1 Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843, 2 Stower's Institute for Medical Research, Kansas City, Missouri 64110 and 3 The Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822, USA.
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Abstract |
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Key words: spermatogenic stage/live germ cell/human spermatogenesis/ICSI/ROSI
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Introduction |
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Following the encouraging work on animal models such as hamster (Ogura and Yanagimachi, 1993), mouse (Ogura et al., 1993
,1994
; Kimura and Yanagimachi, 1995
), rabbit (Sofikitis et al., 1994
), and experimentation in the human (Sousa et al., 1996
), conception by ROSI was proposed for the treatment of sterility in humans (Edwards et al., 1994
). The first human spermatid-derived pregnancies have been achieved by ROSNI (Hannay, 1995
), and the first births have resulted from ELSI (Fishel et al., 1995
) and ROSI (Tesarik et al., 1995
). These works were followed by a large number of reports on ELSI and ROSI treatment cycles (Aslam et al., 1998a, review). However, the results obtained after ROSI remain disappointingly low. The birth of only a few babies has been reported after ROSI (Fishel et al., 1995
,1996
; Tesarik et al., 1995
,1996
; Vanderzwalmen et al., 1997
; Gianaroli et al., 1999
). An important known problem in the use of ROSI is the difficulty in identifying round spermatids within the heterogeneous population of testicular cells in a wet preparation (Vanderzwalmen et al., 1998a
,b
; Verheyen et al., 1998
).
The lack of accurate selection methods may partly explain the low success rate of ROSI (Yamanaka et al., 1997; Silber and Johnson, 1998
; Vanderzwalmen et al., 1998a
,b
; Verheyen et al., 1998
). Notwithstanding, current protocols for correct live selection of round spermatids have been presented (Sousa et al.1998
,1999
; Cremades et al., 1999
), and the low clinical outcome of ROSI has been attributed to other causes, such as genetic anomalies or epigenetic disorders of male germ cells, capability of round spermatids to activate oocytes, and quality of oocytes (Sousa et al., 1998
; Tesarik et al., 1998
).
To improve the identification of various types of live, dispersed, human testicular cells in vitro and place them in the context of their developmental steps at corresponding stages of the spermatogenic cycle, this comparative morphologic study used both live in-vitro cells and fixed cells within the context of the embedded testicular tubules. Our laboratory has used this approach to characterize identifying characteristics of spermatogonia, spermatocytes and spermatids and it was found that the size of nuclei were comparable whether fixed or live germ cells (Johnson et al., 1999).
This study places the live germ cells in the context of specific stages of the human spermatogenic cycle that is comparable with the conventional description (Clermont, 1963; Holstein and Roosen-Runge, 1981
) of human germ cells in each spermatogenic stage. This more detailed description of live cells, of small changes in spermatocytes and spermatids as they progress through subsequent stages of the cycle, will facilitate a more accurate comparison of ROSI results of different laboratories by this detailed description of the spermatids that are injected into unfertilized oocytes. Also the stage specific identity of germ cells will facilitate evaluation of treatment effects or toxicant effects that might influence specific steps of germ cell development in given stages of the cycle. Isolation of a group of germ cells at specific steps of development, identified by this detailed comparison, will facilitate molecular biological studies as different kinds of spermatids might differ in specific types of gene expression.
The live testicular cells were obtained from a brain-dead human male who was an organ donor. The cells were dispersed enzymatically and incubated in vitro (Johnson et al., 1999). The fixed tissue was originally obtained from 10 control men, representing varied spermatogenic potential (Johnson et al., 1981
). The higher production levels represented the live cell donor, and the lower ones represented that of typical clinical patients. It was found that distinct morphologic characteristics of germ cells at different developmental steps in the spermatogenic cycle could be distinguished in live human testicular cells in vitro viewed with Nomarski optics. Hence, this generated a checklist of distinguishing characteristics to allow identification of various types of human germ cells in situ for scientific investigation at various developmental steps throughout spermatogenesis and specific cells in defined stages of the human spermatogenic cycle.
