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RESEARCH |
Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China
Correspondence should be addressed to Daishu Han; Email: daishu{at}public.bta.net.cn
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Many aspects of the mechanisms of spermatogenesis have remained elusive because of a lack of suitable in vitro or in vivo models. Numerous trials have been aimed at mammalian spermatogenesis in vitro by many researchers over decades. But spermatogonial stem cells cannot survive and differentiate out of the microenvironment formed by other testicular cells, especially Sertoli cells. Further refinement of the culture conditions is needed, and hopefully this will lead to in vitro expansion of spermatogenic stem cells.
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 (Brinster & Zimmermann 1994). In the most successful transplantation, the recipient mouse could transmit the haplotype of the donor cells to progeny (Brinster & Avarbock 1994). A significant and surprising extension of these studies was xenogeneic spermatogonial transplantation. 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 the recipient mice (Clouthier et al. 1996). However, defects in spermiogenesis of transplanted hamster testis cells occur in the recipient mouse, which may reflect a limited ability of endogenous mouse Sertoli cells to support fully the larger and evolutionarily distant hamster germ cell (Ogawa et al. 1999). The transplantation of germ cells from phylogenetically more distant species, including rabbits, dogs, pigs, bulls, horses and primates, into mouse testes does not result in spermatogenesis beyond the stage of spermatogonial proliferation (Dobrinski et al. 1999 , 2000, Nagano et al. 2001), probably owing to the incompatibility of microenvironments.
The transplantation of testis within and between individual animals is another experimental endeavour that has a long history in reproductive biology (Goldstein et al. 1983, Johnson et al. 1996a,b, Nagler et al. 2001). In comparison with germ cell culture and spermatogonial transplantation, this approach can maintain microenvironment integrity and provide the accessibility that is essential for studying the function of testes and spermatogenesis. The host can be regarded as a living incubator and a culture system of testicular tissue. In combination with the experimental manipulation of the donor testis in vitro before transplantation to the recipient, testis grafting would be a useful model for studying the molecular mechanisms of spermatogenesis. A model with syngeneic testicular grafts in Fischer rats was established in 1996 (Johnson et al. 1996a,b). In this model, syngeneic prenatal/neonatal testicular grafts were transplanted under the skin of the outer ear in adult inbred Fischer rats that had been castrated and hypophysectomized. Complete spermatogenesis was found in grafts transplanted into the tips of the hosts’ outer ears. It has been reported that different mammalian testicular grafts that were transplanted under the dorsal skin of 6-week-old male immunodeficient NCr mice could produce mature sperm (Honaramooz et al. 2002). Recently, progeny from sperm obtained after ectopic grafting of neonatal mouse testes were produced (Schlatt et al. 2003).
Most testicular transplantation models that have been established were used to study the effects of hormones on the growth and functions of the transplanted testis. In order to analyse the schedule of spermatogenesis after syngeneic testicular transplantation in an effort to establish an in vivo culture system within the same strain for studying the mechanisms of mammalian spermatogenesis, in the present study we have developed a model of grafting testis tissues within the same strain of Balb/c mice to investigate the spermatogenesis in transplanted testes. Meanwhile, in order to look at the effects of host sex on the spermatogenesis of grafts, we used both intact male and female mice as the hosts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Testicular transplantation experimental surgery
Four-week-old Balb/c mice (~10–12 g) were used as hosts. They were divided into two groups (20 mice each group) according to sex. All of them were intact. They were anaesthetized by intraperitoneal injections of sodium pentobarbital (4 mg/100 g body weight), which proves sufficient to anaesthetize them for over 1 h. For a minimally invasive technique, we have developed a syringe needle plunger system for grafting. With this method, a great number of transplantations can be carried out in a short space of time. The procedure for transplantation is shown in Fig. 1
. The testis removed from the 5-day-old donor was placed into the tubing of a 16-gauge needle (ID 1.0 mm, OD 1.2 mm), then the needle penetrated the dorsal skin of the host, and the testis was grafted in the site of destination under the skin by pushing a fine steel wire inside the tubing of the needle. Each host received 4 testicular grafts. The wound in the host is self-sealing, and no sutures are necessary.
