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A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection
FERTILITY AND STERILITY威 VOL. 74, NO. 5, NOVEMBER 2000 Copyright ©2000 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection Catherine Metzler-Guillemain, M.D., and Marie-Roberte Guichaoua, Ph.D. Laboratoire de Biologie de la Reproduction, Hoˆpital de la Conception, Marseille, France
Objective: To develop a reliable and simple method allowing meiotic studies to be performed on testicular samples used for ICSI. Design: Evaluation of meiotic abnormalities in patients with severe spermatogenic impairment. Setting: Centre de Me´decine de la Reproduction, Marseille. Patient(s): Two azoospermic men undergoing testicular biopsy for ICSI and one control individual with normal testicular histology. Intervention(s): The immature germ cells from the patients came from testicular biopsy used for ICSI, after dispersal into a thin cell suspension. Cells were cytocentrifuged to obtain well-spread spermatocytes and then immunocytochemical techniques were performed. We used rabbit polyclonal antibodies against the specific meiotic proteins Cor1 and Syn1 and a human CREST anti-kinetochore antibody. Main Outcome Measure(s): Synapsis abnormalities in patients with severe spermatogenesis impairment. Result(s): Pachytene spermatocytes are easily analyzed with this technique, without damage of the axial core and synaptonemal complex. The loss of germ cells is limited. Conclusion(s): The cytocentrifugation method is the most suitable technique for meiotic studies in patients with severe spermatogenic failure, because it can be used on the testicular cell suspension remaining after ICSI with testicular spermatozoa. (Fertil Steril威 2000;74:916 –9. ©2000 by American Society for Reproductive Medicine.) Key Words: Severe spermatogenic failure, meiosis, cytocentrifugation
Received January 13, 2000; revised and accepted April 28, 2000. Supported by grants from the Association pour la Recherche sur le Cancer (ARC). Reprint requests: Catherine Metzler-Guillemain, M.D., Laboratoire de Biologie de la Reproduction, Hoˆpital de la Conception, 147, Bd Baille, 13385 Marseille cedex 5, France (FAX: 33 1 04 91 38 38 97; E-mail: [email protected] -mrs.fr). 0015-0282/00/$20.00 PII S0015-0282(00)01551-X
916
The role of meiotic abnormalities in the origin of human male infertility has not yet been precisely established. Vendrell et al. (1) reported that meiotic disorders such as meiotic arrest and synaptic failure affect 57.8% of patients with a normal karyotype and a sperm concentration of ⬍1 ⫻ 106/mL. Up to now, the small number of meiotic studies reported for patients with severe spermatogenic defects have been insufficient to allow a precise evaluation of the role of meiotic abnormalities in male infertility (1, 2). Currently, the most reliable technique for the study of meiotic synapsis is the microspreading technique, which is accomplished by placing a drop of sucrose solution onto a slide, followed by the germ cell suspension. The mixture is then gently spread over the surface of another slide and fixed. This technique allows
meiotic analyses using light microscopy, electron microscopy, or immunocytochemistry. Unfortunately, a significant loss of germ cells is inherent with such a technique, making the analysis of a sufficient number of cells difficult. Here, we describe a reliable and simple method based on cytocentrifugation that minimizes the loss of germ cells. Our method is a useful research tool for studying the meiotic process in patients with severe idiopathic oligozoospermia or with azoospermia. It can be applied to testicular material biopsied for an intracytoplasmic sperm injection (ICSI) attempt, or as part of an investigation of infertility. Immunocytochemistry techniques with antibodies directed against meiotic protein can be performed on meiotic spreads obtained with our method.
