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From the * Department of Veterinary Anatomy,
Histology and Embryology, University of Giessen, Germany; the
Department of Urology, University of Marburg,
Germany; the Clinic and Polyclinic of Urology,
University of Muenster, Germany; and the
Institute of Pathology, University of Giessen,
Germany.
| Correspondence to: Prof Dr Martin Bergmann, Justus-Liebig-Universität, Institut für Veterinär-Anatomie, -Histologie und -Embryologie, Frankfurter Straße 98, D-35392 Giessen, Germany (e-mail: Martin.Bergmann{at}vetmed.uni-giessen.de). |
| Received for publication April 27, 2004; accepted for publication August 3, 2004. |
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Abstract |
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Key words: Gap junctional intercellular communication, cx33, testis, spermatogenesis
Therefore, they play a key role in the regulation of spermatogenesis (Risley et al, 1992; Jegou, 1993; Griswold, 1995; Juneja et al, 1999; Risley, 2000; Roscoe et al, 2001; Risley et al, 2002). In rodents, the cells of the seminiferous tubule express a complex panel of cx (Risley, 2000). In human testis, only 2 members of the cx family are described at present: cx26 and cx43 (Steger et al, 1999; Brehm et al, 2002). However, many other cx are probably implicated in the complex cell-cell interaction and communication in human testis. One of them could be cx33, which has already been detected in the rodent seminiferous tubule.
For the first time, cx33 was described in 1992 by Haefliger et al (Haefliger et al, 1992). This cx seems to be a member of the gap junction protein family with a couple of specialties. Haefliger et al noticed, by Northern blot analysis, that the expression pattern in the rat was restricted to testis tissue only, in contrast with most other cx-family members, which are expressed in a wide variety of organs and tissues. With the more sensitive method of reverse transcription-polymerase chain reaction (RT-PCR), it has been shown that rat cx33 mRNA seems to be present predominantly but not only in testis (Chung et al, 1999). Additionally, Chang et al (1996) observed that cx33 was able to inhibit the expression of cx37 and reduced the expression of cx43. Therefore, a possible inhibitory role for this cx was suggested. A model is proposed where cx33 limits the capacity of cells to build functional channels by enabling formation of heterotypic channels with other cells, while formation of homotypic channels is not possible. In immunogold electron microscopy and immunofluorescence, rat cx33 was described to be part of intratubular Sertoli cell gap junctions but not of interstitial Leydig cell gap junctions. Most cx33 immunoreactivity occurred as stage-dependent in a linear pattern near the basal Sertoli cell junctional complexes. Occasionally, it has been found on adluminal surfaces with most strongly immunoreactive tubules at stages II-VII and weak reactivity at stages IX-XIV (Tan et al, 1996). In RT-PCR experiments with isolated rat testis cells, cx33 mRNA was detected mainly in Sertoli cells but also in pachytene spermatocytes and round spermatids (Risley, 2000). More recently, Willecke et al (Willecke et al, 2002) compared the cx genes in the mouse genome with those in human and observed that only 1 mouse cx gene (cx33) and 2 human cx genes (cx25 and cx59) seem to have no orthologs in the other genome, suggesting that no further cx gene would be discovered in the remaining nonsequenced portion of the human genome.
This study investigates the existence and expression of cx33 gene in normal human testis as well as in arrests of spermatogenesis and Sertoli cell-only syndrome. Because of its inhibitory effect on other cx, it is possible that an overexpression of cx33 alters communication defects between Sertoli cells and germ cells or Sertoli cells to each other. Healthy testis tissue from other mammals, including the marmoset monkey as a representative of the primates, was likewise investigated for cx33 gene status.
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Materials and Methods |
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B) Human Testicular Tissue— Investigations were performed on biopsies from 18 human patients with obstructive azoospermia and on testicular tissue from 18 orchiectomized patients treated for prostate carcinoma, both showing histologically normal spermatogenesis (score 10-8; Bergmann and Kliesch, 1998). Additionally, biopsies of 19 infertile patients showing mixed atrophy were used. In these cases, testicular sections showed tubules with an arrest of spermatogenesis at the level of round spermatids, spermatocytes or spermatogonia, or Sertoli cell-only syndrome (SCO syndrome). Informed consent was obtained from all patients providing biopsies for our studies.
