Biomedical subjects

C Y Cheng

Publications and source records attributed to C Y Cheng.

219 records · Page 13Linked to original sources

PubMedDate not supplied

Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro.

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.

Animals↗

PubMedDate not supplied

Haptoglobin is a Sertoli cell product in the rat seminiferous epithelium: its purification and regulation.

Using multiple HPLC steps, a protein of 67 kDa (estimated by gel permeation HPLC) was purified from Sertoli cell-enriched culture medium that consisted of two dissimilar subunits of 9 (alpha chain) and 24 (beta chain) kDa on SDS-polyacrylamide under reducing conditions. Direct protein sequence analysis of the 9-kDa subunit revealed a sequence of NH2-VELGNDATDIEXD, which is identical to the alpha subunit of the rat haptoglobin (Hp). Hp is a 67-kDa tetrameric serum acute-phase protein consisting of two alpha and two beta subunits (alpha2beta2) of 8.5 kDa and 24.5 kDa, respectively. Using a 351-bp cDNA coding for Hp for northerns and two Hp primers for RT-PCR, we have demonstrated the expression of Hp in Sertoli and Leydig cells, germ cells, and the testis, but not in the epididymis. In contrast to the hepatic haptoglobin, an acute-phase protein whose steady-state mRNA level increased by as much as fivefold during induced inflammation, the testicular homolog reduced by fourfold within 24 hours following induced inflammation, suggesting that this gene is regulated differently in the testis and in the liver. Moreover, the testicular steady-state Hp mRNA level increased steadily after birth during maturation, suggesting its involvement in spermatogenesis. Using primary Sertoli cell cultures in vitro, it was found that the Sertoli cell Hp expression was not regulated by either FSH, testosterone, estradiol, dexamethasone, interleukin-1beta (IL-1beta), IL-6, interferon-gamma (INF-gamma), transforming growth factor-beta (TGF-beta), lymphocyte inhibitory factor (LIF), or germ-cell-conditioned medium (GCCM). Since transferrin secreted by Sertoli cells is an important molecule in maintaining the crucial iron level necessary for spermatogenesis, the identification of haptoglobin as a Sertoli and germ cell product adds a new member to the growing family of metal transporters in the testis that are likely to play an important role in iron metabolism in the testis.

Acute-Phase Reaction↗

PubMedDate not supplied

Evidence for cross-talk between Sertoli and germ cells using selected cathepsins as markers.

To examine whether proteases are possibly involved in cellular migration and/or spermiation when developing germ cells translocate across the seminiferous epithelium during spermatogenesis, in situ hybridization was used to localize messenger RNA (mRNA) transcripts of cathepsin L, D, and S in the epithelium at different stages of the spermatogenic cycle in the rat. Cathepsin L mRNA was found to localize almost exclusively near the basal lamina of the epithelium. At stages VI and VII of the cycle before spermiation, the signal of cathepsin L mRNA was so intense that it formed a complete dark precipitate near the basal lamina encircling the entire tubule. At stage VIII, the expression of cathepsin L was completely abolished, and no staining of cathepsin L mRNA was seen in the epithelium. The mRNA of cathepsin D and S was found near the basal lamina, a finding consistent with their localization in Sertoli cells and possibly primary spermatocytes in almost all stages, but peaked at stages VII-IX and VII-VIII of the cycle, respectively, at the time before and during spermiation. These results illustrate the possible involvement of these proteases in facilitating germ cell movement and spermiation. To examine whether germ cells express any of these cathepsin genes, spermatocytes with a purity of greater than 95% were isolated from 15-day-old rat testes by Percoll gradient centrifugation for reverse transcriptase-polymerase chain reaction. It was found that primary spermatocytes expressed multiple cathepsin genes, including cathepsin B, C, D, H, L, and S. Furthermore, the expression of cathepsin L by germ cells isolated from 15-day-old rats (largely spermatocytes and spermatogonia) can be stimulated by Sertoli cell-enriched culture medium in a dose-dependent manner, but not by germ cell-conditioned medium. These results reveal that germ cell function can be regulated by Sertoli cells. Moreover, these results suggest that germ cells may play an active role in the overall testicular protease expression. Also, we present evidence suggesting there is cross-talk between Sertoli and germ cells, since the expression of cathepsin L in each cell type is regulated by one another via either soluble factors or cell-cell contact.

Animals↗