MATERIAL AND METHODS As of 1998, our reproduction center no longer performs testicular biopsy on oligozoospermic or azoospermic males as part of the clinical investigations into the causes of their infertility. The biopsy is systematically coupled with an ICSI attempt. In accordance with the Huriet law, the patient’s informed consent is requested before taking a small testis sample for meiotic studies and before each ICSI attempt. The protocol was approved by the CCPPRB (Comite´ Consultatif de Protection des Personnes dans la Recherche Biome´dicale). In practice, the spermatogenesis of these patients is often extremely impaired so all testicular fragments are used for the assisted fertilization. The meiotic studies are then carried out on the cell suspension that remains after ICSI, which contains the immature germ cells. We emphasize that the technique described here can be used on testicular cells biopsied for an ICSI attempt, as well as on diagnostic testicular biopsies, whether the testis sample is obtained surgically or by needle biopsy.
Patients and Control This technique was developed on two azoospermic patients, one with an idiopathic etiology and one with an obstructive etiology. Azoospermia was diagnosed with two semen analyses performed over 1 year for both patients. The first patient was a 30-year-old man who had a normal phenotype apart from a moderate testicular hypotrophy. His karyotype was normal and his FSH level was 10.5 UI/L. The second patient was a 35-year-old man who had a normal phenotype and an infertility with an obstructive origin. His karyotype was normal and his FSH level was 7.0 UI/L. To obtain spermatozoa for an ICSI attempt, a surgical bilateral testicular biopsy was performed under general anesthesia, and the fragments were teased apart to obtain a thin cell suspension. For the first patient, only eight spermatozoa were found in the suspension with a few immature germ cells. For the second patient, plenty of germ cells were present after biopsy delaceration. After ICSI, the remaining cell suspensions containing the immature germ cells were centrifuged at 1900 rpm for 10 minutes, resuspended in 5 mL Ham F-10/glycerol 10%, aliquoted to five cryotubes for each patient, and frozen in liquid nitrogen for further use. The same protocol was applied to germ cells from a 70-year-old patient on whom was performed an orchidectomy for the management of a prostate carcinoma. Histologic analysis showed abundant normal spermatogenic activity. Small testicular fragments were frozen in Ham F-10/ glycerol 10% at ⫺196°C. After thawing, one fragment was teased apart in Ham F-10/glycerol 10% to obtain a thin cell suspension without fragments; this suspension was used for cytospin spreads.
Cytospin Spreads One cryotube was thawed at 37°C, and the cellular suspension was gently mixed to obtain a homogeneous cell FERTILITY & STERILITY威
suspension; 200 L of the cell suspension was spread onto one silane-coated slide (Sigma) by cytocentrifugation with a cytospin 2 (Shandon) at 500 rpm for 5 minutes. After the spread was stained with 4% Giemsa solution, the quality of the spread and the number of germ cells were assessed. Based on the first slide, the cell concentration was adjusted, if necessary, by dilution in Ham F-10/glycerol and the optimal centrifugation speed from 500 to 800 rpm was determined. The aim was to obtain well-spread spermatocytes. Slides were treated for immunocytochemistry, according to the technique described by Moens and associates (3).
Immunocytochemical Technique All antibodies against meiotic proteins that cross react with human can be applied on these preparations. The quality of the spreads was estimated using a rabbit polyclonal antibody that recognizes the Cor1 protein of the axial/lateral elements of the synaptonemal complex and the Syn1 protein of the central element (kindly provided by P. B. Moens). We also used human CREST anti-kinetochore antibodies, obtained from the patients’ sera with titers reaching at least 1/10000 (Laboratory of Immunology, Hoˆpital de La Conception, Marseilles, France). Immunocytochemistry was performed as described by Moens and colleagues (3), with slight modifications. Each cell spot was overlain with 15 L of the rabbit polyclonal antibody at a dilution of 1/1000 in ADB (antibody dilution buffer: PBS/10% goat serum/3% BSA/0.05% triton), covered with a 22 ⫻ 22 parafilm, and placed cells-down in a moist chamber with PBS for 1 hour at 37°C. The parafilm was removed; 15 L of CREST antikinetochore antibodies, diluted at 1/20th in ADB, were placed on the spot overnight at 37°C. After washes, detection was performed with secondary antibodies purchased from Sigma and used according to the manufacturer’s instructions: FITC conjugated goat anti-rabbit IgG diluted at 1/160th and TRITC conjugated anti-human IgG diluted at 1/16th in PBS were applied on each spot for 1 hour at 37°C in a moist chamber. After washes, the slides were left to dry and were mounted in antifade medium (Vectashield). These antibodies allowed the recognition of leptotene, zygotene, pachytene, and diplotene stages. We essentially studied the pachytene nuclei, as they are the most informative for the synapsis abnormalities.
