Two cell lines have been isolated from testis of immature BALB/c mice. These lines have each been cloned several times and have been established in culture for over 2 years. The cells are epithelial in appearance and do not form tumors when injected into BALB/c - nu/nu mice. l’hc cell lines can be said to be two distinct cell types on the basis of their morphology, hormone response, responses to gonadotropins, and metabolism of steroids. One line (TM4) has been tentatively identified as being of Sertoli cell origin, since it shows a growth response to purified ovFSH and an increase in cAMP in the presence of FSII but not LII. The other line (TM3), believed to be Leydig cells, is not growth-stimulated by either LII or FSII, shows an increase in cAMP in the presence of LII, and a change in the metabolism of Itm4 Cl cholesterol in the presence of LI-I.

Establishment and characterization of two distinct mouse testicular epithelial cell lines.

Several clonal lines of cultured Leydig tumor cells have been established and characterized in terms of gonadotropin receptors and steroid production. Although freshly isolated cells derived from the M5480P tumor have functional hCG receptors, only two of the five clonal lines established were shown to bind significant quantities of hCG. In these clones, steroid production can be stimulated to the same extent by hCG, cholera toxin, and 8-Br-cAMP. The other three clones bind a small amount of hCG and respond to the hormone with a marginal increase in steroidogenesis. Steroid production, however, is significantly stimulated by cholera toxin or 8-Br-cAMP. A comparison of the steroids produced by freshly isolated cells and two of the clones revealed some changes in the steroidogenic pathway. The most obvious change is an increase in the ability of the cultured cells to synthesize 20α-dihydroprogesterone (20α-hydroxypregn-4-en-3-one). These clonal lines may provide a suitable model system for the study of gona...

Characterization of Several Clonal Lines of Cultured Ley dig Tumor Cells: Gonadotropin Receptors and Steroidogenic Responses*

The induction of acetylcholine synthesis in primary cultures of dissociated rat sympathetic neurons: I. Effects of conditioned medium☆

Herein we summarize important discoveries made over many years about Leydig cell function and regulation. Fetal Leydig cells produce the high levels of androgen (testosterone or androstenedione, depending upon the species) required for differentiation of male genitalia and brain masculinization. Androgen production declines with loss of these cells, reaching a nadir at postpartum. Testosterone then gradually increases to high levels with adult Leydig cell development from stem cells. In the adult, luteinizing hormone (LH) binding to Leydig cell LH receptors stimulates cAMP production, increasing the rate of cholesterol translocation into the mitochondria. Cholesterol is metabolized to pregnenolone by the CYP11A1 enzyme at the inner mitochondrial membrane, and pregnenolone to testosterone by mitochondria and smooth endoplasmic reticulum enzymes. Cholesterol translocation to the inner mitochondrial membrane is mediated by a protein complex formed at mitochondrial contact sites that consists of the cholesterol binding translocator protein, voltage dependent anion channel, and other mitochondrial and cytosolic proteins. Steroidogenic acute regulatory protein acts at this complex to enhance cholesterol movement across the membranes and thus increase testosterone formation. The 14-3-3γ and e adaptor proteins serve as negative regulators of steroidogenesis, controlling the maximal amount of steroid formed. Decline in testosterone production occurs in many aging and young men, resulting in metabolic and quality-of-life changes. Testosterone replacement therapy is widely used to elevate serum testosterone levels in hypogonadal men. With knowledge gained of the mechanisms involved in testosterone formation, it is also conceivable to use pharmacological means to increase serum testosterone by Leydig cell stimulation.

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Leydig cells: formation, function, and regulation.

Targeted Disruption of the Peripheral-type Benzodiazepine Receptor Gene Inhibits Steroidogenesis in the R2C Leydig Tumor Cell Line

A cell culture in monolaycr, derived from a mouse testicular interstitial cell tumor of spontaneous origin, persistently produces steroids de novo. The major products are progesterone2 and its reduced derivative, 20αhydroxy-4-pregnen-3-one. The latter accumulates to a concentration as high as 7 μg/ml of culture medium in 4 days. Exogenous pregne:iolone or progesterone is readily converted to this steroid within hours. Added 3′,5 ′-cyclic AMP at A very low concentration (5×10−6M) significantly {stimulates steroidogenesis, a maximal 3-fold increase being obtained at 10−4M. Both TPNH and DPNH at 10−3M also cause 3-fold stimulation in steroid secretion. Cycloheximide (10 ppm) inhibits such stimulation by added factors completely. So far, no trophic hormones have been found to elicit similar effects from the culture. The cultured cells are near-diploid and have a generation time of approximately 3.5 days in the exponential phase of growth. A clonal line retains these functional characteristics after over 14 mo...

Studies on interstitial cells in tissue culture: steroid biosynthesis in monolayers of mouse testicular interstitial cells.

A stable, clonal, monolayer culture has been established from a transplantable tumor of the testicular interstitial cells of rat. The original culture contained 2 distinct cell types. An epithelial-type clone isolated from this culture secretes a high level of steroid hormones, is nearly diploid and is functionally stable through extended serial subcultures. The major steroids synthesized by this clone are 20α-hydroxypregn-4-en-3-one and progesterone. Androgenic steroids could not be detected in significant amounts. A second clone from the original culture was composed of a fibroblasttype cell and showed no steroidogenic capacity. (Endocrinology 82: 614, 1968)

Studies on interstitial cells in tissue culture. II. Steroid biosynthesis by a clonal line of rat testicular interstitial cells.

Inhibition by actinomycin D, cycloheximide and puromycin of steroid synthesis induced by cyclic AMP in interstitial cells.

The transport mechanism of HfO2-based metal-ferroelectric-metal (MFM) capacitors was investigated using low-frequency noise (LFN) measurements for the first time. The current–voltage measurement results revealed that the leakage behavior of the fabricated MFM capacitor was caused by the trap-related Poole–Frenkel transport mechanism, which was confirmed by the LFN measurements. The current noise power spectral densities (SI) obtained from the LFN measurements followed 1/f noise shapes and exhibited a constant electric field (E) × SI/I2 noise behavior. No polarization dependency was observed in the transport characteristics of the MFM capacitor owing to its structural symmetry.

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Seung-Il Shin

Papers

Studies on interstitial cells in tissue culture: steroid biosynthesis in monolayers of mouse testicular interstitial cells.

Studies on interstitial cells in tissue culture. II. Steroid biosynthesis by a clonal line of rat testicular interstitial cells.

Inhibition by actinomycin D, cycloheximide and puromycin of steroid synthesis induced by cyclic AMP in interstitial cells.

Low-Frequency Noise Characteristics in HfO2-Based Metal-Ferroelectric-Metal Capacitors