Association between prepartum vaginal temperature changes and placenta expulsion time in Holstein dairy cattle.

Retained placenta (RP) adversely affects postpartum productivity and reproduction in dairy cattle. Thus, methods to predict the occurrence of RP before calving would be desirable. Herein, we assessed whether vaginal temperature measurements (which have already been applied to detect calving) could be used to predict the occurrence of RP in cattle. A vaginal temperature recording device was inserted into the vagina of 49 pregnant Holstein-Friesian heifers (n = 16) and cows (n = 33); this device recorded the vaginal temperature every 5 min until the device dropped out at calving. Serum was collected 10 days before the expected calving date. The time points of calving and placental expulsion were identified via video recordings. We further calculated calving duration (temperature decrease to calving) and placenta expulsion time (PE time = calving to placenta expulsion). The PE times were divided into four categories (0-4 h, 4-8 h, 8-12 h, and RP at >12 h), while subsequent analysis revealed that an extension of the PE time dependent on the shortening of the calving duration (P < 0.05). The vaginal temperature patterns also differed in a PE time-dependent manner, and cows with RP did not show any re-elevation of vaginal temperature. Serum analyses indicated an energy deficiency in RP cattle. These results suggest that RP may be detected early as a specific change in the vaginal temperature associated with reproductive hormone secretion.

Molecular approaches to mammalian uterine receptivity for conceptus implantation.

Mammalian reproduction is more inefficient than expected and embryo/conceptus implantation into the maternal endometrium is considered to be a rate-limiting process. Although extensive physiological and structural diversity exists among mammalian species, the basic molecular mechanisms underlying successful implantation are conserved. The extensive use of genetically engineered mouse models has provided considerable information on uterine receptivity for embryo implantation. The molecular mechanisms and cellular processes identified thus far require further validation in other mammalian species. In this review, representative ovarian steroid hormone-induced signaling pathways controlling uterine adaptation are presented based on the results of rodent studies. Selected examples of functional conservation in mammals, such as humans and cattle, are briefly described. To date, molecular therapeutic trials for fertility improvement have not been conducted. Considerable efforts are required to provide further understanding of these molecular mechanisms. Such understanding will contribute to the development of reliable clinical diagnostics and therapeutics for implantation failure, leading to reproductive success in a wide variety of mammals in the future.

Changes in immunoglobulin A and immunoglobulin G concentrations in the saliva of Japanese Black beef cows during calving: A pilot study.

Immunoglobulin A (IgA) in saliva, mostly consisting of secretory IgA, plays an important role in the mucosal immune mechanism. This study evaluated changes in salivary IgA and Immunoglobulin G (IgG) concentrations in Japanese Black cows (n = 16) during calving. Individual saliva samples were collected -2, 0, and 2 weeks postpartum. Immunoglobulin concentrations differed significantly among weeks (P < 0.05), but the effect of parity and week × parity was insignificant. Salivary IgA concentrations decreased drastically (P < 0.05) after calving compared with those at -2 weeks postpartum and remained low until 2 weeks postpartum. The salivary IgG concentrations decreased gradually during peripartum and differed at -2 and 2 weeks postpartum (P < 0.05). Considering the immunoglobulin concentrations at -2 weeks postpartum as the reference standard for 100%, the rates of decrease in IgA concentrations (36.7 ± 6.9%) were significantly lower (P < 0.05) than those of IgG (70.3 ± 10.1%) at calving day. To our knowledge, this is the first report indicating that salivary IgA concentrations decreased drastically after calving in Japanese Black cows. Further studies monitoring the secretory functions of IgA in the salivary gland are essential for understanding maternal immunity in cattle.

Steroidal but not embryonic regulation of mucin 1 expression in bovine endometrium.

