Browsing by Subject "Leydig cells"
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Item Function and regulation of CCAAT/enhancer binding protein beta in Leydig cell development and steroidogenesis(Texas Tech University, 2002-08) Nalbant, DemetPituitary luteinizing hormone (LH) is required for the development of multiple cell types in both the male and female reproductive systems. In particular, LH promotes differentiation of testicular Leydig cells and stimulates steroid production by Leydig cells in the testis, theca, granulosa and luteal cells in the ovary. We have been interested in identifying nuclear transcription factors that are targets of LH signaling pathways in Leydig cells and concentrated on CCAAT/enhancer binding protein beta (C/EBPp). Our initial studies showed that C/EBPp is expressed in a differentiation specific pattern whose expression is stimulated by LH/human chorionic gonadotropin (hCG) and cyclic AMP (cAMP) in Leydig cells. We hypothesized that C/EBPp plays an important role in LH regulated Leydig cell development and steroidogenic function. To assess the specific roles of C/EBPp in Leydig cell function we have analyzed the steroidogenic capacity of Leydig cells from C/EBPp-deficient mice generated by gene targeting. This study revealed that testosterone production in male C/EBPp deficient mice is severally compromised suggesting that C/EBPp is essential for complete functional differentiation of Leydig cells. In order to understand how LH effects on Leydig cell differentiation and/or function may be mediated through C/EBPp, we attempted to identify genetic control elements that control C/EBPp transcription in steroidogenic and non-steroidogenic cells. We identified an evolutionarily conserved, steroidogenic cell-specific, distal enhancer element located in the C/EBPp 5'-flanking region. Our studies suggest that the activity of the enhancer may be, at least in part, controlled by as yet uncharacterized nuclear factors specifically detected in nuclear extracts of steroidogenic cells. These studies set the stage for elucidation of the molecular mechanisms controlling C/EBPp expression in steroidogenic cells in general, and may aid in uncovering alternative LH-dependent signaling pathways critical for functional maturation of Leydig cellsItem Leydig cell development in boars(Texas Tech University, 1997-12) Geiger, Tammy LynnThree waves of Leydig cell development are found in the pig testes Clearly defined periods of cell proliferation and differentiation occur during fetal, perinatal and pre-pubertal periods. Luteinizing hormone (LH) regulates Leydig cell proliferation via growth factors in the rat. However, it is not known how LH affects Leydig cell development in the neonatal pig testis. The purpose of the study was to identify growth factors involved in Leydig cell proliferation in neonatal pigs In this study we investigated the effects of hCG and the following growth factors; EGF (TGFa), SEP, IL-lp, IGF-1 and TGFP in vitro on DNA synthesis in neonatal Leydig cells. Leydig cells were isolated from 3- to 5-day-old pigs using coUagenase dispersion and a Percoll® gradient centrifligation. The cells were then cultured for 48 hours in serum free media, washed and treated with growth factors and or hCG for 18 hours. Tritiated thymidine incorporation into DNA was assessed over a subsequent four hour incubation period. Incorporation of ['^Hj-thymidine was stimulated by hCG treatment with a 2.3-fold increase over control cultures. SIP also induced a significant increase ( p <.0001) in the incorporation of ["'HJ-thymidine into Leydig cell DNA. Similarly, EGF and IGF-1 also increased DNA synthesis in porcine Leydig cells. When IGF-1 and SIP were added together, they had an additive effect on Leydig cell DNA synthesis. TGFp had very little, if any, etTect on DN.A synthesis when added alone, but inhibited the stimulatory effects of other growth tactor and mitogens (SIP, hCG, EGF, and IGF-1) on DN.A. synthesis Our results indicate thai these growth factors may play a role in proliferation of Leydig cells during the perinatal period of development.Item Probing the characteristics of constitutive steroid biosynthesis in R2C Leydig tumor cells(Texas Tech University, 2004-12) Jo, YoungahThe Steroidogenic