Differential expression of novel abundant and highly regionalized mRNAs of the canine epididymis

Abstract

Three novel gene products have been cloned by differential screening of a dog epididymis cDNA library as part of a global appraisal of specific gene expression in the epididymis. The predicted proteins were provisionally named CE8-CE10 (for canine epididymal gene products 8-10). Northern blot analyses and in situ transcript hybridization confirmed that the cDNAs were all derived from tissue-specific, moderately to highly abundant mRNAs of the epididymal epithelium, showing a distinct regionalized expression pattern within the epididymal duct. Their sequences predict (i) a novel 19 kDa member of the Ly-6-domain protein superfamily (CE8), (ii) an approximately 30 kDa protein with multiple membrane-spanning regions (CE9), and (iii) a novel approximately 13 kDa single whey acidic protein domain protein (CE10). Closely related, cross-hybridizing gene products were abundant in the epididymis of stallions and bulls, but not in rodents or men. Changes in mRNA frequency were observed that specifically correlated with a cryptorchid situation and with the age of the dogs. Gene products restricted to the caput epididymidis were affected by both conditions, while those with a wider regional distribution were not.

The adult boar testicular and epididymal transcriptomes

“…For the anterior caput, the boar homologue to CE9 (canine epididymal 9 gene) was analyzed. This gene has been found in the upper part epididymis of dog and mouse epididymis (named ME9 ) but not in human epididymis [ 49 , 50 ]. It has been suggested that its expression is regulated by luminal factors from the testis [ 51 ].…”

“…GPX5 gene has also been extensively study in the epididymis where it codes for an androgen-dependent glutathione peroxidase specific in the distal caput epididymis of mouse and rat [ 49 , 52 ]. A role for sperm and epididymis protection against oxidative stress has been suggested for this enzyme.…”

The orthologue of the "acatalytic" mammalian ART4 in chicken is an arginine-specific mono-ADP-ribosyltransferase

“…After denaturation for 10 min at 70°C reverse transcription using RevertAid™ H Minus M-MuLV reverse transcriptase at 37°C for 60 min was performed. An inactivation phase of 10 min at 70°C was followed by second strand synthesis, ring ligation, and inverse nested PCR amplification (primer pairs chART4 inverse fwd1/RT_chART4 and chART4 inverse fwd2/chART4 inverse rev2 as described previously [ 15 ].…”

A Putative 12‐Transmembrane Domain Cotransporter Associated With Apical Membranes of the Epididymal Duct

“…Although the overall nucleotide sequence similarity between CE11 and TSCOT was high (approximately 80%), an RT‐PCR approach based on the homologous human and mouse sequences did not yield the 5′ end of CE11. An inverse RT‐PCR protocol (Gebhardt et al, 1999) using specific CE11 cDNA synthesis primers (compare the Table) was repeatedly applied. After 4 cycles, the cDNA sequence comprised 2280 nucleotides (Figure 1), which is in good agreement with the length of the hybridizing mRNA band (see below).…”

“…Step 5 was a positive ramp at 0.1°C per second to 60°C, followed by 60°C for 2 minutes (step 6), 55°C for 2 minutes (step 7), 60°C for 2 minutes an additional 10 times (step 8), 70°C for 10 minutes (step 9), 4°C for 10 minutes (step 10) until the end (step 11). Second‐strand synthesis, ring ligation, and inverse PCR amplification was performed as previously described (Gebhardt et al, 1999). PCR products were isolated and ligated into the TA‐cloning vectors pCRII (Invitrogen, Heidelberg, Germany) or pGEM‐T Easy (Promega, Mannheim, Germany).…”