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Materials and methods |
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Live, dispersed human germ cells were obtained from an individual in his early twenties listed as an organ donor and diagnosed as brain-dead after a car accident (Johnson et al., 1999). Fragments of testicular tissue were removed and subjected to enzymatic digestion according to a modified method (Bellve et al., 1977
). Testicular parenchyma fragments where placed in HEPES-TC199 medium containing 1.0 mg/ml collagenase for 15 min at 32°C in a shaking water bath. Tubules were separated from the dispersed interstitial cells by unit gravity sedimentation for 34 min, and the supernatant was decanted. This step was repeated 3 times. The tubular fragments were then placed in HEPES-TC199 medium with 0.5 mg/ml trypsin and 1 µg/ml DNase-I for 15 min at 32°C. The tubular fragments were pipetted vigorously to separate spermatogenic cells and were washed in HEPES-TC199 medium with 0.5 mg/ml BSA. The resulting chunks of cells were filtered through a 40 µm mesh wire screen. The resulting cell suspension was washed by centrifugation for 5 min at 400 g in a large volume of HEPES-TC199 medium. The suspended cells were examined unstained on a glass slide using Nomarski's differential-interference optics.
Morphological characteristics of live, dispersed testicular cells as described in our previous paper (Johnson et al., 1999) of this individual were compared with those of previously identified cell types (Clermont, 1963
; Heller and Clermont, 1964
; Johnson et al., 1992
) in fixed, embedded, and thick-sectioned seminiferous tubules in various developmental steps in the six stages of the spermatogenic cycle for the 10 control men.
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Results |
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Stage II of the cycle begins with the appearance of the acrosomic vesicle flattened over the nuclei of young Sa spermatids (Figures 1u and 2u). The flagellum has developed sufficiently to be seen extending beyond the cell diameter and can sometimes be observed (not shown). Before being released from the seminiferous epithelium, Sd2 spermatids have small spear-shaped heads and enlarged middle pieces with mitochondria in place around the tail. A cytoplasmic droplet is often present, located at the anterior end of the middle piece, in the proximal position of the tail (Figures 1aa and 2aa
). The spermatocytes associated with these two generations of spermatids are in the pachytene step of meiosis. These cells show slow, progressive growth (Figures 1n and 2n
). Type A and type B spermatogonia are also present at this stage (Figures 1b, 2b, and 1h, 2h
, respectively).
Stage III of the cycle includes only one generation of spermatids, the older ones having spermiated from the seminiferous epithelium as spermatozoa. The remaining generation of spermatids is composed of Sb1 (cap phase) spermatids. These cells still have a spherical nucleus, but the acrosome has developed a cap over the nucleus (Figures 1v and 2v). Two generations of spermatocytes are found. One is obviously at the midpachytene step of the meiotic prophase (Figures 1o and 2o
) while the second is a new generation of so-called resting primary spermatocytes resulting from the division of type B spermatogonia. The nuclear envelope of these resting spermatocytes is not well defined and chromatin flakes are fine. Their nuclear size is slightly larger than that of type B spermatogonia (Figures 1i and 2i
). Type A spermatogonia are also present at this stage (Figures 1c and 2c
).
Stage IV of the cycle is characterized by Sb2 spermatids which have nuclei showing initial signs of elongation and the presence of the manchette. The acrosomal cap is a predominant feature attached to the nucleus opposite to that of the attached flagellum (Figures 1w and 2w). Whereas the older generation of spermatocytes is always at the midpachytene step of meiosis (Figures 1p and 2p
), the younger generation of spermatocytes is just entering meiotic prophase. The nucleus of these leptotene spermatocytes is finely granular with evenly distributed areas of chromatin condensation (Figures 1j and 2j
). Type A spermatogonia are also present (Figures 1d and 2d
).
