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a School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6009
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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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Spermatogenesis can be divided into three phases. In the first phase, the spermatogonial stem cells divide to provide daughter spermatogonia that undergo differentiation through a series of subsequent divisions. Spermatogonia are located on the basement membrane of the seminiferous tubules, separated from the adluminal compartment of the tubule by tight junctions between Sertoli cells (blood-testis barrier). The second phase of spermatogenesis is the meiotic phase through which four haploid spermatids are produced from each tetraploid preleptotene spermatocyte, the daughter cell of type B spermatogonia. Preleptotene spermatocytes give rise to leptotene spermatocytes, which are located in the adluminal compartment of the seminiferous tubules. During subsequent development to zygotene, pachytene, and diplotene spermatocyte, meiotic recombination occurs. The last phase, spermiogenesis, is characterized by the dramatic morphological transformation of the spermatogenic cell from secondary spermatocyte to spermatid and finally to spermatozoon. Spermiogenesis is subdivided into Golgi, cap, acrosome, and maturation phases, each being subdivided into several steps on the basis of the changes observed in the nucleus and the developing acrosome [3–5]. These final steps are complex and essential for making functional spermatozoa that are highly specialized to carry the haploid genome to the ovum for the creation of a new individual.
Throughout spermatogenesis, interactions between germ cells and somatic cells are essential. In particular, Sertoli cells maintain continuous contact with germ cells, from spermatogonia to spermatozoa. Spermiogenesis appears to be particularly dependent on interaction of germ cells with Sertoli cells. Characteristic structural features, such as ectoplasmic specializations and tubulobulbar complexes, suggest special interactions between Sertoli cells and spermatids [6]. Hormonal control of spermatogenesis also appears to be mediated by Sertoli cells. The complexity of germ cell differentiation, the long time frame for completion of the process, and the close interaction between germ cells and Sertoli cells suggest that spermatogenesis should be highly species specific. The differences among various species in morphology of spermatozoa and time to complete the process support that belief.
In 1994, the technique of spermatogonial transplantation was reported, through which germ cells of a fertile mouse can be transplanted into the seminiferous tubules of an infertile mouse to develop donor cell-derived spermatogenesis [7]. Several months after transplantation, many foci of donor cell colonization were observed in the seminiferous tubules of recipient mice. In the most successful transplantations, the recipient mouse could transmit the haplotype of the donor cells to progeny [8]. A significant and surprising extension of these studies was xenogeneic spermatogonial transplantation [9]. Testis cells from transgenic rats were transplanted to the testes of immunodeficient mice, and complete rat spermatogenesis occurred in the recipient mouse, resulting in the presence of normal-appearing rat spermatozoa in the epididymides of recipient mice. Although there is similarity in the general appearance of rat and mouse spermatozoa, significant differences exist between the spermatozoa of these species. The sperm heads are distinct and the sperm tail is 40% longer in the rat. Furthermore, the process of spermatogenesis takes 50% longer in the rat than in the mouse. Because rat spermatogenesis could occur in the mouse testis in spite of these significant differences in sperm morphology and developmental timing, and in spite of the fact that the two species phylogenetically diverged 10–11 million years ago, it seemed possible that xenogeneic transplantation might be achieved for other species.
