In the present study, we reported that VK1 had the potential to enhance the ant

Further more, we examined the effects of some inhibitors for ErbB, PI3K, and MAPK on EGF induced spermatogonial proliferation to clarify the receptors and signaling selleck chemicals阻害剤 path ways operated by EGF. Finally, in order to explore the molecular mechanism by which spermatogonial prolifer ation was stimulated in response to EGF, we tested the effect of rhEGF on the gene expressions of SCF, c kit, neuregulin1, and ErbB family members, which are impli cated in spermatogonial proliferation, in the organ cul ture. Methods Animals and inhibitors Animal experiments have been carried out under the con trol of the Guideline to Animal Experiment in Kumamoto University. Adult male newts, Cynops pyrrhogaster, were purchased from Hamamatsu Seibutsu Kyozai Ltd. and kept at 8 C.

Prior to be used for all the experiments, newts were transferred to 22 C and fed buy Lenalidomide fro zen Tubifex for 1 week. PD98059, a MAPK inhibitor, and AG879, an ErbB2 inhibitor, were purchased from Calbio chem, AG1478, an EGFR inhibitor, and Wortmannin and LY294002, PI3K inhibitors, from Sigma, and PD153035, a pan ErbB inhibitor, from Biaffin. Organ culture of testicular fragments, histology, and BrdU incorporation assay The immature portions containing late spermatogonial stage were removed from the whole testes and cut transversely and longitudinally into several pieces. They were cultured on floaters of nucleopore filters in 2 ml of Leibovitzs 15 medium supple mented with 10 mM HEPESNaOH, pH 7. 4, at 18 C in a humidified incubator in the absence or presence of either porcine FSH at 200 ngml or rhEGF at various doses as indicated.

The pieces were fixed in Bouins fixa tive, embedded in paraffin wax, and serially sectioned at 5 m. The sections were stained with hematoxylin and eosin. After the fragments were cultured for 2 weeks, dif ferentiation of spermatogonia into primary spermatocytes was evaluated in the sections by histological observation. Proliferation LY2228820 ic50 of spermatogonia was assayed in the sec tions by immunohistochemical detection of BrdU incor porated into replicating DNA in the cells. After cultured for 1 week, the fragments were labeled for 6 hrs by addi tion of BrdU and processed for immunohistochemistry according to manufacturers instructions. For quantification of spermatogonial proliferation, a min imum of 300 500 cysts from at least 3 independent sec tions was examined for BrdU incorporation because all the spermatogonia in a given cyst incorporate BrdU syn chronously into DNA during replication.

The fre quency of proliferation was expressed as a percentage of BrdU positive cysts among live ones in 3 sections obtained from 3 independent experiments. Spermatogenic staging, and fractionation of germ cells and somatic cells The immature portions of testes containing early sperma togonial, late spermatogonial, or primary spermatocyte stage exclusively was cut transversely into several pieces and then each piece was cut longitu dinally into halves as described previ ously. A half of each piece was processed for histology, and the counterpart was used for RNA extrac tion followed by reverse transcription and polymerase chain reaction. The testicular portions containing spermatogonial stage were cut into small fragments and dissociated by incuba tion in 0.