Stage V of the cycle is characterized by further elongation and condensation of the spermatid nuclei to form Sc (acrosome phase) spermatids. The annulus is distinct in its location in the proximal position of the developing tail near the nucleus. Mitochondria have not yet wrapped around the middle piece of the flagellum (Figures 1x and 2x). The older generation of primary spermatocytes has entered the late pachytene step of meiotic prophase. These cells have the largest spherical nuclei of all germ cells, well-defined nuclear envelopes, and one or more large nucleoli (Figures 1q and 2q
). The younger generation of spermatocytes is still at the leptotene step of meiosis (Figures 1k and 2k
). Type A spermatogonia are also present at this stage (Figures 1e and 2e
).
Stage VI of the cycle has diplotene primary spermatocytes (not shown), resulting from the differentiation of late pachytene spermatocytes, that divide to produce secondary spermatocytes. These are slightly larger than Sa spermatids, but much smaller than primary spermatocytes. The interphasic nuclei of secondary spermatocytes show a homogeneous, finely granular chromatin and usually several nucleoli (Figures 1r and 2r). Meiotic telophase figures also characterize this stage as secondary spermatocytes divide to produce spermatids (not shown). Newly formed spermatids have the smallest spherical nuclei of all germ cells (Figures 1s and 2s
). The maturing Sc spermatids show a shorter tapered nuclear profile and distinct tail formation (Figures 1y and 2y
). The younger generation of primary spermatocytes has just entered the zygotene step of meiotic prophase. These cells have larger nuclei, larger evenly dispersed chromatin clumps, and well-defined nuclear envelopes in comparison to leptotene primary spermatocytes (Figures 1l and 2l
). Type A spermatogonia are also seen at this stage (Figures 1f and 2f
).
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Discussion |
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There has been a great deal of effort towards improving the recognition of spermatids within a heterogeneous population of testicular cells in a wet preparation. Tesarik and Mendoza described how to recognize a round spermatid under an inverted microscope with Hoffman modulation (Tesarik and Mendoza, 1996). However, Hoffman modulation contrast optics may not allow reliable identification of the round spermatid (Vanderzwalmen et al., 1997
; Verheyen et al., 1998
). Even the difference between the acrosomal vesicle and a vacuole often seems unclear (Tesarik and Mendoza, 1996
). Several other techniques have been investigated to identify round spermatids, such as immunocytochemistry methods (Mendoza and Tesarik, 1996
), vital staining coupled with morphological features and fluorescence in-situ hybridization (Angelopoulos et al., 1997
), confocal scanning laser microscopy and computer-assisted image analysis (Yamanaka et al., 1997
), fluorescent-activated cell sorting (FACS) (Aslam et al., 1998b
), and epifluorescence microscopy after incubation of dispersed male germ cells with a vital mitochondrion-specific fluorescent probe (Sutovsky et al., 1999
). All these studies provide good methods for round spermatid identification and evaluation, but they are often expensive and cannot be introduced in every laboratory. Moreover, the question should be asked whether these cells remain suitable for therapeutic ICSI after these treatments. As pointed out by Tesarik, it is urgent to define as exactly as possible the developmental stage of the spermatids used in each injection (Tesarik, 1997
). In fact, without a clear terminology for each stage of spermiogenesis, it is difficult to compare the results of the different laboratories as they could be injecting spermatids representing different stages of the cycle. More recently, however, criteria for the correct diagnosis and selection of live round spermatids (Sousa et al., 1998
; Cremades et al., 1999
) and the correct recognition and classification of spermatids along the different spermiogenic steps (Sousa et al., 1999
) have been published.
We offer here a simple, rapid, objective and reliable way to identify male germ cells at each stage of their differentiation from spermatogonia to mature elongated spermatids using Normarski optics. In fact, Normarski differential interference contrast microscopy allows identification of cellular morphology inside whole cells, as it uses a small depth of focus. This enables one to optically section through cells or tissues such as testicular specimens (Saacke and Marshall, 1968; Johnson et al., 1976
,1981
,1983
,1984a
, b
,1987
,1999
; Bellve et al., 1977
; Johnson and Neaves, 1981
; Neaves et al., 1984
; Sutovsky et al., 1999
).
