Identification and Localization of T-type Voltage-operated Calcium Channel Subunits in Human Male Germ Cells
2002; Elsevier BV; Volume: 277; Issue: 10 Linguagem: Inglês
10.1074/jbc.m105345200
ISSN1083-351X
AutoresSuchitra Jagannathan, Emma L. Punt, Yuchun Gu, Christophe Arnoult, Denny Sakkas, Christopher L. R. Barratt, Stephen J. Publicover,
Tópico(s)Ion channel regulation and function
ResumoLow voltage activated, voltage-operated Ca2+ channels are expressed in rodent male germ cells and are believed to be pivotal in induction of the acrosome reaction in mouse spermatozoa. However, in humans, very little is known about expression of voltage-operated Ca2+ channels in male germ cells or their function. We have used reverse transcription-polymerase chain reaction, in situ hybridization, and patch clamp recording to investigate the expression of low voltage activated voltage-operated Ca2+ channels in human male germ cells. We report that full-length transcripts for both α1G and α1H low voltage activated channel subunits are expressed in human testis. Multiple isoforms of α1G are present in the testis and at least two isoforms of α1H, including a splice variant not previously described in the human. Transcripts for all the isoforms of both α1G and α1H were detected by reverse transcription-polymerase chain reaction on mRNA isolated from human spermatogenic cells. In situhybridization for α1G and α1H localized transcripts both in germ cells and in other cell types in the testis. Within the seminiferous tubules, α1H was detected primarily in germ cells. Using the whole cell patch clamp technique, we detected T-type voltage-operated Ca2+ channel currents in isolated human male germ cells, although the current amplitude and frequency of occurrence were low in comparison to the occurrence of T-currents in murine male germ cells. We conclude that low voltage activated voltage-operated Ca2+ channels are expressed in cells of the human male germ line. Low voltage activated, voltage-operated Ca2+ channels are expressed in rodent male germ cells and are believed to be pivotal in induction of the acrosome reaction in mouse spermatozoa. However, in humans, very little is known about expression of voltage-operated Ca2+ channels in male germ cells or their function. We have used reverse transcription-polymerase chain reaction, in situ hybridization, and patch clamp recording to investigate the expression of low voltage activated voltage-operated Ca2+ channels in human male germ cells. We report that full-length transcripts for both α1G and α1H low voltage activated channel subunits are expressed in human testis. Multiple isoforms of α1G are present in the testis and at least two isoforms of α1H, including a splice variant not previously described in the human. Transcripts for all the isoforms of both α1G and α1H were detected by reverse transcription-polymerase chain reaction on mRNA isolated from human spermatogenic cells. In situhybridization for α1G and α1H localized transcripts both in germ cells and in other cell types in the testis. Within the seminiferous tubules, α1H was detected primarily in germ cells. Using the whole cell patch clamp technique, we detected T-type voltage-operated Ca2+ channel currents in isolated human male germ cells, although the current amplitude and frequency of occurrence were low in comparison to the occurrence of T-currents in murine male germ cells. We conclude that low voltage activated voltage-operated Ca2+ channels are expressed in cells of the human male germ line. acrosome reaction zona pellucida zona pellucida glycoprotein 3 voltage-operated calcium channel low voltage activated high voltage activated reverse transcription neoglycoprotein digoxigenin ohm(s) The AR1 of spermatozoa is crucial for fertilization. In humans, male factor infertility is highly correlated with failure of AR and/or the events that activate AR (1Krausz C. Bonaccorsi L. Luconi M. Fuzzi B. Criscuoli L. Pellegrini S. Forti G. Baldi E. Hum. Reprod. 