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,
From the * Institute for the Study of Fertility,
Lis Maternity Hospital, Institute of
Pathology, Tel Aviv Sourasky Medical Center, and
Pediatric Hemato-Oncology Department, Division
of Hematology, Chaim Sheba Medical Center, Tel-Hashomer, Sackler Faculty of
Medicine, Tel Aviv University, Tel Aviv, Israel.
Present address: Immunology and Allergy,
Department of Pediatrics, Infection, Immunity, Injury and Repair Program,
Research Institute, The Hospital for Sick Children and the University of
Toronto, Toronto M5G 1X8, Canada.
| Correspondence to: Dr S. E. Kleiman, Institute for the Study of Fertility, Lis Maternity Hospital, Tel Aviv Sourasky Medical Center, 6 Weizman St, Tel Aviv 64239, Israel (e-mail: ser{at}tasmc.health.gov.il). |
| Received for publication February 10, 2003; accepted for publication April 28, 2003. |
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Abstract |
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Key words: Testicular HGCL expression, markers of spermatogenesis, spermatogenesis impairments, motility impairments, HGCL and chromosome bivalent formation
One gene with a potential role in human fertility is germ cell-less
(GCL). The GCL protein was first described in Drosophila
melanogaster as a crucial factor in embryonic germ cell development
(Jongens et al, 1992,
1994). It was shown that
Drosophila females with reduced GCL function give rise to sterile
adult progeny that lack germ cells. Drosophila with a
gcl genotype is associated with impaired spermatogenesis due
to the failure to establish transcription quiescence necessary for the proper
formation of germ cell precursors
(Robertson et al, 1999;
Leatherman et al, 2002).
Mouse and human orthologues of Drosophila GCL (mGCL, HGCL, and dGCL, respectively) were recently cloned and characterized (de la Luna et al, 1999; Kimura et al, 1999; Nili et al, 2001a). An important feature of all GCL proteins is the presence of an evolutionary conserved BTB/POZ (broad-complex, tram track, and bric-abrac/poxvirus and zinc finger) domain. This domain is an evolutionarily conserved protein-protein interaction domain often found in developmentally regulated genes (Godt et al, 1993; Bardwell and Treisman, 1994; Zollman et al, 1994). It is strongly implicated in the regulation of gene expression through oligomerization and interaction with cofactors, ultimately leading to chromatin remodeling and changes in gene expression (review by Collins et al, 2001).
mGCL was found to be expressed in low levels at the primordial
germ cells and highly expressed in the adult mouse, mainly at the pachytene
spermatocyte stage (Kimura et al,
1999; Leatherman et al,
2000). This protein rescued the dGCL null phenotype,
indicating that mGCL is a functional orthologue of dGCL
(Leatherman et al, 2000). mGCL was demonstrated to interact with the DP3
component of
the E2F-DP heterodimer transcription factor, an interaction that was found to
repress the transcriptional activity of the E2F complex. This repression is
thought to be mediated through anchoring of the E2F complex to the nuclear
envelope, possibly through LAP2ß, a nuclear envelope protein that also
binds GCL (de la Luna et al,
1999; Nili et al,
2001b). Furthermore, overexpression of mGCL was suggested
to cause the accumulation of cells in the G1 phase, suggesting that it has
properties of a negative cell-cycle regulator
(de la Luna et al, 1999).
The HGCL gene was recently isolated and mapped to chromosome 2p13. HGCL expression is not ubiquitous, and the highest levels of messenger RNA were detected in the testis (Nili et al, 2001a).
