We report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP2). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP2 amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue aminoterminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP2. The cDNA insert hybridizes with 3.2-and 1.8-kb mRNA species in human liver poly(A)+ RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP2 gene in the human genome or that the SCP2 gene is very large. Coexpression of the SCP2 cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These min are concordant with the notion that SCP2 plays a role in regulating steroidogenesis, among other possible functions.Sterol carrier protein 2 (SCP2), also known as nonspecific lipid transport protein, is a 13.2-kDa basic polypeptide which is believed to facilitate the movement of sterols and phospholipids within cells (1-3). SCP2 has been purified to homogeneity from rat, bovine, and human liver (4-6). The amino acid sequences ofthe rat and bovine liver polypeptides have been determined, revealing homology (>90% identity) (4-7). The amino acid composition of the human liver polypeptide is very similar to that of the rat and cow, suggesting a high degree of conservation across species (8). Antibodies raised against rat liver SCP2 recognize the 13.2-kDa polypeptide as well as 40-and 58-kDa proteins, which are presumably related (8). The 58-kDa protein localizes to peroxisomes, a finding concordant with the presence of a tripeptide sequence (Ala-Lys-Leu) related to the peroxisomal targeting sequence (Ser-Lys-Leu) in the carboxyl terminus of the SCP2 molecule (9). It is noteworthy that SCP2 is present in low levels in subjects with Zellweger syndrome (cerebrohepatic-renal syndrome), whose cells are deficient in peroxisomes and who have an associated impairment in plasmalogen and bile acid synthesis and catabolism of phytanic acid and very-long-chain fatty acids (8).In steroid hormone producing cells SCP2 is thought to facilitate the transport of cholesterol to mitochondria, where the first committed step in steroidogenesis takes place (1-3). The evidence supporting this line of thinking is primarily based on the analysis of the effects of purified SCP2 on isolated organelles (e.g., mitochondria) and the use of antibodies to neutralize SCP2 activity in cytosolic preparations (10-12).cDNAs encod...

Ovaries were obtained from sows immediately after slaughter and were morphologically assigned to different stages of the ovarian cycle. Follicular fluid contained in the predominating follicles was analysed for ten steroid hormones by a multiple, simultaneous radioimmunoassay technique. The steroids measured were pregnenolone, progesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone, oestrone and oestradiol-17 beta. The concentrations of the steroids remained relatively low during the luteal stages until the mid-follicular stage when ovaries contained predominantly small to medium-sized (less than 5.0 mm in diam.) follicles. With the exception of 5 alpha-dihydrotestosterone concentrations, which remained low regardless of the size of the follicles or the stage of the cycle, the concentrations of all the steroids were significantly elevated in the transition from the mid- to late follicular stage, a period when the ovaries contained mainly large (6-10 mm diam.) follicles. Follicles at the ovulatory stage exhibited a profound decline in the concentrations of androgens and oestrogens. In contrast, the magnitude of decline in the levels of 3 progestagens, i.e. pregnenolone, progesterone and 17 alpha-hydroxyprogesterone, was much less than that for androgens and oestrogens, while the concentration of 20 alpha-dihydroprogesterone was actually elevated in the ovulatory follicles. The present results agree with those of earlier studies which measured fewer steroids in follicles obtained by repeated and sequential laparotomies of sows during spontaneous cycles. In contrast, these hormone results differ from those using the PMSG/hCG-stimulated ovary, suggesting that such ovaries may not be a completely valid model for ovarian steroid hormone metabolism in the normally cycling sow.

The morphologic and functional differentiation of human trophoblast cells culminates in the formation of the terminally differentiated multinucleated syncytial trophoblast. In culture, isolated mononuclear cytotrophoblasts aggregate and then fuse to form syncytia, recapitulating the in vivo process. In the present studies, we investigated the expression of the Ca(2+)-dependent cell adhesion molecule (CAM), E-cadherin, during the morphologic differentiation of trophoblastic cells. Cytotrophoblasts were isolated from human chorionic villi, and JEG-3 and BeWo choriocarcinoma cells, cytotrophoblastic cell lines which under standard culture conditions are not fusion competent, were obtained by dispersion of ongoing cultures. Cultures were terminated at timed intervals and E-cadherin was analyzed by immunocytochemistry and electron microscopy using specific antibodies. In addition, E-cadherin expression was investigated by western and northern blotting. During the aggregation of cytotrophoblasts, E-cadherin was localized on the cell surface at points of cell-cell contact and could not be demonstrated following cellular fusion. In contrast, it remained on the surface of aggregated JEG-3 and BeWo cells throughout the duration of culture. Western blot analysis revealed a time-dependent increase in E-cadherin (120 × 10(3) Mr) which coincided with maximal aggregate formation at 24 h in both normal cytotrophoblasts and JEG-3 cells. A marked reduction of E-cadherin in fusing cytotrophoblasts was subsequently observed as syncytial trophoblasts became the predominant cellular form in culture. In agreement with the immunohistochemical observations, there was no change in E-cadherin levels in the non-fusing JEG-3 cells. Northern blotting demonstrated a significant reduction in the 4.5 kb transcript in fusion-competent cells over the 96 h of culture. Exposure of the normally non-fusing BeWo cells to 1.5 mM 8-bromo cyclic AMP induced cellular fusion and syncytium formation. This process was accompanied by a disappearance of E-cadherin from the cell surface as assessed by immunocytochemistry and western blotting and a parallel reduction in the abundance of the E-cadherin mRNA. Immunoneutralization experiments using an antiserum directed against the extracellular domain of cadherins inhibited syncytium formation in normal trophoblasts compared to an antiserum against the E-cadherin cytoplasmic tail, which had no effect upon aggregation and fusion of these cells. We conclude that E-cadherin exists in a dynamic state in fusion-competent cytotrophoblasts and that down regulation of its gene expression coincides with cellular fusion. In addition, this process appears to be cyclic AMP-mediated in BeWo choriocarcinoma cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of growth factors on regulating gene expression in the preimplantation mouse embryo was examined, since results of previous experiments revealed a stimulatory effect of exogenously-added growth factors on preimplantation development in vitro. Treatment of early cavitating blastocysts with either 250 pM TGF-alpha or TGF-beta results in changes in the pattern of total protein synthesis as assessed by high-resolution two-dimensional gel electrophoresis. In some cases, the synthesis of a particular polypeptide is either up- or downregulated by each growth factor, whereas in other instances the synthesis of a polypeptide is modulated by one but not the other growth factor. Use of the mRNA differential display method permitted the identification of genes whose expression is either up- or downregulated by these growth factors. Treatment of mouse blastocysts with either TGF-alpha or TGF-beta results in the increased expression of the b subunit of the F0ATPase. TGF-beta also stimulates the expression of the DNA polymerase alpha. TGF-alpha treatment results in the increase in expression of a gene homologous to the human HEPG2 cDNA, as well as in a decrease in expression of fibronectin.

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