Acute Regulatory (StAR) protein plays an important role in the acute regulation of steroid hormone biosynthesis through its function in mediating cholesterol transfer to the inner mitochondrial membrane where the cholesterol side chain cleavage enzyme system resides. The StAR protein was identified and cloned in 1994, a process simplified through studies in two different Leydig tumor cell lines: MA-10 and R2C cells. MA-10 cells synthesize steroids and StAR protein only following trophic hormone stimulation, but R2C cells produce abundant steroids and StAR protein in a constitutive manner. In this work, we attempted to identify the differential regulation of steroidogenesis in both cell lines and to explore the mechanism(s) behind the highly active steroidogenic phenotype in R2C cells. In studying the alterations in cholesterol homeostasis found in the R2C cell, we first examined the levels of cholesterol esters and free cholesterol. The results indicated that R2C mitochondria were four-fold enriched in free cholesterol content when compared to MA-10 mitochondria. In support of this observation, in addition to the high levels of StAR protein found in R2C cells, R2C cells express much higher levels of SR-BI (the physiological receptor for the binding and uptake of HDL cholesterol in steroidogenic tissues of rodents), and thus have a higher capacity to take up extracellular cholesteryl esters when compared to MA-10 cells. R2C cells also show enhanced expression of Hormone Sensitive Lipase, the enzyme involved in hydrolysis of cholesteryl esters to release free cholesterol. Therefore, the high level of steroid biosynthesis in R2C cells is a result of the constitutive expression of the components involved in the uptake of cholesteryl esters (SR-BI), their conversion to free cholesterol (HSL) and its mobilization to the inner mitochondrial membrane (StAR). Next, we performed experiments to determine the levels of both positive and negative transcription factors known to bind the StAR promoter in order to determine if one of them might be responsible for the constitutive steroid production observed. Several transcription factors in R2C cells were measured and the results demonstrated that DAX-1 protein, a negative regulator of several steroidogenic genes, was undetectable in R2C cells and therefore might account for the observed increase in StAR synthesis and steroidogenesis. We utilized both a Tet-on inducible gene expression system and a retroviral infection system to determine the effects of DAX-1 expression in R2C cells and observed that overexpression of DAX-1 protein in R2C cells repressed both steroid and StAR synthesis by --60% and -40% respectively. These observations suggest that the high levels of constitutive steroid biosynthesis and StAR expression in R2C cells may be, in part, a result of the absence of the DAX-1 transcription factor. Finally, we focused on the PKA and PKC signal transduction pathways in order to determine whether these pathways were actively driving steroidogenesis and StAR expression. R2C cells had marginally elevated PKA activity and an active PKC pathway under basal conditions since inhibitors of PKA and PKC resulted in decreases in steroid production and StAR levels. To study the roles of both the PKA and PKC pathways in steroid and StAR synthesis we tested other Leydig tumor cell lines, MA-10 and mLTC-1 cells, using PMA (10 nM, a PKC activator) and a cAMP analog ((Bu)2cAMP 0.05 mM, a PKA activator). While neither treatment alone produced significant increases in steroid synthesis, PMA could induce high amounts of StAR protein. Treatment with both PMA and 0.05 mM (Bu)2cAMP resulted in significant increases in steroids. These studies determined that addition of 0.05 mM (Bu)2cAMP was required for the phosphorylation of StAR protein and was necessary to activate the PMA-induced StAR.Item Regulation of steriodogenesis in rodent leydig cells by corticotropin-releasing hormone (CRH)(Texas Tech University, 1995-08) Huang, Bu-Miin; Norman, Reid L.; Stocco, Douglas M.; Beale, Elmus G.; Droms, Kurt A.; Hutson, James C.; Weitlauf, Harry M.Not availableItem Regulation of steroidogenesis in subclones of the Ma-10 mouse leydig tumor cell line(Texas Tech University, 1990-05) Kilgore, Michael Wayne.