Systems biology discoveries using non-human primate pluripotent stem and germ cells: novel gene and genomic imprinting interactions as well as unique expression patterns

The study of pluripotent stem cells has generated much interest in both biology and medicine. Understanding the fundamentals of biological decisions, including what permits a cell to maintain pluripotency, that is, its ability to self-renew and thereby remain immortal, or to differentiate into multiple types of cells, is of profound importance. For clinical applications, pluripotent cells, including both embryonic stem cells and adult stem cells, have been proposed for cell replacement therapy for a number of human diseases and disorders, including Alzheimer's, Parkinson's, spinal cord injury and diabetes. One challenge in their usage for such therapies is understanding the mechanisms that allow the maintenance of pluripotency and controlling the specific differentiation into required functional target cells. Because of regulatory restrictions and biological feasibilities, there are many crucial investigations that are just impossible to perform using pluripotent stem cells (PSCs) from humans (for example, direct comparisons among panels of inbred embryonic stem cells from prime embryos obtained from pedigreed and fertile donors; genomic analysis of parent versus progeny PSCs and their identical differentiated tissues; intraspecific chimera analyses for pluripotency testing; and so on). However, PSCs from nonhuman primates are being investigated to bridge these knowledge gaps between discoveries in mice and vital information necessary for appropriate clinical evaluations. In this review, we consider the mRNAs and novel genes with unique expression and imprinting patterns that were discovered using systems biology approaches with primate pluripotent stem and germ cells

Biological activity of Single chain Choriogonadotropin, hCGαβhCG \alpha \beta is decreased upon deletion of five Carboxyl terminal amino acids of the α\alpha subunit without affecting its receptor binding

The strategy of translationally fusing the two subunits of human Choriogonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C terminus of the a subunit is fused to the N terminus $\beta$ without any linker using Pichia pastoris expression system. The Pichia expressed $hCG\alpha \beta$ $(phCG\alpha \beta)$ attained an overall conformation similar to that of hCG, could bind to the receptor and elicit biological response suggesting that receptor binding and signal transduction can take place even with a molecule having blocked C-terminus of the $\alpha$ subunit. The carboxyl terminal of the $\alpha$ subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the $\alpha$ subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction in its ability to elicit biological response. These data show that these five amino acids at the C-terminus of the $\alpha$ subunit in the single chain hCG are not absolutely essential for attaining a conformation required for receptor binding, but are essential for obtaining a full biological response

Use of \alpha - and \delta -subunit specific antibodies in studying interaction of hCG with Leydig cell receptors

Antisera (a/s) raised to individual \alpha - and \delta -subunits of human chorionic gonadotropin (hCG) [9002-61-3] were characterized for specificity and used to study the disposition of the 2 subunits when intact hCG is complexed with LH [9002-67-9] receptor of the Leydig cells. The ability of the preformed hormone-antibody (H-Ab) complex to bind to receptor and stimulate a response; the ability of the a/s to dissoc. hCG from its complex with the receptor and thereby terminate response; and the ability of the premixed antibody and receptor to compete for binding of labeled hCG were examd. Although the subunit specific a/s were equipotent in binding hCG, their behavior once the receptor prepn. of Leydig cell is introduced into the system was drastically different. The \delta -subunit antibody relative to the \alpha -subunit antibody was poorly effective in preventing hCG from either binding to the receptor or inhibiting the continuation of response. Apparently, hCG upon interaction with the receptor loses the determinants specific to the \delta -region more rapidly than those specific to the \alpha -region suggesting that the initial interaction of hCG with the receptor occurs through sites in the \delta -subunit. Although the \alpha -subunit portion of the hCG mol. is available for binding to the antibody for a relatively longer time, the biol. response of the cell seems very sensitive to such binding with the antibody as it invariably results in loss of response. In the Leydig cell system, the ability of the a/s to bind hCG that is already complexed to the receptor appears dependent upon the time of addn. of the antibody to the incubation medium. The antisera were totally ineffective in inhibiting steroidogenic response to hCG if added 60 min after addn. of hCG. The hormone-receptor complex once formed, perhaps continues to change its orientation with the result that with time relatively less and less of antigenic determinants become available for antibody bindin