Microscope Analysis Nuclei were first observed using a Zeiss Axioplan 2 fluorescent photomicroscope (Zeiss, Germany). Selected nuclei were then examined with a confocal scanner Leica TCS 4D, mounted on a Leica DMIRBE microscope (Leica, Heidelberg, Germany), equipped with a ⫻100 planapochromat objective. One 50 mW argon krypton laser was used, producing two major lines at 488 nm (green) and 568 nm (red). For each nucleus, the Z-series (containing 4 to 5 optical sections) of both labels were obtained at a pitch of 1 m. The 917
FIGURE 1 Pachytene spermatocytes from patient one. (A) Normal nucleus showing 22 autosomal synaptonemal complexes and the sex vesicle in green, and 24 kinetochores (red signals). (B) Nucleus showing fragmented synaptonemal complexes and no identifiable sex vesicle.
Metzler-Guillemain. Cytocentrifugation method for meiotic studies. Fertil Steril 2000.
scanning zoom varied from 2.3 to 2.7. The pixel size x and y was 0.9 mm.
RESULTS Patients For the first patient, a suitable monolayer was obtained with the initial dilution of the suspension recovered after ICSI. Twenty pachytene spermatocytes were analyzed, 17 of which were normal; each of them had 22 entire and unbroken autosomal synaptonemal complexes (SC) and one XY bivalent, which appeared either with a short SC and two fine axial elements, or with a splitting of the X and Y axes. Each autosomal bivalent had one signal for the kinetochores of each homologue at the pachytene stage (Fig. 1A). Three pachytene spermatocytes (15%) showed fragmented SC with asynapsed regions, whereas the sex bivalent was not identifiable (see Fig. 1B). For the second patient, the initial cell suspension was diluted at 1/8th to obtain well-spread spermatocytes. Among 121 pachytene nuclei analyzed, 99 nuclei were normal and 22 nuclei (18%) presented abnormal bivalents.
Control The initial cell suspension was diluted at 1/8th in Ham F10/glycerol to obtain a suitable germ cell monolayer. Of 158 pachytene spermatocytes, 148 showed no visible damage of the analyzed structures. Ten pachytene spermatocytes (6.3%) presented the same abnormalities as described in the patient. This percentage corresponds to the percentage of nuclei with asynapsis and decondensed bivalents observed in 918
Metzler-Guillemain and Guichaoua
normal spermatogenesis when meiosis was studied with the air-drying technique. The difference between the results of the two patients was not significant (Fisher test, P ⫽ 1), but the difference between the patients and the control was significant (chisquare, P ⫽ 0.002). This variation between patients and control may be related to the spermatogenic disorders observed in the patients. The interpretation and the significance of such a variation will be discussed elsewhere (Guichaoua et al., in preparation).
