Effect of Luteinizing Hormone Deprivation In Situ on Steroidogenesis of Rat Leydig Cells Purified by a Multistep Procedure12

Abstract

Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.

Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells

“…Primary Rat Leydig Cell Isolation and Lentiviral InfectionLeydig cells were isolated from 60-to 64-day-old Sprague-Dawley rats (Charles River Laboratories) as described previously (67). In brief, the testis were decapsulated, digested in M-199 medium containing 0.05% collagenase/dispase, 0.005% soy trypsin inhibitor, and 0.001% deoxyribonuclease I (all from Sigma) and shaken at 90 cycles/min for 45 min at 34°C.…”

Orally applied doxazosin disturbed testosterone homeostasis and changed the transcriptional profile of steroidogenic machinery, cAMP/cGMP signalling and adrenergic receptors in Leydig cells of adult rats

“…This suspensions of interstitial cells (Klinefelter et al, 1987) was used to prepare primary cultures of purified Leydig cells (Andric et al, 2007(Andric et al, , 2010aKostic et al, 2008Kostic et al, , 2010Kostic et al, , 2011 by centrifugation on a Percoll gradient consisting of four 2 mL layers of Percoll with densities of 1.090, 1.080, 1.065 and 1.045 g/mL (formed by mixing isotonic Percoll consisting of 109 concentrated DMEM/F12 enriched with 3% of BSA and the corresponding amount of Percoll and distilled water). A crude suspension of interstitial cells (approximately 35-40 9 10 6 cells), containing besides Leydig cells macrophages and endothelial cells (Klinefelter et al, 1987), was applied to each Percoll gradient and centrifuged at 500 g for 28 min at room temperature. Fractions containing Leydig cells were collected from the 1.080/1.065 g/mL and 1.065/1.045 g/mL interfaces, washed in 50 mL M199-0.1% BSA and centrifuged at 200 g for 5 min at room temperature.…”

“…It is important to emphasize that the structural organization of the testis may account for the integration of autocrine and paracrine factors to regulate testicular steroidogenesis and TIF characteristics and/or content. Although testosterone is an exclusive product of Leydig cells, secretory products originated from seminiferous tubule and/or Sertoli cells and/or non-steroidogenic cells in the testicular interstitium (Klinefelter et al, 1987;Saez, 1994;Hutson, 2006) could take place in regulation of TIF characteristics. Accordingly, reported changes in the TIF and testosterone content in testis could be the consequence of the complex effects of secretory contribution of the different cell types including, besides HSD3B-positive Leydig cells, also macrophages, endothelial cells, fibroblasts, peritubular cells, Sertoli cells etc.…”

“…Results of this study on Leydig cells isolated from control or Doxa-treated rats showed different effects: 19Doxa/29Doxa applications significantly increased basal/hCG-stimulated androgens production by Leydig cells ex vivo, whereas 109Doxa decreased. The discrepancy between levels of androgens in serum, TIF, testes, and those produced ex vivo by Leydig cells could be explained by the fact that isolation/ purification/stimulation of Leydig cells takes approximately 12 h when isolated Leydig cells are removed from the biologically active inhibitory and/or stimulatory paracrine compounds released from the seminiferous tubules and/or macrophages, endothelial cells, fibroblasts, peritubular cells, Sertoli cells (Klinefelter et al, 1987;Saez, 1994;Hutson, 2006). In summary, the complex structural organization of the testes and coexistence of multiple regulatory mechanisms that control testicular cells and microvasculature could provide a degree of redundancy in the maintenance of testicular steroidogenesis, a crucial component of the reproductive process.…”

Mouse Leydig cells express multiple P2X receptor subunits

“…Cells were centrifuged again at 900×g for 5 min, resuspended and seeded on coverslips. The efficiency of this process was confirmed by cytochemical detection of the enzyme 3β-HSD as described elsewhere [28].…”