In cow herd management, inadequate embryo implantation leads to pregnancy loss and causes severe economic losses. Thus, it is crucial to understand the molecular mechanisms underlying endometrial receptivity and subsequent embryo implantation. Transmembrane glycocalyx mucin 1 (MUC1) has a large and highly glycosylated extracellular domain known to inhibit embryo implantation via steric hindrance. The role of MUC1 in the bovine endometrium remains to be explored. Herein, we used simple but reliable in vivo and in vitro experiments to investigate the expression and regulation of MUC1 in the bovine endometrium. MUC1 gene expression was analyzed in endometrial epithelial cells collected by the cytobrush technique using reverse transcription-quantitative polymerase chain reaction. MUC1 protein expression was evaluated by immunohistochemical analysis of endometrial samples collected from slaughtered cows. We used an in vitro cell culture model to study the regulation of MUC1 expression by treating cells with sex steroidal hormones or co-culturing cells with a blastocyst. The results revealed that MUC1 was expressed and localized to the apical surface of luminal epithelial cells in the bovine endometrium. MUC1 expression disappeared during the luteal phase of the estrous cycle and during pregnancy. 17ß-estradiol induced MUC1 expression, whereas progesterone inhibited its increase and co-culturing with blastocysts did not affect the expression. A long postpartum interval is a known risk factor for reduced fertility, and MUC1 expression was higher in this compromised condition. Our results demonstrated the MUC1 regulation by steroid hormones in bovine endometrium for embryo implantation, and we observed a negative correlation between MUC1 expression and fertility.

Krüppel-like factor 5 is upregulated and induces cell proliferation in endometrial cancer.

Krüppel-like factor 5 (KLF5) is involved in various cellular processes, such as cell proliferation and survival. KLF5 has been implicated in cancer pathology. The aim of the present study was to investigate the expression levels and function of KLF5 in endometrial cancer. A total of 30 patients, including 12 patients with endometrial cancer and 18 with benign gynecological diseases (controls), were enrolled at Tokyo Medical University (Tokyo, Japan) between March 2017 and May 2018. Endometrial cancer and control endometrium tissues were collected, and the expression levels of KLF5 were determined using reverse transcription-quantitative PCR, western blotting and immunohistochemistry. For the functional analyses of KLF5 in endometrial cancer, the present study employed a loss-of-function strategy in the human endometrial cancer cell lines in vitro. Ishikawa and HEC1 cells were transduced with lentiviral constructs expressing shRNAs targeting KLF5. MTT and TUNEL assays were performed in cells after knockdown to analyze the role of KLF5 in cell proliferation and survival. The results revealed that the mRNA and protein expression levels of KLF5 were increased in endometrial cancer tissues. In vitro analyses demonstrated that depletion of KLF5 inhibited cell proliferation and decreased the expression levels of cyclin E1. However, silencing KLF5 did not induce cell death. Overall, these results indicated that KLF5 may be crucial in the tumorigenesis of endometrial cancer and has potential as a therapeutic target.

SATB1 promotion of trophoblast stem cell renewal through regulation of threonine dehydrogenase.

BACKGROUND: Trophoblast stem (TS) cell renewal and differentiation are essential processes in placentation. Special AT-rich binding protein 1 (SATB1) is a key regulator of the TS cell stem state. In this study, we identified SATB1 downstream targets and investigated their actions. METHODS: RNA-sequencing analysis was performed in Rcho-1 TS cells expressing control or Satb1 short hairpin RNAs (shRNAs) to identify candidate SATB1 targets. Differentially regulated transcripts were validated by reverse transcription-quantitative polymerase chain reaction. The role of a target of SATB1, L-threonine 3-dehydrogenase (TDH), in the regulation of trophoblast cell development was investigated using a loss-of-function approach. RESULTS: Among the differentially regulated transcripts in SATB1 knockdown TS cells, were downregulated transcripts known to affect the TS cell stem state and upregulated transcripts characteristic of trophoblast cell differentiation. Tdh expression was exquisitely responsive to SATB1 dysregulation. Tdh expression was high in the TS cell stem state and decreased as TS cells differentiated. Treatment of Rcho-1 TS cells with a TDH inhibitor or a TDH specific shRNA inhibited cell proliferation and attenuated the expression of TS cell stem state-associated transcripts and elevated the expression of trophoblast cell differentiation-associated transcripts. TDH disruption decreased TS cell colony size, Cdx2 expression, and blastocyst outgrowth. CONCLUSIONS: Our findings indicate that the actions of SATB1 on TS cell maintenance are mediated, at least in part, through the regulation and actions of TDH. GENERAL SIGNIFICANCE: Regulatory pathways controlling TS cell dynamics dictate the functionality of the placenta, pregnancy outcomes, and postnatal health.

Pancreatic prolactin receptor signaling regulates maternal glucose homeostasis.