DISCUSSION Up to now, most information concerning synapsis came from the three-dimensional reconstructions of meiotic prophase nuclei serially sectioned by electron microscopy (4), and from surface spread meiotic nuclei (1). These techniques require either fresh material (electron microscopy techniques) or a sufficient number of cells (whole mount spreading), and are not adapted in cases where the testicular material comes from patients with very poor spermatogenesis. Recently, cytocentrifugations of metaphasic somatic chromosomes have been shown to produce metaphase spreads without methanol/acetic acid fixative (5). We applied the cytocentrifugation technique to meiotic cells and obtained well-spread spermatocytes. This spreading technique, applied to germ cells, is an excellent research tool for the study of meiotic abnormalities in infertile patients. Cytocentrifugation is a very simple way to spread testic-
Cytocentrifugation method for meiotic studies
Vol. 74, No. 5, November 2000
ular cells. As with the standard spreading technique, wellspread spermatocytes can be obtained without impairment of the axial core and synaptonemal complex. Immunocytochemical and in situ hybridization techniques can be easily performed on these preparations, leading to a high-resolution analysis of the meiotic pairing process and genetic recombination (our unpublished results). A small quantity of antibodies is needed on each spot, and microscope analysis can be performed rapidly as the cells are concentrated on a small surface. If the number of germ cells is very low, the quantity of spermatocytes present on one slide is not sufficient to allow evaluation of the patient’s meiosis. Further slides must then be done to analyze the largest possible number of cells. Indeed, it is not recommended to concentrate the initial solution, because of the presence of many cell fragments and somatic cells that may disrupt the quality of the spread. Unlike the conventional air-drying and spreading techniques, this new meiotic technique avoids the loss of numerous germ cells. This is particularly important where meiotic studies have to be conducted on patients with severe spermatogenic defects. Thus, the cytocentrifugation technique can be performed on each testicular cell suspension obtained after biopsy delaceration, whatever method is used for the biopsy. One can avoid taking a testis fragment only for meiotic studies, which is an enormous advantage in cases of testicular hypotrophy and reduced spermatogenesis. The only limiting factor in this technique is the absence of immature germ cells. Another advantage of this technique is that, as the cell suspension is frozen and stored at ⫺196°C, cells can be stored in liquid nitrogen for transport and later analysis. This allows us to perform meiotic studies on testicular cells retrieved far from our laboratory.
FERTILITY & STERILITY威
We would like to emphasize that this technique is applicable in extreme cases of spermatogenesis impairment. We agree with Vendrell et al. (1), who have proposed that meiotic studies must be done when a testicular biopsy is performed on an infertile patient, because the frequency of meiotic abnormalities in these patients is probably high.
Acknowledgments: The authors thank P. Moens, Ph.D., and B. Spyropoulos, Ph.D. (York University, Canada) for the generous gift of the antibodies; J. M. Grillo, Ph.D., for providing us with the testicular cell suspensions; D. Daı¨oglou for her technical assistance; J. Sampol, Ph.D. and R. Pistoresi for the confocal analysis; M. San Marco, Ph.D., for the gift of the antikinetochore antibodies; and Dr. F. Romain for the statistical analysis (Hoˆpital de la Conception, Marseilles, France). We also are grateful to Dr. M. G. Mattei (Ph.D., Inserm U491, Marseilles, France) for her precious advice.
References 1. Vendrell JM, Garcia F, Veiga A, Calderon G, Egozcue S, Egozcue J, Barri PN. Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Hum Reprod 1999;14:375– 8. 2. Lange R, Krause W, Engel W. Analyses of meiotic chromosomes in testicular biopsies of infertile patients. Hum Reprod 1997;12:2154 – 8. 3. Moens PB, Pearlman RE, Heng HHQ, Traut W. Chromosome cores and chromatin at meiotic prophase. In: Handel MA, ed. Current topics in developmental biology 37: meiosis and gametogenesis. Orlando, FL: Academic Press, 1998: 241– 62. 4. Guichaoua MR, de Lanversin A, Cataldo C, Delafontaine D, Alasia C, Fraterno M, Terriou P, Stahl A, Luciani JM. Three dimensional reconstruction of human pachytene spermatocyte nuclei of a 17;21 reciprocal translocation carrier: study of XY-autosome relationships. Hum Genet 1991;87:709 –15. 5. Jeppesen P, Turner M. The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell 1993;74:281–9.
