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a Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The postnatal phase can be divided into three main stages: 1) mitotic proliferation of spermatogonial stem cells and premeiotic differentiation of spermatogonia cells to diploid primary spermatocytes; 2) meiotic differentiation of primary spermatocytes to haploid early round spermatids via two successive divisions—the reductional division, in which homologous chromosomes are separated into two haploid secondary spermatocytes (each chromosome consists of two chromatids), and an equational division in which the two chromatids of each chromosome are separated into two haploid round spermatids; and 3) spermiogenesis, a cellular and nuclear reorganization process that turns spermatids into spermatozoa [3]. This latter stage consists of intensive condensation of the chromatin due to replacement of histones by transition proteins and later by protamines [4–7], a substantial reduction in the cell's volume due to severance of large cytoplasmic fragments, the residual bodies, and formation of a compact head and a long tail.
Although the structural and morphological characteristics of spermatogenesis are well defined, very little is known about the molecular regulation of this key biological process. A major limitation in the study of genes and factors involved in germ cell differentiation has been the lack of an efficient in vitro system that supports this differentiative process. A partial alternative to use of the in vitro system was to study the transcription and protein expression pattern of various genes in isolated populations of spermatogenic cells derived from adult testes. Testicular cell suspensions were separated by velocity sedimentation at unit gravity or by centrifugal elutriation. However, only three main populations of cells could be obtained using this technique: meiotic prophase spermatocytes (predominantly in the pachytene stage of meiosis), postmeiotic early spermatids, and elongated spermatids. An additional fraction of residual bodies and cytoplasmic fragments could also be obtained [8–10]. Flow cytometry of testicular cell suspensions was utilized as another useful tool for studying mammalian spermatogenesis. Various fluorescent dyes were used to stain DNA (and/or other components) of testicular cells, and the stained cells were analyzed or sorted according to the intensity of fluorescence emission, which correlates with DNA content [11–19]. This technique has also been used as a diagnostic tool to assess spermatogenesis as well as development of testicular cancer in human patients [20–22]. However, since the adult testis consists of cells at all stages of spermatogenic differentiation, the approaches mentioned above cannot enable accurate analysis of stage-specific biochemical events and gene expression. An efficient way to address this problem is to analyze the biochemical events and gene expression in postnatal testes following testicular development and germ cell differentiation. This approach, however, requires a detailed predetermination of the testicular developmental schedule. Bellve et al. [23] have determined a developmental timetable in CD-1 mice by separating seminiferous epithelial cells from testes of animals at different postnatal ages, using the sedimentation velocity at unit gravity method, and analyzing them morphologically by electron microscopy. However, although the rat is widely used as a model system for studying spermatogenesis in mammals, only partial information on spermatogenesis in rat during testicular development is available [11, 24]. A detailed developmental schedule has not been characterized.
In this study we used four-parameter flow cytometry to determine the rat's testicular developmental schedule as compared to that of the mouse. This developmental schedule, together with the advantage of efficiently separating the testicular cell subpopulations using the FACS (fluorescence-activating cell sorter) machine, will enable an easy and accurate analysis of the developmental stage-specific biochemical events and gene expression in the rat as well as the mouse.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Balb/C mice, older than 60 days, and Sprague-Dawley rats, older than 80 days, were used as a source of normal adult tissues for all experiments. For the developmental studies, neonatal testes were collected from animals at Days 5–31 and Days 5–60 of postnatal development of mice and rats, respectively. Day of delivery was designated Day 1. Litter size was adjusted to a maximum of 8 pups per mother for the mice and 10 for the rats. Animals were killed by cervical dislocation. Large animals were killed with chloroform prior to dissection. This investigation was conducted in accordance with the Guiding Principles for the Care and Use of Research Animals Promulgated by the Society for the Study of Reproduction.