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RNA isolation and reverse transcription (RT)-PCR
For monitoring spermatogenesis of surviving grafts at the molecular level, the expression of Protamine-2 gene in transplanted testes was examined by RT-PCR at each time point after transplantation. Total RNA was isolated from surviving grafts with TRIzol reagent (Gibco) according to the manufacturer’s instructions. The isolated RNA was treated with RNase-free DNase (Promega, Madison, WI, USA) at 37 °C for 20 min to avoid contamination of genomic DNA, and was then reverse transcribed into cDNA in 50 µl of reverse transcription reaction mixture containing 2 µg RNA, 2 mM random hexamers (Dingguo, Beijing, China), 2 mM dNTPs, 0.01 M dithiothreitol, 5 U RNase inhibitor (Takara, Dalian, China), and 200 U M-MLV reverse transcriptase (Gibco). The reaction was incubated at 37 °C for 1 h, and the enzyme was heat inactivated at 95 °C for 5 min. Two microlitres of cDNA synthesized were then amplified with the following primers for protamine-2 (GeneBank accession number: NM_008933
[GenBank]
): forward, 5'-ATG GTT CGC TAC CGA ATG A-3', reverse, 5'-TGA TGG TGC CTC CTA CAT TTC-3'. PCR cycle conditions were 94 °C for 5 min, 94 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s for 30 cycles, and 72 °C for 10 min. PCR products were subjected to electrophoresis in 1.5% (w/v) agarose gels, stained with ethidium bromide, and photographed.
Statistical analysis
Standard statistical methods were used for analysis of data. Probabilities of 0.05 or less were considered to be statistically significant. The data for the graft size ratio were summarized as means±S.D. In order to stabilize the group-to-group variability, a logarithmic transformation (log10) was performed on the graft size ratios.
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Results |
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and Fig. 2). Most grafts had vascularized after transplantation. The grafts that did not vascularize might disappear later. There was no significant difference in graft survival rates between different sexed hosts. Most of the recovered grafts had increased in volume, some more than 30-fold (Table 1). Surviving graft sizes in male hosts increased significantly faster and were larger than in female hosts from 7 weeks post transplantation (P < 0.05).
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for male hosts, Fig. 3a–h for female hosts). The testes of 5-day-old mice contain abundant interstitial tissue and relatively thin seminiferous tubules which originally contain primitive type A spermatogonia and Sertoli cells (Fig. 3Y). No spermatogenic progression was observed in the 5-day-old mice testes. At one week after transplantation, the seminiferous tubules showed growth, and contained mainly spermatogonia and few preleptotene spermatocytes, indicating the initiation of spermatogenesis after grafting (Fig. 3A and a). We observed the continual growth of the seminiferous tubules and the progression of spermatogenesis from 2 weeks to 5 weeks after transplantation. The presence of more spermatocytes was observed at 2 weeks and 3 weeks respectively, and round spermatids appeared at 5 weeks after transplantation. Two weeks after transplantation, 58.3% of the seminiferous tubules in male hosts and 58.6% in female hosts contained spermatocytes (Table 2). Round spermatids were seen in 6.2% of the seminiferous tubules in male hosts and in 7.9% in female hosts at 5 weeks after transplantation. At 12 weeks after transplantation, the number of seminiferous tubules that contained spermatocytes was 63.9% in male hosts and 90.3% in female hosts, but the number of seminiferous tubules that contained round spermatids was only 11.6% in male hosts and 18.3% in female hosts. Dilation of the seminiferous tubule lumen was found in both male and female hosts starting at week 2 after transplantation; the percentage of seminiferous tubules with dilation of the lumen was 18.3% in males and 6.9% in females. Starting at week 3, dilation of the seminiferous tubule lumen became more prominent in male hosts, whereas much less dilation was observed in female hosts (Table 2). In contrast to male hosts, the architecture of most seminiferous tubules in female hosts appeared to be better. These results indicated that female mice might be more suitable to serve as hosts for testicular transplantation than male mice. However, no complete spermatogenesis was observed in any of the surviving grafts until 15 weeks after transplantation. In most surviving grafts, spermatocytes were the most advanced germ cells, round spermatids were observed only in some seminiferous tubules in both male and female hosts. In contrast to grafts, complete and normal spermatogenesis was observed in all male host testes after transplantation (Fig. 3I–VIII).