Given the involvement of mGCL in spermatogenesis, the present study focused on the role of HGCL in human spermatogenesis and, particularly, at meiosis. The expression of the HGCL gene in testicular biopsy specimens of azoospermic men who were grouped according to their histologic and cytologic findings was evaluated. To overcome the nonhomogeneous nature of the testis, expression of HGCL was also correlated to that of germ cell-specific genes (RBM, DAZ, and CDY1), denoting the presence of germ cells in the biopsy specimen used for RNA extraction. In addition, the expression of HGCL was correlated to meiosis normality as measured by the rate of meiotic bivalent formation in spermatocytes.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Testicular Tissue Evaluation
One biopsy specimen from each testis was divided into 3 pieces: 2 small
pieces (approximately 5 mg each) were taken, one for histopathological
analysis and the other for RNA isolation. The third portion of the biopsy
(approximately 50 mg) was minced for spermatozoa isolation to be used in the
intracytoplasmic sperm injection (ICSI) process. In 22 cases, an additional
small piece was minced, fixed, and further analyzed by fluorescence in situ
hybridization (FISH) for meiosis evaluation
(Yogev et al, 2000). Two
additional biopsy specimens from other locations were taken from each testis
for spermatozoa extraction only in almost all cases
(Hauser et al, 1998).
Quantitative Analysis and Classification of Testicular Biopsy
Specimens
Histological examination was performed on Bouin's fixed paraffin-embedded
biopsy specimens after staining with hematoxylin-eosin. The most advanced
spermatogenetic cell that was identified determined the histological
definition. The terms mature spermatid and spermatozoa were
used for histological and minced biopsy specimen detection, respectively.
Normal spermatogenesis in azoospermic men was classified by the mean number of
mature spermatids per tubule according to the classification proposed by
Silber et al (1997). At least
20 seminiferous tubules were scored in each specimen. The presence of
spermatozoa in minced testicular tissue was assessed as previously described
(Ben-Yosef et al, 1999). Sperm
motility was qualitatively judged in each biopsy specimen immediately after
mincing and at 30-minute intervals of incubation up to 2 hours.
Four groups were established according to the combined findings of histological and cytological examination of the 3 biopsy specimens taken from the respective testis. The control group included 19 biopsy specimens from men with a normal number of mature spermatids per tubule (>17 sperm cells per tubule; Silber et al, 1997) by histological analysis. Fourteen of them were from male carriers of cystic fibrosis mutations/5T polymorphism combined with congenital absence of the vas deferens. The hypospermatogenesis group included 23 men in which at least one biopsy specimen contained spermatozoa or mature spermatids. The spermatocyte maturation arrest group (10 biopsy specimens) was characterized by the absence of spermatozoa or mature spermatids in all locations of both testes. The Sertoli cells only group included 15 men in which an absolute absence of germ cells was found in all biopsy specimens.
The mean ± SE follicular stimulating hormone values in the normal spermatogenesis, hypospermatogenesis, spermatocyte maturation arrest, and Sertoli cell only groups were 5.7 ± 1.4, 19.24 ± 2.6, 16.3 ± 3.3, and 20 ± 2 mIU/mL, respectively.
Genetic Evaluation
Karyotype and Y-chromosome microdeletion tests were performed on peripheral
lymphocytes. Chromosome analysis was performed by the G-banding chromosome
staining technique. The Y-chromosome AZF microdeletion test was performed by
multiplex polymerase chain reaction (PCR) with genomic DNA as previously
described (Kleiman et al,
1999).
Testicular biopsy specimens from one testis (left or right) of each man was
frozen in liquid nitrogen immediately after having been dissected and
cryopreserved until RNA isolation. Total RNA was extracted after
homogenization by the High Pure RNA Tissue kit (Roche, Mannheim, Germany), and
10 µL of the extract was used for complementary DNA (cDNA) synthesis with
AMV reverse transcriptase (Roche) and oligo-dT15. The
oligonucleotide primer sets for CDY1 minor, DAZ, RBM, and
ß-actin were previously described
(Kleiman et al, 2001). The
oligonucleotide primers set for the detection of HGCL expression
(GCL-up CTATTACACATCAGCAGGGAC and GCL-down CTTGAGGCCCCACCTCACTGTCC) were
designed from the published sequence accession XM_031592. These primer sets
for HGCL, CDY1 minor, DAZ, and RBM gave
differential PCR products for cDNA and genomic DNA. The same PCR product for
cDNA and genomic DNA was obtained with ß-actin primers. In view
of this observation, RNA samples were tested with and without the reverse
transcriptase (RT) step to verify that there was no genomic DNA contamination.
