- Email: [email protected]
Morphologic and mitochondrial characterization of human spermatogenic cells dispersed in wet preparation for testicular sperm extraction: establishment of a microscopic diagram of developing human spermatogenic cells
Morphologic and mitochondrial characterization of human spermatogenic cells dispersed in wet preparation for testicular sperm extraction: establishment of a microscopic diagram of developing human spermatogenic cells The successful identification of spermatozoa at microdissection testicular sperm extraction is critical and can depend on the judgment of experienced reproductive clinicians. Therefore, we characterized human spermatogenic cells based on stage-specific mitochondrial location and morphologic change by using a vital mitochondrionspecific fluorescent probe, Mitotracker, and stage-specific antibodies to make a useful microscopic diagram. (Fertil Steril 2011;95:2665–8. 2011 by American Society for Reproductive Medicine.) Key Words: Mitochondria, spermatozoa, microdissection TESE, azoospermia, MitoTracker
The introduction of microdissection testicular sperm extraction (MD-TESE) has significantly improved the sperm retrieval rate (SRR) in nonobstructive azoospermia (NOA) (1). In earlier reports, MD-TESE has proven to be superior to conventional techniques regarding SRR (1–7). However, the reported SRRs by MD-TESE vary from 40% to 63% (1–7). One reason for this variation is differences in the proportion of testicular histologies. The SRR for hypospermatogenesis is 80%–100%, remarkably higher than for Sertoli cell–only syndrome (20%–40%) (3, 4, 6). Another reason is that the successful identification of Hidenobu Okuda, M.D.a Akira Tsujimura, M.D.a Keisuke Yamamoto, M.D.a Shinichiro Fukuhara, M.D.a Jiro Nakayama, M.D.a Hiroshi Kiuchi, M.D.b Tetsuya Takao, M.D.a Yasushi Miyagawa, M.D.a Norio Nonomura, M.D.a Akihiko Okuyama, M.D.a a Department of Urology, Osaka University Graduate School of Medicine, Osaka, Japan b Department of Urology, Osaka Central Hospital, Osaka, Japan Received June 29, 2010; revised April 6, 2011; accepted April 7, 2011; published online May 5, 2011. H.O. has nothing to disclose. A.T. has nothing to disclose. K.Y. has nothing to disclose. S.F. has nothing to disclose. J.N. has nothing to disclose. H.K. has nothing to disclose. T.T. has nothing to disclose. Y.M. has nothing to disclose. N.N. has nothing to disclose. A.O. has nothing to disclose. Reprint requests: Akira Tsujimura, M.D., Department of Urology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.04.026
spermatozoa can depend on the judgment of experienced reproductive clinicians. Reproductive clinicians usually detect spermatozoa morphologically at low magnification owing to the requirements of micromanipulation. To identify spermatozoa precisely, they are required to identify developing spermatogenic cells as well, because seminiferous tubules with sperm production, rather than those with Sertoli cells alone, are likely to contain many more developing spermatogenic cells than tubules without sperm production (1). Therefore, the precise identification of spermatogenic cells by reproductive clinicians is likely to affect the SRR, and therefore a standardized criterion has been needed for a long time. A novel method of spermatid selection using Mitotracker, which is a vital mitochondrion-specific fluorescent probe, has been reported (8). This probe proved to be useful to correctly identify live round spermatids based on the polarized patterns of the mitochondria. Therefore, we considered that Mitotracker staining could be an easy and promising method for identifying spermatozoa precisely, even if observed under low magnification. The aim of the present study was to make a systematic description and diagram of human spermatogenic cells that focuses on the mitochondrial location and morphologic characteristics at each step in development by using Mitotracker to understand spermatogenic cells, and to apply this information to artificial reproductive techniques. We also confirmed the accuracy of our classification by immunostaining with various stage-specific spermatogenic cell antibodies at the same time. Several human testis tissues from consenting subjects with obstructive azoospermia that had been obtained by TESE and were no longer used for intracytoplasmic sperm injection (ICSI) were used as samples for experimentation. Dispersal of spermatogenic cells from seminiferous tubules was carried out as previously described (8).
