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Biology of Reproduction 61, 927-934 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

A Comparative Morphological Study of Human Germ Cells In Vitro or In Situ Within Seminiferous Tubules1

Larry Johnson2,a, William B. Neavesb, Jeffrey J. Barnardc, Genevieve E. Keillora, Steven W. Browna, and Ryuzo Yanagimachid

a Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843 b Department of Cell Biology and Neuroscience and Department of Pathology, c University of Texas Southwestern Medical School, Dallas, Texas 75235 d Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, Hawaii 96822


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For many infertile couples, intracytoplasmic germ cell/spermatozoon injection into unfertilized eggs may be their only hope for producing their own biological children. Thus far, success with injection of pre-spermatozoan germ cells such as round spermatids has not been as great as that of spermatozoon injection. This could be due in part to the difficulty of identifying younger (less mature) male germ cells in testicular biopsy dispersions. To improve the identification of various types of live, dispersed, human testicular cells in vitro, a comparative study of the morphological characteristics of human spermatogenic germ cells in vitro or in situ within seminiferous tubules was conducted. Live human testicular tissue was obtained from an organ-donating, brain-dead person with a high density of various germ cells. A cell suspension was obtained by enzymatic digestion, and cells were cultured for 3 days in an excessive volume (100-fold medium:cells; v:v) of HEPES-TC 199 medium at 5°C and observed live with Nomarski optics (interference-contrast microscopy). For comparative purposes, testes from ten men obtained at autopsy were fixed, embedded in epoxy resin, sectioned at 20 µm, and observed unstained by Nomarski optics. This approach allowed comparison of morphological characteristics of individual germ cells seen in vitro or in situ in the human testis. In both live and fixed preparations from control men with varied daily sperm production rates, Sertoli cells have oval to pear-shaped nuclei with indented nuclear envelopes and large nucleoli, which makes their appearance distinctly different from germ cells. The size, shape, and chromatin pattern of nuclei, and the presence of meiotic metaphase figures, acrosomic vesicles/structures, tails, and/or mitochondria in the middle piece of germ cells are characteristically seen in live cells in vitro and in those cells observed in the fixed seminiferous tubules. Hence, this comparative approach allows verification of the identity of individual germ cells seen in vitro and provides a checklist of distinguishing characteristics of live human germ cells, to be used by scientists and technical staff in infertility clinics when selecting specific germ cells from a testicular aspirate or enzymatically digested biopsy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Using ``testicular sperm'' (mature spermatids) as the source of the male genome injected, intracytoplasmic spermatozoon injection (ICSI) successfully treats some forms of male infertility associated with the absence of spermatozoa or the failure of spermatozoa in the ejaculate or epididymis to fertilize oocytes by conventional in vitro fertilization techniques [1–6]. However, in patients with tubular sclerosis, maturation arrest, or Sertoli cell-only syndrome, mature spermatids/testicular spermatozoa can be recovered in only 50% of the cases [7]. On the basis of encouraging work on animal models [8–10], younger germ cells (round spermatid or spermatocytes) could be a possible source of male genome nuclear material for injection (ROSI, round spermatid injection; SCI, spermatocyte injection). In spite of low success rates, electrofusion of round spermatids into mouse oocytes [11] or ROSI with round spermatids in mice [12] and rabbits [13] produced normal embryonic development and healthy offspring.

Success has been reported with ROSI in humans [14–19]; however, fertilization and pregnancy rates following ROSI procedures have been disappointingly low as compared to ICSI using mature spermatozoa or elongated spermatids [19, 20]. Because published studies using spermatids have not defined the level of spermatogenic development of the injected spermatids, an unequivocal and systematic evaluation of the conception potential of spermatids in different developmental steps of spermiogenesis could not be made [21]. Two human pregnancies have been reported that resulted from assisted fertilization using secondary spermatocytes [22] and in vitro-matured spermatogenic cells [23]. However, no details of identifying characteristics of secondary spermatocytes were given, and the in vitro maturation reported was far more rapid than expected from in vivo timing of spermatogenesis.