C) Testicular Tissue From Different Mammals— Testicular tissue from different mammals, such as rat, dog, cattle, pig, horse, and marmoset monkey with histologically normal spermatogenesis was obtained from routine castrations. Rat testicular tissue was always used as positive control. The samples derived from patients treated in different veterinary clinics belonging to the Department of Veterinary Medicine at the University of Giessen, Germany. Castration methods were performed specifically for each species. All animals were treated humanely.
Immunohistochemistry
A) Cx33: Paraffin Sections and Immunohistochemistry—
For cx33 immunostaining, deparaffinized and rehydrated sections from human,
dog, pig, horse, marmoset monkey, and rat were boiled for 30 minutes in sodium
citrate buffer (pH 6.0), exposed to 20% acetic acid for 15 seconds, blocked
with 5% bovine serum albumin (Fluka Chemie AG, Buchs, Switzerland) for 30
minutes, and incubated with the only commercially available polyclonal
anti-cx33 primary antibody (1:500; rabbit anti-rat; Biotrend, Köln,
Germany) overnight at 4°C. Sections were then exposed to the secondary
antibody (1:50; mouse anti-rabbit, IgG; DAKO, Glostrup, Denmark) followed by
the third antibody (1: 50; rabbit anti-mouse, IgG; DAKO, Glostrup, Denmark)
for 30 minutes each. Sections were incubated with a mouse alkaline phosphatase
antibody complex (1:100; DAKO, Hamburg, Germany) for 30 minutes. Following
each incubation, sections were washed thoroughly with 0.1 M Tris-HCl buffer
(pH 7.4; Sigma, Steinheim, Germany). The immunoreactive product was visualized
using HistoMark Red (KPL, Gaithersburg, Md). Negative controls were performed
by omitting the primary antibody as well as by replacing the primary antibody
with rabbit IgG at the same dilution and under similar incubation conditions
as the primary antibody. The Leydig cells served as internal negative
control.
B) Cx33: Cryostat Sections and Immunofluorescence— In order to demonstrate that the fixation procedure did not change the epitope structure of cx33, we performed immunofluorescence on fresh-frozen tissue of human testis. Cryostat sections from 6 frozen human testis biopsies were attached to microscope slides, fixed with 96% ethanol, blocked with 5% bovine serum albumin for 15 minutes, and incubated with the polyclonal anti-cx33 primary antibody (1:100; rabbit anti-rat; Biotrend, Köln, Germany) for 90 minutes at room temperature. Then sections were exposed to the secondary antibody (1:200; donkey anti-rabbit, fluoresceinisothiocyanate [FITC]-conjugated; Chemicon International, Temecula, Calif) for 30 minutes. Following each incubation, sections were washed with 0.1 M phosphate buffered saline (PBS; pH 7.4; Sigma, Steinheim, Germany). Negative controls were performed by omitting the primary antibody.
C) Cross-Reaction of the cx33 Antibody— In order to clarify if the cx33 antibody cross-reacts with mitochondrial proteins in the human spermatids, 2 assays were performed: first, the mitochondria in human testis tissue were visualized by immunostaining with an anti-cytochrome c oxidase antibody; the signal localization in round spermatids was compared with that obtained for cx33. Second, based on similarities between the cx33 antibody and a protein in human testis, mitochondrial ferritin, the unspecific detection of the latter by our cx33 antibody, was investigated by an immunoabsorbant assay.