919
A simple and reliable method for meiotic studies on testicular samples used for intracytoplasmic sperm injection Catherine Metzler-Guillemain, M.D., and Marie-Roberte Guichaoua, Ph.D. Laboratoire de Biologie de la Reproduction, Hoˆpital de la Conception, Marseille, France
Objective: To develop a reliable and simple method allowing meiotic studies to be performed on testicular samples used for ICSI. Design: Evaluation of meiotic abnormalities in patients with severe spermatogenic impairment. Setting: Centre de Me´decine de la Reproduction, Marseille. Patient(s): Two azoospermic men undergoing testicular biopsy for ICSI and one control individual with normal testicular histology. Intervention(s): The immature germ cells from the patients came from testicular biopsy used for ICSI, after dispersal into a thin cell suspension. Cells were cytocentrifuged to obtain well-spread spermatocytes and then immunocytochemical techniques were performed. We used rabbit polyclonal antibodies against the specific meiotic proteins Cor1 and Syn1 and a human CREST anti-kinetochore antibody. Main Outcome Measure(s): Synapsis abnormalities in patients with severe spermatogenesis impairment. Result(s): Pachytene spermatocytes are easily analyzed with this technique, without damage of the axial core and synaptonemal complex. The loss of germ cells is limited. Conclusion(s): The cytocentrifugation method is the most suitable technique for meiotic studies in patients with severe spermatogenic failure, because it can be used on the testicular cell suspension remaining after ICSI with testicular spermatozoa. (Fertil Steril威 2000;74:916 –9. ©2000 by American Society for Reproductive Medicine.) Key Words: Severe spermatogenic failure, meiosis, cytocentrifugation
Received January 13, 2000; revised and accepted April 28, 2000. Supported by grants from the Association pour la Recherche sur le Cancer (ARC). Reprint requests: Catherine Metzler-Guillemain, M.D., Laboratoire de Biologie de la Reproduction, Hoˆpital de la Conception, 147, Bd Baille, 13385 Marseille cedex 5, France (FAX: 33 1 04 91 38 38 97; E-mail: [email protected] -mrs.fr). 0015-0282/00/$20.00 PII S0015-0282(00)01551-X
916
The role of meiotic abnormalities in the origin of human male infertility has not yet been precisely established. Vendrell et al. (1) reported that meiotic disorders such as meiotic arrest and synaptic failure affect 57.8% of patients with a normal karyotype and a sperm concentration of ⬍1 ⫻ 106/mL. Up to now, the small number of meiotic studies reported for patients with severe spermatogenic defects have been insufficient to allow a precise evaluation of the role of meiotic abnormalities in male infertility (1, 2). Currently, the most reliable technique for the study of meiotic synapsis is the microspreading technique, which is accomplished by placing a drop of sucrose solution onto a slide, followed by the germ cell suspension. The mixture is then gently spread over the surface of another slide and fixed. This technique allows
meiotic analyses using light microscopy, electron microscopy, or immunocytochemistry. Unfortunately, a significant loss of germ cells is inherent with such a technique, making the analysis of a sufficient number of cells difficult. Here, we describe a reliable and simple method based on cytocentrifugation that minimizes the loss of germ cells. Our method is a useful research tool for studying the meiotic process in patients with severe idiopathic oligozoospermia or with azoospermia. It can be applied to testicular material biopsied for an intracytoplasmic sperm injection (ICSI) attempt, or as part of an investigation of infertility. Immunocytochemistry techniques with antibodies directed against meiotic protein can be performed on meiotic spreads obtained with our method.