In order to explore and develop this approach, we chose the Syrian golden hamster as a donor animal because of the widespread interest in hamsters as experimental animals in both reproductive and nonreproductive research. In addition, hamster spermatozoa are distinct in size and shape from those of the mouse or rat. The hamster spermatozoon is larger than the mouse spermatozoon and is characterized by a large, hook-shaped head with a prominent acrosomal cap as well as a long, thick tail [10]. Thus, using the hamster as a donor species represents a logical and important extension of xenogeneic spermatogonial transplantation to a species that is significantly more divergent from the mouse, in physiology and sperm structure, than is the rat. The studies described here were done to investigate whether hamster spermatogenesis from donor cells can occur in the seminiferous tubules of recipient mice.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Syrian golden hamsters (Harlan Sprague Dawley, Inc., Indianapolis, IN), between 1 and 2 mo of age, were used as the source of donor testis cells. Spermatogenesis was fully developed in all donor testes. The animals were maintained in an air-conditioned environment at 22°C on a 14L:10D photoperiod before they were killed. All experimental procedures were approved by the Animal Care and Use Committee at the University of Pennsylvania. Testis cells were collected by a two-step enzymatic digestion previously described [11], with minor modifications. Briefly, the tunica albuginea was manually removed from the testes. The exposed seminiferous tubules were then dissociated with collagenase (1 mg/ml; type IV, Sigma Chemical Co., St. Louis, MO) in 10 ml of Hanks' Balanced Salt Solution without calcium and magnesium (HBSS) at 37°C for 5–10 min. DNase I (7 mg/ml) in HBSS was added as needed (approximately 100 µl) to further dissociate tubules. After rinsing 2–4 times in HBSS, the tissue was digested with 0.25% trypsin and 1 mM EDTA in 10 ml of HBSS at 37°C for 5–10 min. Fetal bovine serum (1–2 ml) was added to stop enzymatic digestion. The resulting cell suspension was filtered through a nylon mesh with 60-µm pore size (Tetko Inc., Kansas City, MO), and the cells were collected by centrifugation, 600 x g for 5 min at 16°C. The pellet was resuspended in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 6 mM lactate, 0.5 mM pyruvate, 30 mg/L penicillin, and 50 mg/L streptomycin (designated DMEM-C) to a final concentration of 100 to 250 x 106 cells/ml. Viability of cells was greater than 95% as determined by trypan blue exclusion.
Donor Cell Freezing
Several aliquots of hamster testis cells collected as described above were frozen and kept in liquid nitrogen for transplantation at a later date. The freezing procedure was essentially the same as for cultured somatic cells [12, 13]. First, collected hamster testis cells were suspended in DMEM-C at a concentration of 56 x 106 cells/ml. Freezing medium (FBS, DMEM-C, DMSO in a ratio of 1:3:1) was added slowly at a volume equal to that of the original cell suspension and mixed. Cells were aliquoted at 1.0 ml per freezing vial, placed in an insulated container at -70°C for at least 12 h, and stored in liquid nitrogen (-196°C). The cells were thawed by swirling in a 37°C water bath, and DMEM-C was added slowly to three times the volume in the vial. This cell suspension was centrifuged at 600 x g for 5 min at 16°C, and the pellet was resuspended in DMEM-C to a cell concentration of 129 x 106 cells/ml for transplantation. Viability of cells after freezing and thawing was 43%.
Recipient Mice and Donor Cell Transplantation
NCr Swiss nude (nu/nu) mice (Taconic, Germantown, NY) 10–20 wk of age were used as recipient animals to avoid immunological rejection of donor cells. The mice were treated with busulfan (40 mg/kg) at least 4 wk before donor cell transplantation to deplete endogenous germ cells in the testes [7]. Because this dose of busulfan is toxic to the hematopoietic system, the mice received bone marrow transplantation from nontreated nude mice. Donor bone marrow cells were collected from a nude mouse by flushing the marrow of femurs and tibiae with DMEM-C. A volume of 0.25 ml, containing 3–6 x 106 cells/ml, was injected into the jugular vein of recipient mice 3 days after busulfan treatment [9]. For transplantation of testis cells, the seminiferous tubules of recipient mice were filled with the donor hamster cell suspension by injection through the efferent ducts as described previously [14].
