Progesterone and gravidity differentially regulate expression of extracellular matrix components in the pregnant rat myometrium

Myometrial growth and remodeling during pregnancy depends on increased synthesis of interstitial matrix proteins. We hypothesize that the presence of mechanical tension in a specific hormonal environment regulates the expression of extracellular matrix (ECM) components in the uterus. Myometrial tissue was collected from pregnant rats on Gestational Days 0, 12, 15, 17, 19, 21, 22, 23 (labor), and 1 day postpartum and ECM expression was analyzed by Northern blotting. Expression of fibronectin, laminin beta2, and collagen IV mRNA was low during early gestation but increased dramatically on Day 23 during labor. Expression of fibrillar collagens (type I and III) peaked Day 19 and decreased near term. In contrast, elastin mRNA remained elevated from midgestation onward. Injection of progesterone (P4) on Days 20-23 (to maintain elevated plasma P4 levels) delayed the onset of labor, caused dramatic reductions in the levels of fibronectin and laminin mRNA, and prevented the fall of collagen III mRNA levels on Day 23. Treatment of pregnant rats with the progesterone receptor antagonist RU486 on Day 19 induced preterm labor on Day 20 and a premature increase in mRNA levels of collagen IV, fibronectin, and laminin. Analysis of the uterine tissue from unilaterally pregnant rats revealed that most of the changes in ECM gene expression occurred specifically in the gravid horn. Our results show a decrease in expression of fibrillar collagens and a coordinated temporal increase in expression of components of the basement membrane near term associated with decreased P4 and increased mechanical tension. These ECM changes contribute to myometrial growth and remodeling during late pregnancy and the preparation for the synchronized contractions of labor.     

Differential temporal and spatial expression of mRNA encoding extracellular matrix components in decidua during the peri-implantation period.

Changes in the temporal and spatial patterns of expression of mRNA encoding uterine extracellular matrix (ECM) proteins were determined during the peri-implantation period. Northern blot hybridization of cDNAs corresponding to laminin (LM) B1, LM B2, entactin, fibronectin, collagen (CL) type IV alpha 1, and CL IV alpha 2 was performed on RNA extracted from either whole mouse uteri or endometrial explants between Day 4, i.e., the day of implantation, and Day 7 of pregnancy, when the decidual response is well established. These analyses revealed a dramatic increase in LM B2, CL IV alpha 1, and CL IV alpha 2 mRNA expression by Day 7 of pregnancy. Relative levels of the mRNA encoding other ECM components, including LM B1, were not altered when compared to changes in the relative level of expression of glyceraldehyde-3-phosphate dehydrogenase mRNA. The differential expression of the B chains of LM appeared to be limited to the stromal cells of the endometrium. In situ hybridization of uterine sections with cRNA probes corresponding to LM B1, LM B2, and CL IV alpha 1 demonstrated that LM B1 was expressed temporally in high amounts in the primary decidual zones (PDZ) and persisted throughout PDZ degeneration. LM B2 mRNA was expressed in both primary and secondary decidual zones and persisted through Day 8 of pregnancy. CL IV alpha 1 mRNA expression mimicked that of LM B2. Oviduct ligation on Day 2 of pregnancy was used to prevent embryo transport to one uterine horn, whereas decidualization and embryo implantation were permitted in the contralateral horn. This experiment demonstrated that the increases in uterine ECM mRNA expression were not due solely to the changing hormonal milieu of the uterus. ECM components, including CL IV, have been shown to bind growth factors such as transforming growth factor-beta (TGF-beta) in an insoluble but biologically active form. The remarkable similarity between the pattern of CL IV and LM B2 expression and previously reported TGF-beta deposition (Tamada et al., Mol Endocrinol 1990; 4:965-972) prompted examination of the effects of this growth factor on blastocyst development in vitro. TGF-beta 1 was tested for its ability to alter embryo outgrowth on LM-coated tissue culture surfaces; however, significant differences in the rate or extent of outgrowth in the presence of TGF-beta were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Recombinant human follicle-stimulating hormone alters maternal ovarian hormone concentrations and the uterus and perturbs fetal development in mice

