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The emergence of the placenta is a revolutionary event in the evolution of therian mammals, to which some LTR retroelement-derived genes, such as PEG10, RTL1, and syncytin, are known to contribute. However, therian genomes contain many more LTR retroelement-derived genes that may also have contributed to placental evolution. We conducted large-scale evolutionary genomic and transcriptomic analyses to comprehensively search for LTR retroelement-derived genes whose origination coincided with therian placental emergence and that became consistently expressed in therian placentae. We identified NYNRIN as another Ty3/Gypsy LTR retroelement-derived gene likely to contribute to placental emergence in the therian stem lineage. NYNRIN knockdown inhibited the invasion of HTR8/SVneo invasive-type trophoblasts, whereas the knockdown of its nonretroelement-derived homolog KHNYN did not. Functional enrichment analyses suggested that NYNRIN modulates trophoblast invasion by regulating epithelial-mesenchymal transition and extracellular matrix remodeling and that the ubiquitin-proteasome system is responsible for the functional differences between NYNRIN and KHNYN. These findings extend our knowledge of the roles of LTR retroelement-derived genes in the evolution of therian mammals., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Endometriosis is characterized by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal hemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, menstruation-related factors, PGE2 and thrombin (P/T), a PAR1 agonist induced epithelial-mesenchymal transition (EMT) of endometrial cells under hypoxia. However, the molecular mechanisms by which P/T induce development of endometriosis have not been fully characterized. To investigate the effects of P/T, RNA extracted from endometrial stromal cells (ESCs) treated with P/T were subjected to RNA sequence analysis, and identified activin A, FOS, and GATA2 as upregulated genes. Activin A increased the expression of connective tissue growth factor (CTGF) and mesenchymal marker genes in ESCs. CTGF induced the expression of fibrosis marker type I collagen, fibronectin, and α-smooth muscle actin (αSMA), indicating fibroblast to myofibroblast transdifferentiation (FMT) of ESCs. In addition, activin A, FOS, GATA2, CTGF, and αSMA were localized in endometriosis lesions. Taken together, our data show that P/T induces changes resembling EMT and FMT in ectopic ESCs derived from retrograde menstruation, and that these are associated with fibrotic changes in the lesions. Pharmacological means that block P/T-induced activin A and CTGF signaling may be strategies to inhibit fibrosis in endometriotic lesions., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Decidualization of human endometrial stromal cells (ESCs) accompanied by the production of prolactin (PRL) and IGF-binding protein (IGFBP) 1 and rounded-cell morphology is indispensable for the establishment and maintenance of pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is known to be crucial for decidualization. We previously reported that activation of a cAMP mediator, called Exchange protein directly activated by cAMP (EPAC) promotes cAMP analog- or ovarian steroid-induced decidualization in cultured human ESCs. In addition, small interfering RNA-mediated knock-down of the EPAC subtypes, EPAC1 or EPAC2, or knock-down of Rap1, a downstream factor of EPAC signaling, blocked functional and morphological decidualization of ESCs. However, factors downstream of EPAC2 other than Rap1 have not been determined. The present study was undertaken to identify additional downstream targets of EPAC2 associated with decidualization. Using proteomic analysis, we identified calreticulin (CRT) as a potential target of EPAC2. Knock-down of CRT expression in cultured ESCs significantly inhibited PKA-selective cAMP analog- or PKA-selective cAMP analog plus EPAC-selective cAMP analog-induced PRL and IGFBP1 expression. Furthermore, CRT knock-down suppressed the ovarian steroid-stimulated PRL and IGFBP1 expression and morphological differentiation, and silencing of EPAC2 or CRT significantly increased senescence-associated β-galactosidase activity with enhanced p21 expression and decreased p53 expression. These results suggest that EPAC2 and CRT are associated with cellular senescence in ESCs. In conclusion, we demonstrate here that EPAC2-mediated CRT expression is essential for the functional and morphological differentiation of ESCs into decidual cells. Furthermore, both EPAC2 and CRT might prevent ESCs from undergoing abnormal cellular senescence during decidualization.

