Human Reproduction, Vol. 15, No. 9, 2058-2059,
September 2000
© 2000 European Society of Human Reproduction and Embryology
Letters to the Editor |
Department of Urology, Tottori University School of Medicine, 36 Nishimachi, Yonago, Japan
Dear Sir,
We thank Schrader and co-workers for discussing the importance of testicular telomerase assay in male infertility clinics, taking into consideration our studies (Mio et al., 1998
; Yamamoto et al., 1999a,b).
Our previous study (Yamamoto et al., 1999a) indicates that Sertoli cell-only syndrome (SCOS) men with foci of active spermatogenesis up to the round spermatid or spermatozoon stage tend to have larger testicular tissue telomerase activity profiles than SCOS men who are negative for testicular haploid cells. Additional experiments in the mouse in that study (Yamamoto et al., 1999a) provide strong evidence that the telomerase assay applied (highly sensitive telomerase assay; Hisatomi et al., 1997) is a quantitative assay and that testicular telomerase assay outcome depends in a quantitative fashion on the amount of testicular tissue protein that exposes telomerase activity and subsequently on the number of testicular cells positive for telomerase activity. The difference in testicular telomerase profiles beween SCOS men positive for foci of haploid cells and SCOS men negative for foci of haploid cells may be due to (i) the larger number of telomerase positive germ cells (i.e. mainly spermatogonia/primary spermatocytes, secondary spermatocytes, and round spermatids) per testis weight unit in SCOS men positive for haploid cells and (ii) the smaller telomerase activity per primary spermatocyte in SCOS men who are positive for primary spermatocytes but negative for haploid cells. The latter two mechanisms are supported by (i) a previous study showing a larger number of primary spermatocytes per testicular weight unit in Klinefelter men who are positive for haploid cells than in Klinefelter men who are positive for primary spermatocyes but negative for haploid cells (Mio et al., 1998) and (ii) a study showing that highly purified fractions of primary spermatocytes recovered from mice with primary testicular damage tend consistently to have smaller telomerase profiles than fractions of primary spermatocytes recovered from healthy mice (Yamamoto et al., 1999b).
Schrader and co-workers misunderstood our comments on the study of Fujisawa et al. (1998). We did not call this study into question. In contrast we emphasized (Yamamoto et al., 1999a) that the results of our study cannot be compared with the findings of the study of Fujisawa and co-workers (1998) for the following reasons: (i) Fujisawa and co-workers did not apply the highly sensitive quantitative telomerase assay but rather applied the colour change-dependent PCR-ELISA assay (semi-quantitative assay); (ii) they did not mince the testicular tissue to evaluate whether some testicles with maturation arrest contained foci of spermatozoa and regions of active spermatogenesis and subsequently their results refer to diagnostic testicular biopsy (tissue processing for fixation and stain) only; and (iii) they assessed telomerase activity in testicles showing obstructive azoospermia with hypospermatogenesis, whereas all the obstructed azoospermic men in our study (Yamamoto et al., 1999a) showed active spermatogenesis with a normal large number of spermatozoa.
Although to perform the highly sensitive telomerase assay a small amount of testicular tissue is necessary (<5 mg), in our study (Yamamoto et al., 1999a), to evaluate testicular telomerase profiles a larger amount (in total >100 mg) of testicular tissue was processed for telomerase assay in each participant. This relatively large amount of testicular tissue was frozen and then divided into 5 mg samples (20–26 aliquots) which were homogenized, incubated, and centrifuged (Yamamoto et al., 1999a). An extract from one 5 mg-sample containing at least 1 µg of protein was used for one TRAP assay. Thus, for each piece of diagnostic testicular biopsy, 20–26 aliquots of extracts were processed for 20–26 TRAP assays (one aliquot was processed for one TRAP assay). The final telomerase activity outcome represented the average of the 20–26 assays performed in the 20–26 5 mg pieces of testicular tissue of each participant. Considering that (i) a large number of aliquots (20–26) containing extracts from one SCOS man were processed for telomerase assay and (ii) telomerase assay is a highly sensitive assay, it appears that if round germ cells were present in any region of the diagnostic testicular biopsy material processed for telomerase assay, increased values of telomerase activity would have been demonstrated. Our previous study (Yamamoto et al., 1999a) suggests that in an SCOS man with foci of active spermatogenesis, (even if most of the seminiferous tubuli within a relatively large fragment of testicular tissue, assayed for telomerase, are negative for germ cells), there is a high probability that few round germ cells are present in that testicular fragment with the overall result being an increased telomerase outcome. This is the reason that a relatively large piece of testicular tissue was processed for telomerase assay and several aliquots of extracts were assayed. Our thesis is that if round germ cells are present somewhere within the testis of an SCOS man, there is a high probability that few round germ cells will be present within a relatively large piece of diagnostic testicular biopsy. Therefore, that diagnostic testicular biopsy fragment will expose telomerase activity. The latter suggestion is consistent with the findings of Silber et al. (1997) who support a distribution of spermatogenesis in men with non-obstructive azoospermia that is multifocal, not of a regional nature, and relatively homogeneous within the testis. Therefore, we believe that if telomerase assay is performed in a relatively large (>100 mg) piece of testicular tissue of a non-obstructed azoospermic man with testicular foci of active spermatogenesis, it will give an increased likelihood of indicating the presence of some round germ cells in that testicular fragment.