Gonadotropins are routinely administered to produce multiple oocytes for clinical in vitro fertilization (IVF) treatment, laboratory research, and livestock industries. Studies in mice have shown gonadotropin stimulation using equine chorionic gonadotropin (eCG) affects the endometrium, implantation, and fetal development. Evidence from clinical studies also indicates that stimulation with recombinant human follicle-stimulating hormone (rhFSH) may be detrimental to the endometrium and implantation rates. We investigated the effect of rhFSH in mice on maternal plasma hormone concentrations and uterine gene and protein expression and the effect of a stimulated maternal environment on pregnancy. Adult females were stimulated with rhFSH or eCG, followed by human chorionic gonadotropin (hCG). On day 4 of pseudopregnancy, mice either had embryos transferred to the uterus or were killed, and blood and uterine samples were collected. Pregnancy outcomes were examined on day 15. Gonadotropin stimulation increased plasma progesterone concentrations on day 4 compared with controls, whereas estradiol concentrations were unaffected. Stimulation also reduced uterine leukemia inhibitory factor (Lif) mRNA, but the expression of estrogen and progesterone receptors (Esr1 and Pgr), homeobox gene Hoxa10, and Vegf mRNA were unchanged. Furthermore, distribution of uterine PGR protein expression was altered by stimulation, but LIF protein was unchanged. Stimulated embryo transfer recipients had lower pregnancy rates than controls, and fetuses from the rhFSH group had reduced weight, length, and maturity. These results demonstrate that gonadotropin stimulation with rhFSH or eCG alters the preimplantation maternal environment, which results in reduced pregnancy rates and fetal development in the mouse.     

Prevention of implantation by [D-Trp6-LH-RH in the rat: comparative study with the effects of large doses of HCG on pregnancy.

Effect of large doses of [D-Trp6]-LH-RH and HCG on implantation of fertilized ova was examined in the rat. Subcutaneous administration of 6ug[D-Trp6]-LH-RH per day from days 1 to 5 of pregnancy by an implanted minipump completely prevented the implantation of fertilized ova associated with a dramatic reduction of plasma progesterone on days 4 and 8, with a slight reduction of estradiol on day 4. There was no balooning of the uterus in the exploratory laparatomy on days 8 and 14. Administration of 1000 U of HCG daily from days 1 to 5 partially prevented implantation and completely terminated gestation. However, plasma progesterone and estradiol levels did not differ from those in the control animals. Ovaries in the [D-Trp6]-LH-RH treated rats appeared to be the same in size as those of the control pregnant rats, whereas the ovaries of the HCG-treated rats were hypertrophied. The results suggest that [D-Trp6]-LH-RH prevents nidation through a mechanism different from that for the adverse effect of excess of HCG on pregnancy.     

Differential expression of prostaglandin E receptor subtype EP2 in rat uterus during early pregnancy

PGE2 is essential for mammalian female reproduction. This study was to examine the expression of EP2 gene in the rat uterus during early pregnancy, delayed implantation and artificial decidualization by in situ hybridization and immunohistochemistry. There was no detectable EP2 mRNA expression in the uterus from days 1 to 4 of pregnancy (day 1 = day of vaginal sperm). A low level of EP2 immunostaining was observed in the luminal and glandular epithelium from days 1 to 4 of pregnancy. Both EP2 mRNA and protein expression were highly detected in the luminal epithelium at implantation sites on day 6 of pregnancy. EP2 expression decreased from day 7 of pregnancy and was undetectable on days 8 and 9 of pregnancy. After delayed implantation was terminated by estrogen treatment and the embryo implanted, both EP2 mRNA and protein expression were strongly observed in the luminal epithelium at the implantation site. There was no detectable EP2 expression in both control and decidualized uteri. In conclusion, these data suggest that EP2 expression at implantation site may play an important role during embryo implantation in rats.     

Endometrial expression of progesterone receptor and uteroglobin genes during early pregnancy in the rabbit

The progesterone receptor (PR) plays a pivotal role in the maturation process of the secretory endometrium, implantation and maintenance of pregnancy in rabbits. To determine the dynamics of PR gene expression and its physiological significance, the endometrial expression of PR and PR mRNA were evaluated and compared with the expression of the progesterone-regulated uteroglobin (UG) gene during 0–5 days post-coitus in rabbits. The results of immunoblot experiments indicated the presence of PR in endometrial cell extracts from days 1–4 of pregnancy with maximum PR immunostaining on day 2, followed by a marked diminution until its complete disappearance on day 5. When endometrial PR mRNA content was assessed by Northern blots, the results were similar to those of PR immunostaining, with maximal concentrations on the second day after mating. However, PR mRNA levels were still high on day 3, despite the concomitant decrease in immunostainable PR. Endometrial UG gene expression, on the other hand, exhibited a different time sequence. Thus, the UG content in uterine flushings progressively increased from day 3 after mating, reaching maximal levels on the fifth day. The endometrial UG mRNA content presented a similar profile, as its maximum concentration occurred on days 4–5. The overall results indicate that endometrial PR is down-regulated at both the mRNA and protein levels, possibly by endogenous progesterone during early pregnancy. The striking observation that maximal expression of endometrial UG gene products occurred when PR and its mRNA are no longer detectable suggests an important role for this progesterone-binding uterine protein during the preimplantation period. © 1993 Wiley-Liss, Inc.     

