Origin of the murine implantation serine proteinase subfamily

The S1 serine protease family is one of the largest gene families known. Within this family there are several subfamilies that have been grouped together as a result of sequence comparisons and substrate identification. The grouping of related genes allows for the speculation of function for newly found members by comparison and for novel subfamilies by contrast. Analysis of the evolutionary patterns of genes indicates whether or not orthologs are likely to be identified in other species as well as potentially indicating that hypothesized orthologs are in fact not. Looking at subtle differences between subfamily members can reveal intricacies about function and expression. Previously, we have described genes encoding two novel serine proteinases, ISP1 and ISP2, which are most closely related to tryptases. The ISP1 gene encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching and invasion in vitro. Additionally both ISP1 and ISP2 are co-expressed in the endometrial gland during the time of hatching, suggesting that they may also both participate in zona lysis from within the uterine lumen. Here, we demonstrate that the ISPs are tandemly linked within the tryptase cluster on 17A3.3. We suggest that remarkable similarities within the 5'-untranslated and first intron regions of ISP1 and ISP2 may explain their intimate co-regulation in uterus. We also suggest that ISP genes have evolved through gene duplication and that the ISP1 gene has also begun to adopt an additional new function in the murine preimplantation embryo.     

Characterization of secretory leukocyte protease inhibitor as an inhibitor of implantation serine proteinases

We have recently identified and characterized two implantation serine proteinase genes, ISP1 and ISP2, which give rise to a dimeric proteinase, ISP that facilitates embryo invasion during peri-implantation period. As many proteinases have cognate serpins that regulate their proteolytic activity, we have been investigating anti-tryptases, expressed during this window of implantation. Here, we report the differential expression of secretory leukocyte protease inhibitor (SLPI) in uterine endometrium around the implantation period. The co-localization of SLPI and ISP suggests the possibility that SLPI is an ISP serpin and that expression of SLPI may lead to a reduction in ISP activity. The expression of SLPI is down regulated during the window of embryo-uterine receptivity. Our results are consistent with a model suggesting that the drop in SLPI expression may help to refine the opening of the window of implantation, by allowing the proteolytic activity of embryo invasive serine proteinases such as the ISPs.     

Implantation serine proteinase 1 exhibits mixed substrate specificity that silences signaling via proteinase-activated receptors

Implantation S1 family serine proteinases (ISPs) are tryptases involved in embryo hatching and uterine implantation in the mouse. The two different ISP proteins (ISP1 and ISP2) have been detected in both pre- and post-implantation embryo tissue. To date, native ISP obtained from uterus and blastocyst tissues has been isolated only as an active hetero-dimer that exhibits trypsin-like substrate specificity. We hypothesised that in isolation, ISP1 might have a unique substrate specificity that could relate to its role when expressed alone in individual tissues. Thus, we isolated recombinant ISP1 expressed in Pichia pastoris and evaluated its substrate specificity. Using several chromogenic substrates and serine proteinase inhibitors, we demonstrate that ISP1 exhibits trypsin-like substrate specificity, having a preference for lysine over arginine at the P1 position. Phage display peptide mimetics revealed an expanded but mixed substrate specificity of ISP1, including chymotryptic and elastase activity. Based upon targets observed using phage display, we hypothesised that ISP1 might signal to cells by cleaving and activating proteinase-activated receptors (PARs) and therefore assessed PARs 1, 2 and 4 as potential ISP1 targets. We observed that ISP1 silenced enzyme-triggered PAR signaling by receptor-disarming. This PAR-disarming action of ISP1 may be important for embryo development and implantation.     

哺乳动物胚胎透明带脱落机制的研究进展

胚胎着床是一个连续的动态过程,其中胚泡从透明带中准时孵出是着床的关键.透明带脱落的机制主要是子宫或(和)胚泡分泌物部分或全部溶解透明带后,胚泡在细胞数量增加及细胞运动的机械压力作用下通过透明带的某一位点孵出.     

