Highly sensitive quantitative telomerase assay of diagnostic testicular ...

Human Reproduction vol.14 no.12 pp.3041–3047, 1999

Highly sensitive quantitative telomerase assay of diagnostic testicular biopsy material predicts the presence of haploid spermatogenic cells in therapeutic testicular biopsy in men with Sertoli cell-only syndrome* Yasuhisa Yamamoto1, Nikolaos Sofikitis1,3 Yasuyuki Mio2 and Ikuo Miyagawa1 1Department

of Urology, Tottori University School of Medicine, 36 Nishimachi, Yonago 683 and 2Reproductive Center, MFC Clinic, Yonago, Japan 3To

whom correspondence should be addressed

The role of a telomerase assay in the recognition of Sertoli cell-only syndrome with testicular foci of haploid cells was evaluated. Men with Sertoli cell-only syndrome (n ⍧ 23) were given a new diagnostic testicular biopsy. Part of the biopsy was stained and the remainder was processed for the quantitative telomerase assay. After 3–13 months, a therapeutic testicular biopsy was performed. This material was minced and then examined using confocal laser scanning microscopy and fluorescent in-situ hybridization. Histology of diagnostic testicular biopsy material confirmed the diagnosis of Sertoli cell-only syndrome in all the participants. All seven men with a telomerase assay value in their diagnostic testicular biopsy of >42 total product generated (TPG) U/µg protein had haploid cells (i.e. spermatozoa and/or spermatids) in their therapeutic testicular biopsy. Among participants with telomerase assay values 42 TPG U/µg protein in the diagnostic biopsy identified 87.5% of the Sertoli cell-only syndrome men with haploid cells in their therapeutic testicular biopsy. Significantly higher values of the telomerase assay were found in men with testicular foci of haploid cells than in men without these foci. The use of a quantitative telomerase assay biopsy appears to be important for identifying those men with Sertoli cell-only syndrome who have foci of haploid cells and can be candidates for assisted reproduction techniques. Key words: fertility/round spermatid/spermatozoon/telomerase/testis

Introduction A significant percentage of men with a histological diagnosis of Sertoli cell-only syndrome (SCOS) following testicular biopsy are subsequently found to have foci of spermatids or spermatozoa in their therapeutic testicular biopsy material (Silbert et al., 1995; Silbert, 1996). Ooplasmic injection of *Presented at The 93rd Annual Meeting of The American Urological Association in San Diego, California, May 30 to June 4, 1998, USA © European Society of Human Reproduction and Embryology

testicular spermatozoa or spermatids is an attractive mode of treatment, enabling these men to father their own children (Silbert et al., 1995; Silbert, 1996; Sofikitis et al., 1998a). One of the most perplexing problems in the therapeutic management of SCOS men is the inability of the histological images of the diagnostic testicular biopsy, peripheral serum hormonal profiles, and testicular size to predict the presence of foci of haploid cells in the therapeutic testicular biopsy. Occasionally, oocyte recovery procedure is cancelled in couples with SCOS because the therapeutic testicular biopsy material is negative for spermatozoa and spermatids. Thus SCOS couples occasionally undergo expensive and unnecessary ovarian stimulation. It appears that it is of great clinical importance to discover/ evaluate testicular biochemical or hormonal parameters in a small amount of diagnostic testicular material that have the ability to predict the presence/absence of foci of haploid cells in the therapeutic testicular biopsy sample. Such parameters of diagnostic testicular biopsy will indicate those SCOS couples who can participate in assisted reproduction programmes, and will give the opportunity to SCOS couples who do not have foci of haploid cells within the testicular tissue to avoid participating in an unnecessary, expensive, and potentially risky ovarian stimulation. Telomeres are repeated DNA sequences located on both ends of individual chromosomes in eukaryotes (Blackburn, 1991, 1992). Telomeres stabilize natural chromosome ends and inhibit aberrant fusions and rearrangements that occur on broken chromosomes. Telomere repeats are synthesized de novo onto chromosome ends by the enzyme telomerase. Telomerase is a transcriptase containing an RNA template. The RNA template allows telomerase to add telomeric sequences to the ends of newly replicated DNA. Telomerase activity is upregulated in a variety of immortal cell lines and tumours in both human and mouse species (Morin, 1989; Counter et al., 1992; Prowse et al., 1993). Telomerase activity is expressed in most human tumour tissues, but not in most normal tissues, tissues adjacent to tumours or benign growths (Kim et al., 1994). The telomerase hypothesis suggests that telomerase activity is high in embryonic cells and that it decreases in somatic tissues during development and differentiation (Eisenhauer et al., 1997). Several studies indicate that mouse, rat, and human spermatogonia, primary spermatocytes, secondary spermatocytes and round spermatids are positive for telomerase activity, whereas testicular and epididymal spermatozoa are negative (Prowse et al., 1993; Eisenhauer et al., 1997; Yamamoto et al., 1999). Results of preliminary studies that were confirmed in the current investigation demonstrate that men with testicles with active spermatogenesis (i.e. men with obstructive azoospermia with normal spermato3041

