AIMIdentification of the rodent counterparts of human and canine epididymal cDNAs HE3, HE4 and Ce8/Ly6G5C by sequence homology and analysis of their expression patterns and regulation level in the rat.

METHODS"Electronic screening" of Expressed Sequence Tag (EST) and genomic databases, followed by RT-PCR and Northern blot analysis.

RESULTSRodent ESTs and genomic sequences homologous to HE3, HE4 and Ce8/Ly6G5C were identified in the public databases and the "full-length" rat cDNAs cloned. To emphasise their homology to the human and canine genes, they were named Me3/Re3, Me4/Re4 and Re8 for mouse and rat counterparts, respectively. mRNA expression patterns were analysed in rats, including rat HE1 and HE5/CD52 counterparts as controls. Re3 and Re8 mRNAs were only found in the rat epididymis, while Re4 showed a broader tissue distribution. Within the epididymis, Re3 and Re4 mRNAs were detected in all regions; Re8, on the other hand, was restricted to the caput. During postnatal development, Re3 and control mRNAs were found from the earliest stages investigated, while Re8 mRNA was observed only from day 24 postnatum, corresponding to the onset of spermatogenesis in the prepubertal testis. Castration and testosterone supplementation of adult male rats suggested that none of the cloned mRNAs was directly androgen-regulated. Efferent duct ligation, however, showed that Re8 mRNA levels depended on testicular factors other than androgens.

CONCLUSIONThe novel rodent cDNAs can now be used to monitor epididymal gene expression more closely and to set up various regulatory and functional studies.

Amino Acid Sequence ; Animals ; Blotting, Northern ; Carrier Proteins ; chemistry ; genetics ; DNA, Complementary ; chemistry ; Dogs ; Epididymal Secretory Proteins ; chemistry ; genetics ; Epididymis ; chemistry ; Glycoproteins ; chemistry ; genetics ; Humans ; Male ; Molecular Sequence Data ; Proteins ; chemistry ; genetics ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Homology ; Tissue Distribution ; beta-Defensins

AIMTo determine the effectiveness of the sk11, sk9 and sk11 TNUA5 Sertoli cell lines in binding germ cells in vitro.

METHODSThe immortalized Sertoli cell lines sk9, sk11 and sk11 TNUA5 were used in co-culture experiments with germ cells in media with or without reproductive hormones and incubated for 44 h at 32 degrees . The number of germ cells bound to Sertoli cells was then determined and statistically analyzed. Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) studies were employed to investigate the presence of cell adhesion proteins and follicle stimulating hormone (FSH) receptor, respectively.

RESULTSNo statistical difference between the number of bound step-8 spermatids and bound pre-step 8 spermatids on Sertoli cells from any of the cell lines existed. After the addition of germ cells, Sertoli cells showed more lipid accumulation in their cytoplasm, indicating active phagocytosis. Western blot analysis in the sk11 TNUA5 line indicated the expression of N-cadherin. FSH-only and testosterone-only treatments increased N-cadherin expression, regardless of germ cell addition. The addition of germ cells to the sk11 TNUA5 Sertoli cells increased the expression of espin, as did the addition of FSH with germ cells. RT-PCR studies of the sk11 TNUA5 cells indicated that the mRNA for FSH receptor decreased with successive passages.

CONCLUSIONIn vitro binding between isolated germ cells and sk9, sk11 or sk11 TNUA5 Sertoli cells is not feasible, and therefore these cell lines are not useful for the in vitro investigation of Sertoli-germ cell interactions and primary Sertoli cell isolates must still be used.

Animals ; Cadherins ; genetics ; metabolism ; Cell Adhesion ; physiology ; Cell Line, Transformed ; cytology ; metabolism ; Coculture Techniques ; Gene Expression ; Image Processing, Computer-Assisted ; Male ; Mice ; Microfilament Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptors, FSH ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sertoli Cells ; cytology ; metabolism ; Spermatids ; cytology ; metabolism