MATERIAL AND METHODS As of 1998, our reproduction center no longer performs testicular biopsy on oligozoospermic or azoospermic males as part of the clinical investigations into the causes of their infertility. The biopsy is systematically coupled with an ICSI attempt. In accordance with the Huriet law, the patient’s informed consent is requested before taking a small testis sample for meiotic studies and before each ICSI attempt. The protocol was approved by the CCPPRB (Comite´ Consultatif de Protection des Personnes dans la Recherche Biome´dicale). In practice, the spermatogenesis of these patients is often extremely impaired so all testicular fragments are used for the assisted fertilization. The meiotic studies are then carried out on the cell suspension that remains after ICSI, which contains the immature germ cells. We emphasize that the technique described here can be used on testicular cells biopsied for an ICSI attempt, as well as on diagnostic testicular biopsies, whether the testis sample is obtained surgically or by needle biopsy.
Patients and Control This technique was developed on two azoospermic patients, one with an idiopathic etiology and one with an obstructive etiology. Azoospermia was diagnosed with two semen analyses performed over 1 year for both patients. The first patient was a 30-year-old man who had a normal phenotype apart from a moderate testicular hypotrophy. His karyotype was normal and his FSH level was 10.5 UI/L. The second patient was a 35-year-old man who had a normal phenotype and an infertility with an obstructive origin. His karyotype was normal and his FSH level was 7.0 UI/L. To obtain spermatozoa for an ICSI attempt, a surgical bilateral testicular biopsy was performed under general anesthesia, and the fragments were teased apart to obtain a thin cell suspension. For the first patient, only eight spermatozoa were found in the suspension with a few immature germ cells. For the second patient, plenty of germ cells were present after biopsy delaceration. After ICSI, the remaining cell suspensions containing the immature germ cells were centrifuged at 1900 rpm for 10 minutes, resuspended in 5 mL Ham F-10/glycerol 10%, aliquoted to five cryotubes for each patient, and frozen in liquid nitrogen for further use. The same protocol was applied to germ cells from a 70-year-old patient on whom was performed an orchidectomy for the management of a prostate carcinoma. Histologic analysis showed abundant normal spermatogenic activity. Small testicular fragments were frozen in Ham F-10/ glycerol 10% at ⫺196°C. After thawing, one fragment was teased apart in Ham F-10/glycerol 10% to obtain a thin cell suspension without fragments; this suspension was used for cytospin spreads.
Cytospin Spreads One cryotube was thawed at 37°C, and the cellular suspension was gently mixed to obtain a homogeneous cell FERTILITY & STERILITY威
suspension; 200 L of the cell suspension was spread onto one silane-coated slide (Sigma) by cytocentrifugation with a cytospin 2 (Shandon) at 500 rpm for 5 minutes. After the spread was stained with 4% Giemsa solution, the quality of the spread and the number of germ cells were assessed. Based on the first slide, the cell concentration was adjusted, if necessary, by dilution in Ham F-10/glycerol and the optimal centrifugation speed from 500 to 800 rpm was determined. The aim was to obtain well-spread spermatocytes. Slides were treated for immunocytochemistry, according to the technique described by Moens and associates (3).
Immunocytochemical Technique All antibodies against meiotic proteins that cross react with human can be applied on these preparations. The quality of the spreads was estimated using a rabbit polyclonal antibody that recognizes the Cor1 protein of the axial/lateral elements of the synaptonemal complex and the Syn1 protein of the central element (kindly provided by P. B. Moens). We also used human CREST anti-kinetochore antibodies, obtained from the patients’ sera with titers reaching at least 1/10000 (Laboratory of Immunology, Hoˆpital de La Conception, Marseilles, France). Immunocytochemistry was performed as described by Moens and colleagues (3), with slight modifications. Each cell spot was overlain with 15 L of the rabbit polyclonal antibody at a dilution of 1/1000 in ADB (antibody dilution buffer: PBS/10% goat serum/3% BSA/0.05% triton), covered with a 22 ⫻ 22 parafilm, and placed cells-down in a moist chamber with PBS for 1 hour at 37°C. The parafilm was removed; 15 L of CREST antikinetochore antibodies, diluted at 1/20th in ADB, were placed on the spot overnight at 37°C. After washes, detection was performed with secondary antibodies purchased from Sigma and used according to the manufacturer’s instructions: FITC conjugated goat anti-rabbit IgG diluted at 1/160th and TRITC conjugated anti-human IgG diluted at 1/16th in PBS were applied on each spot for 1 hour at 37°C in a moist chamber. After washes, the slides were left to dry and were mounted in antifade medium (Vectashield). These antibodies allowed the recognition of leptotene, zygotene, pachytene, and diplotene stages. We essentially studied the pachytene nuclei, as they are the most informative for the synapsis abnormalities.