1995; 10: 120-124Crossref PubMed Scopus (82) Google Scholar, 2Baldi E. Luconi M. Bonaccorsi L. Maggi M. Francavilla S. Gabriele A. 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In both instances, activation of exocytosis requires a multiphasic entry of Ca2+ through plasma membrane ion channels (9Florman H.M. Arnoult C. Kazam I.G. Li C. O'Toole C.M.B. Biol. Reprod. 1998; 59: 12-16Crossref PubMed Scopus (166) Google Scholar, 10Shirakawa H. Miyazaki S. Dev. Biol. 1999; 208: 70-78Crossref PubMed Scopus (37) Google Scholar, 11Kirkman-Brown K.C. Bray C. Stewart P.M. Barratt C.L.R. Publicover S.J. Dev. Biol. 2000; 222: 326-335Crossref PubMed Scopus (74) Google Scholar, 12O'Toole C.M.B. Arnoult C. Darszon A. Steinhardt R.A. Florman H.M. Mol. Biol. Cell. 2000; 11: 1571-1584Crossref PubMed Scopus (192) Google Scholar).Stimulation of mouse spermatozoa with purified ZP3 induces a brief (200 ms) [Ca2+]i transient, which is blocked by antagonists of VOCCs (13Arnoult C. Kazam I.G. Visconti P.E. Kopf G.S. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6757-6762Crossref PubMed Scopus (186) Google Scholar). This transient activates a separate sustained [Ca2+]i influx, probably through store-operated channels incorporating Trp2, which leads to AR (12O'Toole C.M.B. Arnoult C. Darszon A. Steinhardt R.A. Florman H.M. Mol. Biol. Cell. 2000; 11: 1571-1584Crossref PubMed Scopus (192) Google Scholar, 13Arnoult C. Kazam I.G. Visconti P.E. Kopf G.S. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6757-6762Crossref PubMed Scopus (186) Google Scholar, 14Jungnickel M.K. Marrero H. Birnbaumer L. Lemos J.R. Florman H.M. Nat. Cell Biol. 2001; 3: 499-502Crossref PubMed Scopus (277) Google Scholar). Application of the patch clamp technique to the study of ion channels in mature spermatozoa has proved to be very difficult and has so far been limited to cell-attached recordings and use of artificial bilayers (Ref. 15Darszon A. Labarca P. Nishigaki T. Espinosa F. Physiol. Rev. 1999; 79: 481-510Crossref PubMed Scopus (277) Google Scholar, and see “Discussion”). Unfortunately, this approach has (so far) provided little information on expression of VOCCs. However, the whole cell clamp technique has been applied successfully to mouse spermatogenic cells (16Hagiwara S. Kawa K. J. Physiol. 1984; 356: 135-149Crossref PubMed Scopus (73) Google Scholar, 17Arnoult C. Cardullo R.A. Lemos J.R. Florman H.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13004-13009Crossref PubMed Scopus (204) Google Scholar, 18Santi C.M. Darszon A. Hernandez A. Am. J. Physiol. 1996; 40: C1583-C1593Crossref Google Scholar). These cells express a LVA, transient VOCC current with voltage dependence and kinetics very similar to those of expressed, recombinant α1G and α1H LVA channels (19Klöckner U. Lee J.-H. Cribbs L.L. Daud A. Hescheler J. Pereverzev A. Perez-Reyes E. Schneider T. Eur. J. Neurosci. 1999; 11: 4171-4178Crossref PubMed Scopus (150) Google Scholar, 20Lacinová L. Klugbauer N. Hofman F. Gen. Physiol. Biophys. 2000; 19: 121-136PubMed Google Scholar, 21McRory J.E. 