Steroidogenesis in the desensitized rat corpora-lutea

Earlier workers have observed that in the leydig cell desensitization brings results in addition to down regulation of receptors, in leisons in the steroidogenlc pathway. In the present study immature rats having heavily leutinized ovaries were given 50 iu hCG and the desenasitized CL removed 48h later were used. At that time no change in the 5 3MSD activity and CAMP binding activity(a measure of CAMP dependent protein kinase) was observed.Followlng desensitization however,l)a significent increase in phosphodiestrase activity,ii)a 50% reduction in total mitochondrial cholesterol level, iii)a significant reduction in its ability to utilize cholesterol or hydrolyse its ester and iv)a significant lowering(by 66%)in cholesterol side chain clean age activity(by measuring pregnanalone formed) was observed. Pregnanalone production was restored to normalcy if exogenous cholesterol was added to the mitohondrial preparation. The results suggest that luteal desensitization is due in addition to down regulation of LH receptors, to a marked reduction in available cholesterol pool in the mitochondrial compartment. The increase in phosphodiestrase activity, though probably a secondary effect,might effectively contribute to the overall reduction in the steroid out-put by increasing the catabolism of CAMP.(Aided by grants from ICMR,New Delhi and WHO, Geneva)

Hyperexpression of biologically active human chorionic gonadotropin using the methylotropic yeast, Pichia pastoris

Human chorionic gonadotropin (hCG), a heterodimeric glycoprotein hormone, is composed of an alpha subunit noncovalentlv associated with the hormone-specific beta subunit. The objective of the present study was recombinant expression of properly folded, biologically active hCG and its subunits using an expression system that could be used for structure-function studies while providing adequate quantities of the hormone for immunocontraceptive studies. We report here expression of biologically active hCG and its subunits using a yeast expression system, Pichia pastoris. The recombinant hGG alpha and hCG beta subunits were secreted into the medium and the levels of expression achieved at shake culture level were 24 and 2.7-3 mg/l secretory medium respectively. Go-expression of both subunits in the same cell resulted in secretion of heterodimeric hGG into the medium. The pichia-expressed hCG was immunologically similar to the native hormone, capable of binding to the LH receptors and stimulating a biological response in vitro. Surprisingly, the maximal response obtained was twice that obtained with the native hGG. The le level of expression of hCG achieved was 12-16 mg/l secretory medium and is expected to increase several-fold in a fermenter. Thus the Pichia expression system is capable of hyperexpressing properly folded, biologically active hGG and is suitable for structure-function studies of the hormone

Biological activity of single chain chorionic gonadotropin, HCG\alpha \beta, is decreased upon deletion of five carboxyl terminal amino acids of the \alpha subunit without affecting its receptor binding

The strategy of translationally fusing the two subunits of human chorionic gonadotropin (hCG) has been used to produce recombinant single chain hCG in which the C-terminus of the alpha subunit is fused to the N-terminus \beta without any linker using Pichia pastoris expression system. The Pichia-expressed hCG\alpha\beta (phCG\alpha\beta) attained an overall conformation similar to that of hCG, and could bind to the receptor and elicit biological response, suggesting that receptor binding and signal transduction can take place even with a molecule having blocked the C-terminus of the \alpha subunit. The carboxyl terminal of the \alpha subunit has been shown to be involved in hormone binding and signal transduction of all the heterodimeric glycoprotein hormones. However, deletion of five amino acids from the C-terminus of the alpha subunit in the single chain hCG did not alter the overall conformation of the fusion molecule and its receptor binding ability, but led to a significant reduction in its ability to elicit biological response. These data show that these five amino acids at the C-terminus of the alpha subunit in the single chain hCG are not absolutely essential for attaining a conformation required for receptor binding, but are essential for obtaining a full biological response

Specific immunoneutralization of FSH leads to apoptotic cell death of the pachytene spermatocytes and spermatogonial cells in the rat

Although requirement for follicle stimulating hormone (FSH) in the initiation of spermatogenesis is well documented, its role in adult spermatogenesis is still debated. In the present communication, we have investigated the effect of specific immunoneutralization of FSH on apoptotic cell death in the testicular germ cells both in immature and adult rats. The germ cells of control animals showed predominantly high molecular weight DNA while the antiserum (a/s) treated group showed DNA fragmentation characteristic of apoptosis. The pattern could be detected within 24 hours of a/s treatment, and became more pronounced after 48 hours. The germ cells were purified from FSH a/s treated rats by centrifugal elutriation and vulnerability of each cell type to undergo apoptosis on FSH neutralization was investigated. The pachytene spermatocytes were found to be most sensitive to absence of FSH, even in the adult animals suggesting the involvement of FSH in spermatogenesis. The in situ analysis of DNA strand breakage following FSH a/s treatment showed fragmentation of the DNA of the pachytene spermatocytes confirming this observation. The in situ analysis also showed that the spermatogonia undergo apoptosis in addition to the pachytene spermatocytes. These data clearly demonstrate the role of FSH in the adult rat spermatogenesis