Nitric oxide modulates ATP-evoked currents in mouse Leydig cells

Testosterone synthesis within Leydig cells is a calcium-dependent process. Intracellular calcium levels are regulated by different processes including ATP-activated P2X purinergic receptors, T-type Ca2+ channels modulated by the luteinizing hormone, and intracellular calcium storages recruited by a calcium-induced calcium release mechanism. On the other hand, nitric oxide (NO) is reported to have an inhibitory role in testosterone production. Based on these observations, we investigated the interaction between the purinergic and nitrergic systems in Leydig cells of adult mice. For this purpose, we recorded ATP-evoked currents in isolated Leydig cells using the whole cell patch clamp technique after treatment with L-NAME (300 μM and 1 mM), L-arginine (10, 100, 300, and 500 μM), ODQ (300 μM), and 8-Br-cGMP (100 μM). Our results show that NO produced by Leydig cells in basal conditions is insufficient to change the ATP-evoked currents and that extra NO provided by adding 300 μM L-arginine positively modulates the current through a mechanism involving the NO/cGMP signaling pathway. Thus, we report an interaction between the nitrergic and purinergic systems in Leydig cells and suggest that Ca2+ entry via the purinergic receptors can be regulated by NO.

Seasonal and experimental reactivation of Leydig cells of the bat Tadarida brasiliensis

The Leydig cells of the bat Tadarida brasiliensis, exhibit two well-defined periods of secretory activity that are intimately associated to the bat reproductive cycle. During the breeding season (August-September, late Winter and early Spring in the southern hemisphere), the interstitial tissue contains hypertrophic Leydig cells characterized ultrastructurally by the presence of pleomorphic mitochondria, depletion of lipid droplets, proliferation of membranes of agranular endoplasmic reticulum (AER) and enlargement of the Golgi complexes. By contrast, from Spring to Fall concurrent with regression of seminiferous tubules, the Leydig cells acquire a quiescent appearance with reduction in size and volume of AER membranes, atrophy of the Golgi complex and a massive storage of lipid droplets. The changes occurring in Leydig cells during the breeding season can be duplicated experimentally in non-breeding bats with exogenous stimulation with hCG. The gonadotropic treatment induces rapid changes in both interstitial cells and seminiferous tubules. The latter present evident signs of reactivation including proliferation of the spermatogenic cell line, permeation of the tubular lumen and depletion of lipid droplets. The Leydig cells display similar features to those found in the bat during the mating season at the peak of secretory activity. The bat T. brasiliensis is an excellent model to correlate the morphological organization of the Leydig cells with either seasonal fluctuations of its secretory activity or after experimental stimulation with gonadotropins.

Calcium-activated potassium channels ar involved in the response of mouse Leydig cells to human chorionic gonadotropin

The patch-clamp technique was used to investigate the involvement of ion channels in the response of Leydig cells to gonadotropic hormones (viz.hCG). Recordings in the cell-attached configuration (pipette containing 140 mM KCl) showed unitary events with conductance of 187.9 + or - 5.2 pS(N = 24 patches) in about 70 percent of the cells. These channels were potassium selective and the open channel probability (Po) was always about 1 percent for displacemtne of potential from the resting value in the range of -20 to +60 mV. Treatment of the cells with hCG (2 ng/ml) led to a large increase in the frequency of openings, concomitant with a reduction in the mean closed time and there was essentially no effect on the mean open time of the channels. Dibutyryl cAMP (100 µM) produced an effect similar to that of hCG and both required external calcium for their action. No direct effect of either dibutyryl cAMP or hCG were observed in inside-out patches. Reversal potential measurements on excised inside-out patches demonstrated that the channels were highly potassium selective with unitary conductance of about 206.8 + or - 6.36 pS(mean + or - SEM of 6 measurements), and an estimated permeability of 3.6 x 10-13 + or - 0.2 x 10--13 cm3/s (mean + or - SEM for 6 measurements), in symmetrical 140 mM KCl. The activity of the channel in excised paches was very sensitive to the free-calcium concentration on the intracellular surface of the free-calcium concentration on the intracellular surface of the channel. Po evaluated at + 60mV increased from 3 percent at 10 nM to 47 percent at 100 nM free calcium. The Hill coefficient under these conditions was 1.1. These results demonstrate that Leydig cells have a Ca2+ -activated K+ channel of large unitary conductance, which can be activated upon the binding of hCG to receptors in the cell membrane