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genesis) have significantly larger sensitive quantitative telomerase assay (SQTA) profiles than men with SCOS (with or without foci of haploid cells). Thus, we attempted to relate the SQTA outcome in diagnostic testicular biopsy specimens to the results of therapeutic testicular biopsy (i.e. presence or absence of testicular foci of active spermatogenesis; identification of the most advanced sperm cell in the minced therapeutic testicular biopsy) in men with SCOS (Hisatomi et al., 1997; Yamamoto et al., 1999). This assay is the gold standard (Hisatomi et al., 1997, 1999; Kanamaru et al., 1998; Kubota et al., 1998; Onoda et al., 1998; Ogino et al., 1999; Yahata et al., 1999) for quantifying telomerase activity, and this is the first report of its use in human testicular tissue. SQTA has been the most popular method for the quantification of tissue telomerase profiles because: (i) it produces reliable data, since internal and external controls are used (Hisatomi et al., 1997); (ii) it gives good reproducibility (Hisatomi et al., 1997, 1999); (iii) it is now commercially available (TRAPezeTM kit and F-TRAP kit; Intergen Co., New York, NY, USA); (iv) it is very sensitive (Hisatomi et al., 1997, 1999; Kanamaru et al., 1998; Onoda et al., 1998; Ogino et al., 1999; Yahata et al., 1999); and (v) other available methods for evaluation of tissue telomerase are qualitative (Eisenhauer et al., 1997; Yamamoto et al., 1999), semiquantitative (relative telomerase assay; Yamamoto et al., 1999), or their outcome depends on colour changes (PCR/ELISA assay; Fujisawa et al., 1998) that are non-sensitive or less sensitive and subjective.

at –80°C. The concentration of protein was determined and an aliquot of extract containing 1 µg of protein was used for each TRAP assay. Aliquots of the extract were incubated with 0.1 ng Cy-5 labelled TS primer (TRAP-eze™). Following a 30-min incubation at 30°C, a polymerase chain reaction (PCR) was performed at 94°C (30 s), 60°C (30 s) and 72°C (45 s) for 30 cycles. The external control was a TSR8 (TRAP-eze™) as a positive control (Hisatomi et al., 1997). The products were applied to a 10% denaturing gel containing 6 mol/l urea fitted to an automated DNA sequencer (ALFred™ DNA Sequencer; Pharmacia Biotech, Uppsala, Sweden). Data from the ALFred™ DNA Sequencer were collected and analysed automatically by Allele Links software (PharmaciaBiotech). Each peak was quantified in terms of size, peak height, and peak area. The quantification of telomerase activity was determined by a previously described mathematical formula (Hisatomi et al., 1997). SQTA outcome values were expressed as TPG U (/µg protein).