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
2665
The spermatogenic cell suspension was incubated for 15 minutes at 37 C in 200 nmol/L Mitotracker CMXRos (Invitrogen). Thereafter, immunofluorescence was used with four kinds of stage-specific spermatogenic cell antibodies to identify the developmental steps of spermatogenic cells. After the fixation and blocking of cell suspension drops, the drops were incubated with 1:200 diluted spermatogenic cell–specific antibodies VASA, MCA (meichroacidin), h-Tektin-T, and Smrp-1 (spermatid-specific manchette-related protein 1) overnight at 4 C. Thereafter, they were incubated for 90 minutes at room temperature using 1:200 diluted secondary antibody. Developmental stages targeted by each antibody are described as follows. VASA (Santa Cruz Biotechnology) is specifically expressed in spermatogonia, where its expression is the highest. It has reduced expression in spermatocytes and low expression in spermatids and is absent in spermatozoa (9). MCA and Smrp-1 were produced as described previously (10–12). MCA is expressed in cytoplasm from pachytene spermatocytes through to the round spermatid stage and is also located on the axoneme. Smrp-1 is expressed in the caudal side of the cytoplasm of elongated spermatids, and it disappears in mature sperm. h-Tektin-t is located specifically on the axoneme, forming filamentous polymers (13). The spermatogenic cells stained with each antibody were observed by using epifluorescence and differential interference contrast (DIC) microscopy at a constant magnification of 400 and were assigned to each stage of spermatogenesis according to staining status and cell shape. Thereafter, the mitochondrial location and morphologic characteristics in each stage of spermatogenic cells were also analyzed and described. The translocations of mitochondria and morphologic changes in each stage of spermatogenesis were analyzed in reference to the staining by spermatogenic cell–specific antibodies (Fig. 1). Spermatogonia were defined as spherical cells with a very distinct nuclear envelope and stained only with VASA. The average diameters of nuclei and cytoplasm were 7.22 mm and 9.03 mm, respectively. Mitochondria were homogeneous in the cytoplasm. Spermatocytes were divided into two groups according to the stages targeted by the antibodies: preleptotene/leptotene/zygotene (P-Z) spermatocytes and pachytene spermatocytes. The former were stained only with VASA and the latter with VASA and MCA. The nuclei and cytoplasm of P-Z spermatocytes were 8.66 mm and 12.53 mm in diameter, respectively, and had become larger and irregular compared with those of spermatogonia, although they remained spherical. The expression of mitochondria was greatly increased compared with that of the spermatogonia. In pachytene spermatocytes the diameters of the oval nuclei and cytoplasm increased to 12.3 mm and 16.72 mm, respectively, and were the largest of all the stages of spermatogenesis. Mitochondria was greatly increased compared with that of P-Z spermatocytes. Compared with spermatocytes, round spermatids, which were stained with MCA and rarely with VASA, were smaller cells whose nuclear and cytoplasmic diameters were 5.78 mm and 8.44 mm, respectively. In this stage, the acrosomal granule appears initially and the acrosomal cap is formed eventually as a cytoskeletal structure covering the round spermatid nucleus. At the magnification used, only acrosomal caps were visible. As the nucleus and cytoplasm condensed, mitochondria began to polarize and condense to the basal pole of the cytoplasm. None of the round spermatids were stained with h-Tektin-t, because axonemes had not developed yet.
2666
Okuda et al.