Preparation of Testicular Cells
Testes were dissected into a petri dish containing ice-cold sterile separation medium (4 mM L-glutamine, 1.5 mM sodium pyruvate, 10% fetal calf serum, and 75 µg/ml ampicillin in Dulbecco's Modified Eagle's medium containing nonessential amino acids). For each experiment with a given developmental stage, testes from several animals (4–6 pups among early postnatal animals down to 2 mature animals) were pooled and processed. Each testis was decapsulated by making a small incision in the testis and forcing (by sterile tweezers) the content of the testis through the incision into a 15-ml Falcon (Los Angeles, CA) tube containing 5 ml ice-cold separation medium. Then, 0.25 ml collagenase (Calbiochem-Behring, La Jolla, CA) from a 2 mg/ml stock solution (prepared in separation medium) was added to the tube with the decapsulated testes, and incubation was carried out for 5 min at 35–37°C under vigorous shaking. The seminiferous cords were then allowed to sediment to the bottom of the tube while being incubated on ice. The seminiferous cords were washed twice in 10 ml separation medium, resuspended in 12 ml separation medium containing 2.5 µg/ml trypsin and 1 U/ml DNase I (Boehringer Mannheim, Mannheim, Germany), incubated for 2 min at 35–37°C, and transferred to ice. Using a pasteur pipette, the seminiferous cords were disintegrated into single cells and were then filtered through a 50-µm nylon mesh, washed twice with separation medium (centrifugation at 200–300 x g), and counted. Experiments with the various developmental stages were repeated at least three times (using a pool of testes at the specific developmental stage in each experiment, as mentioned above).
FACS Analysis
For FACS analysis, testicular cells were brought to a concentration of 2 x 106 cells/ml in separation medium and diluted 1:1 with propidium iodide solution (10 mM Tris pH 8, 1 mM NaCl, 0.1% Nonidet P-40, 0.7 mg/ml RNase A, and 0.05 mg/ml propidium iodide). Cells were analyzed by a Becton, Dickinson (Rutherford, NJ) FACSort instrument, equipped with an argon laser, within 2 h from staining. Excitation was at 488 nm and emission at 585 nm. Four parameters were measured for each cell: forward scatter (FSC-H); side scatter (SSC-H); total fluorescence emitted from the cell (FL2-A); and the duration of emitted fluorescence from the cell (FL2-W), which correlates to the nucleus diameter. For microscopic analysis, cells from designated populations were sorted into a tube containing 1 ml separation medium, washed (3 times) in PBS, and applied onto 3-aminopropyltriethoxy-silane-treated slides. The cells were then fixed with 4% paraformaldehyde (30 min), washed (3 times) with PBS, and stained with hematoxylin.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the testicular developmental schedule of the rat as compared to that of the mouse by FACS analysis, we followed the development-dependent acquisition pattern of each of the seven subpopulations determined in the mature testis and monitored the percentage of cells with differing DNA content (Table 1). In the mouse (Fig. 4), postnatal (p.n.) Day 7–10 testes exhibited only the two 2d subpopulations (R3 and R4) in the first window. However, in the second and third windows, a small 4d subpopulation (~3%) and a small intermediate subpopulation (~3%), representing cells in S phase of the cell cycle, could be distinguished in addition to the 2d cells (~94%). By p.n. Day 12, a significant number of cells exhibited FSC-H values appropriate for the R1 subpopulation, although they did not appear as a discrete group. At this developmental stage, a significant increase in the portion of 4d cells (~7%, p < 0.0005) was observed for the first time (windows 2 and 3). By p.n. Day 14 there was a discrete R1 group, with a further-increased portion of 4d cells (~15%). By p.n. Days 17–18, the R1 group showed very high intensity, with the first indications of R2 cells and with the 4d cells composing ~40% of all cells. Cells localized to the R6 gate were seen for the first time at p.n. Days 20–21 (Fig. 3e) in accordance with the appearance of 1d cells (~7%) in the second and third windows. At this developmental stage the R1 and R2 subpopulations were well established. Elongating spermatids (R7) started to appear at about p.n. Days 24–25, at which time haploid cells composed ~23% of all cells; and by p.n. Days 27–28, elongated spermatozoa were present, as evidenced by the localization of cells to the R5 region and the apparent fourth level of fluorescence intensity. The haploid cells at this developmental stage represented ~75% of all cells.