Materials and methods

Processing of therapeutic testicular biopsy material Therapeutic testicular biopsy material was washed four times with normal saline. The seminiferous tubules were then washed in Dulbecco’s phosphate buffered saline (DPBS; Sigma Co., St Louis, MO, USA) containing 5.6 mmol/l glucose and 5.8 mmol/l sodium lactate (modified DPBS; Sofikitis et al., 1997) and subsequently minced into small pieces. Samples were maintained at 5°C and observed on a dissecting microscope (Olympus SZ-STS; Olympus, Tokyo, Japan) during the mincing process. Then the samples were centrifuged at 500 g for 30 min, sedimented pieces of tissue and cells were suspended in modified DPBS, and the samples were passed through filter paper of 30–40 µm pore size. The filtrate was collected, centrifuged at 750 g for 30 min, and the majority (major fraction) of sedimented cells were observed on a CLSM-CAS, while a minor fraction was processed for FISH.

Patients Twenty-three azoospermic men who had previously undergone a testicular biopsy in other centres and were diagnosed with SCOS were referred to our facilities [age 31.1 ⫾ 3.9 years; follicle stimulating hormone (FSH) 29.9 ⫾ 10.0 IU/l]. A new diagnostic testicular biopsy was performed. A part of the new diagnostic testicular biopsy material (approximately half by volume) was processed for haematoxylin– eosin staining. The remaining piece was frozen and processed for SQTA. After 3–13 months all men underwent a therapeutic testicular biopsy during assisted reproduction trials. The therapeutic testicular biopsy material was processed for mincing and dispersion/extraction of spermatogenic cells. The major part of the minced testicular tissue was observed on a confocal laser scanning microscope-computer assisted system (CLSM-CAS; Sofikitis et al., 1994, 1996a,b, 1997) and dispersed haploid cells were processed for ooplasmic injections, whereas, a minor part was processed for fluorescent in-situ hybridization (FISH). In additional studies, SQTA was performed on testicular biopsy material from eight obstructed azoospermic men with normal spermatogenesis.

Confocal laser scanning microscope-computer assisted system (CLSM-CAS) CLSM-CAS (Lasertec Co., Yokohama, Japan) is a powerful instrument in the field of microscopy (Sofikitis et al., 1994). Unlike the conventional light microscope, the CLSM-CAS produces sharp images free of out-of-focus artifacts. It also provides an automatic system for the instantaneous measurement of distance. This technique has the capacity to provide three-dimensional images of cells at a high magnification without requiring fixation and staining (Sofikitis et al., 1994, 1997; Yamanaka et al., 1997). Therefore, it is possible to determine morphometric parameters of undisturbed, living cells and subsequently recognize with high accuracy spermatogonia/primary spermatocytes, secondary spermatocytes, and round spermatids applying morphometric criteria for germ cell identification (Sofikitis et al., 1996a,b, 1998a,b; Yamanaka et al., 1997). Round spermatids can also be recognized by CLSM-CAS by the presence of an acrosomal granule attached to the nuclear membrane (Sofikitis et al., 1997; Yamanaka et al., 1997). The stage of the most advanced spermatogenic cell in each sample was recorded.

Sensitive quantitative telomerase assay (SQTA) SQTA was assessed in diagnostic testicular biopsy in duplicate by The Japanese Special Reference Laboratory (Matsue, Japan). A quantitative modification of the telomere repeat amplification protocol (TRAP) method was applied (Hisatomi et al., 1997). Frozen testicular samples (5 mg for each assay) were homogenized in 100 µl of ice cold CHAPS lysis buffer (TRAP-eze™ kit; Oncor Inc.–Kyowa Co., Tokyo, Japan) and were incubated for 30 min on ice. After incubation, the lysates were centrifuged at 12 000 g for 20 min at 4°C. The supernatants were rapidly frozen and were stored

Fluorescent in-situ hybridization (FISH) techniques FISH techniques were applied as an additional methodology to define the most advanced germ cell in the minced testicular samples. Twocolour FISH was performed as previously described (Sofikitis et al., 1998b) using previously described methodology (Harper et al., 1994). The probe kit was obtained from the Fujisawa Company (Osaka, Japan). This probe kit is a combination of X Spectrum Orange and Y Spectrum Green fluorescently labelled DNA probes specific for chromosomes X and Y respectively. As primary spermatocytes were characterized XY-(4C)-DNA cells (4C indicates a tetraploid amount

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Figure 1. Haematoxylin–eosin stain in diagnostic testicular biopsy material showing Sertoli cell-only syndrome (original magnification ⫻400; image from TV monitor).