An important known problem in the use of ROSI is the ability to identify round spermatids within the heterogeneous population of round cells produced by enzymatic dispersion of testicular tissue [24, 25]. Characteristics of elongated spermatids are relatively apparent in dispersed human testicular cells; however, identifying round spermatids (Sa and Sb1) has been very difficult [19, 24, 25]. The mistaken selection of round testicular cells other than spermatids may partly explain the low success rate of ROSI [24, 26].

Verheyen et al. [24] used phase-contrast microscopy to select Sa (without or with acrosomic structures) and Sb1 spermatids, and they used fluorescent in situ hybridization to confirm the numerical chromosomal constitution of select cells. This approach provided a reliable selection of round spermatids of types Sa and Sb1 and the possibility of injecting the identified germ cell into the oocyte cytoplasm. The difficulty in identifying round spermatids in dispersed human testicular cell preparations using phase-contrast or the Hoffman modulation contrast system led to the conclusion that more effort should be given to search for testicular spermatozoa or elongated spermatids rather than trying to locate round spermatids [24, 26]. However, there is merit in obtaining the capability to identify all types of spermatids and younger germ cells within dispersed human testicular tissue for future use as reproduction technology develops and as clinical application follows these developments.

To improve the identification of various types of live, dispersed, human testicular cells in vitro, this comparative study of the morphological characteristics of human live spermatogenic germ cells in vitro (from one individual) and in situ within seminiferous tubules of fixed and embedded tissue (from ten controls) was conducted. In this way, morphological characteristics of live cells seen in cell suspensions could be compared with those of fixed cells of known identity based on their context (location within seminiferous tubules and stage of the spermatogenic cycle). The controls represented varied spermatogenic potential, with higher production levels similar to the live-cell donor and the lower ones similar to that of typical clinical patients. It was found that distinct characteristics of various types of germ cells could be observed in live human testicular cells in vitro, viewed by Nomarski optics, and that a checklist of distinguishing characteristics could be made against Sertoli cells and various types of human germ cells in situ.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In a previously published study [27], testes were obtained at autopsy from 10 men between 26 and 53 years of age; these were the source of unstained germ cells in the cortex of the stage of the spermatogenic cycle in fixed tissue. Tissues were obtained and prepared as described previously for observation with Nomarski optics [27–29]. Daily sperm production per gram of parenchyma (DSP/g) was determined for these men on the basis of the number of round spermatids [27].

Sizes of nuclei of spermatogonia, early primary spermatocytes, late primary spermatocytes, secondary spermatocytes, and round spermatids as well as Sertoli cells and Leydig cells were determined by direct measure of nuclei of each type of germ cell, which were embedded in 20-µm epoxy resin sections and viewed unstained with Nomarski optics in a previously published study on these ten control men [27] or studies on similarly aged men [30–32]. These data are tabulated here (Table 1) and compared to measures of pachytene primary spermatocytes or Sa spermatids made on fixed, embedded, 20-µm sections by Nomarski optics from the live-cell donor. The identity of various germ cell types in these sections was based on their location in the seminiferous epithelium and in various stages of the spermatogenic cycle as described previously [33, 34].


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  		TABLE 1. Diameters and/or volumes of nuclei or nucleoli of human germ cells, Sertoli cells, and Leydig cells as observed and measured under Nomarski optics