Mouse blastocysts hatch in vitro by using a trypsin-like proteinase associated with cells of mural trophectoderm

The mammalian blastocyst must hatch from its extracellular coat, or zona pellucida, to implant in the uterus and continue development normally. Results of experiments described here strongly suggest that a proteinase (74K Mr), called "strypsin," is directly involved in hatching of isolated mouse blastocysts in vitro. Strypsin is a trypsin-like proteinase, based on its substrate specificity and sensitivity to inhibitors, that is present in mouse blastocysts and exhibits certain properties characteristic of membrane-associated enzymes. Histochemical and autoradiographic evidence suggests that, prior to hatching of blastocysts, strypsin is found with cells of mural trophectoderm; not with polar trophectoderm or inner cell mass. Following hatching, strypsin is also found associated with empty zonae pellucidae, specifically at the opening through which the embryo emerged. These and other observations suggest that hatching of mouse blastocysts in vitro is initiated by limited proteolysis of the region of zona pellucida overlying mural trophectoderm.     

Expression of rat uterine serine proteinases homologous to mouse implantation serine proteinase 2

Implantation serine protease (ISP) was first identified in the uteri of pregnant mice. It is thought that ISP may have an important role in the initiation of implantation. However, the expression status and detailed functions of ISP remain unclear. In this study, the expression of ISP was investigated in the rat uterus. The analysis of two rat genes registered in GenBank, accession nos. XM_220240 and XM_577076, exhibited high identities to the mouse ISP2 genes, respectively at an mRNA level. We labeled the former as rISP2a and the latter as rISP2b. Using RT-PCR, we found that both genes were expressed in the uterus. Specifically, rISP2a mRNA was detected in the uterus throughout pregnancy, whereas rISP2b mRNA was only expressed in the uterus from day 5 of pregnancy until the end of gestation. Expression of both genes was observed specifically within the endometrial gland epithelium. Furthermore, rISP2a was also observed to be expressed in the fetus and placenta, whereas rISP2b expression was observed in the fetus but not in the placenta. An expressional signal of the rISP2a gene was observed in the spongiotrophoblasts, giant cells and decidual endometrium in the placenta. In the embryo, the ventral specific region was positive in rISP2a and rISP2b gene expression. These findings indicate the possibility that the presently examined genes with high identity to mouse ISP2 may play some role not only during the implantation phase, but also in the development of the placenta and embryo.     

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.     

Ecotin-like serine peptidase inhibitor ISP1 of Leishmania major plays a role in flagellar pocket dynamics and promastigote differentiation

Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.     

Immunocytochemical localization and messenger ribonucleic acid levels of a progesterone-dependent endometrial secretory protein (cathepsin L) in the pregnant cat uterus.

The purpose of this study was to localize immunocytochemically a progesterone-dependent protein (PDP) and to determine PDP mRNA levels during the initial stage of the implantation period. Uterine tissue was collected from Day 0-18 postcoital animals. The tissue was processed for immunocytochemical localization of PDP, and the endometrial RNA was isolated and analyzed for PDP gene expression by slot-blot hybridization. PDP was detected immunocytochemically as early as Day 5 postcoitus in the epithelial cells of the deep uterine glands, and the intensity of immunostaining appeared to peak by Day 12 postcoitus. PDP was absent in the endometrium obtained from implantation sites after Day 16 postcoitus, but the synthesis of PDP was maintained in the endometrium obtained from nonimplantation sites. Immunogold electron microscopy demonstrated that PDP was present in electron-dense granules of the glandular epithelial cells. PDP mRNA was detectable in the endometrium at Day 5 postcoitus and peaked around Day 10 postcoitus. PDP mRNA was absent in the endometrium from implantation sites after Day 16 postcoitus, but was maintained in the endometrium from nonimplantation sites. In summary, the results of this study illustrate that PDP is synthesized within the epithelial cells of the deep uterine glands, packaged within membrane-bound secretory granules, and released into the uterine lumen. Also, the process of implantation alters the gene expression in a very localized way since PDP mRNA and PDP-positive granules were absent in the endometrial glands obtained from the implantation site within 1-2 days of the onset of implantation, whereas both PDP mRNA and PDP-positive granules were maintained in the endometrial glands from nonimplantation-site regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of progesterone on oocyte quality and embryo development in cows

In cattle, the majority of embryo loss occurs very early during pregnancy (approximately Day 16), around or prior to maternal recognition of pregnancy. The actions of P4 in controlling LH pulsatility and ovarian follicular development may impinge negatively on oocyte quality. A considerable proportion of embryo loss may be attributable to inadequate circulating progesterone (P4) concentrations and the subsequent downstream consequences on endometrial gene expression and histotroph secretion into the uterine lumen. Conceptus growth and development require the action of P4 on the uterus to regulate endometrial function, including conceptus-maternal interactions, pregnancy recognition, and uterine receptivity for implantation. This review summarizes recent data highlighting the role of progesterone in determining oocyte quality and embryo development in cattle.     