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) showed that protamine-2 was activated in grafts from 5 weeks posttransplantation in male and female hosts, suggesting that the spermatogenic cells in grafts could at least differentiate into round spermatids regardless of host sex. The fact that round spermatids could be found only in some grafts from 5 weeks post-transplantation whereas protamine-2 mRNA was detected in almost all grafts from 5 weeks to 15 weeks after transplantation is possibly caused by the section sites of the seminiferous tubules.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The major hurdle in most instances of animal transplantation is tissue rejection by the recipient animal (Nagler et al. 2001). Statter et al.(1988) reported that testicular grafts from fetuses in the latter part of gestation as well as from several-days-postnatal animals grew and differentiated with minimal tissue rejection when they were implanted beneath the renal capsule of an adult male or female allogeneic host for 10 days without the benefit of immunosuppression. Previous studies (Johnson et al. 1996a,b) have claimed greatest success with neonatal (0 day old) donor testes, when the number of spermatogenic stem cells is high. To extend the previous studies, we used 5-day-postnatal Balb/c mouse testes as the donor tissue, which contains only primitive A spermatogonia and Sertoli cells in the seminiferous tubules. After transplantation under the dorsal skin of 4-week-old syngeneic hosts, most grafts could grow and differentiate. Most of the surviving grafts were vascularized quickly after transplantation, as indicated by clearly visible arteries on the surface and interior of the organs (Fig. 1d
). Turner (1938) transplanted whole rat testes into various sites in adult hosts, and proved that the intraocular and intramuscular sites provided the best vascular bed. An extensive vascular supply to the testicular transplants beneath the kidney capsule was also described (Kuopio et al. 1989). Our results indicated that a good vascular bed for grafted testis could be formed under the dorsal skin.
In the present study, most surviving grafts had grown in size, some of them increasing by 30-fold. There was no significant difference in GSR between host sex by 5 weeks after transplantation, but surviving graft size in male hosts increased faster than in female hosts from 7 weeks post-transplantation. The difference in graft size is likely due to different hormone composition between male and female hosts. Further detailed investigation of the relationship between graft development and hormones in the hosts should expand the current understanding of the endocrinology of the testis.
Histological analysis showed that grafts under the dorsal skin were further developed in both male and female hosts. The diameters of the seminiferous tubules in the transplanted organs enlarged gradually, and the spermatogenic cells, which are mainly primitive type A spermatogonia and Sertoli cells in donor testes, were further developed after transplantation. Primary spermatocytes were observed at week 1 after transplantation. This corresponds to the development schedule of testes in normal 2-week-old mice when the primary spermatocytes are the most advanced germ cell type, suggesting that grafts developed immediately after transplantation. Recently, Schlatt et al.(2003) transplanted neonatal mice testes under the dorsal skin of male immunodeficient NCr mice, and found that the first round of spermatogenesis had been completed by 4 weeks after transplantation, but at weeks 12 and 16, the number of seminiferous tubules containing spermatids was only about 40% and the number of fully matured germ cells remained low. In our study, 5-day-old mice transplanted testes could differentiate into round spermatids by week 5 after transplantation. In comparison with the results of Schlatt et al.(2003) the number of seminiferous tubules containing round spermatids was much lower (11.6% in male hosts and 18.3% in female hosts 12 weeks after transplantation) and fully matured germ cells were not found in graft tissues in our study. These differences may be caused by two factors: (1) the temperature of the transplanted sites is lower in nude mice than in Balb/c mice and (2) neonatal donor testes may have greater developmental potential than 5-day donor testes after transplantation, because the spermatogenic stem cells rapidly decrease in volume during the period immediately after birth (Bellvé et al. 1977).