Background: The mononuclear villous cytotrophoblast (CTB) differentiates and fuses to the multinucleated syncytiotrophoblast (STB), which produces hCG and progesterone. cAMP-mediated intracellular pathways are involved in the process of endocrine differentiation and fusion (syncytialization). The exchange protein directly activated by cAMP (Epac) is a mediator of cAMP signaling. We examined the differential roles of Epac and protein kinase A (PKA) signaling in the cell fusion and differentiation of trophoblast-derived BeWo cells., Methods: Epac1 and Epac2 were localized in human placental tissue (n = 9) by immunohistochemistry. The PKA-selective cAMP analog (N(6)-phenyl-cAMP, Phe) or Epac-selective cAMP analog (CPT) was tested for effects on hCG and progesterone production, and syncytialization in BeWo cells. The effect of knockdown of Epac or its downstream target molecule (Rap1) on syncytialization was evaluated., Results: Epac1 and Epac2 proteins were expressed in villous CTB, STB, stroma, blood vessels and extravillous CTB of the placenta. Phe increased the expression of hCG alpha/beta mRNA and secretion of hCG protein in BeWo cells (P < 0.01 versus control). CPT-stimulated production of hCG (P < 0.05), albeit to a lesser extent than Phe. Progesterone production was also enhanced by Phe or CPT (P < 0.01 and P < 0.05, respectively). CPT or a stable cAMP analog (dibutyryl-cAMP: Db) increased the number of syncytialized BeWo cells (P < 0.01), whereas Phe did not stimulate fusion. CPT- or Db-induced syncytialization was observed, even in the presence of a PKA inhibitor. Knockdown of Epac1 or Rap1 repressed the Db-, CPT- or forskolin-induced cell fusion., Conclusions: The Epac signaling pathway may be associated with the cAMP-mediated functional differentiation and syncytialization of human trophoblasts.

Interferon tau gene (IFNT) is expressed only by mononuclear trophectoderm cells in ruminant ungulates. To our knowledge, its epigenetic regulation and interaction with trophectoderm lineage-specific caudal-related homeobox 2 transcription factor (CDX2) have not been characterized. Herein, we studied differences in chromatin structures and transcription of endogenous bovine IFNT in bovine trophoblast BT-1 and CT-1 cells and in nontrophoblast MDBK cells. Transcripts from endogenous IFNT and CDX2 genes were found in BT-1 and CT-1 cells but not in MDBK cells. Chromatin immunoprecipitation study revealed that CDX2 binding sites exist in proximal upstream regions of IFNT (IFN-tau-c1). Endogenous IFNT transcription in BT-1 cells was increased with CDX2 overexpression but was reduced with short interfering RNA specific for the CDX2 transcript. In chromatin immunoprecipitation studies, histone H3K18 acetylation of IFNT was higher in CT-1 cells than in MDBK cells, while histone H3K9 methylation was lower in CT-1 cells than in nontrophoblast cells. In MDBK cells (but not in CT-1 cells), histone deacetylases were bound to IFNT, which was reversed with trichostatin A treatment; treatment with trichostatin A and CDX2 then increased IFNT mRNA levels that resulted from abundant CDX2 mRNA expression. These data provide evidence that significant increase in endogenous IFNT transcription in MDBK cells (which do not normally express IFNT) can be induced through CDX2 overexpression and high H3K18 acetylation, but lowering of H3K9 methylation could also be required for the degree of IFNT transcription seen in trophoblast cells.

Local hypoxia that occurs during menstruation triggers angiogenesis that is crucial for cyclical remodeling of the endometrium during the menstrual cycle. Hypoxia is thought to be important for the expression of vascular endothelial growth factor (VEGF) via its transcriptional factor, hypoxia inducible factor (HIF)-1alpha, in the endometrium. The activation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway may modulate HIF-1alpha protein levels. Stathmin, a microtubule regulatory protein, was expressed in the stroma, glandular epithelium, and vascular endothelium in human endometrium. In this study, we examined a possible role of stathmin in hypoxia-induced HIF-1alpha and VEGF expression in primary isolated and immortalized human endometrial stromal cells, glandular epithelial cells, and human umbilical venous endothelial cells (HUVEC). Knocking down stathmin expression using small interfering RNA caused microtubule stabilization and inhibited hypoxia-induced VEGF mRNA expression via the reduction of HIF-1alpha protein levels in endometrial cells and HUVEC. Treatment of the cells with a PI3K inhibitor, wortmannin, inhibited the expression of VEGF mRNA and the accumulation of HIF-1alpha protein. Silencing of stathmin expression repressed the activation (phosphorylation) of Akt in endometrial cells and HUVEC. These results suggest that endometrial stathmin is linked to HIF-1alpha protein accumulation and VEGF expression through the PI3K/Akt signaling pathway and may be involved in regeneration of the endometrium during the menstrual cycle in human uterine cells.