Uterine and placental expression of TRPV6 gene is regulated via progesterone receptor- or estrogen receptor-mediated pathways during pregnancy in rodents

Transient receptor potential cation channel, subfamily V, member 6 (TRPV6) is an epithelial Ca²⁺ channel protein expressed in calcium absorbing organs. In the present study, we investigated the expression and regulation of uterine and placental TRPV6 during gestation in rodents. Uterine TRPV6 peaked at pregnancy day (P) 0.5, P5.5 and, P13.5 and was detected in uterine epithelium and glands of rats, while placental TRPV6 mRNA levels increased in mid-gestation. Uterine and placental TRPV6 mRNA levels in rats appear to cyclically change during pregnancy, suggesting that TRPV6 may participate in the implantation process. In addition, uterine TRPV6 mRNA is only expressed in placenta-unattached areas of the uterus, and uterine TRPV6 immunoreactivity was observed in luminal and glandular epithelial cells. In the placenta, TRPV6 was detected in the labyrinth and spongy zone. These results may indicate that TRPV6 has at least two functions: implantation of the embryo and maintenance of pregnancy. To investigate the pathway(s) mediating TRPV6 expression in rodents, anti-steroid hormone antagonists were injected prior to maximal TRPV6 expression. In rats, TRPV6 expression was reduced by RU486 (an anti-progesterone) through progesterone receptors, and ICI 182,780 (an anti-estrogen) blocked TRPV6 expression via estrogen receptors in mice. The juxtaposition of uterine and placental TRPV6 expressed in these tissues supports the notion that TRPV6 participates in transferring calcium ions between the maternal and fetal compartments. Taken together, TRPV6 gene may function as a key element in controlling calcium transport in the uterus between the embryo and the placenta during pregnancy.     

Serum progesterone and corpus luteum function in pregnant pigtailed monkeys (Macaca nemestrina)

Corpus luteum (CL) function and control during pregnancy and early lactation in the pigtailed macaque was investigated. Peripheral concentrations of progesterone (P) on day 10 of pregnancy were 12.98 ± 2.21 ng/ml and decreased progressively to 7.96 ± 1.27 ng/ml by day 21 of pregnancy. The concentration of P increased around day 27 of gestation and reached peak levels of 18.48 ± 2.45 ng/ml on day 37, there-after gradually decreasing to a nadir at about midgestation. Ten days before parturition P concentrations increased again (P < 0.05). Concentrations of P decreased from 6.62 ± 1.48 ng/ml on the day of delivery to 2.16 ± 0.43 ng/ml on day 2 of lactation and remained low thereafter. Ovariectomy on day 35 did not affect the normal course of gestation or the patterns of P secretion during pregnancy. However, in these ovariectomized animals, in spite of suckling, P was not detectable after parturition. In intact monkeys, serum concentrations of P in the uteroovarian vein at days 80 and 159 of pregnancy were higher relative to the uterine vein. Incubation studies utilizing ³H-cholesterol as a substrate revealed that the CL were capable of synthesizing P on days 35 and 159 of gestation. Histologically, the CL contained active luteal cells at late pregnancy.Low serum concentrations of chorionic gonadotropin were detected on day 10 of gestation; concentrations of this hormone reached high levels between days 18 and 24 and the titers were nondetectable after day 40 of pregnancy. Luteinizing hormone was present in constant amounts in the circulation during pregnancy and lactation.These data suggest that the CL of pregnancy in the pigtailed monkey is functional or capable of functioning during various stages of pregnancy. However, the fetoplacental unit is the primary source of P during the latter 4.5 months of gestation. As in other primates, a functional CL is not required for maintenance of pregnancy after implantation nor for lactation. Thus, the physiological significance of CL function during pregnancy is unclear.     