Analysis of the Recipient Testes and Epididymides
Between 89 and 457 days after donor cell transplantation, the recipient mice were killed by CO2 inhalation, and both testes and epididymides were removed. In most cases, one testis was fixed in 10% neutral buffered formalin (NBF) for 24 h and processed for paraffin embedding and sectioning. The other testis was snap-frozen in liquid nitrogen and stored at -70°C for use at a later date in a different experiment. Four histological sections with a 25-µm interval between sections were made from the testis of each mouse, and the sections were stained with periodic acid-Schiff (PAS) and hematoxylin. Each slide was examined at x400 magnification for hamster spermatogenesis. To determine the extent of hamster spermatogenesis in recipient mouse testes, the number of tubule cross sections containing hamster, mouse, or no spermatogenesis was recorded for one section from each testis. Spermatozoa were recovered from the epididymis and vas deferens of recipient mice and fixed in NBF. The spermatozoa suspension was stained by addition of an equal volume of 100 µg/ml Hoechst 33258 [15] and examined at x630 magnification by fluorescence and phase-contrast microscopy. Hamster and mouse spermatozoa were identified based on sperm head morphology and tail size, and up to 2000 spermatozoa were analyzed per sample.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the first experiments, testis cells were collected and within 5 h injected into the seminiferous tubules of the recipient nude mice. Approximately 80–90% of the surface tubules of the recipient testes were filled with the donor cell suspension as judged with the aid of trypan blue added to the solution as an indicator [14]. Surface filling of tubules with dye was regarded as reflecting the degree of donor cell suspension distribution throughout the tubules. This experiment was repeated on four separate days using similar procedures to assure repeatability. The animals were killed 99–457 days after the cell transplantation, and testes were examined for the presence of hamster spermatogenesis. In the golden hamster and in the mouse, 35 days are necessary for a type A1 spermatogonium to form a mature spermatozoon. Thus, the interval from transplantation to analysis represented 2.5–13 times the duration of hamster spermatogenesis. This assured that there was adequate time for maturation of hamster spermatids and spermatozoa, which are necessary to distinguish hamster from mouse spermatogenesis. In addition, the longer intervals would reflect the ability of foreign spermatogenesis to persist. The results of these four experiments are shown in Table 1.
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Hamster spermatogenesis was generated from donor stem cells in all recipient mouse testes. In order to estimate the degree of colonization, a cross section of the seminiferous tubules of each testis was examined, and the contents were classified as hamster, mouse, or no spermatogenesis. The morphology of hamster germ cells during spermiogenesis is characterized by round spermatids displaying large acrosomal caps that stain brightly with PAS reagent (Fig. 1E). In addition, elongated spermatids and spermatozoa have a long and hook-shaped head (Fig. 1B). On the basis of these criteria, hamster spermatogenesis was identified on average in 6.3% of tubules examined with a range of 1–18% in individual testes. Clearly, this technique underestimates the extent of hamster spermatogenesis because stages of hamster spermatogenesis that do not contain round or elongated spermatids or spermatozoa would not be identified as hamster and were therefore classified as mouse spermatogenesis. The areas of mouse spermatogenesis resulted from endogenous recipient stem cells that were not destroyed by busulfan treatment. This regeneration of endogenous recipient spermatogenesis is a general phenomenon resulting from the survival ability of the spermatogonial stem cell. In recipient nude mice, regeneration of endogenous spermatogenesis is common, because the dose of busulfan tolerated by the animal is limited due to the compromised immune system.
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Hamster spermatogenesis in recipient mouse testes showed several morphological abnormalities. Spermatogonia, spermatocytes, and round spermatids appeared morphologically normal. However, abnormalities in head shape of elongated spermatids were frequently observed, and in some cases the acrosomal cap of these cells had marked deformities, such as a ballooned shape (Fig. 1F). Furthermore, the positional arrangement of hamster elongated spermatids in mouse seminiferous tubules appeared poorly organized (Fig. 1D) compared to the pattern in hamster testes (Fig. 1B), and the number of elongated spermatids appeared reduced compared to the number of round spermatids present.
Cryopreserved Hamster Testis Cells Generated Xenogeneic Spermatogenesis
To determine whether hamster spermatogenic cells can be stored for long periods, testis cells were collected and cryopreserved in liquid nitrogen for 18 days and then thawed for transplantations. Viability of thawed hamster cells was only 43% compared to more than 95% for freshly collected testis cells. Nonetheless, the cryopreserved hamster testis cells colonized 5 of 6 recipient testes (Table 2). However, only 1% or 2% of the cross sections of recipient seminiferous tubules observed contained evidence of spermatogenesis. This appears to be fewer than the 6.3% observed with freshly collected cells. The morphological appearance of hamster spermatogenesis in the recipient mouse seminiferous tubules appeared identical to that seen after transplantation of fresh cells. Abnormalities in the early differentiation stages of hamster spermatogenesis, spermatogonial differentiation and meiotic stages, were not observed. However, acrosomal and head shape defects in elongated spermatids and spermatozoa were similar to those present after transplantation of freshly collected testis cells described above.