Correspondence
The axoneme was apparent in the next stage, the elongating spermatid, which was well stained with MCA and h-Tektin-t. The manchette is a stage-specific structure that appears around the nuclei of elongating spermatids, and it was stained with Smrp-1. As the axonemes became distinctive, an annulus could be observed at the base of each axoneme. Mitochondria were condensed and located to the base of the axoneme. Spermatozoa showed some specific features, such as more condensed nuclei and the presence of an annulus in the middle piece of the axoneme. As before, the axonemes were stained with MCA and h-Tektin-t. Observed under DIC microscopy, the nuclei of spermatozoa were oval in shape and the smallest of all the stages of spermatogenesis. The diameter of the nuclei was 5.44 mm across the major axis. Some spermatozoa had vacuoles in the nuclei. Mitochondria were arranged from base to middle piece of axoneme. Sertoli cells had a distinctive nuclear envelope and nucleolus, and large spherical nucleus. The diameter of the nuclei was 12.25 mm. The formation of acrosomal granules or acrosomal cap and axoneme elongation have been well studied to identify spermatogenic cells precisely. However, they were not always adequate or convincing. In the present study, we examined mitochondrial and morphologic changes by using Mitotracker, which enabled creation of a diagram of spermatogenic cells at each developmental stage. This diagram has provided one more criterion, namely mitochondrial location, for identification of spermatogenic cells in addition to the morphologic characteristics and can help reproductive clinicians to train themselves, using a standardized criterion, to detect spermatozoa. The precise identification of spermatozoa can result in improvement of fertilization rate in patients with NOA. The nuclear diameter of each spermatogenic cell measured in this study was almost the same as previously reported (14). In addition to the nuclear diameter, the cytoplasmic diameter is considered to be advantageous for identifying spermatogonia, spermatocytes, and spermatids as well. Observation under this magnification is suitable for micromanipulation and enough to identify spermatogenic cells. Furthermore, not only are morphologic characteristics important but spermatogenic cell vitality is also important for ICSI. The vitality of spermatogenic cells should be related to the fertilization rate. There are some methods to assess the vitality of spermatozoa, but their efficacies are limited. In the future, the method using Mitotracker may be applied as a vitality test. On the other hand, there are some concerns about the mitochondrion staining in spermatogenic cells that are destined for use in reproductive therapy. In mice, sperm mitochondria and mitochondrial DNA were reported to be recognized and eliminated by active degradation after fertilization (15). The changes in the polarization of mitochondria are not related to the developmental capacity of subsequently fertilized embryo. Therefore, mitochondrion staining has no harmful effects on embryo development in mice (16). Mitotracker may also accumulate in the nucleus, because it passively diffuses across the plasma membrane and accumulates in active mitochondria, depending on the time and concentration of incubation. However, mural embryos stained with <200 nmol/L Mitotracker were similar to those without staining in embryo development, and normal healthy newborns were gained, although
Vol. 95, No. 8, June 30, 2011
FIGURE 1 Spermatogenic cells from spermatogonia to spermatocytes labeled with Mitotracker CMXRos and spermatogenic cell–specific antibodies as viewed under epifluorescence and differential interference contrast (DIC) microscopy. Some round spermatids have comparatively distinct acrosomal caps even at a magnification of 400 (white arrowhead). The annulus of elongating spermatids could be recognized at the base of the nuclei (black arrow). In some spermatozoa, nuclear vacuoles also could be recognized (white arrow). Scale bar ¼ 10 mm. Constant magnification: 400.
Okuda. Correspondence. Fertil Steril 2011.
those with more than 500 nmol/L developed abnormally and the rate of embryo progression was significantly reduced (16). The present study showed enough staining with 200 nmol/L to identify spermatogenic cells in humans. Further studies on how mitochondrial staining of human germ cells affects fertilization, pregnancy, and neonates are needed before validation in human reproductive technology. Conceptually, it would have been ideal to perform this
in patients with NOA. However, this may be a more challenging patient population to study. In conclusion, the current method of labeling with Mitotracker is simple and efficacious in identification of precise spermatogenic cells. The systematic diagram at each developmental step provided a checklist of mitochondrial relocation and morphologic change.
REFERENCES 1. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14: 131–5. 2. Amer M, Ateyah A, Hany R, Zohdy W. Prospective comparative study between microsurgical and conventional testicular sperm extraction in nonobstructive azoospermia: follow-up by serial ultrasound examinations. Hum Reprod 2000;15: 653–6. 3. Okada H, Dobashi M, Yamazaki T, Hara I, Fujisawa M, Arakawa S, et al. Conventional versus microdissection testicular sperm extraction for nonobstructive azoospermia. J Urol 2002;168: 1063–7. 4. Ramasamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction. Urology 2005;65:1190–4.
Fertility and Sterility
5. Ravizzini P, Carizza C, Abdelmassih V, Abdelmassih S, Azevedo M, Abdelmassih R. Microdissection testicular sperm extraction and IVF-ICSI outcome in nonobstructive azoospermia. Andrologia 2008;40:219–26. 6. Tsujimura A, Matsumiya K, Miyagawa Y, Tohda A, Miura H, Nishimura K, et al. Conventional multiple or microdissection testicular sperm extraction: a comparative study. Hum Reprod 2002;17: 2924–9. 7. Tsujimura A, Matsumiya K, Miyagawa Y, Takao T, Fujita K, Koga M, et al. Prediction of successful outcome of microdissection testicular sperm extraction in men with idiopathic nonobstructive azoospermia. J Urol 2004;172:1944–7. 8. Sutovsky P, Ramalho-Santos J, Moreno RD, Oko R, Hewitson L, Schatten G. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe Mito-
9.
