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Quantification
of human telomerase RNA (hTR) and human telomerase reverse transcriptase
(hTERT) mRNA in testicular tissue of infertile patients
Mark
Schrader, Markus Mller, Rdiger Heicappell, Bernd Straub, Kurt Miller
Department
of Urology, Universitätsklinikum Benjamin Franklin, Freie Uni
Asian J Androl 2001 Dec; 3: 263-270
Keywords:
Abstract
Aim:
1
Introduction
Testicular sperm extraction (TESE) with subsequent ooplasmic injection of haploid germ cells is the therapy of choice for patients with non-obstructive azoospermia[1]. Problematic in connection with this invasive approach is the lack of prognostic parameters for the presence of haploid testicular germ cells. It is undisputed that peripheral FSH or inhibin B serum levels, testicular size and anamnestic data are of low value in predicting the presence of haploid germ cells in the testicles of men with non-obstructive azoospermia[2].
However, even the gold standard for assessing testicular tissue specimens, i.e., diagnostic testicular biopsy with histological workup, is not a highly accurate parameter for detecting and classifying spermatogenesis[3]. It has been demonstrated that foci of spermatogenesis are missed, particularly in hypergonadotropic hypogonadal men, and that focal islands of spermatogenesis are overlooked during the conventional histological workup in up to 30% of the biopsies[4,5]. An approach for improving the diagnostic value of testicular biopsies is the detection of germ-cell-specific gene expression.
A promising diagnostic parameter is the ribonucleoprotein telomerase, which functions as a cellular reverse transcriptase that catalyzes the synthesis and extension of telomeres[6]. Telomeres, the distal ends of eukaryotic chromosomes, protect the encoding DNA sequences from destabilization. Of special interest is their role in DNA replication[7]. Most human somatic cells lose telomeric nucleotides with each cell division, which limits their number of cell divisions[8]. In contrast, germline, stem, and more than 90% of tumor cells are believed to be immortal because telomere length is maintained by the action of telomerase[9], which progressively adds hexamer TACGGGs repeats to the end of human chromosomes. Major components of the active enzyme are the RNA template, known as human telomerase RNA (hTR)[10]; the catalytic subunit human telomerase reverse transcriptase (hTERT)[11-14]; and several telomerase-associated proteins such as TP1 (telomerase-associated protein 1) and tankyrase[15,16]. hTR is expressed in benign and malignant tissue and correlates only loosely with the detection of telomerase expression[10].
On the other hand, hTERT mRNA is expressed almost exclusively in germ cells, stem cells and malignant tumors and correlates closely with the detection of telomerase activity by telomeric repeat amplification protocol assay[11-14].
Several groups have recently shown that the detection of telomerase activity in testicular biopsies is a helpful parameter for excluding SCOS[17-19].
We
have also been able to show that the hTERT mRNA encoding for the catalytic
subunit of telomerase in testicular tissue is a highly sensitive and specific
marker for detecting germ cells in the testicles of men with non-obstructive
azoospermia[18]. The objective of the present study was to
evaluate the quantitative detection of hTERT mRNA and hTR by real-time
fluorescence RT-PCR as new molecular diagnostic
parameters, especially for classifying spermatogenesis disorders, in the
workup of testicular tissue specimens from patients presenting with non-obstructive
azoospermia.
2 Subjects and methods
2.1
Patients
Institutional
Review Board approval was obtained for this study. All patients signed
a consent form approved by the Committee on Human Rights in Research of
the Freie Universität Berlin. Thirty-eight testicular biopsies
were taken from patients presenting with infertility of varying etiology.
All had azoospermia. Test icular volume was determined by ultrasound.
An outpatient testicular biopsy was performed in all cases (n=38).
A small incision was made in the tunica albuginea to remove samples of
the exposed tissue measuring altogether about 333 mm.
2.2
Processing of testicular biopsy material
The
tissue samples were subdivided into 7 fractions, and the largest part
(3 fractions) was immediately placed in 1.0 mL of Sperm-Freeze solution
(Medicult, Hamburg, Germany) and transferred to liquid nitrogen by a computer
guided system (Planer 10, Messer-Griesheim, Griesheim, Germany). One sample
of testicular tissue from each patient was placed in a
Petri dish containing Sperm-Prep solution (Medicult, Hamburg, Germany)
and examined within 10 minutes. Minced tissue was examined by phase-contrast
microscopy at 400 magnification to detect cells of spermatogenesis,
especially mature spermatids. In the case of negative findings, tissue
was treated with collagenase type I
(Sigma, Heidelberg, Germany) following a modified form of the protocol
published by Schulze et al[20]. The samples found to
contain germ cells were likewise cryopreserved.