Prolactin (PRL) signaling has been implicated in the regulation of glucose homeostatic adaptations to pregnancy. In this report, the PRL receptor (Prlr) gene was conditionally disrupted in the pancreas, creating an animal model which proved useful for investigating the biology and pathology of gestational diabetes including its impacts on fetal and placental development. In mice, pancreatic PRLR signaling was demonstrated to be required for pregnancy-associated changes in maternal ß cell mass and function. Disruption of the Prlr gene in the pancreas resulted in fewer insulin producing cells, which failed to expand appropriately during pregnancy resulting in reduced blood insulin levels and maternal glucose intolerance. This inability to sustain normal blood glucose balance during pregnancy worsened with age and a successive pregnancy. The etiology of the insulin insufficiency was attributed to deficits in regulatory pathways controlling ß cell development. Additionally, the disturbance in maternal blood glucose homeostasis, was associated with fetal overgrowth and dysregulation of inflammation and prolactin-associated transcripts in the placenta. Overall, these results indicate that the PRLR, acting within the pancreas, mediates maternal pancreatic adaptations to pregnancy and therefore its dysfunction may increase a woman's chances of becoming glucose intolerant during pregnancy.

Origin of a rapidly evolving homeostatic control system programming testis function.

Mammals share common strategies for regulating reproduction, including a conserved hypothalamic-pituitary-gonadal axis; yet, individual species exhibit differences in reproductive performance. In this report, we describe the discovery of a species-restricted homeostatic control system programming testis growth and function. Prl3c1 is a member of the prolactin gene family and its protein product (PLP-J) was discovered as a uterine cytokine contributing to the establishment of pregnancy. We utilized mouse mutagenesis of Prl3c1 and revealed its involvement in the regulation of the male reproductive axis. The Prl3c1-null male reproductive phenotype was characterized by testiculomegaly and hyperandrogenism. The larger testes in the Prl3c1-null mice were associated with an expansion of the Leydig cell compartment. Prl3c1 locus is a template for two transcripts (Prl3c1-v1 and Prl3c1-v2) expressed in a tissue-specific pattern. Prl3c1-v1 is expressed in uterine decidua, while Prl3c1-v2 is expressed in Leydig cells of the testis. 5'RACE, chromatin immunoprecipitation and DNA methylation analyses were used to define cell-specific promoter usage and alternative transcript expression. We examined the Prl3c1 locus in five murid rodents and showed that the testicular transcript and encoded protein are the result of a recent retrotransposition event at the Mus musculus Prl3c1 locus. Prl3c1-v1 encodes PLP-J V1 and Prl3c1-v2 encodes PLP-J V2. Each protein exhibits distinct intracellular targeting and actions. PLP-J V2 possesses Leydig cell-static actions consistent with the Prl3c1-null testicular phenotype. Analysis of the biology of the Prl3c1 gene has provided insight into a previously unappreciated homeostatic setpoint control system programming testicular growth and function.

Defining the Role of Estrogen Receptor ß in the Regulation of Female Fertility.

Estrogens are essential hormones for the regulation of fertility. Cellular responses to estrogens are mediated by estrogen receptor α (ESR1) and estrogen receptor ß (ESR2). In mouse and rat models, disruption of Esr1 causes infertility in both males and females. However, the role of ESR2 in reproductive function remains undecided because of a wide variation in phenotypic observations among Esr2-mutant mouse strains. Regulatory pathways independent of ESR2 binding to its cognate DNA response element have also been implicated in ESR2 signaling. To clarify the regulatory roles of ESR2, we generated two mutant rat models: one with a null mutation (exon 3 deletion, Esr2ΔE3) and the other with an inframe deletion selectively disrupting the DNA binding domain (exon 4 deletion, Esr2ΔE4). In both models, we observed that ESR2-mutant males were fertile. ESR2-mutant females exhibited regular estrous cycles and could be inseminated by wild-type (WT) males but did not become pregnant or pseudopregnant. Esr2-mutant ovaries were small and differed from WT ovaries by their absence of corpora lutea, despite the presence of follicles at various stages of development. Esr2ΔE3- and Esr2ΔE4-mutant females exhibited attenuated preovulatory gonadotropin surges and did not ovulate in response to a gonadotropin regimen effective in WT rats. Similarities of reproductive deficits in Esr2ΔE3 and Esr2ΔE4 mutants suggest that DNA binding-dependent transcriptional function of ESR2 is critical for preovulatory follicle maturation and ovulation. Overall, the findings indicate that neuroendocrine and ovarian deficits are linked to infertility observed in Esr2-mutant rats.

Effect of organic and inorganic selenium supplementation on semen quality and blood enzymes in buffalo bulls.