In this current study, we provide, with the help of more recent tools, a detailed organized presentation of live human germ cells in the context of their developmental steps and stages of the spermatogenic cycle, as described (Clermont, 1963; Holstein and Roosen-Runge, 1981
), and convincingly considered. This comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing cytologic and morphologic characteristics of live human germ cells to be used by scientists interested in studying live cells at known steps in spermatogenic development characteristic of germ cells in specific stages of the spermatogenic cycle. The most important morphological characteristics noted in this comparative study were size; shape; chromatin pattern within nuclei; number and shape of nucleoli; definition of the nuclear membrane; presence of meiotic metaphase figures; the Golgi apparatus; the acrosomic vesicles or cap; presence of the tail; location of the annulus; and/or mitochondria around the middle piece of the tail.
Previous published studies using spermatids for ROSI procedures have not defined the level of spermatogenic development of injected spermatids, because a systematic evaluation of the conception potential of spermatids in different developmental steps of spermiogenesis could not be made. The influence of the immaturity of the early round spermatids on the development rate of the embryos resulting from ROSI is important. The problem does not appear to be imprinting since it has been shown that expression of imprinted genes in mouse embryos derived by ROSI do not differ from normally-produced animals (Shamanski et al., 1999
). This indicates that paternal genes have undergone proper imprinting by the first step of round spermatid development. However, not all postmeiotic cells have the same chances of producing a high quality embryo (Sousa et al., 1999). A classification (Figure 2
, Table I
) is essential to know if the development rate of ROSI embryos correlates with the development step of the round spermatids injected.
Spermatogenesis is accomplished by an amazing array of gene regulation by hormonal stimulation and local controlling factors (Parvinen, 1982; Skinner, 1991
; Jegou, 1993
; Carreau et al., 1994
; Kierzenbaum, 1994; Lejeune et al., 1996
,1998
; Mauduit and Benahmed, 1996
). To study this complex process in humans, methods need to be developed to isolate and identify individual germ cells in each developmental step throughout spermatogenesis including spermatocytes and spermatogonia. To this end, this study allows an easy identification of human germ cells in the context of their developmental steps and stages of the spermatogenic cycle. This could be useful to study stage specific events of human spermatogenesis. Moreover, after isolating germ cells at the same stage, it allows the study of the expression of genes of germ cells at specific stages. It makes it possible to determine germ cell specific gene expression patterns for each germ cell at any step of its differentiation.
In conclusion, comparison of live and fixed human germ cells in the context of development step and stage of the spermatogenic cycle provides a detailed description of spermatogonia, spermatocytes and spermatids as they progress through subsequent stages. This description facilitates a more precise comparison of ROSI results among laboratories; facilitates molecular biological studies using a few germ cells identified to be at steps in a specific stage; and facilitates evaluation of treatment effects and toxicant effects on specific development steps of live human germ cells in specific stages of the spermatogenic cycle.
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Acknowledgements |
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Notes |
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References |
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Antinori, S., Versaci, C., Dani, G. et al. (1997a) Successful fertilization and pregnancy after injection of frozen-thawed round spermatids into human oocytes. Hum. Reprod., 12, 554556.[ISI][Medline]
Antinori, S., Versaci, C., Dani, G. et al. (1997b) Fertilization with human testicular spermatids: four successful pregnancies. Hum. Reprod., 12, 286291.[Abstract]
Aslam, I., Fishel, S., Green, S. et al. (1998a) Can we justify spermatid microinjection for severe male factor infertility? Hum. Reprod. Update, 4, 213222.
Aslam, I., Robins, A., Dowell, K. et al. (1998b) Isolation, purification and assessment of viability of spermatogenic cells from testicular biopsies of azoospermic men. Hum. Reprod., 13, 639645.[Abstract]
Balaban, B., Urman, B., Isiklar, A. et al. (2000) Progression to the blastocyst stage of embryos derived from testicular round spermatids. Hum. Reprod., 15, 13771382.
Bellve, A.R., Cavicchia, J.C., Millette, C.F. et al. (1977) Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. J. Cell. Biol., 74, 6885.