Microscope Analysis Nuclei were first observed using a Zeiss Axioplan 2 fluorescent photomicroscope (Zeiss, Germany). Selected nuclei were then examined with a confocal scanner Leica TCS 4D, mounted on a Leica DMIRBE microscope (Leica, Heidelberg, Germany), equipped with a ⫻100 planapochromat objective. One 50 mW argon krypton laser was used, producing two major lines at 488 nm (green) and 568 nm (red). For each nucleus, the Z-series (containing 4 to 5 optical sections) of both labels were obtained at a pitch of 1 m. The 917
FIGURE 1 Pachytene spermatocytes from patient one. (A) Normal nucleus showing 22 autosomal synaptonemal complexes and the sex vesicle in green, and 24 kinetochores (red signals). (B) Nucleus showing fragmented synaptonemal complexes and no identifiable sex vesicle.
Metzler-Guillemain. Cytocentrifugation method for meiotic studies. Fertil Steril 2000.
scanning zoom varied from 2.3 to 2.7. The pixel size x and y was 0.9 mm.
RESULTS Patients For the first patient, a suitable monolayer was obtained with the initial dilution of the suspension recovered after ICSI. Twenty pachytene spermatocytes were analyzed, 17 of which were normal; each of them had 22 entire and unbroken autosomal synaptonemal complexes (SC) and one XY bivalent, which appeared either with a short SC and two fine axial elements, or with a splitting of the X and Y axes. Each autosomal bivalent had one signal for the kinetochores of each homologue at the pachytene stage (Fig. 1A). Three pachytene spermatocytes (15%) showed fragmented SC with asynapsed regions, whereas the sex bivalent was not identifiable (see Fig. 1B). For the second patient, the initial cell suspension was diluted at 1/8th to obtain well-spread spermatocytes. Among 121 pachytene nuclei analyzed, 99 nuclei were normal and 22 nuclei (18%) presented abnormal bivalents.
Control The initial cell suspension was diluted at 1/8th in Ham F10/glycerol to obtain a suitable germ cell monolayer. Of 158 pachytene spermatocytes, 148 showed no visible damage of the analyzed structures. Ten pachytene spermatocytes (6.3%) presented the same abnormalities as described in the patient. This percentage corresponds to the percentage of nuclei with asynapsis and decondensed bivalents observed in 918
Metzler-Guillemain and Guichaoua
normal spermatogenesis when meiosis was studied with the air-drying technique. The difference between the results of the two patients was not significant (Fisher test, P ⫽ 1), but the difference between the patients and the control was significant (chisquare, P ⫽ 0.002). This variation between patients and control may be related to the spermatogenic disorders observed in the patients. The interpretation and the significance of such a variation will be discussed elsewhere (Guichaoua et al., in preparation).