The present study aimed to evaluate the effect of organic and inorganic selenium (Se) supplementation on semen quality and blood serum profiles of buffalo bulls. Nine mature buffalo bulls were divided into three groups: control (non-supplemented); organic Se (10 mg Sel-Plex®/head twice weekly) and inorganic Se (10 mg sodium selenite/head twice weekly). Semen was collected twice a week for 3 months during Se supplementation. Semen properties were evaluated from fresh ejaculate. Moreover, fructose concentration, aspartate and alanine transaminase (AST and ALT) activities, total protein and total cholesterol were assayed in seminal plasma. Additionally AST, ALT, testosterone and Se levels were determined in the blood serum. Results showed that Se supplementation significantly (P < 0.05) influences the semen parameters during 3 months of treatment. Organic Se significantly (P < 0.05) increased the percentage of viable sperms compared to inorganic Se and the control group. Fructose concentration was significantly higher (P < 0.05) in the seminal plasma of organic Se-treated bulls. Serum testosterone and Se concentrations were significantly (P < 0.05) increased in the Se supplemented groups than the control group. In conclusion, Se supplementation improved the parameters of buffalo bull semen and more precisely, organic Se was more effective for the improvement of semen quality and some blood components than inorganic Se.

Rethinking progesterone regulation of female reproductive cyclicity.

The progesterone receptor (PGR) is a ligand-activated transcription factor with key roles in the regulation of female fertility. Much has been learned of the actions of PGR signaling through the use of pharmacologic inhibitors and genetic manipulation, using mouse mutagenesis. Characterization of rats with a null mutation at the Pgr locus has forced a reexamination of the role of progesterone in the regulation of the female reproductive cycle. We generated two Pgr mutant rat models, using genome editing. In both cases, deletions yielded a null mutation resulting from a nonsense frame-shift and the emergence of a stop codon. Similar to Pgr null mice, Pgr null rats were infertile because of deficits in sexual behavior, ovulation, and uterine endometrial differentiation. However, in contrast to the reported phenotype of female mice with disruptions in Pgr signaling, Pgr null female rats exhibit robust estrous cycles. Cyclic changes in vaginal cytology, uterine histology, serum hormone levels, and wheel running activity were evident in Pgr null female rats, similar to wild-type controls. Furthermore, exogenous progesterone treatment inhibited estrous cycles in wild-type female rats but not in Pgr-null female rats. As previously reported, pharmacologic antagonism supports a role for PGR signaling in the regulation of the ovulatory gonadotropin surge, a result at variance with experimentation using genetic ablation of PGR signaling. To conclude, our findings in the Pgr null rat challenge current assumptions and prompt a reevaluation of the hormonal control of reproductive cyclicity.

CITED2 modulation of trophoblast cell differentiation: insights from global transcriptome analysis.

Trophoblast stem (TS) cells possess the capacity to differentiate along a multi-lineage pathway yielding several specialized cell types. The regulatory network controlling trophoblast cell differentiation is poorly understood. Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) has been implicated in the regulation of placentation; however, we know little about how CITED2 acts to influence trophoblast cells. Rat Rcho-1 TS cells can be manipulated to proliferate or differentiate into specialized trophoblast lineages and are an excellent model for investigating trophoblast differentiation. CITED2 transcript and protein showed a robust induction during Rcho-1 TS cell differentiation. We used an shRNA knockdown approach to disrupt CITED2 expression in order to investigate its involvement in trophoblast cell differentiation. RNA-sequencing was used to examine the impact of CITED2 on trophoblast cell differentiation. CITED2 disruption affected the differentiating trophoblast cell transcriptome. CITED2 possessed a prominent role in the regulation of cell differentiation with links to several signal transduction pathways and to hypoxia-regulated and coagulation processes. In summary, our findings indicate that CITED2 contributes to the regulation of trophoblast cell differentiation.

Dynamic Regulation of AP-1 Transcriptional Complexes Directs Trophoblast Differentiation.