C1) Mitochondrial Staining Pattern: Paraffin Sections and Immunohistochemistry— We performed immunohistochemical staining of deparaffinized and rehydrated paraffin sections from human using a mouse monoclonal antibody against cytochrome c oxidase (1:500; mouse anti-human; Molecular Probes, Leiden, Netherlands). The sections were boiled in sodium citrate buffer (pH 6.0) for 30 minutes, exposed to 20% acetic acid for 15 seconds, blocked with bovine serum albumin for 30 minutes, and incubated with the primary antibody described above for 90 minutes at room temperature. Sections were then exposed to the secondary antibody (1:50; rabbit anti-mouse, IgG) for 30 minutes. Afterward, the sections were treated with a mouse alkaline phosphatase antibody complex (1:100; DAKO, Glostrup, Denmark). Following each incubation, sections were washed thoroughly with 0.1 M Tris-HCl buffer, ph 7.4. The immunoreactive product was visualized using HistoMark Red (KPL). Negative controls were performed by omitting the primary antibody.
C2) Mitochondrial Ferritin: Paraffin Sections and Immunohistochemistry— Cx33 antibody was incubated with recombinant human mitochondrial ferritin (kindly provided by Prof Arosio, University of Brescia, Italy) for 60 minutes at room temperature (10 µg mitochondrial ferritin/500 µL diluted antibody [1:500]). After centrifugation, we used the supernatant for immunohistochemical staining of human sections as described above.
Protein Extraction and Western Blot Analysis
Proteins were extracted from unfixed frozen human and rat tissue by using
the TRIzol® reagent as recommended by the manufacturer (Life Technologies,
Karlsruhe, Germany). Proteins were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) in 10% resolving gels under
reducing conditions and blotted onto a Westran® polyvinylidene fluoride
transfer membrane (Schleicher and Schuell, Dassel, Germany). The membrane was
blocked with 5% nonfat dry milk in 0.1 M phosphate buffered saline (PBS; pH
7.4) and incubated with polyclonal anti-cx33 antibody (1:500; rabbit anti-rat;
Biotrend, Köln, Germany) overnight. A biotinylated goat anti-rabbit IgG
antibody (1:500; DAKO, Glostrup, Denmark) was used as secondary antibody.
Finally, the membrane was treated with Vectastain Elite ABC Kit (Vector,
Burlingame, Calif) and developed with True Blue Peroxidase Substrate (KPL).
Control Western blots were performed by omitting the primary antibody.
RT-PCR
The RT-PCR assay for detection of cx33 mRNA was first performed on RNA
extract from whole human testis tissue. Because we suggested a weak expression
of cx33, certain fractions of cells of the seminiferous epithelium, where the
cx33 expression was presumptively high, were investigated. The cell isolation
was performed by ultraviolet laser-assisted microdissection leading to a
higher specificity and sensitivity of the assay.
A) RNA Extraction and RT-PCR From Tissue Homogenate— Total RNA from frozen rat and human testicular tissue was extracted by using the TRIzol® reagent (Life Technologies) as recommended by the manufacturer. Then it was incubated with RNase-free DNase I (10 U/µL; Roche, Mannheim, Germany; 1-3 U/1 µg RNA, 40 minutes, 37°C) to digest genomic DNA. The DNase activity was stopped by incubating the probe at 72°C for 10 minutes. First-strand cDNA synthesis was performed using Superscript II RNase H- reverse transcriptase (Invitrogen, Karlsruhe, Germany) and Omniscript reverse transcriptase (Qiagen, Hilden, Germany) according to the manufacturer's protocols, showing identical results. Four different primer sequences were used for PCR: 2 primer pairs for cx33 according to literature defined by Chung et al (Chung et al, 1999; 648-base pair [bp] amplicon; 5' CAG AAA CAT ACA GGA AAG CAT ATC 3pr = sense; 5' AAT GTT AAT GTC CAG TAC CCT