Carreau, S., Foucault, P. and Drosdowsky, M.A. (1994) La cellule de Sertoli : Aspects fonctionnels comparés chez le rat, le porc et l'homme. Annales d'Endocrinologie, 55, 203220.
Clermont, Y. (1963) The cycle of the seminiferous epithelium in man. Am. J. Anat., 112, 3551.[ISI]
Cremades, N., Bernabeu, R., Barros, A. et al. (1999) In-vitro maturation of round spermatids using co-culture on Vero cells. Hum. Reprod., 13, 12871293.
Devroey, P., Liu, J., Nagy, Z. et al. (1994) Normal fertization of human oocytes after testicular sperm extraction and intracytoplasmic sperm injection. Fertil. Steril., 62, 639641.[ISI][Medline]
Devroey, P., Liu, J., Nagy, Z. et al. (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum. Reprod., 10, 14571460.[Abstract]
Edwards, R.G., Tarin, J.J., Hirsch, A. et al. (1994) Are spermatid injections into human oocytes now mandatory? Hum. Reprod., 9, 22172219.[ISI][Medline]
Fishel, S., Aslam, I., and Tesarik, J. (1996) Spermatid conception: a stage too early, or a time too soon? Hum. Reprod., 11, 13711375.
Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic injection of spermatid. Lancet, 345, 16411642.
Gianaroli, L., Selman, H.A., Magli, M.C. et al. (1999) Birth of a healthy infant after conception with round spermatids isolated from cryopreserved testicular tissue. Fertil. Steril., 72, 539541.[ISI][Medline]
Hannay, T. (1995) New Japanese IVF method finally made available in Japan. Nature Med., 1, 289290.[ISI]
Heller, G.C. and Clermont, Y. (1964) Kinetics of the germinal epithelium in man. Recent. Prog. Horm. Res., 20, 545575.[ISI]
Holstein, A.F. and Roosen-Runge, E.C. (1981) Atlas of human spermatogenesis. Grosse Verlag, Berlin, 224 pp.
Jégou, B. (1993) The Sertoli-germ cell communication network in mammals. Int. Rev. Cytol., 147, 2596.[ISI][Medline]
Johnson, L. (1995) Efficiency of spermatogenesis. Microsc. Res. Tech., 32, 385422.[ISI][Medline]
Johnson, L. and Neaves, W.B. (1981) Age-related changes in the Leydig cell population, seminiferous tubules, and sperm production in stallions. Biol. Reprod., 24, 703712.[ISI][Medline]
Johnson, L., Berndtson, W.E. and Pickett, B.W. (1976) An improved method for evaluating acrosomes of bovine spermatozoa. J. Anim. Sci., 42, 951954.[ISI][Medline]
Johnson, L., Petty, C.S. and Neaves, W.B. (1981) A new approach to quantification of spermatogenesis and its application to germinal cell attrition during Human spermiogenesis. Biol. Reprod., 25, 217226.[ISI][Medline]
Johnson, L., Petty, C.S. and Neaves, W.B. (1983) Further quantification of human spermatogenesis: germ cell loss during postprophase of meiosis and its relationship to daily sperm production. Biol. Reprod., 29, 207215.[Abstract]
Johnson, L., Chaturvedi, P.K. and Williams, J.D. (1992) Missing generations of spermatocytes and spermatids in seminiferous epithelium contribute to low efficiency of spermatogenesis in humans. Biol. Reprod., 47, 10911098.[Abstract]
Johnson, L., Petty, C.S., Porter, J.C. et al. (1984a) Germ cell degeneration during postprophase of meiosis and serum concentrations of gonadotropins in young adult and older adult men. Biol. Reprod., 31, 779784.[Abstract]
Johnson, L., Zane, R.S., Petty, C.S. et al. (1984b) Quantification of the human Sertoli cell population: its distribution, relation to germ cell numbers, and age-related decline. Biol. Reprod., 31, 785795.[Abstract]
Johnson, L., Nguyen, H.B., Petty, C.S. et al. (1987) Quantification of human spermatogenesis: germ cell degeneration during spermatocytogenesis and meiosis in testes from younger and older adult men. Biol. Reprod., 37, 739747.[Abstract]
Johnson, L., Neaves, W.B., Barnard, J.J. et al. (1999) A comparative morphological study of human germ cells in vitro or in situ within seminiferous tubules. Biol. Reprod., 61, 927934.