10.
11.
12.
tracker and high resolution differential interference contrast microscopy. Hum Reprod 1999;14: 2301–12. Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP. The human VASA gene is specifically expressed in the germ cell lineage. Proc Natl Acad Sci U S A 2000;97:9585–90. Matsuoka Y, Miyagawa Y, Tokuhiro K, Kitamura K, Iguchi N, Maekawa M, et al. Isolation and characterization of the spermatid-specific Smrp1 gene encoding a novel manchette protein. Mol Reprod Dev 2008;75:967–75. Tokuhiro K, Hirose M, Miyagawa Y, Tsujimura A, Irie S, Isotani A, et al. Meichroacidin containing the membrane occupation and recognition nexus motif is essential for spermatozoa morphogenesis. J Biol Chem 2008;283:19039–48. Tsuchida J, Nishina Y, Wakabayashi N, Nozaki M, Sakai Y, Nishimune Y. Molecular cloning and
2667
characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein). Dev Biol 1998;197:67–76. 13. Iguchi N, Tanaka H, Nakamura Y, Nozaki M, Fujiwara T, Nishimune Y. Cloning and characterization of the human tektin-t gene. Mol Hum Reprod 2002;8:525–30.
2668
Okuda et al.
Correspondence
14. Johnson L, Neaves WB, Barnard JJ, Keillor GE, Brown SW, Yanagimachi R. A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules. Biol Reprod 1999;61:927–34. 15. Kaneda H, Hayashi J, Takahama S, Taya C, Lindahl KF, Yonekawa H. Elimination of paternal
mitochondrial DNA in intraspecific crosses during early mouse embryogenesis. Proc Natl Acad Sci U S A 1995;92:4542–6. 16. Hikichi T, Kishigami S, Thuan NV, Ohta H, Mizutani E, Wakayama S, et al. Round spermatids stained with mitotracker can be used to produce offspring more simply. Zygote 2005;13:55–61.
Vol. 95, No. 8, June 30, 2011
The introduction of microdissection testicular sperm extraction (MD-TESE) has significantly improved the sperm retrieval rate (SRR) in nonobstructive azoospermia (NOA) (1). In earlier reports, MD-TESE has proven to be superior to conventional techniques regarding SRR (1–7). However, the reported SRRs by MD-TESE vary from 40% to 63% (1–7). One reason for this variation is differences in the proportion of testicular histologies. The SRR for hypospermatogenesis is 80%–100%, remarkably higher than for Sertoli cell–only syndrome (20%–40%) (3, 4, 6). Another reason is that the successful identification of Hidenobu Okuda, M.D.a Akira Tsujimura, M.D.a Keisuke Yamamoto, M.D.a Shinichiro Fukuhara, M.D.a Jiro Nakayama, M.D.a Hiroshi Kiuchi, M.D.b Tetsuya Takao, M.D.a Yasushi Miyagawa, M.D.a Norio Nonomura, M.D.a Akihiko Okuyama, M.D.a a Department of Urology, Osaka University Graduate School of Medicine, Osaka, Japan b Department of Urology, Osaka Central Hospital, Osaka, Japan Received June 29, 2010; revised April 6, 2011; accepted April 7, 2011; published online May 5, 2011. H.O. has nothing to disclose. A.T. has nothing to disclose. K.Y. has nothing to disclose. S.F. has nothing to disclose. J.N. has nothing to disclose. H.K. has nothing to disclose. T.T. has nothing to disclose. Y.M. has nothing to disclose. N.N. has nothing to disclose. A.O. has nothing to disclose. Reprint requests: Akira Tsujimura, M.D., Department of Urology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan (E-mail: [email protected]).