DISCUSSION Up to now, most information concerning synapsis came from the three-dimensional reconstructions of meiotic prophase nuclei serially sectioned by electron microscopy (4), and from surface spread meiotic nuclei (1). These techniques require either fresh material (electron microscopy techniques) or a sufficient number of cells (whole mount spreading), and are not adapted in cases where the testicular material comes from patients with very poor spermatogenesis. Recently, cytocentrifugations of metaphasic somatic chromosomes have been shown to produce metaphase spreads without methanol/acetic acid fixative (5). We applied the cytocentrifugation technique to meiotic cells and obtained well-spread spermatocytes. This spreading technique, applied to germ cells, is an excellent research tool for the study of meiotic abnormalities in infertile patients. Cytocentrifugation is a very simple way to spread testic-
Cytocentrifugation method for meiotic studies
Vol. 74, No. 5, November 2000
ular cells. As with the standard spreading technique, wellspread spermatocytes can be obtained without impairment of the axial core and synaptonemal complex. Immunocytochemical and in situ hybridization techniques can be easily performed on these preparations, leading to a high-resolution analysis of the meiotic pairing process and genetic recombination (our unpublished results). A small quantity of antibodies is needed on each spot, and microscope analysis can be performed rapidly as the cells are concentrated on a small surface. If the number of germ cells is very low, the quantity of spermatocytes present on one slide is not sufficient to allow evaluation of the patient’s meiosis. Further slides must then be done to analyze the largest possible number of cells. Indeed, it is not recommended to concentrate the initial solution, because of the presence of many cell fragments and somatic cells that may disrupt the quality of the spread. Unlike the conventional air-drying and spreading techniques, this new meiotic technique avoids the loss of numerous germ cells. This is particularly important where meiotic studies have to be conducted on patients with severe spermatogenic defects. Thus, the cytocentrifugation technique can be performed on each testicular cell suspension obtained after biopsy delaceration, whatever method is used for the biopsy. One can avoid taking a testis fragment only for meiotic studies, which is an enormous advantage in cases of testicular hypotrophy and reduced spermatogenesis. The only limiting factor in this technique is the absence of immature germ cells. Another advantage of this technique is that, as the cell suspension is frozen and stored at ⫺196°C, cells can be stored in liquid nitrogen for transport and later analysis. This allows us to perform meiotic studies on testicular cells retrieved far from our laboratory.
FERTILITY & STERILITY威
We would like to emphasize that this technique is applicable in extreme cases of spermatogenesis impairment. We agree with Vendrell et al. (1), who have proposed that meiotic studies must be done when a testicular biopsy is performed on an infertile patient, because the frequency of meiotic abnormalities in these patients is probably high.
Acknowledgments: The authors thank P. Moens, Ph.D., and B. Spyropoulos, Ph.D. (York University, Canada) for the generous gift of the antibodies; J. M. Grillo, Ph.D., for providing us with the testicular cell suspensions; D. Daı¨oglou for her technical assistance; J. Sampol, Ph.D. and R. Pistoresi for the confocal analysis; M. San Marco, Ph.D., for the gift of the antikinetochore antibodies; and Dr. F. Romain for the statistical analysis (Hoˆpital de la Conception, Marseilles, France). We also are grateful to Dr. M. G. Mattei (Ph.D., Inserm U491, Marseilles, France) for her precious advice.
References 1. Vendrell JM, Garcia F, Veiga A, Calderon G, Egozcue S, Egozcue J, Barri PN. Meiotic abnormalities and spermatogenic parameters in severe oligoasthenozoospermia. Hum Reprod 1999;14:375– 8. 2. Lange R, Krause W, Engel W. Analyses of meiotic chromosomes in testicular biopsies of infertile patients. Hum Reprod 1997;12:2154 – 8. 3. Moens PB, Pearlman RE, Heng HHQ, Traut W. Chromosome cores and chromatin at meiotic prophase. In: Handel MA, ed. Current topics in developmental biology 37: meiosis and gametogenesis. Orlando, FL: Academic Press, 1998: 241– 62. 4. Guichaoua MR, de Lanversin A, Cataldo C, Delafontaine D, Alasia C, Fraterno M, Terriou P, Stahl A, Luciani JM. Three dimensional reconstruction of human pachytene spermatocyte nuclei of a 17;21 reciprocal translocation carrier: study of XY-autosome relationships. Hum Genet 1991;87:709 –15. 5. Jeppesen P, Turner M. The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell 1993;74:281–9.
919