In
one part of the sample, the expression of hTERT and hTR was quantitatively
determined by fluorescence real-time RT-PCR. The part of biopsy material
intended for this was shock-frozen immediately after removal (3-5 min)
and then stored in liquid
itrogen. The tissue were thawed to -20,
and frozen serial tissue sections were performed. The first and last cryosections
were histologically examined after HE staining where as the major part
was used for RNA extraction. In this way, the
molecular-diagnostic work-up was paralled by an additional histological
work up of the examined sample.
Another
part of the sample was placed in Stieve's solution (formaldehyde DAB 1020.0
g, acetic acid 100% DAB 104.0 g, aqueous saturated 7% mercuric (II) chloride
solution 76.0 g), paraffin-embedded and prepared in 5 m slices. The
slices were stained using hematoxylin-eosin (HE). The biopsy material
was histologically evaluated according to the Johnsen score[21].
When assessment of the HE slices did not correspond to that of the wet preparation and/or the germ-cell-specific hTERT expression, tissue samples were also prepared using the semithin sectioning technique[22]. This procedure was also performed in all samples with spermatogenetic arrest and SCOS.
2.4 Quantitative detection of human telomerase catalytic subunit (hTERT) messenger RNA
Quantitative
detection of hTERT mRNA was performed with the commercially available
LightCycler Telo TAGGG hTERT Quantification Kit® (Roche
Diagnostics GmbH, Mannheim, Germany) using the LightCycler®
instrument (Roche Molecular Systems, Alameda, CA) for on-line PCR and
all subsequent quantification steps according to the manufacturer's instructions.
The
recently introduced LightCycler®[24] is a thermocycler
for on-line monitoring of PCR. According to the hybridization protocol
the amplicon is using two oligonucleotides that hybridize internally to
the amplicon during the annealing phase within each PCR cycle. One probe
is labeled with a fluorescent dye at the 5'end, the other with fluorescein
at the 3' end. The probes are designed to hybridize to the target strand
so that the two dyes are in close proximity and fluorescence resonance
energy transfer (FRET) takes place between the two fluorophores. This
leads to the emission of fluorescence, which is continuously detected
on-line during the PCR.
A
typical 20 µL one-tube RT-PCR reaction contained 200 ng of total
RNA (sample) or standard
RNA templates provided with the kit. mRNA was reverse-transcribed for 10
min at 60, PCR
amplifications were performed in separate tubes for 40 cycles (0.5 sec
at 95; 10 sec
at 60; 10 sec
at 72) using
manufacturer-supplied reaction mixtures specific for hTERT or the housekeeping
gene PBGD, respectively. The PBGD reaction product served as a control
for RT-PCR and as a reference for relative quantification of hTERT mRNA.
In
addition, all measurements included the detection of 5 in-vitro-transcribed
hTERT standards representing 1.3106, 9.8104,
8.0103, 7.2102 and 1.4102 copies
of hTERT mRNA. Probes without the addition of template that otherwise
fulfilled the same requirements were used as negative controls. Each sample
was normalized on the basis of its PBGD content according to the formula
NhTERT= hTERT mRNA copies sample/ (PBGD mRNA copies sample
/1000). The graph of the linear regression and calculation of the regression
coefficient r served to confirm the accuracy and reproducibility
of this approach.
Probes
were evaluated as hTERT mRNA-positive when the measurement of standard
probes and controls yielded adequate results and >400 copies of PBGD
mRNA were detected, suggesting an appropriate initial quantity and quality
of total RNA. They were assessed as negative when no hTERT mRNA was detected
in the presence of >400 copies of PBGD mRNA.
The
linear measuring range of the assay was set at 102-106
copies by the manufacturer in an exemplary system using in vitro transcribed
hTERT mRNA. Our use of the
provided in vitro transcribed hTERT RNA as a reference showed the applied
kit to have a sensitivity of approximately 100 hTERT mRNA copies. This
high sensitivity of real-time fluorescence RT\PCR with the LightCycler
has also been described
by many other groups[25-30] and are comparable to results obtained on
other real time PCR equipment[31].
Figure
1. Control electrophoretic separation of RT-PCR products for
hTERT mRNA detection.