The expression of ß-actin was evaluated as an internal control
for the quality of the RNA isolation and efficiency of the RT-PCR method. A
positive control (cDNA from the castrated man) and blank controls were
included in each PCR run. Whenever PCR results were negative in 2 independent
reactions, RT and PCR steps were
redone.
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Meiotic Evaluation
Spermatocyte evaluation with triple-color FISH analysis using centromeric
DNA probes for chromosomes X, Y, and 18 (Vysis, Downers Grove, Ill) was
performed (Yogev et al, 2000).
The rate of bivalent formation was scored as previously described
(Yogev et al, 2002).
Generally, at least 350 primary spermatocytes were analyzed in each specimen,
according to statistical calculations for the required sample size. The FISH
technique used for bivalent evaluation had certain advantages over other
techniques mainly because it facilitates the evaluation of meiosis, even in
pathological cases with an extremely low number of spermatocytes that reflect
serious testicular damage (Metzler-Guillermain and Guichaoua, 2000).
Statistical Analysis
The association between the groups with various impairments of
spermatogenesis (determined by the combined results of the histological and
cytological evaluations) and the presence of HGCL expression was
checked using the Pearson 
2 test. Fisher's exact test was
performed to assess the significance of the relationship between the presence
of expression of HGCL and the spermatogenic impairment. The
significance of HGCL association to the overall expression of
DAZ, RBM, and CDY1 and to the normal motility of sperm cells
was assessed by the same test. Pearson's test was performed to assess the
correlation between percentage of biopsy specimens with the spermatozoa and
chromosomes bivalent formation rate. The difference in the percentage of
chromosome bivalents formation between groups expressing and not expressing
HGCL was calculated by the t test.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Characteristics of Biopsy Specimens With Impairment of HGCL
Expression
Specimens with hypospermatogenesis showed significant differences in the
sperm motility between HGCL-expressing and
non-HGCL-expressing ones (P = .039). Although motility is
difficult to assess when performing ICSI, testicular specimens with weakly
motile, nonmotile, or a low percentage of motile spermatozoa were clearly
detected among 6 (75%) of 8 men in the hypospermatogenesis group with the
absence of HGCL expression. These findings were different from men in
the same group who expressed HGCL, among whom only 4 (27%) of 15 had
motility impairments. Yet, there were 2 biopsy specimens from the normal group
with no detectable HGCL who had normal sperm motility and
morphology.
HGCL Expression and Chromosome Bivalents Formation in
Spermatocytes
As a transcription repressor expressed mainly at the pachytene
spermatocytes in mouse, GCL might regulate meiosis. We therefore tested for a
possible correlation between HGCL expression and meiosis bivalent
formation in the testicular biopsy specimens.
Table 2 depicts individual
results of the bivalent formation rate of homologous chromosomes X-Y and 18,
the HGCL expression findings, and the presence of testicular
spermatozoa in at least 1 of the 3 biopsy samples of the testis. There was a
significantly higher rate of bivalent formation of homologous chromosomes
(both X-Y and 18) wherever spermatozoa were found in at least one location of
the testis (Pearson correlation, P < .001). The rate of chromosome
bivalent formation was not affected by the presence or absence of
HGCL expression (Table
2). The rate of bivalent X-Y chromosomes (mean ± SE) was
66.8 ± 6.43 and 75 ± 9.52 in the groups with and without
GCL expression, respectively, and the rate of 18 chromosome bivalent
was 87.1 ± 4.26 and 91 ± 5.71, respectively.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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A significant association between HGCL expression in the testis and the presence of germ cells or mature spermatid or spermatozoa was demonstrated. The detection of HGCL expression in 40% of the biopsy specimens with Sertoli cell only was intriguing, because its homologous mouse gene mGCL was detected in germ cells but not in somatic (eg, Sertoli) cells of the testis (Kimura et al, 1999; Leatherman et al, 2000). It is possible that HGCL expression might reflect abnormal events in the somatic cells of the testis. Studies using immunohistochemical analysis with anti-HGCL antibodies may clarify these findings. Unfortunately, polyclonal anti-mGCL antibodies did not work on paraffin-embedded human testicular biopsy specimens, so the possibility that HGCL expression might serve as a marker for testicular abnormalities could not be tested.