Live dispersed human germ cells and Sertoli cells were obtained from an organ donor with good spermatogenesis (high density of spermatogonia, spermatocytes, and spermatids). He was in his early 20s, diagnosed as brain dead after a car accident. Fragments of testicular tissue were removed and subjected to enzymatic digestion according to a modified method of Bellve et al. [35]. Testicular parenchyma fragments were placed in HEPES-TC 199 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 3–4 min, and the supernatant was decanted. The wash by unit gravity sedimentation was repeated 3 times. The tubular fragments were placed in HEPES-TC 199 medium with 0.5 mg/ml trypsin and 1 µg/ml DNase I (Sigma) for 15 min at 32°C. The tubular fragments were pipetted vigorously to separate spermatogenic cells and were washed in HEPES-TC 199 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 x g, resuspended and cultured in an excessive volume (100-fold medium:packed cells; v:v) of HEPES-TC 199 medium incubated in vitro on ice or at 5°C for 3 days, and examined unstained on a glass slide with Nomarski differential-interference optics. Morphological characteristics of live, dispersed testicular cells of this individual were compared with the cell types identified [33, 36] in the fixed, embedded, and thick-sectioned seminiferous tubules at various stages of the spermatogenic cycle for the 10 control men.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The isolation of seminiferous tubules by unit gravity sedimentation from fragments of human testicular tissue yielded cell preparations that were largely Sertoli cells and spermatogenic germ cells (Fig. 1). The interstitial cells and myoid cells were removed during isolation and were not a major contaminant of these enriched germ cells. The live, dispersed human cells of the seminiferous epithelium retained morphological characteristics in vitro that allowed direct comparison with cells of similar characteristics within the context of fixed seminiferous tubules during spermatogenic development (Figs. 1–3). It is not clear whether the in vitro maturation occurred during the 3-day incubation at 5°C in HEPES-TC 199 or whether the maturational steps of these were present at the time of germ cell harvest. Germ cells of all developmental steps were present after the 3-day incubation (Figs. 1–3). The size, shape, and chromatin pattern of nuclei; the presence of meiotic metaphase figures (chromosomes), acrosomic vesicles or cap, and tails (flagella); and/or the presence of mitochondria in the middle piece of the tail of germ cells were the main characteristics that were visible in live human germ cells. Nomarski optics allowed observation of identifying characteristics of live, testicular cells in suspension. Spermatogonia were identified on the basis of their nuclear size, very distinct nuclear envelope, and chromatin pattern (Fig. 2). Primary spermatocytes in vitro portrayed the typical differences in chromatin pattern and varying nuclear size during progression of meiosis (Fig. 2). Preleptotene primary spermatocytes had the smallest nuclei of primary spermatocytes, chromatin clumps were the finest, and the nuclear envelope was least defined. The zygotene step of meiosis was marked by slightly longer nuclei and an enlargement of the chromatin clumps. Pachytene primary spermatocytes were the largest of all germ cells in the testis and had one or more large spherical nucleoli. Meiotic metaphase figures from dividing primary or secondary spermatocytes could be identified by their figure size, lack of nuclear envelopes, and the clumping of chromosomes (Fig. 2). Secondary spermatocytes were rare but could be seen in the dispersed-cell suspension (Fig. 2). They have fine chromatin and an indistinct nuclear envelope, and were intermediate in nuclear size between primary spermatocytes and round spermatids.



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  		FIG. 1. Cells of the human seminiferous epithelium viewed under Nomarski optics in vitro (a, b) or in situ (c) within seminiferous tubules in stages I (I), V (V), or II (II). Enzymatically dispersed, live germ cells (a, b) represent a host of sizes from the smallest (round spermatid, Sa) to the largest (pachytene primary spermatocytes, PPS), and shapes from spherical nuclei (of spermatogonia, preleptotene [Pl] or pachytene [PPS], and round spermatids [Sa]) to meiotic figures (MF), elongating (Sc) or elongated (Sd2) spermatids, and testicular spermatozoa (TS). Live Sertoli cells (SC) have oval to pear-shaped nuclei and indented nuclear envelopes. In 20-µm epoxy resin sections (c), Sertoli cells (SC), spermatogonia (Sg), preleptotene primary spermatocytes (Pl), pachytene primary spermatocytes (PPS), round spermatids (Sa), elongating spermatids (Sc), elongated spermatids (Sd2), and testicular spermatozoa (TS) can be identified in the context of the seminiferous epithelium and the cellular association or stage of the spermatogenic cycle. Bar = 10 µm



