| To study the regulation of germ cell function during spermatogenesis, centrifugal elutriation was used to separate spermatocytes and spermatids from chicken testis. Cell suspensions were prepared by enzymatic digestion of testis, with the resultant mixed cells being separated by elutriation based on size. The purity of cells in the collected fractions was assessed by flow cytometric DNA analysis. Round spermatids (5 × 108) with an enrichment to 86% and spermatocytes (1 × 108) with purity of 61% were obtained following elutriation, proteins in the two cell fraction did not reflect significant degradation.; The ontogeny of expression of the LH receptor gene was studied in chicken testis from hatch to sexual maturity. Northern bolts showed the presence of three transcripts of LH receptor, a major 2.7 Kb band and minor 7 Kb and 1.5 Kb bands. The expression of estrogen receptor (ERα and ERβ) was investigated in chicken hypothalamus, ovary, pituitary and testis by Northern blots. Both ERα and ERβ were expressed in all four tissues. A single 7.8 Kb transcript was detected for ERα, however, two transcripts with sizes of 7.5 Kb and 4.5 Kb were observed for ERβ, with both bands being observed in hypothalamus, pituitary and testis but not the ovary, where only the 4.5 Kb species appeared. Neither ERα nor ERβ expression was detected by RT-PCR in enriched spermatocyte or spermatid fractions produced by centrifugal elutriation.; In order to identify genes differentially expressed during spermatogenesis, mRNA differential display was used to compare the mRNA expression profiles in chicken testis before and after the initiation of spermatogenesis, which was triggered by photostimulation. Two cDNA fragments, with homologies to the human Bub3 and RNase L inhibitor (RLI) genes, were cloned Bub3 has bee shown by others to encode for a mitotic checkpoint protein, while RLI is involved in regulating RNA turnover and stability in cells. Subsequent Northern blots revealed that both chicken Bub3 and RLI were expressed in testes before and after photostimulation. Their mRNAs also were identified in chicken brain, granulosa cells, liver, pituitary and spleen, their existence in multiple tissues suggest more global roles, not spermatogenesis specific, for cBub3 and cRLI in cell development and/or differentiation. |
