Alpha-tocopherol transfer protein is specifically localized at the implantation site of pregnant mouse uterus

Alpha-tocopherol transfer protein (alpha-TTP) was first described to play a major role in maintaining alpha-tocopherol levels in plasma, while alpha-tocopherol was primarily reported to be a factor relevant for reproduction. Expression of alpha-TTP is not only seen in the liver, from where it was first isolated, but also in mouse uterus, depending on its state of pregnancy, stressing the importance of alpha-TTP for embryogenesis and fetal development. The cellular localization of alpha-TTP in mouse uterus is reported here. By immunohistochemistry, alpha-TTP could be localized in the secretory columnar epithelial cells of the pregnant uterus on Days 4.5 and 6.5 postcoitum as well as in the glandular epithelial cells and the inner decidual reaction zone surrounding the implantation site. On Days 8.5 and 10.5 postcoitum (midterm of mouse pregnancy), alpha-TTP could still be detected in the uterine secretory columnar epithelial cells, while in alpha-TTP knockout mice, no immunostaining was visible. It is suggested that alpha-TTP plays a major role in supplying the placenta and consecutively the fetus with alpha-tocopherol throughout pregnancy. We conclude that alpha-tocopherol plays a role in the process of implantation and that alpha-TTP may be necessary for adequate alpha-tocopherol status of the fetus.     

Angiopoietin-like gene expression in the mouse uterus during implantation and in response to steroids

The purpose of this work was to determine if and where Angiopoietin-like genes are expressed in the mouse uterus during the implantation period of pregnancy and to determine if uterine expression of such genes is controlled by estrogen or progesterone. We found that all six known murine angiopoietin-like genes were expressed in the mouse uterus during implantation. The expression of four genes was controlled by either estrogen or progesterone. Only the levels of angiopoietin-like 4 (Angptl4) mRNA dramatically increased in implantation segments of the uterus during decidualization and was conceptus-independent. Due to this increased expression and the fact that angiopoietin-like 4 protein plays a role in lipid metabolism and angiogenesis in other tissues, only the expression of Angptl4 was further examined in the uterus and developing placenta. Angptl4 mRNA was localized to subpopulations of the endometrial stromal fibroblast and endothelial cell populations during decidualization. It was also localized to the ectoplacental cone, trophoblast giant cells and parietal endoderm of the conceptus at this time. By mid-pregnancy, Angptl4 mRNA was localized mainly to the mesometrial lymphoid aggregate region plus mesometrial endothelial cells of the uterus, as well as in various cell types of the conceptus. Additional work showed that Angptl4 expression increases in mouse endometrial stromal cells as they undergo decidualization in vitro. As in other cell types, the expression of Angptl4 in endometrial stromal cells was increased in response to an agonist of the peroxisome proliferator activated receptors. Taken together, the results of this work support the hypothesis that locally expressed Angptl4 might play a role in local uterine/placental lipid metabolism and vascular changes during implantation and thus provide a basis for future research.     

GRP78 expression and regulation in the mouse uterus during embryo implantation

The aim of this study was to investigate the spatiotemporal expression and regulation of GRP78 in the mouse uterus during the peri-implantation period. The GRP78 protein was mainly detected in the luminal and glandular epithelia on days 1–4 of pregnancy. On day 5 of pregnancy, the GRP78 protein was more highly observed around the implanted embryo at the implantation site. There was no detectable GRP78 protein signal on day 5 of pseudopregnancy. GRP78 mRNA and protein levels gradually increased on days 6–8 of pregnancy, and the expression pattern was also expanded, coinciding with the development of decidua. Similarly, GRP78 expression was also strongly expressed in decidualised cells following artificial decidualisation. Compared with the results obtained with the delayed uterus, a high level of GRP78 expression was detected in the implantation-activated uterus. In the uteri of ovariectomised mice, GRP78 expression increased and reached its highest level after injection of oestrogen, and progesterone seemed to have an antagonistic effect on oestrogen up-regulation of GRP78 expression. Our data indicate that GRP78 might play an important role during the process of mouse embryo implantation, and GRP78 expression was mainly regulated by active blastocysts and maternal oestrogen.     

Steroids modulate the expression of alpha4 integrin in mouse blastocysts and uterus during implantation

Blastocyst implantation and successful establishment of pregnancy require delicate interactions between the embryo and the maternal uterine milieu, which are controlled at the embryo-maternal interface by the coordinated interplay of a variety of growth factors, cytokines, hormones, and cell adhesion molecules expressed by both the decidualized endometrium and the trophoblast cells. Proper implantation of the embryo is solely dependent on the initial endometrial receptivity and the preparation of the blastocyst to glue itself to the uterine wall. Both these events are considered to be mediated by cell adhesion molecules and integrins expressed by the blastocyst as well by as the maternal endometrium. Integrin expression by the blastocyst and the uterus is a dynamic process. However, reports on the expression and the hormonal modulation of integrins and their role in blastocyst activation and uterine receptivity during implantation are meager. The present study investigates the expression and hormonal regulation of alpha4beta1 integrin by steroid hormones in the blastocyst and the receptive uterus using an in vivo, delayed-implantation mouse model system. The dormant and activated blastocysts as well as the uteri were recovered from ovariectomized mice after progesterone-alone and progesterone-plus-estrogen therapy, respectively. Immunolocalization of protein expression of alpha4 and beta1 integrin subunits indicate that steroids modulate the expression of alpha4beta1 integrin receptor in the mouse blastocyst as well as the uterus and that a differential expression is observed with exposure to progesterone and estrogen. Intrauterine blocking of alpha4 integrin by specific antibody resulted in implantation failure in normal as well as in delayed-implantation mice. Based on our data, we propose here, to our knowledge for the first time, that alpha4beta1 integrin, which is responsible for binding to fibronectin and vascular cell adhesion molecule-1, is induced by estradiol and is down-regulated by progesterone in mice during implantation. Furthermore, the results also indicate the direct role of alpha4 integrin in the process of implantation.     