Estrogen induces expression of secretory leukocyte protease inhibitor in rat uterus

In rodents, the steroid hormone estrogen (E) profoundly influences the early events in the uterus leading to embryo implantation. It is thought that E triggers the expression of a unique set of genes in the endometrium that in turn control implantation. To identify these E-induced genes, we employed a delayed implantation model system in which embryo attachment to rat endometrium is dependent upon E administration. Using a gene expression screen method, we isolated a number of cDNAs representing mRNAs whose expression is either turned on or turned off in response to an implantation-inducing dose of E. We identified one of these cDNAs as that encoding secretory leukocyte protease inhibitor (SLPI), an inhibitor of serine proteases. The expression of SLPI mRNA was induced in the uteri of ovariectomized rats in response to E, confirming the hormonal regulation of this molecule. Spatiotemporal analysis revealed a biphasic pattern of expression of SLPI mRNA during early pregnancy. A considerable amount of SLPI mRNA was detected in the uterine epithelium on Day 1 of pregnancy. The level of this mRNA, however, declined sharply on Days 2 and 3 of gestation. Interestingly, on Day 4 of gestation, there was a marked resurgence in SLPI mRNA expression in the uterine epithelium. This second burst of SLPI expression diminished by Day 6 of pregnancy. The transient induction of SLPI mRNA during Days 4 and 5 overlapped with the window of implantation in the rat. Although the precise function of SLPI in the uterus eludes us presently, its known effects as a serine protease inhibitor in other tissues and its hormone-induced expression in the rat uterus immediately preceding implantation lead us to propose that this gene plays an important role in controlling excessive proteolysis and inflammation during a critical phase of early pregnancy.     

Restraint Stress Inhibits Mouse Implantation: Temporal Window and the Involvement of HB-EGF,Estrogen and Progesterone

It is known that psychological stress affects reproduction in women, but it is unknown whether the effect is by impairing implantation. Although studies suggest that long periods of auditory or restraint stress may inhibit implantation in rats and mice, the exact stage of pregnancy at which stress impairs implantation is unclear. Furthermore, whether stress impairs implantation by decreasing the heparin-binding epidermal growth factor-like growth factor (HB-EGF), estrogen and/or progesterone and whether by acting on embryos or on the uterus need further investigations. In this study, a 24-h restraint stress was initiated at 15:30 of day 3 (regimen 1) or at 07:30 (regimen 2) or 15:30 of day 4 (regimen 3) of pregnancy (vaginal plug  =  day 1) to observe effects of restraint stress applied at different peri-implantation stages on implantation. Among the three regimens, whereas regimens 1 and 3 affected neither term pregnancy nor litter size, regimen 2 reduced both. Further observations indicated that regimen 2 of restraint stress also delayed blastocyst hatching and the attachment reaction, decreased serum concentrations of progesterone and estradiol, and down regulated the expression of HB-EGF in both the endometrium and blastocysts. Taken together, the results suggested that restraint stress inhibited mouse implantation in a temporal window-dependent manner and by impairing blastocyst activation and hatching and uterine receptivity via down-regulating HB-EGF, estrogen and progesterone. Thus, the stress applied within the implantation window impaired implantation by acting on both embryos and the uterus.     

ICAM1 and fibrinogen-γ are increased in uterine epithelial cells at the time of implantation in rats

Uterine epithelial cells transform into a receptive state to adhere to an implanting blastocyst. Part of this transformation includes the apical concentration of cell adhesion molecules at the time of implantation. This study, for the first time, investigates the expression of ICAM1 and fibrinogen‐γ (FGG) in uterine epithelial cells during normal pregnancy, pseudopregnancy and in hormone‐treated rats. An increase (P < 0.05) in ICAM1 was seen at the apical membrane of uterine epithelial cells at the time of implantation compared with day 1 of pregnancy. ICAM1 was also increased (P < 0.05) on day 6 of pseudopregnancy as well as in ovariectomized rats treated with progesterone plus oestrogen. These results show that ICAM1 up‐regulation at the time of implantation is under the control of progesterone, and is not dependent on cytokine release from the blastocyst or in semen. FGG dimerization increased (P < 0.05) on day 6 of pregnancy compared with day 1, and was not up‐regulated in day 6 pseudopregnant animals, suggesting this increase is dependent on a developing blastocyst. The presence of ICAM1 and FGG in the uterine epithelium at the time of implantation in the rat is similar to that seen in lymphocyte–endothelium adhesion, and we suggest a similar mechanism in embryo–uterine epithelium adhesion is utilized. Mol. Reprod. Dev. 78:318–327, 2011. © 2011 Wiley‐Liss, Inc.     

Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.     

Progesterone and placentation increase secreted phosphoprotein one (SPP1 or osteopontin) in uterine glands and stroma for histotrophic and hematotrophic support of ovine pregnancy

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.     

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.