Background: The microtubule-destabilizing protein stathmin is expressed by the villous cytotrophoblasts and invasive extravillous trophoblasts (EVTs) in the first-trimester human placenta. Here, we evaluated the significance of stathmin expression in terms of the functions of trophoblasts., Methods: We employed two choriocarcinoma cell lines (BeWo and JEG-3), an EVT cell line (HTR-8/SVneo) and isolated first-trimester trophoblast cells. The effects of small-interfering (si) RNA-mediated stathmin knockdown on trophoblast proliferation and migration were measured by WST-1 and Transwell assays, respectively. Trophoblast differentiation was induced by dibutyryl (db)-cAMP treatment and evaluated by measuring human chorionic gonadotrophin beta (hCGbeta) and syncytin expression and cell fusion. We examined the effect of knockdown and induced stathmin expression on db-cAMP-induced differentiation., Results: siRNA-induced silencing of stathmin expression had a marked inhibitory effect on BeWo, JEG-3 and HTR-8/SVneo cell migration and also suppressed their proliferation, albeit to a lesser extent. db-cAMP-enhanced hCGbeta and syncytin expression and cell fusion in BeWo cells was inhibited by stathmin knockdown. However, induced expression of stathmin reversed the hCGbeta and syncytin expression and cell fusion in the Tet-On BeWo cells. Suppression of stathmin expression also inhibited the migration of and hCGbeta production by first-trimester trophoblasts., Conclusions: Stathmin expression may be closely associated with early trophoblast migration and differentiation into syncytiotrophoblasts during placentation.

Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E(2)) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E(2) levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E(2) secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.

IGF binding protein-related protein 1 (IGFBP-rP1) is highly expressed in the rat uterus around the time of implantation. In the present study, we determined the periimplantation localization of IGFBP-rP1 mRNA and assessed the effects of recombinant IGFBP-rP1 on the proliferative and prostacyclin (PGI(2))-producing abilities of cultured endometrial cells early in pregnancy. IGFBP-rP1 mRNA was detected at high levels in endometrial stromal cells close to the smooth muscle of interimplantation sites around the time of implantation but absent from decidual zones surrounding the embryo. Differential uterine IGFBP-rP1 expression was also recognized in the delayed implanting pregnant model, but the level of mRNA decreased as decidual tissues formed in the decidualization model. Recombinant IGFBP-rP1 inhibited the proliferation of endometrial stromal cells in vitro and arrested them in the G(1) phase of the cell cycle. Furthermore, IGFBP-rP1 significantly stimulated PGI(2) synthesis and cyclooxygenase II mRNA expression in myometrial cells, both of which are essential molecules for successful implantation. These data suggest that IGFBP-rP1 is an implantation-associated protein and that it modulates the proliferation of rat uterine cells and their production of PGI(2) during the periimplantation period.

The development of the corpus luteum (CL), which involves angiogenesis, is essential for the establishment of early pregnancy. We investigated the roles of the prostaglandin synthases cyclooxygenase (COX) I and COX-II in angiogenesis and progesterone production in the newly formed CL, using inhibitors of the COX enzymes and the gonadotropin-induced pseudopregnant rat as a model. Injection of indomethacin, a nonselective COX inhibitor, on the day of ovulation and the following day decreased serum levels of progesterone, as did injection of the selective COX-II inhibitor NS-398. In contrast, a selective COX-I inhibitor, SC-560, had no effect on serum progesterone concentrations. None of the inhibitors had any effect on the weight of the superovulated ovaries or on the synthesis of progesterone by cultured luteal cells. To determine whether changes in angiogenesis are responsible for the decrease in progesterone synthesis, we measured hemoglobin and CD34 levels in luteinized ovaries following injection of COX inhibitors and measured the relative frequency of cells positive for platelet-endothelial cell adhesion molecule as a specific marker for endothelial cells. All of these parameters were reduced by the COX-II inhibitors, suggesting that changes in the vasculature are responsible for the decrease in serum progesterone. Histological examination of ovarian corrosion casts indicated that NS-398 inhibited the establishment of luteal capillary vessels following the injection of hCG. The results are consistent with the hypothesis that the activity of COX-II is associated with the formation of functional CL via its stimulation of angiogenesis.

We used the library subtraction technique to identify genes specifically expressed in the rat uterus during early pregnancy. One such gene was that for stathmin, a factor that is associated with tubulin binding and the destabilization of microtubules. Stathmin was expressed at higher levels in implantation sites than in interimplantation sites on d 6 and 7 of pregnancy; the levels on d 6 and 7 were higher in implantation sites than in the entire uterus on d 3-5 of pregnancy or in nonpregnant uteri. Intense expression of stathmin mRNA was primarily limited to the subluminal stromal cells at the implantation site. Expression was also detected in the decidual zones and was accentuated during the period of decidualization (d 7-12). In the delayed implantation pregnant rat model, uterine stathmin expression was low, but increased after implantation induced by administration of 17beta-estradiol to the progesterone-primed animal. Further, decidualization in the pseudopregnant rat, induced by intrauterine infusion of oil, enhanced stathmin expression. Stathmin expression clearly increases in the uterus when stimulated by embryo implantation and decidualization and may play a role in the early stages of pregnancy.