Line 1-5 internal standard: 1.3106, 9.8104,
8.0103, 7.2102 and 1.4102 copies
hTERT mRNA; Line 6 positive control hTERT mRNA; Line 7 negative
control hTERT (reaction mix without control hTERT RNA); Line 8 positive
control PBGD mRNA; Line 9 negative control PBGD (reaction mix without
control PBGD RNA); Line 10 sample with detection of hTERT mRNA (198
bp); Line 11 sample with detection of PBGD mRNA (153 bp).
2.5 Quantitative detection of human telomerase RNA (hTR)
The
quantitative detection of hTR was performed using the same procedures
described for hTERT, however in combination with the LightCycler®
Telo TAGGG hTR Quantification Kit® (Roche Diagnostics GmbH,
Mannheim, Germany) since the hTR-encoding gene is intron-free, the PCR
products could result from contaminating
genomic DNA. To rule this out, an additional RT-PCR for each sample wa
performed without the addition of reverse transcriptase (minus-RT controls).
To carry out a positive control and establish an external standard curve,
all measurements included the determination of 5 standard RNA templates
with in vitrotranscribed hTR containing 8.6106, 5.8105,
6.9104, 6.1103 and 4.4102 copies
as well as total RNA purified from an hTR-expressing cell line provided
by the detection kit.
2.6 Statistical analysis
Statistical analysis was performed using the Kruskal Wallis test for nonparametric analysis of variance to compare the histological subgroups. The correlation of NhTERT and NhTR was evaluated by Spearman's test. A discriminant analysis of NhTERT and NhTR was also performed for the different histological subgroups. Values were expressed as follows: mean, standard deviation, median, 25th percentile, 75th percentile and range. Statistical analysis was performed using SPSS Software Version 10.0.
3
Results
3.1 Histopathology of the tissue samples
The histopathological examination of testicular biopsy material revealed Sertoli-cell-only syndrome (SCOS) in 12 cases. In 14 others, biopsy findings showed partial tubular atrophy with maturation arrest (MA). These included 10 samples exhibiting spermatogenesis with primary and secondary spermatocytes, corresponding to a Johnsen score of 4-5. Spermatogonia only (Johnsen score 3) were present in 4 cases. Biopsy results in the remaining 12 cases were within normal histological limits.hTERT expression (NhTERT range 30.8-11.2 copies) was detected in three tissue specimens even though they evidenced SCOS at the histological workup. The subsequent workup by semithin sectioning revealed spermatocytes in one case (NhTERT=30.8 copies) and spermatogonia only in two cases (NhTERT=11.2 and 14.2 copies). Two specimens with an hTERT expression of NhTERT=100.5 and 80.5 showed maturation arrest at the conventional histological workup. Subsequent assessment by semithin sectioning disclosed focal islands with full spermatogenesis in both of these specimens.
3.2 Testicular biopsy of patients with normal spermatogenesis
In 12 patients, testicular biopsy revealed histological evidence of full spermatogenesis. Tissue samples in all these cases were characterized by high hTERT mRNA expression with a mean NhTERT=131.948.0 copies (range 246.7-80.5). The mean hTR expression was NhTR=295.5181.8 copies (range 540.0-119.5). Ten of 12 tissue samples with evidence of full spermatogenesis had an hTERT mRNA expression of NhTERT>100 copies. Two tissue samples with only focally full spermatogenesis had an hTERT mRNA expression of NhTERT=80.5 and 82.6 copies, which was in the range of tissue samples with maturation arrest (Figure 2).3.3 Testicular biopsy of patients with maturation arrest
Fourteen
testicular biopsies revealed partial tubular atrophy with maturation arrest.Among
these were 10 tissue samples in which semithin sectioning provided histological
evidence of spermatogenesis arrest at the primary and secondary spermatocyte
level. In these cases, tissue samples showed a mean NhTERT
expression of 55.818.6
copies (range 83.9-30.0). The mean NhTR expression was 109.1248.99
copies (range 181.0-62.6).
Four tissue samples evidenced spermatogenesis arrest at the level of the spermatogonia. In these cases, tissue samples had a mean NhTERT expression of 17.95.3 copies (range 25.0-13.1). The mean NhTR expression was 112.1244.80 copies (range 176.0-81.0) and thus above that of patients with maturation arrest at the primary and secondary spermatocyte level (Figure 2).
Figure 2.
