Damayanti Chakraborty

Natural killer cells direct hemochorial placentation by regulating hypoxia-inducible factor dependent trophoblast lineage decisions

Natural killer (NK) cells are recruited into the uterine stroma during establishment of the hemochorial placenta and are proposed regulators of uterine spiral artery remodeling. Failures in uterine spiral artery remodeling are linked to diseases of pregnancy. This prompted an investigation of the involvement of NK cells in placentation. NK cell depletion decreased the delivery of proangiogenic factors and delayed uterine spiral artery development, leading to decreased oxygen tension at the placentation site, stabilized hypoxia-inducible factor 1A protein, and redirected trophoblast differentiation to an invasive phenotype. Trophoblast cells replaced the endothelium of uterine spiral arteries extending the depth of the placental vascular bed and accelerating vessel remodeling. Hypoxia-regulated trophoblast lineage decisions, including expansion of invasive trophoblast, could be reproduced in vitro by using rat trophoblast stem cells and were dependent on hypoxia-inducible factor signaling. We conclude that NK cells guide hemochorial placentation through controlling a hypoxia-sensitive adaptive reflex regulating trophoblast lineage decisions.

FGF4-DEPENDENT STEM CELLS DERIVED FROM RAT BLASTOCYSTS DIFFERENTIATE ALONG THE TROPHOBLAST LINEAGE

Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines not only possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation.

SATB homeobox proteins regulate trophoblast stem cell renewal and differentiation

Background: Trophoblast cells, the functional components of the placenta, are derived from multipotent trophoblast stem (TS) cells. Results: SATB homeobox proteins regulate the TS cell stem state through up-regulation of a stem-specific transcription factor, EOMES, and inhibition of trophoblast differentiation. Conclusion: SATB proteins regulate TS cell development. Significance: Understanding TS cell biology is crucial to determining processes underlying placental development. 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.

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 17β-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.

OVO-like 1 regulates progenitor cell fate in human trophoblast development

Significance Epithelial barrier integrity is dependent on progenitor cells that either divide to replenish themselves or differentiate into a functional epithelium. In the placenta, cytotrophoblast cells comprise this progenitor population, but the differentiation program they undertake is unlike any other in human tissues: acquisition of hormonogenesis and cell fusion to form a syncytialized (syncytio)trophoblast. Syncytiotrophoblast forms the primary epithelial barrier separating maternal and fetal tissue and performs functions vital for pregnancy. In the present study, we found that OVO-like 1 (OVOL1), a transcription factor homolog of Drosophila ovo, regulates the transition between progenitor and differentiated cytotrophoblast. It does so by repressing genes that maintain cytotrophoblast progenitor traits. This study provides insight into the role of OVOL1 in human trophoblast development. Epithelial barrier integrity is dependent on progenitor cells that either divide to replenish themselves or differentiate into a specialized epithelium. This paradigm exists in human placenta, where cytotrophoblast cells either propagate or undergo a unique differentiation program: fusion into an overlying syncytiotrophoblast. Syncytiotrophoblast is the primary barrier regulating the exchange of nutrients and gases between maternal and fetal blood and is the principal site for synthesizing hormones vital for human pregnancy. How trophoblast cells regulate their differentiation into a syncytium is not well understood. In this study, we show that the transcription factor OVO-like 1 (OVOL1), a homolog of Drosophila ovo, regulates the transition from progenitor to differentiated trophoblast cells. OVOL1 is expressed in human placenta and was robustly induced following stimulation of trophoblast differentiation. Disruption of OVOL1 abrogated cytotrophoblast fusion and inhibited the expression of a broad set of genes required for trophoblast cell fusion and hormonogenesis. OVOL1 was required to suppress genes that maintain cytotrophoblast cells in a progenitor state, including MYC, ID1, TP63, and ASCL2, and bound specifically to regions upstream of each of these genes. Our results reveal an important function of OVOL1 as a regulator of trophoblast progenitor cell fate during human trophoblast development.

NK cells, hypoxia and trophoblast cell differentiation

Hemochorial placentation is characterized by extensive remodeling of the maternal vasculature, converting them to flaccid low resistance vessels. This process greatly facilitates exchange of nutrients and gases between the mother and the fetus. Two key modulators that orchestrate these vascular changes have been identified at the maternal fetal interface, natural killer (NK) cells and invasive trophoblast cells. Hypoxia-inducible factor (HIF) transcription factors direct cellular responses to low oxygen, influencing trophoblast lineage commitment and promoting development of the invasive trophoblast lineage. This short review focuses on role of NK cells on uterine spiral artery development and subsequent modulation of oxygen tensions at the maternal fetal interface.

HIF-KDM3A-MMP12 regulatory circuit ensures trophoblast plasticity and placental adaptations to hypoxia

Significance The hemochorial placenta is a dynamic structure endowed with responsibilities controlling the extraction of maternal resources, ensuring fetal development and preserving maternal health. A healthy placenta exhibits plasticity and can adapt to environmental challenges. Such adaptations can be executed through instructive actions on trophoblast stem cells, influencing their abilities to expand and differentiate into specialized cells that accommodate the challenge. Hypoxia, when appropriately timed, promotes invasive trophoblast-directed uterine spiral artery remodeling. Hypoxia activates hypoxia inducible factor-dependent expression of lysine demethylase 3A, modifying the histone landscape on key target genes, including matrix metallopeptidase 12, which acts to facilitate trophoblast invasion and uterine vascular remodeling. Plasticity and adaptations at the maternal–fetal interface safeguard placental development and the healthy progression of pregnancy. The hemochorial placenta develops from the coordinated multilineage differentiation of trophoblast stem (TS) cells. An invasive trophoblast cell lineage remodels uterine spiral arteries, facilitating nutrient flow, failure of which is associated with pathological conditions such as preeclampsia, intrauterine growth restriction, and preterm birth. Hypoxia plays an instructive role in influencing trophoblast cell differentiation and regulating placental organization. Key downstream hypoxia-activated events were delineated using rat TS cells and tested in vivo, using trophoblast-specific lentiviral gene delivery and genome editing. DNA microarray analyses performed on rat TS cells exposed to ambient or low oxygen and pregnant rats exposed to ambient or hypoxic conditions showed up-regulation of genes characteristic of an invasive/vascular remodeling/inflammatory phenotype. Among the shared up-regulated genes was matrix metallopeptidase 12 (MMP12). To explore the functional importance of MMP12 in trophoblast cell-directed spiral artery remodeling, we generated an Mmp12 mutant rat model using transcription activator-like nucleases-mediated genome editing. Homozygous mutant placentation sites showed decreased hypoxia-dependent endovascular trophoblast invasion and impaired trophoblast-directed spiral artery remodeling. A link was established between hypoxia/HIF and MMP12; however, evidence did not support Mmp12 as a direct target of HIF action. Lysine demethylase 3A (KDM3A) was identified as mediator of hypoxia/HIF regulation of Mmp12. Knockdown of KDM3A in rat TS cells inhibited the expression of a subset of the hypoxia–hypoxia inducible factor (HIF)-dependent transcripts, including Mmp12, altered H3K9 methylation status, and decreased hypoxia-induced trophoblast cell invasion in vitro and in vivo. The hypoxia-HIF-KDM3A-MMP12 regulatory circuit is conserved and facilitates placental adaptations to environmental challenges.

Defining the Role of Estrogen Receptor β 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 β (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.

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.