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  		FIG. 2. Sertoli cells and germ cells of spermatocytogenesis and meiosis as viewed under Nomarski optics as dispersed live cells in vitro-cultured in HEPES-TC 199 for 3 days at 5°C (a–i) or within fixed seminiferous epithelium in 20-µm epoxy resin sections (j–q). Live Sertoli cells in suspension (a) are noted by their large size, elongated contour, well-defined and indented nuclear envelope (arrow), and distinctive nucleolus. The cytoplasm contained granules of various sizes. Similar nuclear features characterize fixed Sertoli cells in situ within tubules (j). The cytoplasm : nucleus ratio, nuclear size, and nuclear shape appear to be similar in germ cells in vitro (b–i) or in situ (k–q). Spermatogonia with well-defined nuclear envelopes and granular chromatin with one or more distinctive nucleoli characterize these cells in vitro (b) or in situ (k). Primary spermatocytes seen in vitro (c–e) or in situ (l–n) exhibit the typical nuclear expansion and enlargement of chromatin flakes. The nuclei remain spherical during development preceding meiosis. In preleptotene/leptotene primary spermatocytes (c and l), the nuclear envelope is not well defined, chromatin flakes are fine, and the nuclear size is slightly larger than that of spermatogonia. Often, the chromatin is slightly displaced (less dense) on one side (arrow) of the preleptotene/leptotene nucleus than the on other side. Zygotene primary spermatocytes (d, m) have larger nuclei, larger evenly dispersed chromatin clumps, and well-defined nuclear envelopes in comparison to leptotene primary spermatocytes. Pachytene primary spermatocytes (e, n) have the largest spherical nuclei of all germ cells, well-defined nuclear envelopes, and one or more very large spherical nucleoli. Large metaphase plates in cells undergoing meiosis in vitro (f ) are similar to both the large metaphase plates of the first meiotic division seen in situ (o). Secondary spermatocytes, both in vitro (g) and in situ (p), have spherical nuclei of a slightly smaller size and slightly finer chromatin flakes than seen in preleptotene primary spermatocytes. Chromatin clumps of secondary spermatocytes are not uniformly distributed next to the nuclear envelope. In vitro newly formed daughter cells (h, i) resulting from the second meiotic division still have the condensed chromatin (arrow) resulting from completion of cell division typical of cells forming from dividing secondary spermatocytes seen in situ (g). These cells (technically spermatids) are observed in vitro, but it is not known whether they divided in vitro per se. These cells may be slightly smaller than, or similar to, typical round spermatids (see Fig. 3). Bar = 10 µm

Live spermatids in suspension retained identifying characteristics typical of those seen in the seminiferous tubule (Fig. 3). Very few Sa spermatids were without some acrosomic structure, but others had a spherical or flattened acrosomic vesicle. With development, the chromatin pattern became finer, yet two or more nuclear clumps were distinct. The Sb1 spermatid had an apparent acrosomal cap and a nucleus that had begun to lose its spherical shape. The Sb2 spermatids in vitro had elongating nuclei, but the manchette was not as distinctly visible as that seen in fixed tissue (compare g–i to u–v of Fig. 3). However, the tail had begun developing and had an annulus located in its proximal position (near the head). The tail, located opposite the developing acrosome, was apparent in cultured cells. The elongating (Sc) spermatid had an annulus around its developing tail located in the proximal position (near its head) or distal position (at the bottom of the middle piece of the tail). The distal position is the location of the annulus in the released spermatozoon. The Sd1 spermatids had shortened nuclear head length and a migrated annulus of its developing tail. The elongated (Sd2) spermatids had mitochondria around its middle piece of the tail, and had a developing cytoplasmic droplet located in the proximal position (next to the head) of the tail. Some testicular spermatozoa had their tails coiled around their heads or coiled within their plasma membranes where other cytoplasmic components were located (see o, z of Fig. 3).