0015-0282/$36.00 doi:10.1016/j.fertnstert.2011.04.026
spermatozoa can depend on the judgment of experienced reproductive clinicians. Reproductive clinicians usually detect spermatozoa morphologically at low magnification owing to the requirements of micromanipulation. To identify spermatozoa precisely, they are required to identify developing spermatogenic cells as well, because seminiferous tubules with sperm production, rather than those with Sertoli cells alone, are likely to contain many more developing spermatogenic cells than tubules without sperm production (1). Therefore, the precise identification of spermatogenic cells by reproductive clinicians is likely to affect the SRR, and therefore a standardized criterion has been needed for a long time. A novel method of spermatid selection using Mitotracker, which is a vital mitochondrion-specific fluorescent probe, has been reported (8). This probe proved to be useful to correctly identify live round spermatids based on the polarized patterns of the mitochondria. Therefore, we considered that Mitotracker staining could be an easy and promising method for identifying spermatozoa precisely, even if observed under low magnification. The aim of the present study was to make a systematic description and diagram of human spermatogenic cells that focuses on the mitochondrial location and morphologic characteristics at each step in development by using Mitotracker to understand spermatogenic cells, and to apply this information to artificial reproductive techniques. We also confirmed the accuracy of our classification by immunostaining with various stage-specific spermatogenic cell antibodies at the same time. Several human testis tissues from consenting subjects with obstructive azoospermia that had been obtained by TESE and were no longer used for intracytoplasmic sperm injection (ICSI) were used as samples for experimentation. Dispersal of spermatogenic cells from seminiferous tubules was carried out as previously described (8).
Fertility and Sterility Vol. 95, No. 8, June 30, 2011 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc.
2665
The spermatogenic cell suspension was incubated for 15 minutes at 37 C in 200 nmol/L Mitotracker CMXRos (Invitrogen). Thereafter, immunofluorescence was used with four kinds of stage-specific spermatogenic cell antibodies to identify the developmental steps of spermatogenic cells. After the fixation and blocking of cell suspension drops, the drops were incubated with 1:200 diluted spermatogenic cell–specific antibodies VASA, MCA (meichroacidin), h-Tektin-T, and Smrp-1 (spermatid-specific manchette-related protein 1) overnight at 4 C. Thereafter, they were incubated for 90 minutes at room temperature using 1:200 diluted secondary antibody. Developmental stages targeted by each antibody are described as follows. VASA (Santa Cruz Biotechnology) is specifically expressed in spermatogonia, where its expression is the highest. It has reduced expression in spermatocytes and low expression in spermatids and is absent in spermatozoa (9). MCA and Smrp-1 were produced as described previously (10–12). MCA is expressed in cytoplasm from pachytene spermatocytes through to the round spermatid stage and is also located on the axoneme. Smrp-1 is expressed in the caudal side of the cytoplasm of elongated spermatids, and it disappears in mature sperm. h-Tektin-t is located specifically on the axoneme, forming filamentous polymers (13). The spermatogenic cells stained with each antibody were observed by using epifluorescence and differential interference contrast (DIC) microscopy at a constant magnification of 400 and were assigned to each stage of spermatogenesis according to staining status and cell shape. Thereafter, the mitochondrial location and morphologic characteristics in each stage of spermatogenic cells were also analyzed and described. The translocations of mitochondria and morphologic changes in each stage of spermatogenesis were analyzed in reference to the staining by spermatogenic cell–specific antibodies (Fig. 1). Spermatogonia were defined as spherical cells with a very distinct nuclear envelope and stained only with VASA. The average diameters of nuclei and cytoplasm were 7.22 mm and 9.03 mm, respectively. Mitochondria were homogeneous in the cytoplasm. Spermatocytes were divided into two groups according to the stages targeted by the antibodies: preleptotene/leptotene/zygotene (P-Z) spermatocytes and pachytene spermatocytes. The former were stained only with VASA and the latter with VASA and MCA. The nuclei and cytoplasm of P-Z spermatocytes were 8.66 mm and 12.53 mm in diameter, respectively, and had become larger and irregular compared with those of spermatogonia, although they remained spherical. The expression of mitochondria was greatly increased compared with that of the spermatogonia. In pachytene spermatocytes the diameters of the oval nuclei and cytoplasm increased to 12.3 mm and 16.72 mm, respectively, and were the largest of all the stages of spermatogenesis. Mitochondria was greatly increased compared with that of P-Z spermatocytes. Compared with spermatocytes, round spermatids, which were stained with MCA and rarely with VASA, were smaller cells whose nuclear and cytoplasmic diameters were 5.78 mm and 8.44 mm, respectively. In this stage, the acrosomal granule appears initially and the acrosomal cap is formed eventually as a cytoskeletal structure covering the round spermatid nucleus. At the magnification used, only acrosomal caps were visible. As the nucleus and cytoplasm condensed, mitochondria began to polarize and condense to the basal pole of the cytoplasm. None of the round spermatids were stained with h-Tektin-t, because axonemes had not developed yet.
