Gbolagade O. Babalola

Aggregation of dispersed human cytotrophoblastic cells: lessons relevant to the morphogenesis of the placenta.

The syncytial trophoblast of the human placenta forms by the fusion of mononuclear cytotrophoblast cells. Cytotrophoblast cells only fuse with other trophoblastic cells, indicating a specificity to this interaction. To explore the cellular aggregation which precedes fusion, we examined the association of cytotrophoblast cells isolated from term placentae and JEG-3 choriocarcinoma cells, a cytotrophoblast-like cell line, in suspension culture. Cytotrophoblast cells were isolated by dispersion of chorionic villi in trypsin-DNase in Ca2+/Mg2(+)-free medium. JEG-3 cells were released from culture flasks by trypsinization in Versene-EDTA buffer. In suspension culture, each cell type aggregated forming tissue-like masses over a 24-hr period. Transmission electron microscope analysis demonstrated the formation of numerous desmosomes between the aggregated cells. In outgrowth culture, the aggregates created in suspension were maintained as microvilli-covered multicellular structures with hollow cores. The extent of aggregation was dependent upon the concentration of cells in the incubations with greater aggregation occurring with higher cell densities. Aggregation of both cytotrophoblast cells and JEG-3 cells progressed rapidly during the initial 10 hr of incubation and then continued at a slower rate. Aggregation took place in serum-containing and serum-free medium, but was impeded in Ca2+/Mg2(+)-free medium. Incubation of JEG-3 and cytotrophoblast cells in the presence of the protein synthesis inhibitor, cycloheximide, prevented aggregation, whereas the inhibitor of N-linked glycosylation, tunicamycin, did not. The inhibitor of RNA synthesis, actinomycin D, had no effect on the aggregation of the cells during the initial 6 hr of aggregation. These findings suggest that trypsin treatment in Ca2+/Mg2(+)-poor medium removed a protein(s) from the trophoblast cell surface which must be resynthesized for cell-cell association to take place.

Differentiation of Human Trophoblasts: Structure-Function Relationships

The human placenta and the chorion laeve are derived from the trophectoderm of the implanting blastocyst (1). During the process of implantation, the trophoblast cells replicate and invade into the uterine endometrium, initiating the formation of a hemochorial placenta. The trophoblast cells differentiate along several different pathways, becoming extravillous trophoblasts (sometimes called intermediate trophoblasts), extravillous multinucleated giant cells, columns of cytotrophoblasts that anchor the conceptus to the uterus, and floating chorionic villi. The chorionic villi form from avascular buds of cytotrophoblasts that develop into multilayered ramifications. The villi comprise an outer layer of syncytiotrophoblast overlying mononucleate cytotrophoblasts that are connected to each other and the syncytiotrophoblast by desmosomes. The cytotrophoblasts sit upon a basement membrane that encapsulates the villus core that contains blood vessels, macrophages, and mesenchymal elements. Each of the trophoblast phenotypes noted above displays characteristic functional properties that have been elucidated by immuno-cytochemical studies, in situ hybridization histochemistry, and analysis of isolated tissues and cells in vitro.

Sex steroid changes in porcine cystic ovarian disease

Ten sex steroids were measured in the peripheral serum and ovarian follicular fluid of female pigs with or without cystic ovarian disease. In general, progestin, especially progesterone, accumulated excessively in the fluid contained in cystic compared with normal follicles. Nonluteinized cystic follicles contained up to four times the progesterone concentration found in large normal preovulatory follicles. Levels of this steroid increased with luteinization of cystic follicles to as much as 10 times those found in large preovulatory follicles. In contrast, the concentration of follicular fluid androgens and estrogens in cystic follicles were, at best, barely detectable (5 to 10 pg/ml). These results are indicative of a steroidogenic blockade in the conversion of C21 progestin to C19 androgens and C18 estrogens in the cystic follicles. In spite of an enormous accumulation of follicular progestin and subnormal concentration of androgens and estrogens, circulating levels of these hormones in pigs bearing cystic ovaries were in the normal range for cycling sows. Clearly, the hormonal abnormalities in the cystic follicles are not reflected in the serum profiles of these steroids.

Developmental profile of serum androgens and estrous cyclicity of male and female rats exposed, perinatally, to maternally administered phenytoin

Pregnant rats were treated, daily, with either 10, 50 or 100 mg/kg of phenytoin-Na from day 17 of gestation through postpartum day 7. The male and female offspring exposed to the 2 higher doses of phenytoin had smaller body weights at birth than the diluent-treated rats, and this subnormal body weight gain persisted throughout the life of the affected animals. In contrast, the anticonvulsant produced no adverse effects on the developmental profile of serum androstenedione, testosterone and dihydrotestosterone or estrous cyclicity in the exposed male and female offspring, respectively. In spite of the normal concentrations of serum androgens, the seminal vesicles of the adult rats exposed to the 50 and 100 mg/kg doses of phenytoin were significantly smaller than the diluent-treated males.

