Rupasri Ain

Gestation stage-dependent intrauterine trophoblast cell invasion in the rat and mouse: novel endocrine phenotype and regulation

Trophoblast cell invasion into the uterine wall is characteristic of hemochorial placentation. In this report, we examine trophoblast cell invasion in the rat and mouse, the endocrine phenotype of invasive trophoblast cells, and aspects of the regulation of trophoblast cell invasion. In the rat, trophoblast cells exhibit extensive interstitial and endovascular invasion. Trophoblast cells penetrate through the decidua and well into the metrial gland, where they form intimate associations with the vasculature. Trophoblast cell invasion in the mouse is primarily interstitial and is restricted to the mesometrial decidua. Both interstitial and endovascular rat trophoblast cells synthesize a unique set of prolactin (PRL)-like hormones/cytokines, PRL-like protein-A (PLP-A), PLP-L, and PLP-M. Invading mouse trophoblast cells also possess endocrine activities, including the expression of PLP-M and PLP-N. The trafficking of natural killer (NK) cells and trophoblast cells within the mesometrial uterus is reciprocal in both the rat and mouse. As NK cells disappear from the mesometrial compartment, a subpopulation of trophoblast cells exit the chorioallantoic placenta and enter the decidua. Furthermore, the onset of interstitial trophoblast cell invasion is accelerated in mice with a genetic deficiency of NK cells, Tg epsilon 26 mice, implicating a possible regulatory role of NK cells in trophoblast cell invasion. Additionally, the NK cell product, interferon-gamma (IFNgamma), inhibits trophoblast cell outgrowth, and trophoblast cell invasion is accelerated in mice with a genetic deficiency in the IFNgamma or the IFNgamma receptor. In summary, trophoblast cells invade the uterine wall during the last week of gestation in the rat and mouse and possess a unique endocrine phenotype, and factors present in the uterine mesometrial compartment modulate their invasive behavior.

Interleukin-11 signaling is required for the differentiation of natural killer cells at the maternal–fetal interface

Interleukin‐11 (IL‐11) is a multifunctional hematopoietic growth factor that has been implicated in the control of reproduction. Studies on IL‐11 receptor‐α (IL‐11Rα) ‐deficient mice showed that female mice are infertile due to defective decidualization. In this report, we evaluated the development of decidual cells, immune cells, and the vasculature associated with the implantation site of IL‐11Rα–deficient mice; with the aim of better understanding the nature of the fertility defect. Messenger RNAs for decidual differentiation, such as decidual prolactin‐related protein and prolactin‐like protein‐J are expressed in the IL‐11Rα mutant. However, the number of decidual cells expressing these genes is decreased in the mutant compared with the wild‐type control. Although, trophoblast cells differentiate and express placental lactogen‐I in the IL‐11Rα–deficient uterine environment, they fail to progress and expand in number. Defects in the organization of the decidual vasculature were also apparent in the IL‐11Rα mutant uterus. The most dramatic effect of IL‐11 signaling was on the hematopoietic environment of the uterine decidua. Differentiated/perforin‐expressing uterine natural killer (NK) cells were virtually absent from implantation sites of IL‐11Rα mutant mice. NK cell precursors were capable of homing to the IL‐11Rα–deficient uterus and a known regulator of NK cell differentiation; IL‐15 was expressed in the IL‐11Rα mutant uterus. Splenic NK cells from IL‐11Rα mutant mice were also able to respond to IL‐15 in vitro. Thus, the defect in NK precursor cell maturation was not intrinsic to the NK precursor cells but was dependent upon the tissue environment. In summary, IL‐11 signaling is required for decidual‐specific maturation of NK cells. Developmental Dynamics 231:700–708, 2004.

Prolactin-like protein-A is a functional modulator of natural killer cells at the maternal-fetal interface.

Natural killer (NK) cells are the predominant lymphocytes present in healthy rodent and human implantation sites. In the rat, the expansion, differentiation and subsequent migration of NK cells away from the developing chorioallantoic placenta coincide with the expression of a novel pregnancy- and trophoblast cell-specific cytokine, prolactin (PRL)-like protein A (PLP-A). PLP-A specifically binds to uterine NK cells but does not appear to utilize receptor systems for PRL. In the present report, we show that PLP-A interactions with NK cells are not mediated by receptors utilized by known modulators of NK cell function, including interleukin-2, interleukin-7, interleukin-12, and interleukin-15 (IL-15). Uterine NK cells respond to PLP-A or IL-15 with an increase in intracellular calcium mobilization. In contrast, PLP-A, unlike IL-15, effectively suppresses the ability of NK cells to produce interferon-gamma (IFNgamma), a key mediator of NK cell function. Placental PLP-A expression is reciprocal to mesometrial decidua expression of IFNgamma. Increased expression of PLP-A by the placenta coincides with the decline of IFNgamma content in the mesometrial decidua adjacent to the placenta. In summary, trophoblast cell-derived PLP-A contributes to the regulation of NK cells at the maternal-fetal interface to ensure appropriate embryonic growth and development.