A review of the literature strongly suggests that mammalian chromosomally haploid cells in the male (secondary spermatocytes and round spermatids) demonstrate telomerase activity (Prowse et al.,1995; Eisenhauer et al., 1997; Yamamoto et al.,1999b). Ravindranath et al. (1997) support the lack of telomerase activity at the spermatozoon stage. We have presented consistent findings (Yamamoto et al., 1999b). In the latter study we provided evidence that spermatogonia/primary spermatocytes are the main source of telomerase activity in the testis. Although (i) there is a decrease in telomerase activity at the round spermatid stage (Yamamoto et al., 1999b) and (ii) spermatozoa are negative for telomerase activity (Yamamoto et al., 1999b), men with SCOS positive for haploid cells have larger testicular telomerase profiles than SCOS men positive for primary spermatocytes but without haploid cells (Yamamoto et al., 1999a). This is probably because there is a larger number of primary spermatocytes per testicular tissue weight unit in the men with SCOS positive for haploid cells (Mio et al.,1998; Sofikitis and Yamamoto, unpublished observations).
Concerning the comparison in testicular tissue telomerase profiles between men in whom the most advanced germ cell in the therapeutic testicular biopsy material is the primary spermatocyte stage and men with focally complete spermatogenesis, we have provided adequate evidence that the former men have less marked telomerase activity profiles (Yamamoto et al., 1999a).
Schrader et al. (2000) report in their letter preliminary findings showing that few patients with a Johnsen score of 2–5 exposed more marked testicular telomerase activity than men with focal spermatogenesis. We believe that the above finding is of limited clinical value because telomerase assay is very sensitive and can give high values of activity in men with a low Johnsen score if round germ cells are present in the therapeutic testicular biopsy material (tissue mincing and processing for assisted reproduction; observation of single cells) (Yamamoto et al., 1999a). A major problem in the interpretation of results presented by Schrader et al. is that they did not perform therapeutic testicular biopsy to disperse the testicular cells and observe single, dispersed cells. Their results refer simply to diagnostic testicular biopsy techniques. Therefore, it is unknown if some men with Johnsen score 2–5 have had foci of primary spermatocytes and haploid cells. If the latter hypothesis is true, high values of telomerase are justified/expected in the latter men. It should be emphasized that Johnsen score refers to tissue exposure to various detergents during fixation/staining and subsequently a number of cells are degenerated and their identity cannot be identified. In contrast therapeutic testicular biopsy refers to isolation and mincing of testicular tissue and its exposure to iso-osmotic mediums containing energy substrates (Sofikitis et al., 1998). The most important point is that, in contrast to Johnsen score, therapeutic testicular biopsy allows observation of single, dispersed cells. We have abandoned the use of the Johnsen score in our University since it cannot predict accurately the outcome of therapeutic testicular biopsy. Furthermore, comparison of any qualitative or quantitative parameter of germ cells between populations with different Johnsen scores may result in confusing conclusions misorientating the reader because (i) a Johnsen score of 1–5 cannot rule out the probability that few spermatids or even spermatozoa are present in the testicular tissue; the latter haploid cells will be evident after tissue mincing and dispersing the cells, and (ii) occasionally a larger number of primary spermatocytes or haploid cells are found in regions of the therapeutic testicular biopsy material of men with Johnsen score 1–2 than in some of the men with Johnsen score 3–4 (Yamamoto and Sofikitis, unpublished observations).
Additional studies are necessary on the outcome of telomerase assay in subpopulations of non-obstructed azoospermic men with differences at the level of spermatogenetic arrest. However, in order for such studies to be meaningful and clinically important, differences in spermatogenesis should be established by observations of dispersed cells from the therapeutic testicular biopsy material.
Notes
1 To whom correspondence should be addressed E-mail: yamamoto{at}grape.med.tottori-u.ac.jp 
References
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Hisatomi, H., Naga, K., and Kamatsu, H. (1997) Quantification of telomerase activity in human liver tissues by fluorescence based TRAP anatology. Hepat. Res., 7, 35–42
Mio, Y., Yamamoto, Y., Sofikitis, N. et al. (1998) Employment of a highly sensitive quantitative telomerase assay in intracytoplasmic sperm sperm injection programs for the treatment of 4, XXY non-mosaic Klinefelter men. Fertil. Steril., (Suppl), S19.
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Sofikitis, N., Miyagawa, I., Yamamoto, Y. et al. (1998) Micro- and macro-consequences of ooplasmic injections of early haploid male gametes. Hum. Reprod. Update, 4, 197–212.
Yamamoto, Y., Sofikitis, N., Mio, Y. et al. (1999a) Highly sensitive quantitative telomerase assay of diagnostic testicular biopsy material predicts the presence of haploid spermatogenic cells in therapeutic testicular biopsy I men with Sertoli cell-only syndrome. Hum. Reprod., 14, 3041–3047.
Yamamoto, Y., Sofikitis, N., Ono, K.. et al. (1999b) Postmeiotic modifications of spermatogenic cells are accompanied by inhibition of telomerase activity. Urol. Res., 27, 336–345.[Web of Science][Medline]
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