Regulation of polypeptide growth factor synthesis and growth factor-related gene expression in the rat and mouse uterus before and after implantation

It is apparent that the uterus is a rich source of growth factors, the synthesis of which may be induced by female sex steroids. These growth factors appear to be involved in complex autocrine/paracrine regulatory circuits which in turn interact with steroid hormones. With the exception of CSF-1, however, detailed studies of the appearance of these growth factors and their receptors during pregnancy and under different hormonal regimens have yet to be performed. Furthermore, causative roles have not been established for any of these growth factors in uterine biology and pregnancy. In the near future we can expect that, by using in-situ techniques, the producing and responding cells will be identified. Cell culture models will also have to be established to investigate specific growth factor-induced functions. In the longer term, once more is known about the regulation of uterine growth factors, imaginative new experiments need to be designed, perhaps involving transgenic animals, to establish causative roles for growth factors in uterine biology.     

Serial analysis of gene expression in the silkworm, Bombyx mori

The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmental life cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.     

Estrogen regulates the expression of the small proline-rich 2 gene family in the mouse uterus

Small proline-rich (SPRR) proteins are structural components of the cornified cell envelope (CE), a specialized structure beneath the plasma membrane of stratified squamous epithelia. They are divided into four families, of which SPRR2 is the most complex consisting of 11 members (2a-2k) in the mouse. To assess the possible influence of estrogen on expression of the SPRR2 family in the uterus, we examined the effect of 17b-estradiol (E2) on SPRR2 mRNA levels on ovariectomized (OVX) adult mice. We employed a combination of laser capture microdissection (LCM) and semiquantitative RT-PCR to examine expression in particular uterine cell types - luminal epithelia, and stromal and muscle cells. We also used quantitative real-time PCR to measure levels of the mRNA of several SPRR2 proteins in the mouse uterus over the estrous cycle and during early pregnancy. Expression of SPRR2a, 2b, 2c, 2d, 2e, 2f and 2g mRNA was increased by estrogen treatment. SPRR2a, 2b, 2d and 2e were highly expressed on day 1 and 2 of pregnancy, but decreased markedly by days 3-6. Interestingly, several members of the SPRR2 family were preferentially up-regulated at implantation sites compared to inter-implantation sites around day 4 of pregnancy. They were abundant during proestrus and estrus but declined rapidly during metestrus. These results indicate that estrogen is a key regulator of the expression of the SPRR2 family in the mouse uterus during the estrous cycle and early pregnancy. In addition, they suggest that some members of the family play an important role in uterine processes such as the estrous cycle, early pregnancy and implantation.     

Characterization and expression of the mouse pregnant specific uterus protein gene and its rat homologue in the intestine and uterus

The pregnant specific uterus protein gene (Psup) is a novel mouse gene expressed in pregnant uterus. This paper describes the identification and expression of the rat homologue of Psup. The gene is highly expressed in the duodenum. Expression decreases in a proximal-distal gradient in the small intestine and was not detected in the cecum and colon. The pattern of expression in the mouse was similar. Expression of Psup in the mouse was localized to the epithelial cells in the intestine and pregnant uterus by in situ hybridization. The data show tissue-specific expression of Psup.     

Mutation at the folate receptor 4 locus modulates gene expression profiles in the mouse uterus in response to periconceptional folate supplementation

Periconceptional supplementation of folic acid to the diet of women is considered a great success for a public health intervention. Higher folate status, either by supplementation, or via the mandatory fortification of grain products in the United States, has led to significant reduction in the incidence of neural tube defects. Besides birth defects, folate deficiency has been linked to a variety of morbidities, most notably to increased risk for cancer. However, recent evidence suggests that excess folate may be detrimental — for birth defect incidence or in the progression of cancer. How folate mediates beneficial or detrimental effects is not well understood. It is also unknown what molecular responses are elicited in women taking folate supplements, and thus experience a bolus of folate on top of the status achieved by fortification. To characterize the response to a periconceptional regimen of supplementation with folinic acid, we performed gene expression profiling experiments on uterus tissue of pregnant mice with either wildtype alleles or targeted disruption at the folate receptor 4 locus. We observed that, depending on the genetic background, folinic acid supplementation affects expression of genes that contribute to lipid metabolism, protein synthesis, mitochondrial function, cell cycle, and cell activation. The extent of the response is strongly modulated by the genetic background. Finally, we provide evidence that folinic acid supplementation in the mutant paradigm affects histone methylation status, a potential mechanism of gene regulation in this model.