Placentation is a process that establishes the maternal-fetal interface and is required for successful pregnancy. The epithelial component of the placenta consists of trophoblast cells, which possess the capacity for multilineage differentiation and are responsible for placenta-specific functions. FOS-like antigen 1 (FOSL1), a component of AP-1 transcription factor complexes, contributes to the regulation of placental development. FOSL1 expression is restricted to trophoblast giant cells and invasive trophoblast cells. In the present study, we characterized the FOSL1 regulatory pathway in rat trophoblast cells. Transcriptome profiling in control and FOSL1 knockdown cells identified FOSL1-dependent gene sets linked to endocrine and invasive functions. FOSL1 was shown to occupy AP-1 binding sites within these gene loci, as determined by chromatin immunoprecipitation (ChIP). Complementary in vivo experiments using trophoblast-specific lentiviral delivery of FOSL1 short hairpin RNAs (shRNAs) provided in vivo validation of FOSL1 targets. FOSL1 actions require a dimerization partner. Coimmunoprecipitation, coimmunolocalization, and ChIP analyses showed that FOSL1 interacts with JUNB and, to a lesser extent, JUN in differentiating trophoblast cells. Knockdown of FOSL1 and JUNB expression inhibited both endocrine and invasive properties of trophoblast cells. In summary, FOSL1 recruits JUNB to form AP-1 transcriptional complexes that specifically regulate the endocrine and invasive trophoblast phenotypes.

Neonatal Progesterone Programs Adult Uterine Responses to Progesterone and Susceptibility to Uterine Dysfunction.

In this report, we investigated the consequences of neonatal progesterone exposure on adult rat uterine function. Female pups were subcutaneously injected with vehicle or progesterone from postnatal days 3 to 9. Early progesterone exposure affected endometrial gland biogenesis, puberty, decidualization, and fertility. Because decidualization and pregnancy success are directly linked to progesterone action on the uterus, we investigated the responsiveness of the adult uterus to progesterone. We first identified progesterone-dependent uterine gene expression using RNA sequencing and quantitative RT-PCR in Holtzman Sprague-Dawley rats and progesterone-resistant Brown Norway rats. The impact of neonatal progesterone treatment on adult uterine progesterone responsiveness was next investigated using quantitative RT-PCR. Progesterone resistance affected the spectrum and total number of progesterone-responsive genes and the magnitude of uterine responses for a subset of progesterone targets. Several progesterone-responsive genes in adult uterus exhibited significantly dampened responses in neonatally progesterone-treated females compared with those of vehicle-treated controls, whereas other progesterone-responsive transcripts did not differ between female rats exposed to vehicle or progesterone as neonates. The organizational actions of progesterone on the uterus were dependent on signaling through the progesterone receptor but not estrogen receptor 1. To summarize, neonatal progesterone exposure leads to disturbances in endometrial gland biogenesis, progesterone resistance, and uterine dysfunction. Neonatal progesterone effectively programs adult uterine responsiveness to progesterone.

Expression and regulation of Foxa2 in the rat uterus during early pregnancy.

The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.

Adaptive mechanisms controlling uterine spiral artery remodeling during the establishment of pregnancy.

Implantation of the embryo into the uterus triggers the initiation of hemochorial placentation. The hemochorial placenta facilitates the acquisition of maternal resources required for embryo/fetal growth. Uterine spiral arteries form the nutrient supply line for the placenta and fetus. This vascular conduit undergoes gestation stage-specific remodeling directed by maternal natural killer cells and embryo-derived invasive trophoblast lineages. The placentation site, including remodeling of the uterine spiral arteries, is shaped by environmental challenges. In this review, we discuss the cellular participants controlling pregnancy-dependent uterine spiral artery remodeling and mechanisms responsible for their development and function.

Generation of Esr1-knockout rats using zinc finger nuclease-mediated genome editing.

Estrogens play pivotal roles in development and function of many organ systems, including the reproductive system. We have generated estrogen receptor 1 (Esr1)-knockout rats using zinc finger nuclease (ZFN) genome targeting. mRNAs encoding ZFNs targeted to exon 3 of Esr1 were microinjected into single-cell rat embryos and transferred to pseudopregnant recipients. Of 17 live births, 5 had biallelic and 1 had monoallelic Esr1 mutations. A founder with monoallelic mutations was backcrossed to a wild-type rat. Offspring possessed only wild-type Esr1 alleles or wild-type alleles and Esr1 alleles containing either 482 bp (Δ482) or 223 bp (Δ223) deletions, indicating mosaicism in the founder. These heterozygous mutants were bred for colony expansion, generation of homozygous mutants, and phenotypic characterization. The Δ482 Esr1 allele yielded altered transcript processing, including the absence of exon 3, aberrant splicing of exon 2 and 4, and a frameshift that generated premature stop codons located immediately after the codon for Thr157. ESR1 protein was not detected in homozygous Δ482 mutant uteri. ESR1 disruption affected sexually dimorphic postnatal growth patterns and serum levels of gonadotropins and sex steroid hormones. Both male and female Esr1-null rats were infertile. Esr1-null males had small testes with distended and dysplastic seminiferous tubules, whereas Esr1-null females possessed large polycystic ovaries, thread-like uteri, and poorly developed mammary glands. In addition, uteri of Esr1-null rats did not effectively respond to 17ß-estradiol treatment, further demonstrating that the Δ482 Esr1 mutation created a null allele. This rat model provides a new experimental tool for investigating the pathophysiology of estrogen action.