Figure 2. (A) Confocal scanning laser microscope-computer assisted system image.The round cell (black arrow) satisfies the quantitative criteria for round spermatids (Yamanaka et al., 1997). In addition, the slightly peripheral position of the nucleus (pink/red arrow) is consistent with the round spermatid stage. A testicular spermatozoon can also be observed. Scale bar ⫽ 10 µm. (B) When a round cell (black arrow) that has been characterized as a round spermatid by morphometric analysis is observed at higher magnification on the confocal scanning laser microscope-computer assisted system, an acrosomal granule (pink/red arrow) can be observed, confirming that the round cell is a round spermatid. Scale bar ⫽ 5 µm. of DNA; Alberts et al., 1994). Secondary spermatocytes were considered X-(2C)-DNA cells or Y-(2C)-DNA cells (2C indicates a diploid amount of DNA; Alberts et al., 1994; Sofikitis et al., 1998b). Round spermatids were characterized as X-(C)-DNA cells or Y-(C)-DNAcells (C indicates a haploid amount of DNA; Alberts et al., 1994; Sofikitis et al., 1998b). Primary spermatocytes are chromosomally diploid cells; however, they contain a tetraploid amount of DNA. Secondary spermatocytes are chromosomally haploid cells and they have a diploid amount of DNA. Spermatids are chromosomally haploid cells and have a haploid amount of DNA. Secondary spermatocytes differ from normal diploid cells in two ways: (i) both of the two DNA copies of each chromosome derive from only one of the two homologous chromosomes in the original cell (except where there has been genetic recombination), and (ii) these two copies are inherited as closely associated sister chromatids, as if they were a single chromosome (Alberts et al., 1994). Since it was found that none of the participants’ secondary spermatocytes were the most advanced germ cells in their therapeutic testicular biopsy sample (i.e. all men positive for secondary spermatocytes also had testicular spermatids or spermatozoa) the term ‘haploid cell’ refers to spermatids and spermatozoa below.

Statistical analysis Statistical analysis was performed using the Wilcoxon’s test for comparisons of two groups and analysis of variance followed by Duncan’s test for comparisons of three groups. Values were expressed as mean ⫾ SD. A probability of P ⬍ 0.05 was considered to be statistically significant.

Results The diagnostic testicular biopsy confirmed the SCOS pathophysiology (Figure 1) in all participants. However, germ cells were found in the therapeutic testicular biopsy material of 17 men. CLSM-CAS and FISH techniques indicated that the most advanced spermatogenic cells were primary spermatocytes or haploid cells (spermatids or spermatozoa) in nine and eight men respectively (Tables I and II, Figures 2 and 3). Among the latter eight men with foci of haploid cells, seven men had SQTA ⬎42 TPG U (range: 42.29–82.27), whereas one man only had SQTA ⬍42 TPG U (SQTA value ⫽ 34.14 TPG U). Thus, using a cut-off value equal to 42 TPG U, the sensitivity, 3043

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Table I. Focal active spermatogenesis up to the stage of primary spermatocytes (PS), round spermatids (RS) or spermatozoa (most advanced germ cell) in therapeutic testicular biopsy (TTB) material in men with Sertoli cellonly syndrome Tissue/cell processing (type of biopsy)

Number of men SCOS without germ cells (DTB)

Staining of DTB 23 CLSM-CAS observation of TTB FISH of TTB

(TTB) 6 6

Number of men with PS-arrest (TTB) RS-arrest (TTB) Spermatozoa (TTB) 9 9

2 7

6 1

DTB ⫽ diagnostic testicular biopsy. CLSM-CAS ⫽ confocal scanning laser microscope-computer assisted system. FISH ⫽ fluorescent in-situ hybridization.