The dilation of the lumen was observed in seminiferous tubules of grafts in both male and female hosts starting at week 2 after transplantation, and many seminiferous tubules contained disorganized epithelium. In contrast to female hosts, dilation of the seminiferous tubule lumen was more prominent in male hosts. Interestingly, the architecture of the seminiferous epithelium of grafts in female hosts appeared better than in male hosts and there was less dilation of the lumen in the grafts in female hosts. Honaramooz and collegues have reported the dilation of the lumen in grafts. They postulated that the obvious absence of efferent ducts in the grafted tissue and the accumulation of fluid secreted into the seminiferous tubules account for the defects leading to disturbance of spermatogenesis (Honaramooz et al. 2002, Schlatt et al. 2003). The reason why grafts in female hosts had less dilation of the lumen in our study is unknown. It may be due to the fact that fluid flow secreted by Sertoli cells is decreased in female hosts but it is difficult to interpret. Nevertheless, female mice could be considered as more suitable hosts for testicular transplantation than male mice. In addition, the spermatogenesis of host testes was also assessed. Histological analysis indicated that testicular grafts had no significant effects on the spermatogenesis of the host testes.
In this study, we did not observe complete spermatogenesis until 15 weeks after transplantation in all grafts. Round spermatids were the most advanced germ cells observed in the seminiferous tubules of transplanted grafts. Turner (1938) transplanted whole testes from 1- to 21-day-old rats into various sites (anterior chamber of the eye, under the skin of the thoracic region and scrotum) in adult hosts. It was proved that testes transplanted into the anterior chamber of the eye and under the skin of the thoracic region resulted in high recovery of viable testicular tissues. However, mature sperm were only found in the testes transplanted to the scrotum. The only difference between the scrotum and the other sites is the temperature, suggesting that the temperature of the transplanted site could be the factor permitting later stages of spermatogenesis to appear in grafts (Chan et al. 1969). Several studies subsequently indicated that testicular grafts under the ear skin where the temperature is approximately the same as that of the scrotum did produce mature sperm (Johnson et al. 1996a,b). According to these reports, the failure of complete spermatogenesis in the present study should be due to the unfavourable temperature under the dorsal skin. Since round spermatids were observed in the grafts, the temperature probably has a greater effect on spermiogenesis. It should be noted that different newborn mammalian testicular grafts that were transplanted under the dorsal skin of 6-week-old male immunodeficient NCr mice could produce mature sperm (Honaramooz et al. 2002). The lack of hair in the nude mice hosts might result in a lower temperature under the dorsal skin, resulting in complete spermatogenesis in grafts. Considering the fact that spermatogenesis fails in cryptorchid testes, temperature might be a key factor during spermatogenesis, particularly in the transition from round spermatids to elongating spermatids.
In order to confirm the developmental stages of germ cells in grafts, we employed RT-PCR to examine expression of protamine-2 gene in transplants. The protamine-2 gene is expressed exclusively in postmeiotic, haploid spermatids (Oliva & Dixon 1991), and can be used as a molecular marker to monitor spermatogenesis. In this study, protamine-2 mRNA was detected in some grafts from 5 weeks posttransplantation, confirming that the spermatogenic cells in the grafts did differentiate into round spermatids. It was reported that size changes of protamine-1 mRNA could be used as a molecular marker to monitor spermatogenesis in wild-type and mutant mice (Hecht et al. 1985). Our present study indicated that the protamine-2 transcript was also a useful molecular marker to monitor spermatogenesis in mouse. In addition, there was no significant difference in expression of protamine-2 between different host sexes.
In summary, we have developed a reliable technique for the rapid transplantation of mouse testes, and with this method we transplanted the testes from 5-day-old Balb/c mice under the dorsal skin of syngeneic mice and analysed the development of the grafts. The results demonstrated that most grafts undergo further development, and that the spermatogenic cells can be differentiated to the round spermatid stage in some grafts. The development of the grafts in female hosts appeared to be better compared with male hosts, with less dilation of the lumen and less disorganized epithelium. Therefore, it is suggested that the female host may be more suitable for testicular transplantation. This is the first report of spermatogenesis in testes transplanted into Balb/c mice, and the first report of syngeneic grafting compared in both intact male and female hosts. This model could be useful in the study of the endocrinology of the testis and the mechanisms of spermatogenesis in combination with treatment of the donor testis in vitro before transplantation.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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