The FOS transcription factor family differentially controls trophoblast migration and invasion.

Extravillous trophoblast invasion is a fundamental component of human placentation. Invading trophoblast cells promote blood flow to the conceptus by actively remodeling the uterine vasculature. The extent of trophoblast invasion is tightly regulated; aberrant invasion is linked with several obstetrical complications. However, the transcriptional networks responsible for controlling the extent of trophoblast invasion are not well defined. Previous studies have identified high levels of FOS (FOS, FOSB, FOS-like (FOSL) 1, and FOSL2) proteins in extravillous trophoblast cells. These proteins form part of the activating protein-1 (AP-1) transcription factor complex and are implicated in regulating gene networks controlling cellular invasion in diverse biological systems. Therefore, we hypothesized that FOS family proteins play a role in regulating trophoblast invasion. We assessed expression of FOS family proteins in trophoblast cell lines and human placentae at different gestational ages. FOS, FOSB, and FOSL1 proteins were robustly increased in trophoblast cells subject to wound-based migration assays as well as Matrigel-based invasion assays. FOS knockdown resulted in cessation of proliferation and an induction of migration and invasion concomitant with robust expression of matrix metalloproteinase (MMP) 1, MMP3, and MMP10. Conversely, FOSL1 knockdown abrogated trophoblast migration and invasion and inhibited the production of MMP1, MMP3, and MMP10. In human placenta, FOS was expressed in proximal anchoring villi in conjunction with phospho-ERK. FOSL1 was temporally expressed only in the distal-most extravillous trophoblast cells, which represent a migratory cell population. Therefore, FOS and FOSL1 exert opposing effects on trophoblast invasion.

Regulatory pathways controlling the endovascular invasive trophoblast cell lineage.

Hemochorial placentation is characterized by trophoblast-directed uterine spiral artery remodeling. The rat and human both possess hemochorial placentation and exhibit remarkable similarities regarding the depth of trophoblast invasion and the extent of uterine vascular modification. In vitro and in vivo research methodologies have been established using the rat as an animal model to investigate the extravillous/invasive trophoblast lineage. With these research approaches, two signaling pathways controlling the differentiation and invasion of the trophoblast cell lineage have been identified: i) hypoxia/hypoxia inducible factor and ii) phosphatidylinositol 3-kinase/AKT/Fos like antigen 1. Dissection of these pathways has facilitated identification of fundamental regulators of the invasive trophoblast cell lineage.

SATB homeobox proteins regulate trophoblast stem cell renewal and differentiation.

The morphogenesis of the hemochorial placenta is dependent upon the precise expansion and differentiation of trophoblast stem (TS) cells. SATB homeobox 1 (SATB1) and SATB2 are related proteins that have been implicated as regulators of some stem cell populations. SATB1 is highly expressed in TS cells, which prompted an investigation of SATB1 and the related SATB2 as regulators of TS cells. SATB1 and SATB2 were highly expressed in rat TS cells maintained in the stem state and rapidly declined following induction of differentiation. SATB proteins were also present within the rat placenta during early stages of its morphogenesis and disappeared as gestation advanced. Silencing Satb1 or Satb2 expression decreased TS cell self-renewal and increased differentiation, whereas ectopic expression of SATB proteins promoted TS cell expansion and blunted differentiation. Eomes, a key transcriptional regulator of TS cells, was identified as a target for SATB proteins. SATB knockdown decreased Eomes transcript levels and promoter activity, whereas SATB ectopic expression increased Eomes transcript levels and promoter activity. Electrophoretic mobility shift assay as well as chromatin immunoprecipitation analyses demonstrated that SATB proteins physically associate with a regulatory site within the Eomes promoter. We conclude that SATB proteins promote TS cell renewal and inhibit differentiation. These actions are mediated in part by regulating the expression of the TS cell stem-associated transcription factor, EOMES.