Table II. Sensitive quantitative telomerase assay (SQTA) outcome in subpopulations of men with Sertoli cell-only syndrome Most advanced spermatogenic cell in TTB

n

SQTA (TPG U/µg protein) (mean ⫾ SD)

No germ cell Primary spermatocyte Round spermatid or spermatozoon

6 9 8

3.52 ⫾ 5.04a 23.65 ⫾ 9.74b 52.01 ⫾ 15.46c

Values not sharing the same superscript a, b, or c are significantly different (P ⬍ 0.05). TTB ⫽ therapeutic testicular biopsy, TPG ⫽ total product generated.

Figure 3. Relationship between quantitative telomerase assay of diagnostic testicular biopsy and the most advanced spermatogenic cell in therapeutic testicular biopsy material (as seen using confocal laser scanning microscope-computer assisted system). TPG ⫽ total product generated; SCOS ⫽ sertoli cell only syndrome; PS ⫽ primary spermatocyte; RS ⫽ round spermatid; SZ ⫽ spermatozoon.

specificity, positive predictive value and negative predictive value of SQTA assay in diagnostic testicular biopsy for the detection of haploid cells in the therapeutic testicular biopsy 3044

material were 87.5, 100, 100, and 93.8% respectively. Significantly larger values of SQTA were found in SCOS men with haploid cells (mean: 52.01 TPG U; range 34.14–82.27) (Table II, Figure 3) than in SCOS men negative for haploid cells; however, they were positive for primary spermatocytes (mean: 23.65 TPG U; range 11.30–39.42). In addition, significantly larger values of SQTA were found in SCOS men with foci of haploid cells than in SCOS men negative for foci of germ cells (Table II; Figure 3). All six SCOS men without germ cells in their therapeutic biopsies had SQTA ⬍13 TPG U (0, 0, 0, 3.41, 4.82 and 12.90). All eight obstructed azoospermic men had SQTA values ⬎92.00 TPG U (92.18, 112.33, 163.31, 159.31, 159.45, 182.52, 182.89, and 223.06 TPG U; mean ⫽ 159.44). The mean SQTA outcome in the eight obstructed azoospermic men (159.44 TPG U) was significantly larger than in the 23 SCOS men taken together (28.26 TPG U). Discussion Diagnostic testicular biopsy is of definite value in distinguishing between reproductive tract obstruction and primary testicular damage in infertility clinics. In contrast, the importance of diagnostic testicular biopsy in the therapeutic management of non-obstructed azoospermic men has been questioned, since a significant percentage of men with the testicular histology of SCOS or spermatogenic arrest at the primary spermatocyte stage in their diagnostic testicular biopsy demonstrate testicular foci of active spermatogenesis up to the secondary spermatocyte, elongating spermatid, round spermatid or spermatozoon stage in their therapeutic testicular biopsy material (for review see Yamanaka et al., 1997; Sofikitis et al., 1998a,b,c). Therefore, diagnostic testicular biopsy cannot indicate the SCOS men who are positive for testicular foci of haploid cells and subsequently can be candidates for assisted reproduction programmes. In addition, peripheral serum concentrations of FSH and testicular size are not considered reliable parameters to predict the presence of foci of haploid cells in therapeutic testicular biopsy of SCOS men and generally non-obstructed azoospermic men (Sofikitis et al., 1998a). In the current study, we evaluated the role of SQTA in the diagnostic testicular biopsy in recognizing the subpopulation of SCOS men with foci of haploid cells seen in therapeutic testicular biopsy. The existence of a significant and large difference in SQTA