TGC 3' = antisense) and Risley (Risley, 2000; 878-bp amplicon; 5' ATG AGT GAT TGG AGT GCC TTA CAC 3' = sense; 5' ACA TGG TTC TGT TCT CTT TAC 3' = antisense) and 2 self-designed pairs of primers: 5' ATG AGT GAT TGG AGT GCC TTA CAC 3' = sense, 5' GGC CAT GTA GGT (C/T)AG CAG CAA (C/T)CT 3' = antisense (cx33W, 484-bp amplicon) and 5' GGT GTC AIT GGT GTC TTT TGT C 3' = sense, 5' CTA GAA IIT ATG GTG GTG AGA AC 3' = antisense (cx33E, 165-bp amplicon). These 2 pairs of primers amplify smaller amplicons and, by using wobbles, respectively, Inosin, they are more unspecific for rat and mouse cx33, more specific for the human genome, and were finally chosen to exclude amplification of highly homologous cx43. The sequences were based on the NCBI accession numbers AK040693 and M76534. The synthesized primers were purchased from MWG Biotech (Ebersberg, Germany). For PCR, 1 µL of cDNA was added to 5 µL 10x PCR buffer (10x Buffer; Promega, Mannheim, Germany), 2 µL MgCl2 (25 mmol/L; Promega), 1 µL dNTPs (10 mmol/L; Promega), 0.5 µL polymerase (5 U/µL; Taq DNA polymerase; Promega), 1 µL of each primer (10 µmol/L), and DEPC-H2O to a final volume of 50 µL. Then different PCR conditions were used: 1) according to Chung et al (1999) and Risley (2000) and 2) the following programs for the primer pair cx33W: 1x 95°C for 3 minutes, 10x 94°C for 40 seconds, 55°C (down to 50°C) for 1 minute, 72°C for 2 minutes, 25x 94°C for 40 seconds, 52°C (down to 47°C) for 1 minute, 72°C for 2 minutes, and 1x 72°C for 10 minutes; and for the primer pair cx33E: 1x 95°C for 3 minutes, 10x 94°C for 40 seconds, 59°C (down to 48°C) for 1 minute, 72°C for 2 minutes, 25x 94°C for 40 seconds, 56°C (down to 45°C) for 1 minute, 72°C for 2 minutes, and 1x 72°C for 10 minutes. PCR products were finally separated on a 2% agarose gel, visualized by ethidium bromide (Sigma, Steinheim, Germany), and the sequences analyzed by Qiagen sequencing service.
For each primer pair, control RT-PCR was performed using rat cDNA. Negative controls were performed by omitting reverse transcriptase. For internal control and evaluation of RNA quality, we used RT-PCR for the housekeeping gene ß-actin from the same RNA samples that were used for cx33 amplification. The used primer sequences and PCR conditions were those defined by Hess et al (1992).
B) Ultraviolet Laser-Assisted Microdissection and Laser Pressure Catapulting— Microdissection was performed using the PALM® MicroBeam system (PALM, Bernried, Germany), consisting of a nitrogen laser of high-beam precision (wavelength 337 nm), which was coupled to an inverted microscope (Axiovert 135; Zeiss, Jena, Germany) via the epifluorescence illumination path.
The PALM® Membrane Slides (PALM, Benried, Germany) were irradiated with an ultraviolet lamp for 30 minutes to achieve better adhesion of the sections. For histological evaluation, the sections were stained with hematoxylin and dehydrated.
Sertoli cells and different generations of germ cells of human and rat testicular tissue were identified by light microscopy and separated from the adjacent tissue by laser microdissection. With a laser shoot, the chosen cells were catapulted into the cap of a test tube (laser pressure catapulting) filled with mineral oil. Per tube, either 15 seminiferous tubules containing all germ and Sertoli cells (rat and human) or only round spermatids of 15 seminiferous tubules (according to the immunohistochemical staining of round spermatids in human, see below) were collected and dissolved in 350 µL lysis buffer (RNeasy Micro Kit; Qiagen) with ß-mercaptoethanol (Serva, Heidelberg, Germany; 10 µL ß-mercaptoethanol/mL lysis buffer) and 5 µL carrier RNA (4 ng/µL; RNeasy Micro Kit, Qiagen). After centrifugation and vortexing, specimens were immediately frozen in liquid nitrogen.
RNA from both human and rat testicular tissue was extracted using the RNeasy Micro Kit (Quiagen) according to the manufacturer's protocol, including the use of carrier RNA and DNase-I digestion. First-strand cDNA synthesis was performed using Sensiscript reverse transcriptase as recommended by the manufacturer (end volume = 20 µL; Qiagen). For PCR from micro-dissected cells, we used the primer pair named cx33E (165-bp amplicon, described above). Because of the instability of nucleic acids in microdissected cells, the amplicon should not be longer than 200 bp. Ten microliters of cDNA were added to 5 µL 10x PCR-buffer (including 15 mmol/L MgCl2; Qiagen), 10 µL 5x Q-Solution (Qiagen), 1 µL dNTPs (Promega, Mannheim, Germany), 0.5 µL polymerase (5 U/µL; Taq polymerase; Qiagen), 1 µL of each primer (10 µmol/L), and DEPC-H2O to a final volume of 50 µL. PCR conditions were as follows: 1x 95°C for 3 minutes, 15x 94°C for 40 seconds, 59°C (down to 48°C) for 1 minute, 72°C for 2 minutes, 40x 94°C for 40 seconds, 56°C (down to 45°C) for 1 minute, 72°C for 2 minutes, and 1x 72°C for 10 minutes. For internal control due to low amounts of mRNA, RT-PCR was performed again for ß-actin from the same RNA samples used for cx33 amplification. The used primer sequences for ß-Actin were 5' TTC CTT CCT GGG CAT GGA GT 3' = sense and 5' TAC AGG TCT TTG CGG ATG TC 3' = antisense (90-bp amplicon). The primers were purchased from MWG Biotech (Ebersberg, Germany). PCR conditions were 1x 95°C for 2 minutes, 50x 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, and 1x 72°C for 10 minutes. PCR products were finally separated on a 2% agarose gel, visualized by SYBR-Green (Sigma-Aldrich, Munich, Germany) and the sequences were analyzed by Qiagen.
DNA Extraction and PCR
For detecting the cx33 gene, DNA from frozen human, rat, dog, cattle, pig,
horse, and marmoset monkey normal testicular tissue was extracted by using the
QIAamp DNA Mini Kit as recommended by the manufacturer (Qiagen). The extracted
DNA was precipitated by mixing with 3 M sodium acetate and 96% ethanol and
incubated overnight at -20°C. After centrifugation and washing with 70%
ethanol, DNA was dissolved in 10 mM Tris buffer (pH 7.5). Identical PCR
conditions and primers were used as described above (RNA extraction and RT-PCR
from tissue homogenate). PCR products were finally separated on a 2% agarose
gel, visualized by ethidium bromide (Sigma, Steinheim, Germany), and the
sequences analyzed (Qiagen). In each case, negative controls were performed by
omitting the DNA.
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In human testis, the cx33 signal occurred differently. We found a signal only in the adluminal compartment in round and elongated spermatids (Figure 2A through C). The staining within round spermatids was noticed as a spotted and not stage-dependent signal in the periphery of the cytoplasm, similar to mitochondrial localization in germ cells (Russell et al, 1990). This indicates that the used antibody may cross-react with a mitochondrial protein. A staining in the basal compartment of the tubules in the cytoplasm of Sertoli cells, as described for rat tissue, was not detected in human. In human testicular tissue showing histologically impaired spermatogenesis (arrest of spermatogenesis at the level of round spermatids, spermatocytes, and spermatogonia, or SCO syndrome), we only could find weak isolated signals in round spermatids in tubules with arrest of spermatogenesis at the level of round spermatids, indicating a specific staining in spermatids. In all other tubules missing round and elongated spermatids, the immunostaining was absent (data not shown). In addition, immunohistochemical experiments using testicular tissue from dog, pig, horse, and marmoset monkey with histologically normal spermatogenesis did not show any specific signal (data not shown).
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Immunofluorescence experiments using frozen human testicular tissue with histologically normal spermatogenesis supported the above described findings for paraffin immunohistochemical staining. Here, we also detected a signal in the adluminal compartment of the seminiferous tubule in spermatids (Figure 2D).
