Isolation, culture, and characterization of canine Sertoli cells.

Primary Sertoli cell cultures have been established from several animals including the sheep and rhesus monkey; however, not for the domestic dog, Canis familiaris. Sertoli cells are the only readily accessible cell type in the body which expresses all six type IV collagens. These collagens play key roles in tissue structure, basement membrane formation, and filtration. The study of these genes is necessary to determine their exact roles and regulation in the aforementioned functions and to investigate diseases associated with mutations in these genes. For such studies, a cell culture system is a requisite tool. Therefore, Sertoli cells were targeted, and a culture was established from cells isolated from canine testes. Cultures maintained consistent morphology and steady growth for up to seven passages. Cultured cells were identified as Sertoli cells through positive Western blot results for SOX9 and Clusterin B proteins and transcript sequence verification of SOX9 as well as the presence of type IV collagen transcripts. Primary cultures of canine Sertoli cells will provide a useful tool for study of the function and regulation of collagen genes and will permit new research pertaining to canine health while also serving as a model for the study of human diseases.

Genetic cause of autosomal recessive hereditary nephropathy in the English Cocker Spaniel.

BACKGROUND: Autosomal recessive hereditary nephropathy (ARHN) in the English Cocker Spaniel is caused by a type IV collagen defect, but the underlying mutation is unknown. ANIMALS: One hundred thirty-four English Cocker Spaniels (12 with ARHN, 8 obligate carriers, and 114 others), 3 mixed breed dogs with X-linked hereditary nephropathy (XLHN), and 7 other dogs without hereditary nephropathy were included. METHODS: Diagnosis of ARHN was based on transmission electron microscopy and immunostaining of kidney. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was used to compare COL4A3, COL4A4, and COL4A5 mRNA concentrations in the renal cortex from ARHN-affected English Cocker Spaniels, XLHN-affected dogs, and dogs without hereditary nephropathy. The entire coding region of COL4A4 was sequenced in 2 ARHN-affected dogs, 2 obligate carriers, 2 English Cocker Spaniels of unknown status, and 2 healthy mixed breed dogs. The exon containing the mutation was sequenced for all 134 English Cocker Spaniels. RESULTS: Quantitative real time RT-PCR implicated COL4A4 as the gene harboring the mutation, and sequencing identified a single nucleotide substitution at base 115 as the cause of ARHN in English Cocker Spaniels. This mutation, which causes a premature stop codon in exon 3 of COL4A4, was segregated with clinical status in all affected dogs and obligate carriers. The mutation also was identified in 39 of 114 other English Cocker Spaniels with previously unknown status. CONCLUSIONS AND CLINICAL IMPORTANCE: The cause of this disease has been identified, and use of a test for the mutation will permit eradication of ARHN in the English Cocker Spaniel.