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However, male germ cells of α1E knockout mice possess normal T-currents, indicating that α1E channels do not mediate the currents seen in these cells (32Sakata Y. Saegusa H. Zong S. Osanai M. Murakoshi T. Shimizu Y. Noda T. Aso T. Tanabe T. Biochem. Biophys. Res. Commun. 2001; 288: 1032-1036Crossref PubMed Scopus (21) Google Scholar). Espinosa et al. (28Espinosa F. López-González I. Serrano C.J. Gasque G. de la Vega-Beltran J.L. Trevino C.L. Darszon A. Dev. Genet. 1999; 25: 103-114Crossref PubMed Scopus (46) Google Scholar) used RT-PCR to investigate expression of LVA channel transcripts in mouse germ cells. Appropriate products were generated using primers directed against the -COOH termini of α1H and α1G. However, Jacob et al. (33Jacob A. Benoff S. J. Androl. 2000; 56: 48Google Scholar), using primer pairs against various regions of α1G, could obtain products only with primers encoding domain IV and the -COOH terminus in rat testis mRNA. Antibodies for LVA channels are not available.Very little is known about the expression and roles of VOCCs in human male germ cells. The effects of VOCC antagonists on progesterone-induced [Ca2+]isignaling in spermatozoa have been studied extensively, but findings have been both variable and contradictory (34Blackmore P.F. Beebe S.J. Danforth D.R. Alexander N. J. Biol. Chem. 1990; 265: 1376-1380Abstract Full Text PDF PubMed Google Scholar, 35Foresta C. Rossato M. di Virgilio F. Biochem. J. 1993; 294: 279-283Crossref PubMed Scopus (101) Google Scholar, 36Aitken R.J. Buckingham D.W. Irvine D.S. Endocrinology. 1996; 137: 3999-4009Crossref PubMed Scopus (60) Google Scholar, 37Blackmore P.F. Eisoldt S. Mol. Hum. Reprod. 1999; 5: 498-506Crossref PubMed Scopus (69) Google Scholar, 38Garcia M.A. Meizel S. Biol. Reprod. 1999; 60: 102-109Crossref PubMed Scopus (76) Google Scholar, 39Publicover S.J. Barratt C.L.R. Hum. 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Frontiers Biosci. 1998; 3: d1220-d1240Crossref PubMed Google Scholar) have provided evidence for the presence of a number of isoforms of the HVA α1C subunit in human testis and spermatozoa. However, only preliminary data are available on expression of LVA channels. Sonet al. (46Son W.-Y. Lee J.-H. Lee J.-H. Han C.-T. Mol. Hum. Reprod. 2000; 6: 893-897Crossref PubMed Google Scholar) obtained a 489-bp RT-PCR fragment of α1H with human testicular mRNA. Transcripts for α1G were not detected in human testicular mRNA (46Son W.-Y. Lee J.-H. Lee J.-H. Han C.-T. Mol. Hum. Reprod. 2000; 6: 893-897Crossref PubMed Google Scholar) or mRNA isolated from human spermatozoa (33Jacob A. Benoff S. J. Androl. 2000; 56: 48Google Scholar).To understand the processes that underlie AR in human spermatozoa, and to assess the accuracy of the mouse model of AR, it is vital that the nature of the Ca2+ channels involved is elucidated. As a vital, first step, we have undertaken the detection, sequencing, and localization of T-channel subunit transcripts from the human testis and have applied the patch clamp technique to immature human germ cells.MATERIALS AND METHODSPCRPCR forward and reverse primers were designed to sequences of α1G from the human brain (GenBank™ accession no.AF029229) and α1H from the human heart (GenBank™ accession no. AF051946). PCR products were amplified on human, adult, normal testis cDNA (Invitrogen, Groningen, Netherlands; Origene Technologies, Inc., Rockville, MD) using in-house primer pairs (TableI) and the Expand High Fidelity PCR System containing thermostableTaq DNA polymerase and a proofreading enzyme (Roche Diagnostics, Mannheim, Germany). A human brain cDNA library provided by the Medical Research Council, Human Genome Mapping Project Resource Center, United Kingdom, was used as positive control. Touchdown PCR was carried out on a Primus 25 legal thermocycling PCR system (MWG Biotech Ltd., Ebersberg, Germany). The cycling conditions were 94 °C for 3 min 30 s, followed by six cycles of 94 °C, 30 s; 64 °C, 30 s; 72 °C, 3 min; with a touchdown temperature change of −0.5 °C/cycle. A further 40 cycles were carried out at 94 °C, 30 s; 61 °C, 30 s; 72 °C, 3 min, followed by a final extension at 72 °C for 10 min. The annealing temperatures for different primer pairs were altered in the range of 64 to 58 °C depending upon the Tm (melting temperature) of the primer pairs in use. Corresponding changes in touchdown temperatures were made. The annealing temperature of the second set of PCR cycling conditions was kept 3 °C below the annealing temperature of the first set of cycling conditions.Table ISense and antisense primers used in the amplification of transcripts for α1G and α1H subunits and the leukocyte common antigen precursorNo.α1G primersSense primerAntisense primerNameSequenceNameSequence1HGS45′-ATGGACGAGGAGGAGGAT-3′HGAS25′-TGTCTCCTCTGCTCATCC-3′2HHS85′-TGCGTGACCCTGGGCATG-3′HGAS35′-GGGAGGCTGAAATTCTCAG-3′3HGS65′-CTGAGCGTGGACCTGGAG-3′HGAS55′-ATGATGTCGACCCAGCCC-3′4HGS75′-GCCTGGATCGCCATCTTC-3′HGAS65′-TGGTGGTGATGGTGGTGG-3′5HGS95′-AGCTGCTCTCGCTCCCAC-3′A1G10R5′-GGCTGGTGAAGACGATGTTG-3′6A1G9F5′-TTGGCCGGGGAATCATGATC-3′HGAS75′-TCGGGCTCTGATTCGGAC-3′7HGS125′-TGACCTTCGGCAACTACGTGC-3′HGAS125′-TCCAGCTGCTTGCAGCGCTC-3′8HGS185′-AAGCAGCTGGACCAGCAGG-3′HGAS185′-GACAAGGACCACGTGGTCG-3′9HGS175′-TCCTGTGTCACCGATCATC-3′HGAS175′-ATGTTCAGGACAAAGAAGGCC-3′10HGS115′-CTGAACATGTTTGTGGGTGTGG-3′HGAS115′-TGACACCTGTGATGAAGAGGTC-3′11HGS115′-CTGAACATGTTTGTGGGTGTGG-3′HGBAS15′-TCAGCCATCTGCTTCTCCTTAC-3′12HGS165′-CACTTGTGCACCAGCCACTAC-3′HGAS165′-CTCTCCTCCAGGTGCTTCATC-3′13HGS135′-ACTTTGTGTCCTTCGTGCTGAC-3′HGAS135′-ACAAGACGGAGCCTGACTGAG-3′14HGS145′-TGTCGGCATGGGAGCACTG-3′HGAS145′-CTCCTCTGCTCATCCAGCC-3′15HGS15′-GAAGTGCTACAGCGTGGAGGC-3′HGAS15′-CCAGGTCTGCTGGGTCAGAGG-3′No.α1H primersSense primerAntisense primerNameSequenceNameSequence1HHS75′-ATGACCGAGGGCGCACG-3′HHAS95′-CTCGGAGCCGCACTCAAC-3′2HHS85′-TGCGTGACCCTGGGCATG-3′HHAS105′-CCTGACAAAGGCACTGTCC-3′3HHS95′-GGACAGTGCCTTTGTCAG-3′HHAS85′-CGGCACACGTTGTAGTAC-3′4HHS25′-CCAGTACTACAACGTGTGCC-3′HHAS25′-GATCTTCTCGTAGGGATCAG-3′5HHS15′-GGCTGGGCACCATGAACTAC-3′HHAS15′-GTGAATTCATAGACGCCACG-3′6HHS45′-GGTGAGCTCAGCGGCTC-3′HHAS135′-CAGCAGCCTGTGCAGAAC-3′7HHS105′-TACCATGAGCAGCCCGAG-3′HHAS145′-CGTGTCGGATCTGTTGGC-3′8HHS145′-CAACTATGTGCTCTTCAACCT-3′HHAS215′-ACAGGGAGAACTTCGGAGGC-3′9HHS145′-CAACTATGTGCTCTTCAACCT-3′HHAS205′-TGGTCTGGAGTTCTCTGAGC-3′10HHS175′-GCGATGCCAACAGATCCGAC-3′HHAS185′-ATCATCTCCGCCACGAAGATG-3′11HHS125′-GTGGCGGAGATGATGGTG-3′HHAS175′-TAGTGGCTGGTGCACAGC-3′12HHS155′-TTCATCTCCTTCCTGCTCATCG-3′HHAS195′-CATGGTGATGACGTTGACACAG-3′13HHS155′-TTCATCTCCTTCCTGCTCATCG-3′HHAS225′-GCGAGTAGTCGGCATAGTAGG-3′14HHS155′-TTCATCTCCTTCCTGCTCATCG-3′HHAS235′-CGAAGGTCACGAAGTAGACGG-3′15HHS165′-TCTACTTCGTGACCTTCGTGC-3′HHAS255′-GAGCTGGTGATGTGGCTGAC-3′16HHS195′-ACGAGGAGGTCAGCCACATC-3′HHAS245′-AGAGGAAGCTCTGGATTCTGG-3′17HHS185′-TCTTGGACGGTAGCCACAGTG-3′HASUTR15′-TCTCCTGACGGGAGGCTGAC-3′No.Leukocyte primersSense primerAntisense primerNameSequenceNameSequenceSLEUKO15′-ACACCCATTTTGTTATACTTATGC-3′ASLEUKO15′-AAGTTGTTCTAAGTCAGTAGAATG-3′ Open table in a new tab The PCR products were run on agarose gels and purified using a QIAquick™ Spin gel extraction kit (Qiagen GmbH, Hilden, Germany) and either sequenced directly (MWG Biotech. Ltd.) or cloned into a pGEM®-T Easy cloning vector (Promega).Isolation of Germ CellsHuman CellsCells were obtained by two methods.For RT-PCR and for the first series of electrophysiological recordings, testicular tissue was removed from patients who were attending for treatment at the Assisted Conception Unit, Birmingham Women's Hospital, Birmingham, United Kingdom (Human Fertilization and Embryology Authority Center 0119). Mature, motile spermatozoa were found in all biopsies used for cell isolation. Sufficient testicular tissue was initially isolated for the treatment of patients by intracytoplasmic sperm injection, whereas the remainder was used to isolate cells undergoing spermatogenesis. Ethical approval was obtained from the local ethics committee (0374 and 0420). A total of six biopsies were used for isolation of cells, three of which had been frozen in sperm freezing medium (MediCult Ltd., Copenhagen, Denmark) after selection of cells for intracytoplasmic sperm injection and subsequently thawed for isolation of germ cells and patching. The extracted testicular tissue was placed in a Petri dish containingin vitro fertilization medium (Scandinavian IVF, Gothenburg, Sweden) and the seminiferous tubules teased out using needles. Following this, the tissue was then passed through a series of smaller gauge needles starting at 26 through to 18. Once the cells were sufficiently dissociated, a 50-μl droplet of the cell suspension was placed in a Petri dish along with two 50-μl droplets of clean medium. The droplets were covered with oil (OvOil, Scandinavian IVF). The Petri dish was then placed on a Nikon Microscope under 200× magnification, and individual cells were removed from the cell suspension using a Narashige micromanipulator. Micropipettes with a 10–15-μm inner diameter were used to individually select germ cells (spermatocytes and spermatids). The classification of germ cells was the same as that previously published by Johnson and colleagues (47Johnson L. Neaves W.B. Barnard J.J. Keillor G.E. Brown S.W. Yanagimachi R. Biol. Reprod. 1999; 61: 927-934Crossref PubMed Scopus (20) Google Scholar). Once an individual germ cell was selected, it was placed in a clean medium droplet. This procedure was repeated until sufficient germ cells were isolated. The droplet was then aspirated using a pipette, and the contents were either placed in an Eppendorf tube for RT-PCR (see above) or incubated overnight in a Petri dish for attachment to gelatin-coated slides prior to electrophysiological recording (see below). For RT-PCR, a total of 75 cells from the six biopsies were pooled before extraction of mRNA. For patch clamping, cells from five of the biopsies were used.For the second series of electrophysiological recordings, seminiferous tubules were isolated from the testes of a patient undergoing an orchidectomy (ethical authorization number DGS 2001/0211) and incubated at 37 °C for 30 min in 3 ml of solution containing (mm): NaCl (150), KCl (5Wassarman P.M. Cell. 1999; 96: 175-183Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), CaCl2 (2Baldi E. Luconi M. Bonaccorsi L. Maggi M. Francavilla S. Gabriele A. Properzi G. Forti G. Steroids. 1999; 64: 143-148Crossref PubMed Scopus (45) Google Scholar), MgCl2 (1Krausz C. Bonaccorsi L. Luconi M. Fuzzi B. Criscuoli L. Pellegrini S. Forti G. Baldi E. Hum. Reprod. 1995; 10: 120-124Crossref PubMed Scopus (82) Google Scholar), NaH2PO4 (1Krausz C. Bonaccorsi L. Luconi M. Fuzzi B. Criscuoli L. Pellegrini S. Forti G. Baldi E. Hum. Reprod. 1995; 10: 120-124Crossref PubMed Scopus (82) Google Scholar), NaHCO3 (12O'Toole C.M.B. Arnoult C. Darszon A. Steinhardt R.A. Florman H.M. Mol. Biol. Cell. 2000; 11: 1571-1584Crossref PubMed Scopus (192) Google Scholar),d-glucose (11Kirkman-Brown K.C. Bray C. Stewart P.M. Barratt C.L.R. Publicover S.J. Dev. Biol. 2000; 222: 326-335Crossref PubMed Scopus (74) Google Scholar), pH 7.3, and collagenase type IA (1 mg/ml; Sigma). Tubules were rinsed twice in collagenase-free medium and cut into 2-mm sections. Spermatogenic cells were obtained by manual trituration and attached to culture dishes coated with Cell-Tak (Collaborative Biomedical Products, Bedford, MA). The cells obtained were primarily pachytene spermatocytes and round spermatids.Mouse CellsTestes were dissected in phosphate-buffered saline and transferred to a Petri dish containing Earle's balanced salts medium (Sigma) containing 1% (w/v) trypsin (Invitrogen) in RNase-free water. Seminiferous tubules were teased out using a sterile needle and the sample incubated at 37 °C for 2 h with intermittent shaking. The aqueous medium was then pipetted out from the Petri dish, and mineral oil (Sigma) was overlaid. Germ cell isolation, including overnight incubation for attachment, was then carried out as described above for isolation of cells from biopsies taken for intracytoplasmic sperm injection (method 1 above). For patch clamp, untrypsinized tissue was used to isolate germ cells. After isolation, cells were allowed to attach to gelatinized slides before recording (see below).RT-PCR on Germ CellsFor both human and mouse preparations, total RNA was isolated from 75–100 germ cells using the StrataPrep® Total RNA Microprep kit (Stratagene) as per the manufacturer's protocol (48Steuerwald N. Cohen J. Herera R.J. Brenner C.A. Mol. Hum. Reprod. 1999; 5: 1034-1039Crossref PubMed Scopus (83) Google Scholar). The expected yield of RNA was 50–100 ng. RNA was eluted in a total volume of 60 μl of elution buffer. 30 μl was used for reverse transcription. The RT reaction was carried out in a total volume of 60 μl in presence of 5 mm MgCl2, 0.8 mm dNTP, 1.5 units of recombinant RNasin® (1 unit/μl), 1× RT buffer (10 mm Tris-HCl (pH 9.0 at 25 °C), 50 mm KCl, 0.1% (v/v) Triton®X-100), 1.5 μg of random hexamers (0.5 μg/μl), and 48 units of avian myeloblastosis virus reverse transcriptase enzyme (15 units/μl) (Promega). The reaction mix was incubated for 10 min at room temperature, followed by 60 min at 42 °C. The sample was placed in a boiling water bath for 5 min and on ice for 5 min to inactivate the enzyme. The RT mix was stored at −70 °C for further use or used immediately in PCR.PCR reaction was carried out using 5 μl of the RT mix from RNA isolated from human or mouse germ cells, 1× RT buffer, 0.64 μm primer pairs (A1G9F-A1G10R specific to α1G and HHS1-HHAS1 specific to α1H, respectively), and nuclease-free water to make up the total reaction volume to 25 μl.Touchdown PCR, similar to that described under “PCR,” was carried out in the presence of Taq DNA polymerase enzyme (Promega). Primers internal to the region amplified were used to confirm the PCR products obtained. Control PCR reactions employed (i) human or mouse β actin primers (Origene) to amplify 614 and 575 bp, respectively, and (ii) primer pairs matching the human T200 leukocyte common antigen precursor gene sequence (GenBank™ accession no. AH007396; see TableI). PCR was carried out using RT mix from germ cell RNA and testis cDNA (Invitrogen) as template. Amplification conditions