The rat prolactin gene family locus: species-specific gene family expansion

In the rat there is a large family of paralogous genes related to prolactin (PRL). Members of the PRL family are expressed in cell- and temporal-specific patterns in the anterior pituitary, uterus, and placenta. An overriding feature of the PRL family is its association with pregnancy. In this investigation, we used information derived from the public rat genome database as a tool for identifying new members of the rat PRL family. The entire rat PRL gene family locus spans approximately 1.7 megabases (Mb) on Chromosome 17. Genes possessed either 5- or 6-exon organization patterns. We provide information on three newly identified genes orthologous to previously identified members of the mouse PRL gene family [placental lactogen-Iα (PL-Iα), PL-Iβ, and proliferin (PLF)] and a new member of the PRL family, termed PRL-like protein-P (PLP-P). Information is also presented on the existence of multiple PLP-M transcripts, which are generated by alternative splicing. Expansion of the PRL family has occurred independently in rodents versus the cow and does not exist in the human and dog. Elucidation of the rat PRL gene family locus provides tools for studying the genetics and biology of the rat PRL family and new insights into species-specific gene family expansion.

Phenotypic Analysis of the Rat Placenta

The rat is an important model for studying the biology of trophoblast-uterine development. This chapter describes methods that are useful for the characterization of the rat uteroplacental compartment.

Intrauterine Fate of Invasive Trophoblast Cells

Invasion of trophoblast cells into the uterine spiral arteries and the uterine wall is characteristic of hemochorial placentation. In the rat, trophoblast cells penetrate through the uterine decidua and well into the metrial gland. In this report, we examined the fate of these invasive trophoblast cells following parturition. Invasive trophoblast endocrine cells were retained in the postpartum mesometrial uterus in the rat. The demise of invasive trophoblast cells was followed by the appearance of differentiated smooth muscle cells surrounding blood vessels previously lined by invasive trophoblast cells and an infiltration of macrophages. Regulation of intrauterine trophoblast cell fate was investigated following premature removal of the fetus or removal of the fetus and chorioallantoic placenta. The presence of the fetus affected the distribution of invasive trophoblast cells within the uterus but did not negatively impact their survival. Premature removal of all chorioallantoic placentas and associated fetuses from a uterus resulted in extensive removal of intrauterine trophoblast cells. In summary, the postpartum demise of intrauterine invasive trophoblast cells is a dynamic developmental event regulated in part by the removal of trophic signals emanating from the chorioallantoic placenta.

Noggin maintains pluripotency of human embryonic stem cells grown on Matrigel

Objective:  Spontaneous differentiation of human embryonic stem cell (hESC) cultures is a major concern in stem cell research. Physical removal of differentiated areas in a stem cell colony is the current approach used to keep the cultures in a pluripotent state for a prolonged period of time. All hESCs available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Under experimental conditions, stem cells are grown on various matrices, the most commonly used being Matrigel.

A simple method for the in situ detection of eosinophils.

A simple and rapid method for identifying eosinophils in tissue sections is described. The assay is based on the incubation of frozen tissue sections with phenol red and the detection of cellular fluorescence. A one-to-one relationship was observed between cells exhibiting fluorescence following exposure to phenol red and eosinophils as identified by histochemical detection of eosinophil peroxidase (EPO) activity. Using the phenol red assay, eosinophils were detected in various eosinophil-infiltrated tissues, including uteri of rats from day 1 of pregnancy and uteri of prepubertal estrogen-treated rats. Intensity of the fluorescing eosinophils was dependent upon phenol red concentration and duration of incubation. The phenol red method of eosinophil identification was disrupted by co-incubation with resorcinol, an EPO inhibitor, or catalase, a hydrogen peroxide scavenger. The merits of this assay are its simplicity and compatibility with other procedures, such as histochemistry, immunocytochemistry, and in situ ligand-receptor binding.

Hypobaric Hypoxia as a Tool to Study Pregnancy-Dependent Responses at the Maternal-Fetal Interface

Establishment of proper oxygen and nutrient supply to the fetus is essential for a successful pregnancy. The maternal-fetal interface is the site of vascular modifications, providing a conduit for the delivery of essential nutrients to the developing fetus. Pregnancy-dependent adaptive vascular responses within the uteroplacental compartment can be exaggerated by exposure to a physiological stressor such as hypoxia. A simple procedure for exposing pregnant rats and mice to hypobaric hypoxia is presented.

Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling

Endothelial nitric-oxide synthase (eNOS) and its bioactive product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. Moreover, genetic disruption or pharmacological inhibition of eNOS attenuates angiogenesis during tissue repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRINs function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of secreted cytokines involved in inflammatory responses, and ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFκB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF-α-induced NFκB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRIN-TRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression leading to deactivation of the NFκB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression.