Apoptosis in uterine epithelium and decidua in response to implantation: evidence for two different pathways

During the initial steps of implantation, the mouse uterine epithelium of the implantation chamber undergoes apoptosis in response to the interacting blastocyst. With progressing implantation, regression of the decidual cells allows a restricted and coordinated invasion of trophoblast cells into the maternal compartment. In order to investigate pathways of apoptosis in mouse uterine epithelium and decidua during early pregnancy (day 4.5–7.0 post coitum), we have investigated different proteins such as TNFalpha, TNF receptor1, Fas ligand, Fas receptor1, Bax and Bcl2 as well as caspase-9 and caspase-3 using immunohistochemistry. To detect cells undergoing apoptosis the Tunel assay was performed. Immunoreactivity for TNFalpha as well as for TNF receptor1 was observed exclusively in the epithelium of the implantation chamber and the adjacent luminal epithelium from day 4.5 post coitum onwards. In the developing decidua the Fas ligand, but not the Fas receptor, was expressed. Bax and Bcl2 revealed a complementary expression pattern with Bax in the primary and Bcl2 in the adjacent decidual zone. Strong immunolabelling for the initiator caspase-9 was restricted to the decidual compartment, whereas caspase-3 expression characterized the apoptotic uterine epithelium. Only some caspase-3 positive decidual cells were found around the embryo which correlated to the pattern of Tunel staining. Taken together, the apoptotic degeneration of the uterine epithelium seems to be mediated by TNF receptor1 followed by caspase-3, whereas the very moderate regression of the decidua did not show the investigated death receptor, but Bax and Blc2 instead and in addition caspase-9, which indicates a different regulation for epithelial versus decidual apoptosis

A suppressive antagonism evidences Progesterone and Estrogen receptor pathway interaction with concomitant regulation of Hand2, Bmp2 and ERK during early decidualization

Progesterone receptor and estrogen receptor participate in growth and differentiation of the different rat decidual regions. Steroid hormone receptor antagonists were used to study steroid regulation of decidualization. Here we describe a suppressive interaction between progesterone receptor (onapristone) and estrogen receptor (ICI182780) antagonists and their relation to a rescue phenomenon with concomitant regulation of Hand2, Bmp2 and p-ERK1/2 during the early decidualization steps. Phenotypes of decidua development produced by antagonist treatments were characterized by morphology, proliferation, differentiation, angiogenesis and expression of signaling molecules. We found that suppression of progesterone receptor activity by onapristone treatment resulted in resorption of the implantation sites with concomitant decrease in progesterone and estrogen receptors, PCNA, KI67 antigen, DESMIN, CCND3, CX43, Prl8a2, and signaling players such as transcription factor Hand2, Bmp2 mRNAs and p-ERK1/2. Moreover, FGF-2 and Vegfa increased as a consequence of onapristone treatment. Implantation sites from antagonist of estrogen receptor treated rats developed all decidual regions, but showed an anomalous blood vessel formation at the mesometrial part of the decidua. The deleterious effect of onapristone was partially counteracted by the impairment of estrogen receptor activity with rescue of expression levels of hormone steroid receptors, proliferation and differentiation markers, and the induction of a probably compensatory increase in signaling molecules Hand2, Bmp2 and ERK1/2 activation compared to oil treated controls. This novel drug interaction during decidualization could be applied to pathological endometrial cell proliferation processes to improve therapies using steroid hormone receptor targets.Fil: Mestre Citrinovitz, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Kleff, Veronika. Universaetsklinikum Duisburg-Essen. Institut für Anatomie; AlemaniaFil: Vallejo, Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Winterhager, Elke . Universaetsklinikum Duisburg-Essen. Institut Für Molekularbiologie; AlemaniaFil: Saragüeta, Patricia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Gap junction formation in rabbit uterine epithelium in response to embryo recognition

Gap junction formation was studied in the uterine epithelium of nonpregnant, pregnant, and pseudopregnant rabbits in the periimplantation phase (6, 7, 8 days post coitum/post human gonadotropin injection) using freeze-fracture and immunocytochemistry as well as intracellular Lucifer yellow injection. At implantation (7 days post coitum) the uterine epithelial cells of the implantation chamber become junctionally coupled as evidenced by all three methods used. Gap junction protein (26K) becomes detectable immunocytochemically with a monoclonal antibody at 6 days post coitum in the epithelium surrounding the blastocyst, i.e., in the forming implantation chamber. The same sequence of events, starting with the presence of the gap junction protein before cell-to-cell coupling becomes evident, was observed in the blastocyst-free segments 1 day later. In contrast, uterine epithelium of nonpregnant and pseudopregnant animals in comparable phases shows an extremely low degree of coupling. The presence of the blastocyst is a necessary condition for the induction of gap junctions as demonstrated by unilateral pregnancy produced by tubal ligation. Thus, gap junction formation is one of the first maternal responses to a locally acting signal of the blastocyst

Uterine NK cells are critical in shaping DC immunogenic functions compatible with pregnancy progression.

Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression

Replacement of connexin43 by connexin26 in transgenic mice leads to dysfunctional reproductive organs and slowed ventricular conduction in the heart

BACKGROUND: In order to further distinguish unique from general functions of connexin43, we have generated mice in which the coding region of connexin43 was replaced by that of connexin26. RESULTS: Heterozygous mothers showed impaired mammary gland development responsible for decreased lactation and early postnatal death of the pups which could be partially rescued by wild type foster mothers. Only about 17% of the homozygous connexin43 knock-in connexin26 mice instead of 25% expected according to Mendelian inheritance, were born and only 6% survived to day 21 post partum and longer. Neonatal and adult connexin43 knock-in connexin26 mice exhibited slowed ventricular conduction in their hearts, i.e. similar but delayed electrophysiological abnormalities as connexin43 deficient mice. Furthermore, connexin43 knock-in connexin26 male and female mice were infertile and exhibited hypotrophic gonads. In testes, tubuli seminiferi were developed and spermatogonia as well as some primary spermatocytes were present, but further differentiated stages of spermatogenesis were absent. Ovaries of female connexin43 knock-in connexin26 mice revealed only few follicles and the maturation of follicles was completely impaired. CONCLUSION: The impaired gametogenesis of homozygous males and females can explain their infertility

Human sFLT1 Leads to Severe Changes in Placental Differentiation and Vascularization in a Transgenic hsFLT1/rtTA FGR Mouse Model

The anti-angiogenic soluble fms-like tyrosine kinase 1 (sFLT1) is one of the candidates in the progression of preeclampsia, often associated with fetal growth restriction (FGR). Therapeutic agents against preeclampsia with/without FGR, as well as adequate transgenic sFLT1 mouse models for testing such agents, are still missing. Much is known about sFLT1–mediated endothelial dysfunction in several tissues; however, the influence of sFLT1 on placental and fetal development is currently unknown. We hypothesize that sFLT1 is involved in the progression of FGR by influencing placental differentiation and vascularization and is a prime candidate for interventional strategies. Therefore, we generated transgenic inducible human sFLT1/reverse tetracycline-controlled transactivator (hsFLT1/rtTA) mice, in which hsFLT1 is ubiquitously overexpressed during pregnancy in dams and according to the genetics in hsFLT1/rtTA homozygous and heterozygous fetuses. Induction of hsFLT1 led to elevated hsFLT1 levels in the serum of dams and on mRNA level in all placentas and hetero-/homozygous fetuses, resulting in FGR in all fetuses at term. The strongest effects in respect to FGR were observed in the hsFLT1/rtTA homozygous fetuses, which exhibited the highest hsFLT1 levels. Only fetal hsFLT1 expression led to impaired placental morphology characterized by reduced placental efficiency, enlarged maternal sinusoids, reduced fetal capillaries, and impaired labyrinthine differentiation, associated with increased apoptosis. Besides impaired placental vascularization, the expression of several transporter systems, such as glucose transporter 1 and 3 (Glut-1; Glut-3); amino acid transporters, solute carrier family 38, member one and two (Slc38a1; Slc38a2); and most severely the fatty acid translocase Cd36 and fatty acid binding protein 3 (Fabp3) was reduced upon hsFLT1 expression, associated with an accumulation of phospholipids in the maternal serum. Moreover, the Vegf pathway showed alterations, resulting in reduced Vegf, Vegfb, and Plgf protein levels and increased Bad and Caspase 9 mRNA levels. We suggest that hsFLT1 exerts an inhibitory influence on placental vascularization by reducing Vegf signaling, which leads to apoptosis in fetal vessels, impairing placental differentiation, and the nutrient exchange function of the labyrinth. These effects were more pronounced when both the dam and the fetus expressed hsFLT1 and ultimately result in FGR and resemble the preeclamptic phenotype in humans

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS) 2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.Peer reviewe

Transplacental Nutrient Transport Mechanisms of Intrauterine Growth Restriction in Rodent Models and Humans

Although the causes of intrauterine growth restriction (IUGR) have been intensively investigated, important information is still lacking about the role of the placenta as a link from adverse maternal environment to adverse pregnancy outcomes of IUGR and preterm birth. IUGR is associated with an increased risk of cardiovascular, metabolic, and neurological diseases later in life. Determination of the most important pathways that regulate transplacental transport systems is necessary for identifying marker genes as diagnostic tools and for developing drugs that target the molecular pathways. Besides oxygen, the main nutrients required for appropriate fetal development and growth are glucose, amino acids, and fatty acids. Dysfunction in transplacental transport is caused by impairments in both placental morphology and blood flow, as well as by factors such as alterations in the expression of insulin-like growth factors and changes in the mTOR signaling pathway leading to a change in nutrient transport. Animal models are important tools for systematically studying such complex events. Debate centers on whether the rodent placenta is an appropriate tool for investigating the alterations in the human placenta that result in IUGR. This review provides an overview of the alterations in expression and activity of nutrient transporters and alterations in signaling associated with IUGR and compares these findings in rodents and humans. In general, the data obtained by studies of the various types of rodent and human nutrient transporters are similar. However, direct comparison is complicated by the fact that the results of such studies are controversial even within the same species, making the interpretation of the results challenging. This difficulty could be due to the absence of guidelines of the experimental design and, especially in humans, the use of trophoblast cell culture studies instead of clinical trials. Nonetheless, developing new therapy concepts for IUGR will require the use of animal models for gathering robust data about mechanisms leading to IUGR and for testing the effectiveness and safety of the intervention among pregnant women