Here, we studied this response as a way to elucidate in extra detail the molecular factors concerned and also the physiological impli cations of their activation. We discovered that uridine sensi tive P2Y2 and P2Y6 receptors are expressed from the TIC membrane and that P2Y activation promoted 3 crucial responses in these cells. one elicited Ca2 mobili zation from intracellular reservoirs, expanding the con centration of this important 2nd messenger inside the cytoplasm, two enhanced cell proliferation by way of a mechanism dependent to the activation of protein kinase C as well as MAPK p44 and p42, and, 3 down reg ulated hCG dependent phosphorylation of CREB, a crucial element in steroidogenesis cascade manage.
selelck kinase inhibitor Techniques Resources ATP, UTP, UDP, suramin, human chorionic gonadotropic hormone, porcine follicle stimulating hormone, and PPADS had been obtained from Sigma Chemical Co, and staurosporin, wortmanin, and phorbol twelve myristate 13 acetate were from Cal biochem, DMEM F12 medium, fetal bovine serum, antibiotic antimycotic mix, as well as other cell culture reagents have been from Gibco Invitrogen Co, Antibodies towards mouse complete or phospho rylated MAPK p44 and p42 and complete or phosphorylated CREB had been from Cell Signaling, and anti physique towards poly polymerase one was from Santa Cruz Biotechnology, Oligo nucleotides, reverse transcriptase, oligo dT, taq poly merase, along with other molecular biology reagents have been purchased from Invitrogen Co, and Fluo4 AM was from Molecular Probes Invitrogen Co, Automatic sequencing was performed during the Molecular Biology Unit of the Instituto de Neurobiolog?a, UNAM. Theca cell isolation and culture Mouse theca interstitial cells have been purified by a discon tinuous Percoll gradient, Immature mice were utilised to avoid cultures enriched in luteal cells.
Hence, intact four to 5 week outdated mice on the strain C57 have been sacri ficed by cervical dislocation, a process approved through the ethical committee of Instituto de Neurobiolog?a UNAM. The ovaries have been dissected and incubated in collagenase for 20 min, as well as ovarian epithelium was removed by passing the full ovary MK-0752 repeatedly in and out by the orifice of the Pasteur pipette. Most granu losa cells have been then eliminated by puncturing the iso lated, epithelium cost-free ovaries by using a fine hypodermic needle. The ovary, free of epithelium and most granulosa, was cut into fine pieces that have been then incubated in the mix of collagenase, DNase I, and BSA for 30 min. The homogenate was fractioned on a discontinuous gradient. bottom layer 44% Percoll, top rated layer Percoll of density one. 055 g ml. The cells have been centrifuged for twenty min at 400 g, and TIC have been collected in the interface by aspiration, then cultured in DMEM F12 medium containing 10% fetal bovine serum supple mented with antibiotic antimycotic at 37 C within a humidi fied ambiance with 5% CO2.