were hot start at 94 °C for 3 min 30 s, followed by 35 cycles of 94 °C, 30 s; 64 °C, 30 s; 72 °C, 1 min. The PCR product obtained using cDNA as template was sequenced to confirm its identity.In Situ HybridizationA digoxigenin (DIG) labeling and detection kit (Roche; Ref. 49Meehan T. Schlatt S. De O'Bryan M.K. Kretser D.M. Loveland K.L. Dev. Biol. 2000; 220: 225-237Crossref PubMed Scopus (162) Google Scholar) was used to make DNA probes with PCR-amplified products. The products used were a 395-bp product from the COOH-terminal end of α1G (PCR product no. 15; Fig. 1) and a 373-bp product from the I-II linker region of α1H (PCR product no. 5; Fig. 1). The PCR product was cloned into pGEM®-T Easy vector and DIG label incorporated into the cloned DNA template by PCR carried out for 25 cycles at 94 °C, 1 min 45 s; 55 °C, 1 min 30 s; 72 °C, 2 min. The annealing temperature was 59 °C for α1H. 0.4 μl of Expand High Fidelity PCR enzyme (1 unit/μl; Roche) was used in a 50-μl reaction containing 5 μl each of 10× DIG DNA labeling mix, 10× HF buffer, 1.6 pmol of primer, and 0.1 μg of plasmid DNA. DIG labeled DNA was run on a 1.5% (w/v) agarose gel. Labeled probe, migrating at a higher molecular weight than unlabeled cDNA, was eluted from the gel using QIAquick spin gel extraction kit. In situ hybridization was carried out on adult human testis sections (Novagen; Peterborough Hospital Tissue Bank, United Kingdom). Probe mixture and hybridization conditions were as described in the Roche manual with some modifications. Sections were de-waxed (Histoclear) for 10 min, rehydrated, and digested with 0.1 μg/ml proteinase K (Sigma) for 5 min at 37 °C. Sections were hybridized overnight at 42 °C and sequentially washed twice for 15 min at 20 °C with 5× SSC and once for 10 min at 42 °C with 1× SSC. Probe hybrids were localized using 1:500 dilution of alkaline phosphatase anti-DIG antibody (Fab fragments; Roche). Alkaline phosphatase activity was finally stained with freshly prepared solutions of 45 μl of nitro blue tetrazolium chloride (3 mg/ml) and 35 μl of 5-bromo-4 chloro-3 indolyl phosphate (3 mg/ml) in 1 ml of detection solution (0.1 m Tris-HCl, pH 8.5, 0.05m MgCl2, 0.1 m NaCl; Ref. 50Fragale A. Aguanno S. Kemp M. Reeves M. Price K. Beattie R. Craig P. Volsen S. Sher E. D'Agostino A. Mol. Cell. Endocrinol. 2000; 162: 25-33Crossref PubMed Scopus (16) Google Scholar). The reaction was stopped with 10 mm Tris-HCl, pH 8.1, and 1 mm EDTA. Sections were mounted using DABCO (Sigma), an aqueous mounting medium and observed under bright-field illumination (Zeiss Axioskop 2). Control hybridization and localization reactions were carried out using non-DIG-labeled probe, using unconjugated DIG (blind probe), without the anti-DIG antibody and without use of nitro blue tetrazolium chloride and 5-bromo-4 chloro-3 indolyl phosphate. Positive reactions were also carried out on other tissues to confirm localization of hybridization.The distributions of α1G and α1Htranscripts within seminiferous tubules, were compared by counting stained cells. For each of the two probes, 20 tubule profiles derived from two different samples were examined. The numbers of stained spermatogenic cells and Sertoli cells were counted, and the distributions of staining (spermatogenic:Sertoli) for the two transcripts were compared using a chi-square contingency table.ElectrophysiologyTwo series of electrophysiological recordings were made. For the first series, cells were isolated from human testicular biopsies taken
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