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outcome between obstructed azoospermic men with normal spermatogenesis and all SCOS men taken together in the current study and in preliminary studies (N.Sofikitis et al., unpublished data) suggests that testicles with normal, active spermatogenesis have a higher telomerase activity than testicles with the histology of SCOS (even if SCOS men are positive for local foci of germ cells). This difference in SQTA results may be attributable to the larger number of telomerasepositive germ cells (i.e. spermatogonia/primary spermatocytes, secondary spermatocytes, and round spermatids) per testis weight unit in obstructed azoospermic men. In addition, a large and significant difference was established in SQTA outcome of the diagnostic testicular biopsy between SCOS men with local foci of spermatogenesis up to the primary spermatocyte stage and SCOS men with foci of haploid cells in the therapeutic testicular biopsy. Similarly, the latter difference may be explained by the fact that the SCOS men with foci of haploid cells have focally more active spermatogenesis and subsequently a larger number of stem cell mitoses, greater stem cell renewal, and enhanced generation of primary spermatocytes compared with SCOS men with foci of spermatogenesis up to the primary spermatocyte stage. The overall result is that SCOS men with foci of haploid cells have a larger number of primary spermatocytes per tissue weight unit and subsequently larger SQTA outcome than SCOS men with foci of germ cells up to the primary spermatocyte stage. It should be emphasized that primary spermatocytes are considered to be the main source of testicular testosterone activity (Eisenhauer et al., 1997; Yamamoto et al., 1999). In addition, SCOS men with foci of haploid cells have higher SQTA outcome due to the presence of round spermatids (telomerase positive cells, as well). Furthermore, since SCOS men with foci of advanced spermatogenesis up to the primary spermatocyte stage have a defect in the primary spermatocyte nuclear capacity to undergo the first meiotic division, the probability that an additional qualitative or quantitative defect is present in the primary spermatocyte nucleus telomerase cannot be ruled out. Thus, the diminished SQTA outcome in SCOS men with foci of spermatogenesis up to the primary spermatocyte stage compared with SCOS men with foci of haploid cells may be additionally due to lower telomerase activity per primary spermatocyte. The latter speculation is supported by a recent study (Yamamoto et al., 1999) showing that highly purified fractions of primary spermatocytes recovered from healthy mice tend consistently to have larger SQTA values than fractions of primary spermatocytes recovered from mice with primary testicular damage. The minimal values of SQTA outcome (⬍13 TPG U) in SCOS men without testicular foci of germ cells in therapeutic testicular biopsy (Table II; Figure 3), the larger values of SQTA outcome in obstructed azoospermic men than in all SCOS men taken together and the larger values of SQTA in SCOS men with foci of haploid cells than SCOS men with foci of germ cells up to the primary spermatocyte stage raised the probability of using the SQTA in the diagnostic testicular biopsy as a novel assay to recognize SCOS men with foci of haploid cells in their therapeutic testicular biopsy. Taking into consideration that (i) all men with SCOS without foci of