Changes in the expression of cytochrome P450c17 associated with ovarian cystic follicles. An immunocytochemical and enzymatic analysis of porcine ovaries.

The steroidogenic activity of normal preovulatory and cystic follicles, and corpora lutea of porcine ovaries was investigated by immunocytochemical and radioenzymatic techniques. Using a specific antibody to porcine cytochrome P450c17, immunocytochemical staining was specifically localized in the theca interna layer of normal follicles and undetectable in the granulosa layer. The theca interna layers of non-luteinized cystic follicles were immunoreactive while those of luteinized follicles were not. Corpora lutea cells were essentially negative. The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase activity was similar in luteinized cystic follicular and corpora lutea tissues, which had 8 times higher activity than found in normal preovulatory follicles. The formation of either corpora lutea or luteinized cysts led to a profound decline (12- to 15-fold) in 17 alpha-hydroxylase and 17,20 lyase activities compared to normal preovulatory follicles. In agreement with these enzyme findings, radioimmunoassays revealed very high levels of progesterone with nearly undetectable levels of androgens in the luteinized cysts. These studies demonstrate the functional similarities between cells of luteinized cysts and those of normal corpora lutea and suggest a pathology associated suppression of P450c17 expression in porcine cystic follicles.

Purified Human Cytotrophoblasts: Surrogates for The Blastocyst in In Vitro Models of Implantation?

Although extensive studies on implantation have been performed with laboratory and domestic animals, it is difficult to extrapolate from these observations to the process of nidation in the human, because there are marked interspecies differences. Schlafke and Enders (1975), in an attempt to put some order in the diverse and oftentimes conflicting reports, proposed three possible modes for trophoblast implantation: fusion implantation, which is thought to take place in the rabbit, in which trophoblasts fuse with endometrial epithelial cells as the initial step in embryo-maternal interaction; displacement implantation which takes place in rodents, involves destruction of the uterine epithelium allowing the embryo to come into contact with the basal lamina prior to initiation of frank stromal invasion. The epithelial destruction is independent of die presence of the embryo and appears to be controlled by specific signals arising from the endometrial stoma. The third mode of implantation, intrusive implantation, appears to be operative in species with hemochoreal placentation, in which trophoblastic cells are markedly invasive. Intrusive implantation has been extensively examined in the ferret, where trophoblasts insinuate between endometrial epithelial cells prior to invading the basal lamina and endometrial stroma (Enders and Schlafke, 1972; Gulamhusein and Beck, 1973). Based on existing morphological observations, intrusive implantation has been proposed to be the mode of nidation in the human. However, lack of in vitro models for human embryo implantation has hindered our ability to study this process at the cellular and molecular level.

Human Trophoblast Differentiation

The human placenta is a dynamic organ which performs vital structural and metabolic functions during pregnancy. The trophoblastic component of this organ, which is derived from the trophectoderm of the blastocyst (1,2), is responsible for many of these specialized roles. Trophoblasts assume different morphological forms and functions during pregnancy and their phenotype appears to be linked, at least in part, to their physical location. Trophoblastic cells invading the uterus can be either mononucleate (X cells or intermediate trophoblasts; 3,4,5) or multinucleated (giant wandering cells). These cells display a distinctive pattern of gene expression which has been mapped to some extent by immunocytochemical analysis of placental bed biopsies (6,7). Trophectoderm of the implanting blastocyst which faces the uterine lumen evolves into the chorion laeve, which contains mononucleate trophoblast cells. These cells also have an unique pattern of gene expression. The chorionic villi of the placenta consist of an outer layer of syncytial trophoblast overlying a layer of mononuclear cytotrophoblasts which sit ontop of a basement membrane encapsulating the core of the villus which contains capillaries, supporting cells and macrophages (Hoffbauer cells). The functional characteristics of the cytotrophoblasts and syncytial trophoblasts differ markedly as revealed by immunocytochemical and in situ hybridization histochemistry analysis of placental tissue (8). For example, cytotrophoblasts appear to be the site of synthesis of a variety of neural peptides including somatostatin, gonadotropin releasing hormone and corticotropin releasing hormone, whereas the syncytial trophoblast is enriched in enzymes participating in steroid hormone synthesis and is the primary site of chorionic gonadotropin (CG), chorionic somatomammotropin (CS) (9,10) and variant growth hormone production.