Detection of hTERT mRNA () in testicular tissue samples by realtime
on-line RT-|PCR analysis using the LightCycler®. Two hundred
nanograms of total
RNA of testicular tissue were analyzed. Concomitant detection of PBGD
mRNA () served as a reference for relative quantification. The copy
numbers of the starting template were calculated by comparing the relative
fluorescence signals of samples to external hTERT mRNA standards. The
standards used contained 1106, 0.9105, 0.8104,
0.8103 and 0.8102 copies/reaction of in vitro
transcribed hTERT mRNA. Relative expression levels were calculated according
to the following formula:
NhTERT=hTERT mRNA copies/(PBGD mRNA copies /1000).
X axis: cycle number, Y axis: fluorescence emission; F2=Lightcycler-Red
640, F1=fluorescein
Representative tissue specimens:
Top: complete spermatogenesis, NhTERT=119.2 copies; Middle:
maturation arrest,
NhTERT=36.2 copies; Bottom: Sertoli-cell-only syndrome, NhTERT=1.2
copies.
3.4 Testicular biopsy of patients with Sertoli-cell-only syndrome
In 12 patients, histology revealed germ cell aplasia. Tissue samples with SCOS showed only a minimal hTERT expression (NhTERT=2.72.4 copies; range 0.00-5.20). The NhTR expression was 61.2437.15 copies (range 119.2-32.0). Table 1 gives a summary of histological findings and the expression of NhTR and NhTERT in testicular biopsy (Figure 2) .Table 1. Quantification of hTERT mRNA and hTR expression in testicular biopsies by real-time fluorescence RT-PCR.
3.5 Correlation of NhTERT and NhTR and discriminate analysis
The Kruskal Wallis test showed significant differences between the NhTR and NhTERT copies expressed in the histological subgroups (P<0.001). Spearman's test yielded a coefficient of 0.599 (P<0.0001) for the correlation of NhTERT and NhTR. Discriminant analysis showed that the combination of NhTERT and NhTR had a predictive value of 89.5% for correct classification in one of the three histological subgroups. We found that 86.8% of the tissue samples would have been correctly classified by NhTERT alone but only 55.3% by NhTR alone.4
Discussion
A
molecular diagnostic parameter that is suitable for supplementing conventional histopathological
diagnostics in the assessment of testicular biopsies is the ribonucleoprotein
telomerase, which was first detected in testicular tissue by Kim et
al in 1994[9]. Prowse and Greider then demonstrated that
telomerase in testicular tissue is attributable solely to germ cells[36].
For stem cells and
tumor cells, telomerase activity was found to be inhibited with increasing
cell differentiation[37].Analogously, a down regulation of
telomerase activity during
gametogenesis has been demonstrated for testicular germ cells. Telomerase
activity was found to be high during spermatogenesis of spermatogonia
to round spermatids, whereas mature spermatids and epididymal spermatozoa
were telomerase-negative[36,38-40].
Several
groups have recently shown that the detection of telomerase activity in
testicular biopsies is helpful for detecting germ cells, particularly
in patients with hypergonadotropic hypogonadism with a predominant Sertoli-cell-only
histology[17,19,41].
Although
the telomeric repeat amplification protocol (TRAP) assay is still considered
the gold standard for detection of telomerase activity it is influenced
by numerous variables that impede an exact quantification, e.g., it does
not check for degradation of the RNA template, variances in the telomerase
yield due to cell lysis or instability of the enzyme activity during storage.
Moreover, the TRAP assay only partially detects enzyme inhibition by tissue
inhibitors and enzyme inactivation with heat or time. The latter two factors
may have been problematic in clinical studies due to the possibly nonuniform
temporal course of tissue preservation
and the contamination of tissue samples. In addition, it should be noted
that the TRAP assay can currently only quantify the PCR products but not
the initial generation of telomeric repeats.
Recently,
we were able to demonstrate that the hTERT mRNA coding for the catalytic
enzyme component in testicular tissue is highly specific and highly sensitive for
the presence of germ cells[18]. In contrast, we found that
the non-quantitative determination of hTR revealed no correlation between
testicular hTR expression and the presence of testicular germ cells.
This
study was the first to quantify hTERT mRNA and hTR expression by real-time
fluorescence RT-PCR in testicular tissue specimens from patients with
various spermatogenesis disorders for which we intended to establish a
molecular diagnostic subclassification. We chose this assay to avoid the
above-mentioned problem in achieving
an exact telomerase quantification by the TRAP assay. The advantage of
this detection procedure is that it accounts for varying tissue degradation
with amplification of a housekeeping gene, and primary purification of
RNA rules out tissue inhibitors of the PCR.
