Cloning and differential expression of steroid 5 alpha-reductase type 1 (Srd5a1) and type 2 (Srd5a2) from the Harderian glands of hamsters.
In hamsters, the Harderian glands (HGs) exhibit a marked sexual dimorphism which is thought to depend on dihydrotestosterone (DHT); however, it is unclear whether hamster HGs contain one or more 5 alpha-reductases and whether these enzymes are differentially expressed in males and females. In this study, we isolated specific cDNAs for 5 alpha-reductase 1 (Srd5a1) and 5 alpha-reductase 2 (Srd5a2), determined their sequences and investigated their expression in the HG of both sexes. Isozyme 1, cloned from liver mRNA, encodes a protein of 255 amino acids (aa); isozyme 2 cDNA, isolated from the epididymis encodes a 254-aa protein. When assayed in transfected HEK-293 cells, the type 1 isozyme displayed activity over a broad pH range (6.5-8), while isozyme 2 had a pH optimum of 5.5. Both isoenzymes efficiently catalyzed the in vitro transformation of T into DHT, with apparent K(m) values of 7.1 and 1.9 micromol/L for Srd5a1 and Srd5a2, respectively. Real-time PCR analysis revealed higher mRNA levels for Srd5a1 than for Srd5a2. Expression of both isoenzymes increased slightly in HGs of castrated males and showed variations during the estrous cycle in females. Hormonal replacement with 17beta-estradiol administered to spayed females induced the up-regulation of Srd5a2 mRNA levels. Altogether, our results demonstrated that both Srd5a1 and Srd5a2 are expressed in HGs without clear differences between males and females. The biochemical characteristics and relative expression of these 5 alpha-reductases support the view that both isozymes may play a relevant role in modulating androgen signaling in HG.
Ramos
Luis
L
Department of Reproductive Biology, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, México City, Mexico.
Chávez
Bertha
B
Vilchis
Felipe
F
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D006224:Cricetinae; D004822:Epididymis / Q000201:enzymology; D030762:Estrous Cycle; D005260:Female; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006243:Harderian Gland / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008297:Male; D008647:Mesocricetus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009919:Orchiectomy / Q000662:veterinary; D010052:Ovariectomy / Q000662:veterinary; D012333:RNA, Messenger / Q000032:analysis; D017385:Sequence Homology; D012727:Sex Characteristics
D007527:Isoenzymes; D012333:RNA, Messenger; D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase