP12 normalized to PDI. Data are represented as Mean ROD ± SE. N = 3 for each time point. Data labeled with different letters are significantly different from each other (P<0.05).</p

List of Primers Used for QPCR.

<p>A list of the primers used for mRNA expression analysis of genes in the ER Stress and Unfolded Protein Response in the pregnant mouse uterus across gestation.</p

Examination of uterine CASP8 cleavage across gestation from E6–E19.

<p>A) Western blot analysis of the cytoplasmic fraction in a gestational series of mouse uteri immunoblotted for full length (FL) and cleaved (CL) CASP8. Recombinant active CASP8 was utilized as a positive control (+) and PDI was used as a loading control B) Graphs of ROD of full length CASP8 normalized to PDI. Data are represented as Mean ROD ± SE. N = 3 for each time point.</p

Localization of ERS and UPR markers in the pregnant mouse uterus.

<p>Immunoflouresence studies were used to determine the localization of A) active CASP3 (Red) B) DDIT3 (Red) C) and HSPA5 (Green) at E 6, 8, 11, 13, 15, 17 and 19.</p