haploid cells (spermatids or spermatozoa) had SQTA values ⬍42 TPG U and (ii) seven out of the eight SCOS men with foci of haploid cells had SQTA values ⬎42 TPG U, we evaluated the role of the 42 TPG U-value as a cut-off value for the prediction of foci of haploid cells in therapeutic testicular biopsy material of SCOS men. Thus, using the latter value as a cut-off value, it was found that the sensitivity, specificity, positive predictive value and negative predictive value of SQTA in the diagnostic testicular biopsy for recognizing the subpopulation of SCOS men with foci of active spermatogenesis up to the round spermatid stage or spermatozoon in the therapeutic testicular biopsy were 87.5, 100, 100 and 93.8% respectively. It appears that SQTA in diagnostic testicular biopsy has a role in the detection of SCOS men with foci of haploid cells in the therapeutic testicular biopsy. SCOS men with SQTA ⬎42 TPG U in the diagnostic testicular biopsy may have higher chances of success when they undergo therapeutic testicular biopsy and participate in assisted reproduction techniques. In contrast, men with SQTA ⬍13 TPG U have lower chances of success in assisted reproduction and if they choose to participate, their wives will undergo unnecessary ovarian stimulation because the assisted reproduction cycle is going to be cancelled due to the lack of testicular haploid cells. Since SQTA is a highly sensitive assay, a very small amount of diagnostic testicular biopsy material (艋5 mg) is necessary to calculate testicular telomerase activity. Thus it is of clinical importance that a minor amount of diagnostic testicular biopsy can recognize with high accuracy those SCOS men who can be candidates for assisted reproduction techniques. In another recent study (Mio et al., 1998), seven men with Klinefelter syndrome underwent therapeutic testicular biopsy. Three men were positive for spermatozoa. All these men had had SQTA values ⬎42 TPG U in the diagnostic testicular biopsy. All four men with Klinefelter syndrome who were negative for foci of haploid cells in the therapeutic testicular biopsy had had SQTA values in their diagnostic testicular biopsy ⬍25 TPG U. All the above observations suggest that SQTA of a minor amount of diagnostic testicular biopsy is a promising, novel assay, important for the therapeutic management of SCOS men. Additional studies are necessary to clarify the role of SQTA in the management of a general population of non-obstructed azoospermic men. Telomerase activity is present in testicular round germ cells up to the stage of the round spermatid (Eisenhauer et al., 1997; Yamamoto et al., 1999). Telomerase is expected to remain active in primary spermatocytes and secondary spermatocytes to ensure the transmission of full-length chromosomes to the round spermatid and subsequently to the spermatozoon. We have previously shown (Yamamoto et al., 1999) that telomerase activity is inhibited during the transformation of the round spermatid to a spermatozoon. To assess testicular tissue telomerase activity, a qualitative assay (Eisenhauer et al., 1997; Yamamoto et al., 1999), a relative telomerase assay (Yamamoto et al., 1999), or a PCR/ ELISA assay evaluating colour changes (Fujisawa et al., 1998) have been previously applied. The above three assays are nonsensitive and subjective. The current study is the first report of the application of the SQTA for quantifying telomerase 3045

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activity in human testicular tissue. SQTA is highly sensitive and is considered to be the gold standard for telomerase quantification (Hisatomi et al., 1997, 1999; Kanamaru et al., 1998; Kubota et al., 1998; Onoda et al., 1998; Ogino et al., 1999; Yahata et al., 1999; Yamamoto et al., 1999). The latter studies described in detail the quantification of tissue telomerase activity (applying SQTA) against standard control samples. To further confirm that SQTA is a quantitative assay an additional experiment was performed. Aliquots from a mixture of mature B6D2F1 mouse testicular extracts were processed for SQTA (aliquot A). Additional aliquots B and C from the same mixture were processed for SQTA after having been mixed with an equal volume of extracts of frozen– thawed B6D2F1 mouse epididymal spermatozoa. The latter spermatozoal extracts had been proven to have SQTA values of 0 TPG U/µg protein (i.e. spermatozoa do not express telomerase activity; Eisenhauer et al., 1997; Yamamoto et al., 1999). The ratio of protein weight of testicular extracts to protein weight of sperm extracts was set as 1:1 and 1:9 within aliquots B and C respectively. Aliquots A, B and C were processed for SQTA as previously described (Hisatomi et al., 1997). Mean SQTA outcome in aliquot A was 67.31 TPG U/µg protein. The mean SQTA values in aliquots B and C were 32.04 and 7.21 TPG U/µg protein respectively. Comparing the SQTA values in aliquots B and C with the SQTA value in aliquot A (pure testicular extracts), it appears that the SQTA values in aliquots B and C correspond well with the proportion of testicular extract-protein weight/sperm extract-protein weight in aliquots B (1:1) and C (1:9) respectively (i.e. the mean SQTA outcome in aliquot B was ~48% of that in aliquot A and the mean SQTA value in aliquot C was ~11% of that of aliquot A). This experiment shows that SQTA outcome depends in a quantitative fashion on the amount/fraction of tissue protein that is exposed to telomerase activity. It should also be mentioned that SQTA is quite a complex assay and subsequently its application may be limited to institutes having molecular biology facilities. Processing the diagnostic testicular biopsy material for SQTA plus other assays evaluating the expression of genes indicating specific stages in spermatogenesis may increase the accuracy of the diagnostic testicular biopsy for predicting the stage of the most advanced germ cell in the therapeutic testicular biopsy sample. A previous study (Fujisawa et al., 1998) showed absence of significant difference in telomerase activity between testes demonstrating maturation arrest and testes of obstructed azoospermic men with hypospermatogenesis. However, the results of that previous study cannot be compared with the results of the current study since Fujisawa et al. (i) did not apply the highly sensitive SQTA but rather applied the colour change-dependent PCR-ELISA assay; and (ii) did not mince the testicular tissue to evaluate whether some testes with maturation arrest contained foci of spermatozoa and subsequently locally active spermatogenesis. Their observations refer to diagnostic testicular biopsy only. In addition, Fujisawa et al. assessed telomerase activity in testes showing obstructive azoospermia with hypospermatogenesis, whereas all the obstructed azoospermic men in the current study 3046