Influence of progesterone on oocyte quality and embryo development in cows

In cattle, the majority of embryo loss occurs very early during pregnancy (approximately Day 16), around or prior to maternal recognition of pregnancy. The actions of P4 in controlling LH pulsatility and ovarian follicular development may impinge negatively on oocyte quality. A considerable proportion of embryo loss may be attributable to inadequate circulating progesterone (P4) concentrations and the subsequent downstream consequences on endometrial gene expression and histotroph secretion into the uterine lumen. Conceptus growth and development require the action of P4 on the uterus to regulate endometrial function, including conceptus–maternal interactions, pregnancy recognition, and uterine receptivity for implantation. This review summarizes recent data highlighting the role of progesterone in determining oocyte quality and embryo development in cattle.     

Stanniocalcin (STC) in the endometrial glands of the ovine uterus: regulation by progesterone and placental hormones

Stanniocalcin (STC) is a hormone in fish that regulates calcium levels. Mammals have two orthologs of STC with roles in calcium and phosphate metabolism and perhaps cell differentiation. In the kidney and gut, STC regulates calcium and phosphate homeostasis. In the mouse uterus, Stc1 increases in the mesometrial decidua during implantation. These studies determined the effects of pregnancy and related hormones on STC expression in the ovine uterus. In Days 10-16 cyclic and pregnant ewes, STC1 mRNA was not detected in the uterus. Intriguingly, STC1 mRNA appeared on Day 18 of pregnancy, specifically in the endometrial glands, increased from Day 18 to Day 80, and remained abundant to Day 120 of gestation. STC1 mRNA was not detected in the placenta, whereas STC2 mRNA was detected at low abundance in conceptus trophectoderm and endometrial glands during later pregnancy. Immunoreactive STC1 protein was detected predominantly in the endometrial glands after Day 16 of pregnancy and in areolae that transport uterine gland secretions across the placenta. In ovariectomized ewes, long-term progesterone therapy induced STC1 mRNA. Although interferon tau had no effect on endometrial STC1, intrauterine infusions of ovine placental lactogen (PL) increased endometrial gland STC1 mRNA abundance in progestinized ewes. These studies demonstrate that STC1 is induced by progesterone and increased by a placental hormone (PL) in endometrial glands of the ovine uterus during conceptus (embryo/fetus and extraembryonic membranes) implantation and placentation. Western blot analyses revealed the presence of a 25-kDa STC1 protein in the endometrium, uterine luminal fluid, and allantoic fluid. The data suggest that STC1 secreted by the endometrial glands is transported into the fetal circulation and allantoic fluid, where it is hypothesized to regulate growth and differentiation of the fetus and placenta, by placental areolae.     

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.     

The Antiapoptotic Effect of Galectin-3 in Human Endometrial Cells under the Regulation of Estrogen and Progesterone

Galectin-3 (Gal-3), a ubiquitously expressed gene involved in many cellular processes, has been recently recognized as a factor related to endometrial receptivity. However, the precise biological function of Gal-3 in the endometrium and its regulation is still unclear. In this study, we detected the antiapoptotic role of Gal-3 in endometrial cells and the expression of Gal-3 regulated by estrogen and progesterone. We found that expression of Gal-3 increased when exposed to the apoptosis inducer staurosporine. Gal-3-silenced endometrial cells were more sensitive to the apoptosis inducer. Estradiol (E2) and progesterone (P4) up-regulated Gal-3 expression, which in turn decreased the apoptotic rate of endometrial cells. Our results strongly suggested that hormonal activation of Gal-3 by E2 and P4 is involved in inhibiting endometrial cell apoptosis, playing key roles in embryo implantation.