2666
Okuda et al.
Correspondence
The axoneme was apparent in the next stage, the elongating spermatid, which was well stained with MCA and h-Tektin-t. The manchette is a stage-specific structure that appears around the nuclei of elongating spermatids, and it was stained with Smrp-1. As the axonemes became distinctive, an annulus could be observed at the base of each axoneme. Mitochondria were condensed and located to the base of the axoneme. Spermatozoa showed some specific features, such as more condensed nuclei and the presence of an annulus in the middle piece of the axoneme. As before, the axonemes were stained with MCA and h-Tektin-t. Observed under DIC microscopy, the nuclei of spermatozoa were oval in shape and the smallest of all the stages of spermatogenesis. The diameter of the nuclei was 5.44 mm across the major axis. Some spermatozoa had vacuoles in the nuclei. Mitochondria were arranged from base to middle piece of axoneme. Sertoli cells had a distinctive nuclear envelope and nucleolus, and large spherical nucleus. The diameter of the nuclei was 12.25 mm. The formation of acrosomal granules or acrosomal cap and axoneme elongation have been well studied to identify spermatogenic cells precisely. However, they were not always adequate or convincing. In the present study, we examined mitochondrial and morphologic changes by using Mitotracker, which enabled creation of a diagram of spermatogenic cells at each developmental stage. This diagram has provided one more criterion, namely mitochondrial location, for identification of spermatogenic cells in addition to the morphologic characteristics and can help reproductive clinicians to train themselves, using a standardized criterion, to detect spermatozoa. The precise identification of spermatozoa can result in improvement of fertilization rate in patients with NOA. The nuclear diameter of each spermatogenic cell measured in this study was almost the same as previously reported (14). In addition to the nuclear diameter, the cytoplasmic diameter is considered to be advantageous for identifying spermatogonia, spermatocytes, and spermatids as well. Observation under this magnification is suitable for micromanipulation and enough to identify spermatogenic cells. Furthermore, not only are morphologic characteristics important but spermatogenic cell vitality is also important for ICSI. The vitality of spermatogenic cells should be related to the fertilization rate. There are some methods to assess the vitality of spermatozoa, but their efficacies are limited. In the future, the method using Mitotracker may be applied as a vitality test. On the other hand, there are some concerns about the mitochondrion staining in spermatogenic cells that are destined for use in reproductive therapy. In mice, sperm mitochondria and mitochondrial DNA were reported to be recognized and eliminated by active degradation after fertilization (15). The changes in the polarization of mitochondria are not related to the developmental capacity of subsequently fertilized embryo. Therefore, mitochondrion staining has no harmful effects on embryo development in mice (16). Mitotracker may also accumulate in the nucleus, because it passively diffuses across the plasma membrane and accumulates in active mitochondria, depending on the time and concentration of incubation. However, mural embryos stained with <200 nmol/L Mitotracker were similar to those without staining in embryo development, and normal healthy newborns were gained, although
Vol. 95, No. 8, June 30, 2011
FIGURE 1 Spermatogenic cells from spermatogonia to spermatocytes labeled with Mitotracker CMXRos and spermatogenic cell–specific antibodies as viewed under epifluorescence and differential interference contrast (DIC) microscopy. Some round spermatids have comparatively distinct acrosomal caps even at a magnification of 400 (white arrowhead). The annulus of elongating spermatids could be recognized at the base of the nuclei (black arrow). In some spermatozoa, nuclear vacuoles also could be recognized (white arrow). Scale bar ¼ 10 mm. Constant magnification: 400.
