title: Persistent expression of a truncated form of the luteinizing hormone receptor messenger ribonucleic acid in the rat testis after selective Leydig cell destruction by ethylene dimethane sulfonate.
abstract: To correlate the process of ethylene dimethane sulfonate (EDS)-induced disappearance and repopulation of Leydig cells with LH receptor (LHR) expression, testicular messenger RNA (mRNA) and binding of LHR were analyzed in male rats, 5, 15, 20, and 40 days after treatment with 75 mg EDS/kg BW. Five and 15 days after EDS treatment, the serum testosterone level was reduced by 90% (P < 0.01) and testicular [125I]iodo-hCG binding was nearly undetectable (P < 0.01). Multiple splice variants of the LHR mRNA, with sizes of 6.8, 4.2, 2.7, and 1.8 kilobases, were detected in control testes upon Northern hybridization. Interestingly, 5 and 15 days after EDS injection, only the 1.8-kilobase band, previously reported to correspond to a truncated form of the LHR mRNA and encoding its extracellular part, remained, whereas the other mRNA species disappeared. On days 20 and 40 after EDS treatment, the pattern of hybridization gradually returned to that resembling the control pattern. To increase the sensitivity of mRNA detection, testicular RNA was reverse transcribed and amplified by polymerase chain reaction, using primers complementary to various parts of the LHR complementary DNA. The specificity of the complementary DNAs generated was verified by Southern hybridization with nested oligonucleotide primers. Five and 15 days after EDS treatment, only truncated mRNA forms, encoding regions of the extracellular part of the LHR, could be amplified. At 20 and 40 days, the pattern of amplification was similar to that in control testes, with amplification of the whole coding sequence. In situ hybridization was performed on day 5 after EDS treatment, when the interstitial space was devoid of morphologically discernible Leydig cells. An antisense complementary RNA probe, corresponding to the extracellular domain of the receptor, hybridized in the interstitial space to apparent precursor Leydig cells. Taken together, these results strongly suggest that the precursor Leydig cells, resistant to the cytotoxic action of EDS, express truncated forms of the LHR mRNA in the early stages of their differentiation. The full-length mRNA of LHR gradually appears when the functional Leydig cells recover and attain their differentiated functions. These data are analogous with our previous findings on testicular ontogeny; the fetal Leydig cell precursors constitutively express a truncated form of the LHR gene as well, and a similar change occurs in its alternative splicing during testicular maturation. Hence, the truncated form of the LHR mRNA is an early sign of Leydig cell differentiation, whether it occurs during ontogeny or in adulthood upon recovery from cytotoxic treatment.
authors.affiliation: Department of Physiology, University of Turku, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Tena-Sempere
authors.forename: F P
authors.initials: FP
authors.lastname: Zhang
authors.forename: I
authors.initials: I
authors.lastname: Huhtaniemi
mesh_terms: D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016923:Cell Death; D007985:Leydig Cells / Q000187:drug effects*; D008297:Male; D008698:Mesylates / Q000494:pharmacology*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011974:Receptors, LH / Q000235:genetics*; D012680:Sensitivity and Specificity; D013737:Testis / Q000166:cytology / Q000378:metabolism*; D014018:Tissue Distribution; D014158:Transcription, Genetic
chemical_list: D008698:Mesylates; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011974:Receptors, LH; C002994:ethylene dimethanesulfonate