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Hamster Spermatozoa in Mouse Epididymides
To determine whether normal hamster spermatozoa were produced, liberated into the tubules, and transported to the epididymis, the contents of the vas deferens and epididymis were collected and carefully examined. In many cases, the number of spermatozoa examined was limited because spermatogenesis was inefficient. Although tubules in the testis may show histological evidence of spermatogenesis, production of spermatozoa may be low because the process is inefficient or not widespread throughout the testis. Nonetheless, hamster spermatozoa were found in approximately one half of the samples examined: 7 of 13 from fresh testis cell transplantations and 2 of 4 from injection of cryopreserved hamster cells (Tables 1 and 2).
The morphological characteristics of the hamster spermatozoa observed (31 in total) were not completely normal. In Figure 2, the first four panels show mouse (Fig. 2, A and B) and hamster (Fig. 2, C and D) spermatozoa to illustrate normal morphology. Figure 2, E–G, shows spermatozoa collected from recipient mouse epididymides. Basically, the general head and tail morphology of the hamster spermatozoa was characteristic of the species. The head size and shape were clearly identifiable, and the tail was large and distinct from that of mouse spermatozoa. Two main abnormalities were noted. First, most hamster spermatozoa observed had head and tail separated. This was not seen in the mouse spermatozoa from the same samples and therefore was not related to a general disturbance of the testicular or epididymal environment. Second, the acrosome of the hamster spermatozoa was absent or was poorly formed and abnormal in appearance. While the number of hamster spermatozoa examined was small, the consistency of observations among the samples strongly suggests that they represent specific cellular defects. No differences in the abnormalities were observed between hamster spermatozoa arising from frozen and fresh testis cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Successful spermatogenesis is highly dependent on structural associations and functional interactions between Sertoli cells and all stages of germ cells. It has been reported that up to 50 different germ cells, which may have developed from several separate stem cells, can be supported by a single Sertoli cell that rests on the basement membrane and extends to the lumen of the seminiferous tubule [20]. Furthermore, most germ cells interact with more than one Sertoli cell. Specialized junctional complexes between germ cells and Sertoli cells are common, and spermatids occupy a cavity or crypt in the Sertoli cell [21]. Thus, the interaction among the cells is intimate and is believed to be essential for the differentiation process. This relationship assumes an interesting and important significance in xenogeneic transplantation. It has been clearly documented that rat germ cells transplanted to mouse seminiferous tubules are supported by mouse Sertoli cells [9, 22]. In ultrastructural observations of spermatogenesis in mouse tubules, rat germ cells were always found with mouse Sertoli cells, and no evidence of rat Sertoli cells was ever identified in recipient mouse testes [22]. Furthermore, the time necessary for rat spermatogenesis completion in mouse recipients is 52 days, identical to that found for donor rat testes [23]. Therefore, the germ cell dominates and controls the germ cell differentiation process; the Sertoli cell appears to function as a relatively passive somatic supporting cell. Because the hamster donor cell population contains Sertoli cells, they have the opportunity to colonize the recipient mouse seminiferous tubule. However, the very strong evidence from rat-to-mouse transplantations suggests that this does not occur. Furthermore, the donor cell population contains only Sertoli cells that have stopped dividing, because of the donor animal's age [24], and they would therefore have little opportunity to colonize the mouse basement membrane already occupied by endogenous Sertoli cells. Thus, it is likely that most if not all xenogeneic hamster spermatogenesis represents differentiation of hamster germ cells supported by mouse Sertoli cells. A similar situation in rat-to-mouse transplantations results in relatively normal rat spermatogenesis and the production of normal-appearing rat spermatozoa [22].
The situation in hamster cell transplantations appears to be different. While normal spermatogonial division and differentiation were observed, and while spermatocyte meiosis proceeded without obvious defects, spermiogenesis of hamster germ cells showed clear abnormalities of head and acrosome development. Furthermore, no normal hamster spermatozoa were found in the epididymal content analysis. Prominent defects were the absence of normal acrosomes and the separation of head and tail. These findings indicate abnormalities in the xenogeneic hamster spermiogenesis process and suggest that the Sertoli cell plays a significant supportive and perhaps species-specific role in its interaction with germ cells that may influence final spermatozoa differentiation stages. Ultrastructural analyses are planned to elucidate in more detail the abnormalities that occur during spermiogenesis in recipient mice at several time intervals following transplantation of hamster testis cells. It seems that the greater evolutionary divergence time and/or structural difference between hamster and mouse as compared to rat and mouse allows the role of the Sertoli cell to become more obvious. Perhaps it is not surprising that the first abnormalities observed with xenogeneic transplantation should be in spermiogenesis, since the process is extremely complicated and species specific [4, 5]. As the phylogenetic separation of donor and recipient is increased and the spermatozoal characteristics reflect a greater difference, abnormalities may be seen in earlier stages of spermatogenesis. Inadequate or incorrect growth factors, cytokines, or surface molecule configurations [25] in a recipient testis are likely to play a critical role in determining whether donor stem cells can survive and undergo differentiation. It is impossible to predict which donor-recipient combinations may succeed, because inadequate information exists about the complex processes involved.
The successful colonization of recipient seminiferous tubules by cryopreserved hamster donor cells is an encouraging extension of studies performed with mouse and rat transplantations. In each of these three species, donor spermatogonial stem cells can be preserved at -196°C for long periods, and spermatogenesis resulted after transplantation ([13]; unpublished results). However, the extent of colonization from cryopreserved donor cells appears to be lower than with freshly collected cells. The results presented in Tables 1 and 2 suggest that this is true for hamster cells, and similar observations have been made for the mouse and rat (unpublished results). The important aspect of the cryopreservation studies is that the evidence is now extremely strong that spermatogonial stem cells of most, if not all, mammalian species can be preserved for long periods. The cryopreservation of mature spermatozoa from a number of species is routinely performed for subsequent use in artificial insemination or in vitro fertilization [26–28]. However, the procedure varies for each species and must be determined empirically in each case. Furthermore, frozen spermatozoa cannot replicate, and each cell is only one haploid combination of the male gamete complement. In contrast, cryopreservation of spermatogonial stem cells follows a simple procedure established for cultured somatic cells, and the same protocol appears to be similarly applicable to all the species examined [12, 13]. More importantly, cryopreserved spermatogonial stem cells can proliferate and undergo meiotic recombination during their development in the recipient testis, thereby reestablishing the entire genetic potential of the donor male. Thus, the stem cells of valuable or unique males could now be frozen for later use after techniques of culture and gene modification are developed.
The present study suggests that the cell-cell interactions between spermatogenic cells and Sertoli cells are rather flexible and can be functionally competent between cells from different species, but with limitations as species divergence increases. This is surprising considering the many structural and functional relationships that have been described [6, 29]. One would expect these intricate interactions to be rigidly controlled and very specific, thereby not allowing minor mismatch of cell surface recognition molecules. The extent of difference that will be tolerated between germ cells and Sertoli cells in cell-cell interactions and allow successful spermatogenesis is not known, nor is it known which specific molecules are responsible for these interactions. However, on the basis of the success of the studies reported here, it seems worthwhile to explore the technique of xenogeneic spermatogonial transplantation in a wide range of animals. With the development of efficient culture systems for spermatogonial stem cells, it should eventually be possible to expand the stem cells in vitro and modify their genetic composition before transplantation to host testes. Using a smaller, more readily available animal as xenogeneic recipient would facilitate the generation of spermatozoa from larger farm animals or endangered species. The resulting spermatozoa could be used for in vitro fertilization or intracytoplasmic sperm injection. This approach to the generation of transgenic animals through manipulation of the male germ line would be useful in animal species in which embryonic stem cell and nuclear transfer technology have not been perfected. Furthermore, xenogeneic spermatogonial transplantation will provide a new avenue for the study of unique aspects of spermatogenesis. As techniques improve, the combination of spermatogonial stem cell culture, genetic manipulation, cryopreservation, and transplantation will represent a powerful approach in biology, medicine, and agriculture.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: R.L. Brinster, School of Veterinary Medicine, University of Pennsylvania, 3850 Baltimore Avenue, Philadelphia, PA 19104–6009. FAX: 215 898 0667.
Accepted: September 25, 1998.
Received: August 18, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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