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  		FIG. 3. Spermatids and testicular spermatozoa as viewed under Nomarski optics as dispersed live cells in vitro (a–o) or fixed cells within the seminiferous epithelium of 20-µm epoxy resin sections (p–z). Newly formed nuclei of Sa spermatids are smaller with a more defined nuclear envelope than those of secondary spermatocytes (Fig. 2), but the chromatin flake size is similar in both live cells in vitro (a, b) and those in situ within the tubule (p). No acrosomic vesicle or acrosomic structures are present in these newest spermatids. The acrosomic vesicle (arrow) is distinctly visible (d, g). Profiles of the Golgi apparatus (arrow) are visible in vitro (e) or in situ (s), and the acrosomic vesicles are flattened over the nuclei of these Sa spermatids. The flagellum has developed sufficiently to be seen extending beyond the cell diameter in vitro (c) and in cells in situ (r). As the Sb1 developmental step occurs (f, t), the acrosomic cap (arrow) can be seen extending over a nucleus that has begun to lose its spherical shape. In both live cells in vitro (g, i) and cells in situ (u, r), the Sb2 spermatids are characterized by the elongating process and the presence of the manchette (arrow). The acrosomal cap (open arrow) is a predominant feature attached to the nucleus opposite to that of the attached flagellum. The manchette of spermatids is not as obvious in vitro (g, i) as that of cells in seminiferous tubules (u, y). In both in vitro (j) and in situ cells (w), Sc spermatids have lost the predominant acrosomal protrusion and the manchette. Sc spermatids have greatly extended flagella; however, the annulus (arrow) is still located around the tail near the nucleus, and mitochondria have not yet wrapped around the middle piece of the flagellum. Sd1 spermatids have shortened nuclei with an annulus (arrow) that has migrated to the end of the middle piece both in vitro (k, i) and in situ (x). Sd2 spermatids (m, y) have the smallest spear-shaped heads of the elongated spermatids, have extended tails with enlarged middle pieces (arrow), and often have a cytoplasmic droplet located at the anterior end of the middle piece. In both in vitro (m) and in situ (y) situations, these Sd2 spermatids are similar in head and tail size to testicular spermatozoa. In both in vitro (n, o) and in situ (z), sometimes the tails may be wrapped around the head in a coil, or the head and coiled tail may be contained within the plasma membrane of the sphere-shaped cytoplasm. Elongated spermatids (Sc-Sd2) often have spaces in the nuclear material that appear to be vacuoles within the nucleus (j–o, w–z). The steps of spermatid development during spermiogenesis in humans as observed in live cells in vitro (a–o) or within seminiferous tubules (p–z) are distinguished by initial elongation, condensation, and then shortening of the nucleus, development of the acrosome from the acrosomic vesicle, development of the tail, and shedding of excess cytoplasm. Abbreviations as in Figure 1 legend. Bar = 10 µm

The group of ten control men had a wide range of values for daily sperm production (DSP) when expressed as DSP/g testicular parenchyma (efficiency of spermatogenesis) or DSP/testis (Fig. 4). Hence, live dispersed cells were compared with fixed cells of control men who had varied spermatogenic activity (DSP/testis ranging from 5 x 106 to 221 x 106, and DSP/g ranging from 0.23 x 106 to 10.77 x 106). In spite of variation in spermatogenic efficiency and production level of these control men, the sizes of nuclei of round spermatids were similar (P > 0.05) among men, with diameters ranging from 6.67 µm to 7.18 µm (Fig. 4), and were similar (P > 0.05) to the 6.81 ± 0.07 µm for fixed, embedded in situ cells in the live-cell donor. Likewise, the diameters of pachytene primary spermatocytes were similar (ranging from 11.05 µm to 12.17 µm) among men with varied levels of spermatogenesis and were similar to the 11.77 ± 0.15 µm of the live-cell donor. The diameters and volumes of various types of human germ cells reported in published studies are listed with their references in Table 1. The variation in nuclear size of germ cells in various steps of development in culture or in situ seen in Figures 1–3 correspond to differences in nuclear size measured previously in fixed, unstained, 20-µm epoxy resin sections by Nomarski optics (Table 1). Hence, variation in nuclear size seen in vitro was confirmed by actual measurements made in fixed tissue in situ from the ten control men. Given that the live-cell donor had complete spermatogenesis at the time of sampling, it was difficult to determine whether the cell types noted in the in situ preparations resulted from in vitro maturation over the 3-day period or simply were present at the time of sampling. Although no vital stain test was conducted and a few cells had swollen and vacuolated cytoplasm or were missing cytoplasm, the general appearance of most cells did not change from an apparently normal morphology over the 3-day incubation.



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  		FIG. 4. Graphic representation of daily sperm production (spermatogenic activity) and nuclear sizes of round spermatids (Sa plus Sb1) and pachytene primary spermatocytes of the ten control men, previously determined [27]. DSP/g (a) (measure of efficiency of spermatogenesis) and daily sperm production per testis (b) are varied for the ten control men. However, the sizes of nuclei of round spermatids (c) and pachytene primary spermatocytes (d) are similar regardless of spermatogenic efficiency


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Round spermatids in ejaculates have been identified on the basis of their size and shape, and are distinct from lymphocytes on the basis of the higher ratio of cytoplasm to nucleus in spermatids [37]. Dispersed, human germ cells of all types have been systematically described in homogenized fixed human testes viewed by phase-contrast microscopy [27]. Verheyen et al. [24] considered published micrographs of round spermatids in wet preparations of humans [18, 37, 38] or animals [8, 12] to be unclear or unconvincing. Vanderzwalmen et al. [19] noted it to be difficult to identify round spermatids under the inverted microscope with the Hoffman modulation contrast system that is most commonly used with ICSI.