Kahraman, S., Ozgur, S., Alatas, C. et al. (1996) High implantation and pregnancy rates with testicular sperm extraction and intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia. Hum. Reprod., 11, 673676.[Abstract]
Kierszenbaum, A.L. (1994) Mammalian spermatogenesis in vivo and in vivo: a partnership of spermatogenic and somatic cell lineages. Endocrine Rev., 15, 116134.[ISI][Medline]
Kimura, Y. and Yanagimachi, R. (1995) Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development, 121, 23972405.
Lejeune, H., Habert, R. and Saez, J.M. (1998) Origin, proliferation and differentiation of Leydig cells. J. Molec. Endocrinology, 20, 125.
Lejeune, H., Jégou, B., Carreau, S. et al. (1996) Régulation paracrine et autocrine des fonctions testiculaires. In Drosdowsky,M.A., Belaisch,J. and Vermeulen,A. (eds), Endocrinologie masculine. Doin Editeurs, Paris, pp. 75101.
Levran, D., Nahum, H., Farhi, J., et al. (2000) Poor outcome with round spermatid injection in azoospermic patients with maturation arrest. Fertil. Steril., 74, 443449.[ISI][Medline]
Mauduit, C. and Benahmed, M. (1996) Growth factors in the testis development and function. In Hamamah, S. and Mieusset, R. (eds) Male gametes production and quality. Les Editions INSERM, Paris, France, pp. 345.
Mendoza, C. and Tesarik, J. (1996) The occurrence and identification of round spermatids in the ejaculate of men with nonobstructive azoospermia. Fertil. Steril., 66, 826829.[ISI][Medline]
Nagy, Z., Liu, J., Cecile, J. et al. (1995) Using ejaculated, fresh, and frozen-thawed epididymal and testicular spermatozoa gives rise to comparable results after intracytoplasmic sperm injection. Fertil. Steril., 63, 808815.[ISI][Medline]
Neaves, W.B., Johnson, L., Porter, J.C. et al. (1984) Leydig cell numbers, daily sperm production, and serum gonadotropin levels in aging men. J. Clin. Endocrinol. Metab., 59, 756763.[Abstract]
Ogura, A. and Yanagimachi, R. (1993) Round spermatid nuclei injected into hamster oocytes form pronuclei and participate in syngamy. Biol. Reprod., 48, 219225.[Abstract]
Ogura, A., Yanagimachi, R. and Usui, N. (1993) Behaviour of hamster and mouse round spermatid nuclei incorporated into mature oocytes by electrofusion. Zygote, 1, 18.[Medline]
Ogura, A., Matsuda, J. and Yanagimachi, R. (1994) Birth of normal young after electrofusion of mouse oocytes with round spermatids. Proc. Natl Acad. Sci. USA, 91, 74607462.[Abstract]
Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, 1718.[ISI][Medline]
Parvinen, M. (1982) Regulation of the seminiferous epithelium. Endocrine Rev., 3, 404417.[ISI][Medline]
Saacke, R.G. and Marshall, C.E. (1968) Observations on the acrosomal cap of fixed and unfixed bovine spermatozoa. J. Reprod. Fertil., 16, 511514.[Medline]
Schamanski, F.L., Kimura, Y., Lavoir, M.C. et al. (1999) Status of genomic imprinting in mouse spermatids. Hum. Reprod., 14, 10501056.
Schoysman, R., Vanderzwalmen, P., Nijs, M. et al. (1993) Pregnancy after fertilisation with human testicular spermatozoa. Lancet, 342, 1237.
Silber, S.J. and Johnson, L. (1998) Are spermatid injections of any clinical value? ROSNI and ROSI revisited. Hum. Reprod., 13, 509515.