Immunohistochemistry, Cross-Reaction of the Anti-cx33 Antibody
Immunohistochemical experiments using the antibody against human cytochrome
c oxidase for detecting mitochondrial staining pattern in round spermatids in
human tissue with normal spermatogenesis exhibited a comparable spotted signal
in the periphery of the cytoplasm of round spermatids as described above using
the anti-cx33 antibody (Figure
2E).
Using the recombinant human mitochondrial ferritin for blocking the antibody against cx33 followed by immunohistochemical staining of human testicular tissue with normal spermatogenesis showed total disappearance of the described staining pattern of cx33 (Figure 2F and G).
Western Blot Analysis
Western blot experiments for cx33 using whole-testis protein extracts from
rat and human revealed a single band between 27 and 33 kD in rat testis
tissue. In contrast, no immunoreactive band using human testicular tissue with
normal spermatogenesis in Western blot analysis was detectable
(Figure 3). No cross-reaction
of the anti-cx33 antibody with cx43 was observed.
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PCR
For detecting the cx33 gene in human and different mammals, PCR with human,
dog, cattle, pig, horse, and marmoset monkey testis tissue and rat testis
tissue as positive control showing normal spermatogenesis was performed. The
results for each used primer pair were comparable. The rat testis tissue
showed the expected cx33 amplicon. This identity was confirmed by sequencing.
Although the cattle and dog testis tissues showed also bands in the expected
position, these amplicons were not specific by sequencing. All other species,
including human, showed no amplicon with the cx33 specific primers
(Figure 4A).
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RT-PCR (Testis Homogenate and Microdissected Cells)
RT-PCR of human and rat testis homogenate with normal spermatogenesis using
4 different pairs of cx33 primers with varying specificity and different size
of amplicons resulted each in 1 clear signal of the expected size in rat used
as positive control. The identity of the amplicons was confirmed by
sequencing. No specific signal in human was found. The housekeeping gene
ß-actin, used for internal control and evaluation of RNA quality, showed
a single band of the expected size in all human samples
(Figure 4B).
RT-PCR using microdissected cells showed comparable results. Rat samples containing RNA from whole seminiferous tubules resulted in a cx33-specific band confirmed by sequencing. No specific or only unspecific amplicons could be detected in human testis RNA extracts. The identity of the amplicons was confirmed by sequencing. Additional experiments using only dissected round human spermatids did not show any signal in human as well. The housekeeping gene ß-actin used for internal control due to a low amount of mRNA in microdissection showed a single band of the expected size in all human samples (Figure 4C and D).
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Discussion |
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Immunohistochemistry of normal human testis with a commercially available antibody against cx33 revealed signals situated in round and elongated spermatids. Therefore, a possible hypothesis for the signal in the adluminal compartment of the rat seminiferous epithelium therefore could have been the necessity of cx33 to modulate gap junctional communication if elongating spermatids in the adluminal compartment change their contact pattern to Sertoli cells by the differentiation of tubulo-bulbar complexes before spermiation. However, a comparison between the immunohistochemical staining pattern of human and rat testis tissue questioned the specificity of the signals in human. The localization of the immunoreactive signal in the rat testis demonstrated the same results as described with a different anti-cx33 antibody (Tan et al, 1996). The signals were present both in the basal and adluminal compartment. However, the latter were not stage specific and due to an unspecific reaction of the antibody with residual bodies. Because the signals situated in the cytoplasm of rat Sertoli cells were absent in human, a direct comparison of these 2 stainings was not possible. However, the spotted signal in the adluminal compartment detected in human testis tissue was comparable with the location of mitochondria in spermatids (Russell et al, 1990). We approved this hypothesis by demonstrating the localization of mitochondria in human germ cells by cytochrome c oxidase immunostaining. By comparing the sequence of the only commercially available peptide antibody against 17 amino acids of the carboxy-terminal of cx33 (confidential information of the manufacturer, therefore not specified) with human proteins in testis, we noticed that a protein named mitochondrial ferritin, which has been described in literature to be located in the mitochondria of round and elongated spermatids (Drysdale et al, 2002) as a possible candidate for cross-reaction containing 6 identical amino acids. We were able to confirm this assumption by successful preabsorbtion studies with recombinant human mitochondrial ferritin leading to a complete disappearance of cx33 immunohistochemical staining. The results indicate that the used anti-cx33 antibody cross reacts in human testis with mitochondrial ferritin. Therefore, we were not able to confirm the suspicion that cx33 protein exists in human testis with normal spermatogenesis.