Nomarski interference contrast microscopy allows identification of cellular morphology inside whole cells, as it has a small depth of focus. This enables one to optically section through cells or tissues such as testicular specimens [27, 30–32, 35, 39–41]. Hence, nuclear diameter/volume estimates can be made repeatedly by focusing up and down on the spherical nucleus of various testicular cells and measuring its maximal diameter as illustrated in Table 1. Likewise, optical sectioning coupled with serial sectioning has allowed observation of spermatogenic stages along the length of human seminiferous tubules [34] or whole mounts of tubules [42, 43]. Optical sectioning of embedded tubules has enabled observation of rare events such as the process of migration of residual bodies (left behind at spermiation) down through the Sertoli cell into its basal cytoplasm [42, 44].

In this current study, Nomarski optics has enabled identification of human male live germ cells (Figs. 1–3). Again, the optical sectioning of a cell facilitates observation of organelles and other structures inside the cell (tail, Golgi apparatus, acrosomic vesicle, developing acrosome, annulus, and chromatin profiles). Nomarski optics have long been used to evaluate the acrosomal status (intact or deteriorated) of live bull spermatozoa [45–47]. Likewise, Nomarski optics have been used to morphologically characterize spermatogenic cells isolated in vitro from prepubertal mice [35]. Both Nomarski optics and phase-contrast microscopy allow observation of cellular detail without fixing or staining, such that cells can be observed in the live state. However, phase-contrast microscopy does not have the small depth of focus of Nomarski optics and cannot effectively optically section a specimen or cell.

Morphological characteristics of human germ cells within spermatogenic stages of the cycle of seminiferous epithelium was first described under brightfield microscopy of thick (7–10-µm), stained, paraffin sections by Clermont [36] and Heller and Clermont [48]. To quantify the germ cell population in fixed human testes by the homogenate method (dispersal of cells/nuclei of cells and counting them in a hemocytometer), phase contrast microscopy was used to describe different developmental steps of human spermatogenesis using isolated cells [27]. Brightfield microscopy and toluidine blue-stained thin sections (0.5 µm) were used to place the images representing different developmental steps of germ cells in the context of the spermatogenic stage [33] and the overall process of spermatogenesis in humans [49, 50].

Although the six stages of the cycle of seminiferous epithelium have been described using Nomarski optics in 20-µm epoxy resin sections [34], the current paper extends these observations to place live dispersed human male germ cells in the context of different developmental steps in human spermatogenesis (Figs. 2 and 3). 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, and tail; location of the annulus; and/or mitochondria around the middle piece of the tail. Given that size of nuclei is highly important, Table 1 illustrates variation in the size of various germ cells from spermatogonia to spermatids in control men or in the live-cell donor that correspond to differences in nuclear size of live human germ cells (Figs. 2 and 3).

Figures 2 and 3 provide a useful checklist for identifying round spermatids for ROSI or secondary spermatocytes currently, or for identifying even younger germ cells should such an approach prove feasible for intracytoplasmic injection into ooplasm of an unfertilized egg in the future. This list is possible because human male live germ cells (from enzymatically dispersed testicular tissue examined under Nomarski optics) portray morphological features that characterize specific developmental steps in human spermatogenesis.


   ACKNOWLEDGMENTS
 
The authors wish to thank Hamo Meguerditchian for obtaining specimens and Penny Churchill for expert secretarial assistance with the manuscript.


   FOOTNOTES
 
1 Supported in part by NIH Funding K04AG00465, N01HD-83281, P30E09106, T32ES07273, and R01HD34362. Back

2 Correspondence. FAX: 409 847 8981; ljohnson1{at}tamu.edu Back

Accepted: May 13, 1999.

Received: January 28, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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