showed active spermatogenesis with a normal large number of spermatozoa. It is of interest that all six men in whom neither CLSMCAS nor FISH demonstrated foci of germ cells in therapeutic testicular biopsy showed minimal SQTA profiles in the diagnostic testicular biopsy (⬍13 TPG U). Three of the above men had SQTA values equal to 0 TPG U, whereas the three remaining men demonstrated minor telomerase activity in the testis, probably due to a very limited number of spermatogenic cells in the diagnostic testicular biopsy (although histology of the diagnostic testicular biopsy showed absence of germ cells and no germ cells were found in the therapeutic testicular biopsy fragment). Apparently, a minor number of germ cells in the diagnostic testicular biopsy was the source of the telomerase activity in the diagnostic testicular biopsy. Although the latter activity was minimal, it was detectable because SQTA is a highly sensitive assay. Observation of different fractions of therapeutic testicular biopsy via CLSM-CAS and FISH indicated six SCOS men without foci of germ cells (26%) and nine SCOS men (39%) with foci of active spermatogenesis up to the primary spermatocyte stage. The same number of SCOS men with foci of haploid cells in their therapeutic testicular biopsy material (spermatids and spermatozoa) was indicated by CLSM-CAS and FISH (eight men; 34%). However, in five of these men the minor therapeutic testicular biopsy fragment that had been processed for FISH revealed round spermatids but did not show spermatozoa, whereas the respective major therapeutic testicular biopsy fragment that had been processed for CLSMCAS demonstrated few spermatozoa (Table I). This difference may be due to the large number of cells processed for CLSMCAS and the small number of cells processed for FISH. Thus the probability of identifying a very limited number of existed spermatozoa was higher during observation by CLSM-CAS. Application of the highly sensitive SQTA to diagnostic testicular biopsy in the current study showed that: (i) obstructed azoospermic men with normal spermatogenesis have greater testicular telomerase activity than SCOS men; (ii) SCOS men with testicular foci of haploid cells have higher SQTA values than SCOS men with foci of spermatogenesis up to the primary spermatocyte stage; (iii) SCOS men without foci of germ cells in therapeutic testicular biopsy material have low SQTA values in their diagnostic testicular biopsy; and (iv) when the value of 42 TPG U is used as a cut-off value, SQTA in the diagnostic testicular biopsy has a high sensitivity, specificity, positive predictive value and negative predictive value for the identification of SCOS men with foci of haploid cells in therapeutic testicular biopsy. Considering that the amount of diagnostic testicular biopsy necessary for the performance of SQTA is very small, it appears that application of SQTA in the diagnostic testicular biopsy has a role in the therapeutic management of men with SCOS. References Alberts, B., Dennis, B., Lewis, J. et al. (1994) Molecular Biology of the Cell. Garland Publishing Inc., New York, pp. 1–1273. Blackburn, E.H. (1991) Structure and function of telomerase. Nature, 350, 569–573.

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Yamanaka, K., Sofikitis, N. and Miyagawa, I. (1997) Ooplasmic round spermatid nuclear injections as an experimental treatment of non-obstructive azoospermia. J. Assist. Reprod. Genet., 14, 55–62. Received on January 5, 1999; accepted on September 21, 1999

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