Moreover,
it has repeatedly been shown that hTERT mRNA is rate-limiting for telomerase
and that hTERT mRNA expression correlates well with telomerase activity[11-14].
For hTR expression, on the other hand, non-quantitative determination
revealed only a weak correlation with telomerase activity, indicating
the need for a quantitative
approach to assess hTR[10, 42].
In
12 cases, the histological examination disclosed complete spermatogenesis.
All specimens showed high hTERT expression of NhTERT=131.948.0
copies. Two of the 12 specimens with reduced spermatogenesis and only
focally complete spermatogenesis had a hTERT expression of only NhTERT=80.5
and 80.7 copies and were thus within the range of tissue samples with
maturation arrest. A cutoff value of NhTERT=100 copies correlated
with the histomorphological picture of complete spermatogenesis, while
one specimen with histologically diagnosed MA as well as two specimens
with focally complete spermatogenesis would also have been detected with
a cutoff of NhTERT=80 copies.
Maturation
arrest was diagnosed in 14 specimens. Tissue samples with a JS of 4-5
had a mean hTERT expression of NhTERT=55.818.6 copies. Four
tissue samples with maturation arrest at the level of spermatogonia (JS
3) had a markedly lower hTERT expression (NhTERT=17.95.3
copies) than those with a JS of 4-5.
Two tissue specimens with histologically diagnosed maturation arrest (JS 4-5) showed an hTERT expression of NhTERT=80.5 and 105.0 copies, which was far above the mean value in the other specimens with maturation arrest. Other sections of the same tissue examined by the semithin sectioning technique showed focal islands of complete spermatogenesis, which indicates that quantitative hTERT determination could contribute to a validation of histopathological findings.
Tissue
specimens with SCOS showed only minimal hTERT expression with a mean of NhTERT=2.72.4
copies. In three tissue specimens that evidenced SCOS in the conventional
histological workup but had hTERT mRNA expression (NhTERT=11.2-14.5
copies), spermatogonia were found in a renewed histological workup by
the semithin sectioning technique. This indicates that quantitative hTERT
determination is highly sensitive and highly specific for detecting germ
cells in testicular tissue specimens. Discriminant analysis showed
that combined determination of NhTERT and NhTR in
tissue samples had a predictive value of 89.5% for correct classification
in one of the three histological subgroups. It was shown that 86.8% of
the tissue samples would have been correctly classified by NhTERT
alone.
The
mean hTR expression was NhTR=295.5181.8 copies in specimens
with normal findings,
NhTR=112.144.8 copies in those with maturation arrest and
NhTR=61.237.1 copies in those with SCOS. NhTR
determination alone correctly classified
only 55.3% of the samples and thus had a markedly poorer diagnostic value
than NhTERT.
These results are in line with previous studies showing that high hTR expression is detectable in both telomerase-activity-positive and -negative tissues and also correlates only weakly with hTERT and telomerase activity[10,13,42,43].
The results of the present study show that hTERT mRNA expression in testicular tissue is highly sensitive and specific for germ cell activity and that its quantitative determination by realtime fluorescence RT-PCR enables a molecular-diagnostic classification of spermatogenesis disorders. Thus quantitative determination of hTERT mRNA expression in testicular tissue appears to be well suited for predicting successful sperm recovery in patients with non-obstructive azoospermia and is a useful molecular diagnostic parameter for supplementing the histopathological diagnostics. Our investigations show that an hTERT expression of NhTERT>100 copies indicates full spermatogenesis, while maturation arrest without complete spermatogenesis may be assumed at values of NhTERT<70 copies. In the gray range of NhTERT=70-100 copies, the diagnosis of maturation arrest should be checked by a further workup of the specimens.
Acknowledgements
The authors wish to thank Ms. Angelika Schneller, Ms. Petra von Kwiatkowski and Ms. Antonia Maas from the Department of Urology for their excellent technical support in assessing the samples. They are also grateful to Thomas Emrich, Ph.D. (Roche Diagnostic, Penzberg, Germany) for technical assistance with the LightCycler® and to Werner Hopfenmller, Ph.D., M.D. (Department of Medical Statistics) for help in performing the statistical analysis.References
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Correspondence
to: Dr.
Mark Schrader, M.D., Department of Urology, Universitätsklinikum
Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm 30, 12200
Berlin, Germany.
Fax: +49-30-8445
4448
E-mail: schrader@medizin.fu-berlin.de
Received 2001-09-24
Accepted 2001-11-20