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A similar acquisition pattern for the various testicular subpopulations was obtained with rats, although the pace was slower (Fig. 5). Until p.n. Days 13–14, only cells corresponding to the R3 and R4 regions could be detected in the first window, and the percentage of 4d cells was ~3% for p.n. Day 6 and Day 13. A statistically significant increase in the portion of the 4d cells was obvious for the first time at p.n. Days 17–18 (6%, p < 0.0005), although R1 cells, in the first window, could not be determined as a group until p.n. Days 19–20. At this developmental stage, the 4d cells totaled up to ~19% of all cells (whereas the 2d cells constituted ~77%). By p.n. Days 22–23 it was possible to discriminate between the R1 and R2 subpopulations, and the percentages of the 4d and 2d cells were almost equal, ~47% and ~48%, respectively. The first signs of haploid cells were obtained in all three windows by p.n. Days 24–25 (~2%); and by p.n. Days 30–31, ~29% of the cells in the testicular cell suspension were haploid cells. Of those, a small portion seemed to localize to the R7 region, indicating the presence of elongating spermatids. The smear that defines the R5 region in the FSC-H/SSC-H plot (first window) started to appear at p.n. Days 36–37, indicating that by this developmental stage, the first condensed elongated spermatozoa are present in the testis. This was also evidenced by the appearance of the fourth fluorescence level in the second and third windows. By p.n. Days 42–44, in the rat, the FACS pattern was very similar to that for mature testis (Fig. 6), indicating that the complete spermatogenic complement is present in the seminiferous epithelium at this stage.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A flow cytometric follow-up on the development of the postnatal testis has been executed in the past in both the mouse [16] and rat [11]. However, these studies focused on the changes in the DNA content of the cells without further staging within each DNA containing group, especially within the 4d group. Concerning changes in the DNA content, the results reported in the present study on the mouse are in accordance with those of Janca et al. [16]. Regarding rat spermatogenesis, Zhengwei et al. [24] reported that primary spermatocytes first appear on Day 15 and round spermatids on Day 25. Our results are consistent with these findings, although we could not detect a significant increase in the 4d cells until Day 17, since leptotene and early zygotene primary spermatocytes appear prior to the increase in the 4d cells (see discussion below and Table 2). However, there are slight differences between our results and those reported by Clausen et al. [11], especially with regard to the time when haploid cells first appear. These differences might be attributed to the enhanced ability to discriminate signal from background today compared to 20 yr ago.
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A morphologically based determination of the mouse testicular developmental schedule was documented by Bellve et al. [23, 30]. According to this developmental schedule, seminiferous epithelium from p.n. Day 6 pups contains only primitive type A spermatogonia and Sertoli cells. At p.n. Day 8, type A and type B spermatogonia are present, and by p.n. Day 10, cells from the first spermatogenic wave can be found at preleptotene and leptotene stages of the first meiotic prophase. Zygotene primary spermatocytes are first detected on p.n. Day 12, and early pachytene and late pachytene spermatocytes first appear on p.n. Days 14–15 and Days 17–18, respectively. Haploid round spermatids first appear at about p.n. Day 21, and at about p.n. Day 30 the complete spermatogenic complement is present in the seminiferous epithelium. The results obtained with the mouse in the present study fit this morphologically based developmental schedule and could be divided into eight developmental stages (Fig. 4, Tables 1 and 2). Testes of p.n. Day 6-7 pups (stage I) exhibited 2d cells with few 4d cells, the latter of which consisted of spermatogonia as well as somatic cells at the G2 stage of a mitotic cell cycle. At p.n. Day 10 (stage II), although the FACS pattern seemed unchanged in both the FSC-H and the FL2 parameters, the constant portion of 4d cells (~3%)—despite the greatly reduced frequency of mitotically dividing somatic cells—is consistent with cells of the first spermatogenic wave reaching the leptotene stage of prophase I. There are no R1 cells (in the first window) in this developmental stage, since leptotene spermatocytes are small cells [23]. As the developmental process progresses, more cells from the first spermatogenic wave enter prophase I. As a result, by p.n. Day 12 (stage III), when zygotene cells are first seen, a significant number of cells exhibit FSC-H values appropriate for the R1 subpopulation, although they do not appear as a discrete group. Additionally, a statistically significant