Silber, S.J., Van Steirteghem, A.C., Liu, J. et al. (1995) High fertilization and pregnancy rate after intracytoplasmic sperm injection with spermatozoa obtained from testicle biopsy. Hum. Reprod., 10, 148152.[Abstract]
Silber, S.J., Johnson, L., Verheyen, G. et al. (2000) Round spermatid injection. Fertil. Steril., 73, 897900.[ISI][Medline]
Skinner, M.K. (1991) Cell-cell interactions in the testis. Endocrine Rev., 12, 4577.[ISI][Medline]
Sofikitis, N.V., Miyagawa, I., Agapitos, E. et al. (1994) Reproductive capacity of the nucleus of male gamete after completion of meiosis. J. Assist. Reprod. Genet., 11, 335341.[ISI][Medline]
Sousa, M., Barros, A. and Tesarik, J. (1998) Current problems with spermatid conception. Hum. Reprod., 13, 255258.
Sousa, M., Mendoza, C., Barros, A. et al. (1996) Calcium responses of human oocytes after intracytoplasmic injection of leucocytes, spermatocytes and round spermatids. Mol. Hum. Reprod., 2, 853857.[Abstract]
Sousa, M., Barros, A., Takahashi, K. et al. (1999) Clinical efficacy of spermatid conception: analysis using a new spermati classification scheme. Hum. Reprod., 14, 12791286.
Sutovsky, P., Ramalho-Santos, J., Moreno, R.D. et al. (1999) On-stage selection of single round spermatids using a vital, mitochondrion-specific fluorescent probe MitoTrackerTM and high resolution differential interference contrast microscopy. Hum. Reprod., 14, 23012312.
Tesarik, J. (1997) Sperm or spermatid conception? Fertil. Steril., 68, 214216.[ISI][Medline]
Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes. I. Laboratory techniques and special features of zygote development. Hum. Reprod., 11, 772779.[Abstract]
Tesarik, J., Mendoza, C. and Testart, J. (1995) Viable embryos from injection of round spermatids into oocytes. N. Engl. J. Med., 333, 525.
Tesarik, J., Rolet, F., Brami, C. et al. (1996) Spermatid injection into human oocytes. II. Clinical application in the treatment of infertility due to non-obstructive azoospermia. Hum. Reprod., 11, 780783.[Abstract]
Tesarik, J., Sousa, M., Greco, E. et al. (1998) Spermatids as gametes: indications and limitations. Hum. Reprod., 13 (Suppl. 3) , 89111.[Medline]
Van Steirteghem, A.C., Liu, J., Joris, H. et al. (1993a) Higher success rate by intracytoplasmic sperm injection than subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum. Reprod., 8, 10551060.[Abstract]
Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993b) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 10611066.[Abstract]
Vanderzwalmen, P., Zech, H., Birkenfeld, A. et al. (1997) Intracytoplasmic injection of spermatids retrieved from testicular tissue: influence of testicular pathology, type of selected spermatids and oocyte activation. Hum. Reprod., 12, 12031213.[ISI][Medline]
Vanderzwalmen, P., Nijs, M., Stecher, A. et al. (1998a) Is there a future for spermatid injections? Hum. Reprod., 13 (Suppl. 4) , 7184.[Abstract]
Vanderzwalmen, P., Nijs, M., Schoysman, R. et al. (1998b) The problems of spermatid microinjection in the human: the need for accurate morphological approach and selective methods for viable and normal cells. Hum. Reprod., 13, 515519.[ISI][Medline]
Verheyen, G., Crabbe, E., Joris, H., et al. (1998) Simple and reliable identificaion of the human round spermatid by inverted phase-contrast microscopy. Hum. Reprod., 13, 15701577.[Abstract]
Yamanaka, K., Sofikitis, N.V., Miyagawa, I. et al. (1997) Ooplasmic round spermatid nuclear injection procedures as an experimental treatment for nonobstructive azoospermia. J. Assist. Reprod. Genet., 14, 5562.[ISI][Medline]
Submitted on November 17, 2000; accepted on April 25, 2001.