Cronología de la diferenciación genital en octodon degus: octodontidae, rodentia / Cronology of genital differentation in octodon degus: octodontidae, rodentia

En atención al interés por manipular la ontogenia y obtener la expresión de fenotipos atávicos o nuevos, nos preocupó precisar la cronología de la diferenciación genital en óctodon degus, un roedor de larga y peculiar gestación, verificando además, si este desarrollo corresponde a un modelo que ejemplifique el mecanismo de heterocronía. Se estudiaron esbozos pregonádicos, gónadas y genitales externos de embriones y fetos de edades conocidas, mediante técnicas histológicas: H-E, azul de alcian, P.A.S. y técnicas convencionales para microscopía electrónica. Se observó que entre 37 a 40 días de edad postcoital, se inicia la diferenciación testicular, dos a tres días después, se observan las primeras células intersticiales y externamente la aparición del tubérculo genital. A los 60 ñ 1,5 días, el testículo está más desarrollado presentando cordones seminíferos, entre los cuales se observaron células intersticiales abundantes y voluminosas, con claros signos de secreción esteroidal. El tubérculo genital ya se ha diferenciado en pene. Transcurrido el 80 por ciento del período gestacional, en los cordones seminíferos se observaron espermatogonios troncales y además, una población de células peritubulares circundando cada cordón. Las células intersticiales presentaban signos evidentes de regresión. El avanzado estado de diferenciación que alcanzan la línea germinal, compartimento peritubular y células intersticiales , separa a este roedor de los otros subordenes miomorfos y sciuromorfos, en los que la diferenciación ocurre durante la vida postnatal. El momento del inicio de las interacciones celulares para el desarrollo gonadal, es similar en los distintos subórdenes de roedores, si se toma como parámetro de comparación el análisis de sus caracteres externos, pero posteriormente, durante la ontogenia de octodon degus, se disocia la tasa de desarrollo de la gónada progresando hasta etapas más avanzadas, lo cual se puede reconocer como una expresión de heterocronía. Esta diferencia temporal podría ser responsable de las particulares características que definen a este caviomorfo como más especializado reproductivamente, largas gestaciones, camada con crías bien desarrolladas, órganos reproductivos con sofisticados dispositivos que aseguran la reproducción, todo lo cual asegura el éxito de la especie en ambientes xéricos

Célula de Leydig: ultra-estrutura e síntese de testosterona / Leydig cell: ultrastucture and testosterone synthesis

A Gonadotrofina LH prende-se a receptores específicos, localizados em microvilos da membrana citoplasmática, formando o complexo hormonio-receptor, que ativa o sistema adenilato ciclase e consequente sintese de AMPc. O nucleolite ativa fosfoquinases que fosforilam e sintetizam proteinas, necessárias para que seja produzida a testosterona. Gotículas de lipídeos, situadas no citoplasma da CL contêm a maior parte do colesterol esterificado que, após hidrólise, sob a forma de colesterol livre, é transportado por proteinas até a mitocôndria e nela, depois de ser levado até a membrana interna da crista, entra em contato com enzimas, que fazem a clivagem da cadeia lateral, transformando-o em pregnenolona. A pregnenolona é transportada para o REA, onde nova bateria de enzimas a transformará em testosterona, se o animal for adulto e suas células, consequentemente, maduras, ou em hormônios reduzidos, se a célula for imatura. Cada etapa do processo tem seus próprios mecanismos de autocontrole, secretando quantidades exatas de testosterona necessária à manutençäo da espermatogênese e caracteres sexuais secundários.

Modulatory effects of adrenergic agonists on testosterone secretion from rat dispersed testicular cells or Percoll-purified Leydig cells

Freshly dispersed testicular interstitial cells as well as Percoll-purified Leydig cells were studied in vitro in order to evaluate the effect of adrenergic agonists on testosterone (T) secretion. Epinephrine and phenylephrine did not change the rate of T release under basal conditions in freshly dispersed interstitial cells, but enhanced it during human chorionic gonadotropin (hCG) stimulation. Norepinephrine and clonidine had no effect on T secretion. In contrast, in Percoll-purified Leydig cells epinephrine increased T release both under basal and hCG-stimulated conditions. These data demonstrate that neurotransmitters may participate in T secretion from isolated Leydig cells