increase in the proportion of 4d cells is observed. The progress of cells from the first spermatogenic wave to the early and late pachytene stages (stages IV and V) is characterized by a well-defined R1 group at p.n. Day 14 and acquisition of the R2 subpopulation at p.n. Days 17–18, respectively. Haploid round spermatids first appear (group R6) at p.n. Days 20–21 (stage VI) as expected, and elongating spermatids become apparent by p.n. Days 24–25 (R7, stage VII), whereas elongated condensed spermatids are apparent (R5) from p.n. Day 27–28 (stage VIII). It is noteworthy that although the mouse testicular developmental schedule obtained in the present study is similar to the morphologically based developmental schedule reported by Bellve et al. [23, 30], there is an advantage to determining the FACS pattern of each developmental stage. This enables an efficient separation of the various subpopulations at each developmental stage (with purity more than 90%) and hence analysis of stage-specific events. For example, we were able to obtain a very pure population of early round spermatids from p.n. Day 21 (subpopulation R6, Fig. 3e), whereas in testes of adult mice, this R6 group is "contaminated" with elongating and elongated spermatids. It is noteworthy, however, that if intact cells from specific subpopulations are to be obtained by sorting, pretreatment of the cells with a detergent-containing solution (e.g., the propidium iodide solution) is not recommended. That is, sorting according to the FSC-H/SSC-H parameters should be performed without prestaining with a fluorescent dye or, alternatively, with use of a fluorescent dye such as Hoechst 33342 that does not require detergents to penetrate the cells.
In the rat, a very similar acquisition pattern for the various subpopulations, composed of the same eight developmental stages, was found. As in the mouse, rat testis at p.n. Days 6–7 contains only mitotically dividing spermatogonia cells and somatic cells, some of which are still undergoing mitotic divisions. The developmental stage of p.n. Days 13–14 in the rat correlates to p.n. Day 10 in the mouse in the sense that it is the last postnatal age showing no increase in the proportion of 4d cells, although mitotic activity of the somatic cells either ceased [25] or at least dramatically declined [26, 28]. This suggests that, as in the mouse, the rather constant portion of 4d cells is attributable to primary spermatocytes that have entered the leptotene stage of prophase I. The appearance of zygotene cells in the mouse, at p.n. Day 12, was characterized by significant increase in the proportion of 4d cells, for the first time, and by the appearance of a substantial number of cells in the R1 region in the first window. This very exact pattern was found in rats at p.n. Days 17–18, suggesting the first appearance of zygotene cells at that developmental stage. At p.n. Days 19–20, the 4d cells show up as a discrete R1 group for the first time, and by p.n. Days 22–23, a distinct R2 group begins to appear. This correlates to p.n. Day 14 and p.n. Days 17–18 in the mouse, meaning acquisition of early and late pachytene spermatocytes, respectively. Haploid round spermatids were first detected by p.n. Days 24–25, corresponding to p.n. Days 20–21 in the mouse; elongating spermatids started to accumulate by p.n. Days 30–31 (as evidenced by cells that began to occupy the R7 region in the first window), corresponding to p.n. Days 24–25 in the mouse; and rat pups older than 36 days seem to have condensed spermatids in their testes (as evidenced by the first signs of cells within the R5 group), corresponding to pups older than 27–28 days in the mouse. This rat testicular developmental schedule in comparison to that for the mouse is summarized in Table 2. It is noteworthy that the similarity between the rat and mouse along the eight developmental stages is not confined to the flow cytometric picture but includes also the proportion of the various cell types within each developmental stage (Table 1). Nevertheless, the stoichiometry of the various cell types varies among reports ([17, 23, 24] and this study), suggesting that the specific analysis procedure is an important parameter that should first be normalized when quantitative assessment is made. This is important, for example, when FACS is used to determine the efficiency of spermatogenesis in response to hormonal manipulation.
In conclusion, we believe that identification of the subpopulations that represent the various differentiative stages in the postnatal development of the testis, together with the advantage of sorting and separating each subpopulation with the FACS, gives us a powerful tool for studying mammalian spermatogenesis at the molecular level.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 972-3-5351824; don{at}ashur.cc.biu.ac.il
Accepted: February 19, 1998.
Received: June 3, 1997.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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