Okuda. Correspondence. Fertil Steril 2011.
those with more than 500 nmol/L developed abnormally and the rate of embryo progression was significantly reduced (16). The present study showed enough staining with 200 nmol/L to identify spermatogenic cells in humans. Further studies on how mitochondrial staining of human germ cells affects fertilization, pregnancy, and neonates are needed before validation in human reproductive technology. Conceptually, it would have been ideal to perform this
in patients with NOA. However, this may be a more challenging patient population to study. In conclusion, the current method of labeling with Mitotracker is simple and efficacious in identification of precise spermatogenic cells. The systematic diagram at each developmental step provided a checklist of mitochondrial relocation and morphologic change.
REFERENCES 1. Schlegel PN. Testicular sperm extraction: microdissection improves sperm yield with minimal tissue excision. Hum Reprod 1999;14: 131–5. 2. Amer M, Ateyah A, Hany R, Zohdy W. Prospective comparative study between microsurgical and conventional testicular sperm extraction in nonobstructive azoospermia: follow-up by serial ultrasound examinations. Hum Reprod 2000;15: 653–6. 3. Okada H, Dobashi M, Yamazaki T, Hara I, Fujisawa M, Arakawa S, et al. Conventional versus microdissection testicular sperm extraction for nonobstructive azoospermia. J Urol 2002;168: 1063–7. 4. Ramasamy R, Yagan N, Schlegel PN. Structural and functional changes to the testis after conventional versus microdissection testicular sperm extraction. Urology 2005;65:1190–4.
Fertility and Sterility
5. Ravizzini P, Carizza C, Abdelmassih V, Abdelmassih S, Azevedo M, Abdelmassih R. Microdissection testicular sperm extraction and IVF-ICSI outcome in nonobstructive azoospermia. Andrologia 2008;40:219–26. 6. Tsujimura A, Matsumiya K, Miyagawa Y, Tohda A, Miura H, Nishimura K, et al. Conventional multiple or microdissection testicular sperm extraction: a comparative study. Hum Reprod 2002;17: 2924–9. 7. Tsujimura A, Matsumiya K, Miyagawa Y, Takao T, Fujita K, Koga M, et al. Prediction of successful outcome of microdissection testicular sperm extraction in men with idiopathic nonobstructive azoospermia. J Urol 2004;172:1944–7. 8. Sutovsky P, Ramalho-Santos J, Moreno RD, Oko R, Hewitson L, Schatten G. On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe Mito-
9.
10.
11.
12.
tracker and high resolution differential interference contrast microscopy. Hum Reprod 1999;14: 2301–12. Castrillon DH, Quade BJ, Wang TY, Quigley C, Crum CP. The human VASA gene is specifically expressed in the germ cell lineage. Proc Natl Acad Sci U S A 2000;97:9585–90. Matsuoka Y, Miyagawa Y, Tokuhiro K, Kitamura K, Iguchi N, Maekawa M, et al. Isolation and characterization of the spermatid-specific Smrp1 gene encoding a novel manchette protein. Mol Reprod Dev 2008;75:967–75. Tokuhiro K, Hirose M, Miyagawa Y, Tsujimura A, Irie S, Isotani A, et al. Meichroacidin containing the membrane occupation and recognition nexus motif is essential for spermatozoa morphogenesis. J Biol Chem 2008;283:19039–48. Tsuchida J, Nishina Y, Wakabayashi N, Nozaki M, Sakai Y, Nishimune Y. Molecular cloning and
2667
characterization of meichroacidin (male meiotic metaphase chromosome-associated acidic protein). Dev Biol 1998;197:67–76. 13. Iguchi N, Tanaka H, Nakamura Y, Nozaki M, Fujiwara T, Nishimune Y. Cloning and characterization of the human tektin-t gene. Mol Hum Reprod 2002;8:525–30.
2668
Okuda et al.
Correspondence
14. Johnson L, Neaves WB, Barnard JJ, Keillor GE, Brown SW, Yanagimachi R. A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules. Biol Reprod 1999;61:927–34. 15. Kaneda H, Hayashi J, Takahama S, Taya C, Lindahl KF, Yonekawa H. Elimination of paternal
mitochondrial DNA in intraspecific crosses during early mouse embryogenesis. Proc Natl Acad Sci U S A 1995;92:4542–6. 16. Hikichi T, Kishigami S, Thuan NV, Ohta H, Mizutani E, Wakayama S, et al. Round spermatids stained with mitotracker can be used to produce offspring more simply. Zygote 2005;13:55–61.
Vol. 95, No. 8, June 30, 2011