Although cx33 expression in human testis could not be demonstrated in healthy tissue, we decided to investigate its expression in human testicular tissue with impaired spermatogenesis. One demanding example is cx26, of which expression is very weak to almost undetectable in normal spermatogenesis but obviously stronger in arrest of spermatogenesis at the level of spermatogonia (Brehm et al, 2002). However, the expected immunohistochemical staining of Sertoli cells remained negative in all patients investigated.
Additionally, our attempts to verify the immunohistochemical findings in Western blot analysis failed as well as the detection of any specific signal for cx33 in human. Human mitochondrial ferritin could not be detected by Western blot analysis using the commercially available anti-cx33 antibody. A possible explanation is the capacity of the antibody to cross-react only with a tissue-preserved epitope of mitochondrial ferritin, which probably changes its steric features in Western blot analysis.
In our investigations on the DNA level, we initially did not focus on a special chromosome. But it is well known that cx33 is mapped to the X chromosome of rodents (Haefliger et al, 1992; Schwarz et al, 1994) and the map localization of the cx genes in mouse seems to be consistent with their map localization in human (Haefliger et al, 1992; Sohl and Willecke, 2004). Therefore, we focused in the X chromosome of the human genome. For this purpose, we used different data bases and data programs, splitting the rat cx33 gene in smaller segments, bearing in mind that parts of the X chromosome in human have not been sequenced yet. Because all investigations on the DNA level, including PCR experiments and additionally at RNA level using RT-PCR for both tissue homogenate and microdissected cells did not show any specific signal in human, we extended our search for the cx33 gene and protein to different mammals, keeping evolutionary aspects in mind. Because specific results could not been detected on the DNA or protein levels, we conclude that cx33 seems to be a rodent-specific member of the gap junction protein family. Our data indicate that cx33 is not implicated in the impairment of cell-cell communication associated with spermatogenic defects in human testis.
The complete sequencing of the murine genome allows us, for the first time, to make good comparisons of the most closely related available genome sequences, those of mouse and human. If we compare the genome of these species, the overall impression is one of similarity because 80% of the genes have 1-to-1 corresponding counterparts in the other's genome. In contrast, differences among genomes have received less attention. It was noticeable that the differences cluster in 3 regions: in genes that are relevant for immunity, in genes for host defence, and also in reproductive-typical genes due to the high selective pressure (Emes et al, 2003). Many reproductive genes are found among the 10% most divergent genes. This widespread phenomenon might have important consequences, such as the establishment of barriers to fertilization that might lead to speciation (Swanson and Vacquier, 2002). In conclusion, testis-specific cx33 could be 1 more member of the rapidly evolving genes in the reproductive system presenting important functions for rodent spermatogenesis but not in other mammals, including human. This hypothesis is sustained by the results of a DNA sequence comparison showing a very high level of divergence in male reproductive proteins between closely related species, for example, mouse and rat (Sutton and Wilkinson, 1997; Wyckoff et al, 2000). Comparing the cx33 protein sequence of rat and mouse, various differences are also visible (Figure 5). The sequences were based on the NCBI accession numbers BAC30669and P28233. The physiological role of cx33 in spermatogenesis remains an open question. The results of this study raise the question of whether cx33 really plays an important role in spermatogenesis. Maybe further investigations using transgenic mice will give hints for the precise role of cx33 in rodent spermatogenesis. Additionally, further investigations will clarify if there is another cx in testis responsible for the inhibitory function of cx33 or which other mechanisms or molecules may lead to inhibition of gap junctional conductance.
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Acknowledgments |
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Footnotes |
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