Patients with germinal failure might have minute foci of spermatogenesis sparse throughout the entire testis (Silber et al, 1997). Side by side, different histological and minced tissue findings such as Sertoli-cell-only and complete spermatogenesis might be detected in such patients. In view of the nonhomogeneous nature of the testis, we compared the expression of HGCL to the expression of DAZ, RBM1, and CDY1 minor genes within the same biopsy specimen. This was done in view of the fact that expression of DAZ and RBM1 confirms the presence of spermatogenetic cells (Menke et al, 1997; Elliot et al, 1998, Lee et al, 1998; Bar-Shira Maymon et al, 2001), whereas CDY1 minor, expressed in spermatids, reflects the presence of haploid germ cells (Kleiman et al, 2001; Lahn et al, 2002). The expression of HGCL correlates with that of DAZ, RBM1, and CDY1 minor. Nevertheless, exceptions were detected, suggesting that HGCL expression is more frequently impaired compared with the other markers tested. Further mutation analysis of the HGCL gene might help to clarify the source of the impairment, either mutations on the gene or impaired regulation of gene expression.
It was estimated that approximately 2000 different genes are involved in a variety of testicular functions, including testicular development, germ cell differentiation, meiosis, and spermiogenesis (Bhasin et al, 1998), suggesting that the genetic basis of male infertility might be highly heterogeneous. We classified our biopsy specimens based on the overall histological and cytological testicular findings: these groups could include subgroups with different unknown mutations that lead to similar impairment, a feature that might complicate the interpretation of our results.
Most biopsy specimens with spermatozoa were found to express HGCL, and defective sperm motility was observed very frequently whenever no HGCL was expressed, suggesting that HGCL might be involved in the regulation of differentiation during spermiogenesis. Larger numbers of patients and a quantitative analysis of motility would be needed to test this assumption. Many factors might influence the testicular sperm motility detected. However, the recently published findings in mgcl-1-null male mice model support our observation (Kimura et al, 2003). Structural abnormalities, decrease of sperm motility, and reduction of path velocity of motile sperm were observed in mgcl-1-null male mice.
Recent studies performed on transfected H1299 cells have reported an intrinsic and additive ability of mGCL to repress transcription (Nili et al, 2001b). Other proteins (eg, LAP2ß) might compensate for the absence of GCL in certain cells or under specific circumstances and might explain the lack of critical impairments of spermatogenesis found whenever HGCL was absent.
Lessons from other species (mice, flies, etc) are usually helpful in understanding human infertility in general and for the function of HGCL in particular. Studies on mGCL suggested that GCL might play a role in cell cycle, particularly at meiosis (de la Luna et al, 1999, Kimura et al, 1999; Leatherman et al, 2000). No particular difference, however, was observed in the rate of X-Y and 18 chromosome bivalents in spermatocytes between biopsy specimens that expressed or failed to express HGCL, suggesting that HGCL was not involved or, at least, was not essential in the normal process of chromosome bivalent formation during meiosis. Recently, it was published that the first abnormality observed during spermatogenesis in mgcl-1-null male mice was abnormal nuclear envelope structure in spermatocytes, affecting the appropriate nuclear-laminar organization. This in turn is essential for normal sperm morphogenesis and chromatin remodeling during spermiogenesis (Kimura et al, 2003).
In conclusion, HGCL by itself seems to play a minor role in spermatogenesis, probably during spermiogenesis. HGCL expression was affected mainly when spermatogenesis was dramatically impaired. It did not appear to be involved in men with a prominent meiotic impairment. HGCL does not seem to be essential for spermatozoa production: it might merely affect the overall normal spermatogenesis process and spermiogenesis in a specific way, by influencing the activity of genes that are directly involved in these processes.
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Acknowledgments |
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Footnotes |
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References |
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