Spermatogonial Stem Cell Isolation, Storage, and Transplantation

Herkanwal Khaira, Derek McLean, Dana A. Ohl, Gary D. Smith
{"title":"Spermatogonial Stem Cell Isolation, Storage, and Transplantation","authors":"Herkanwal Khaira,&nbsp;Derek McLean,&nbsp;Dana A. Ohl,&nbsp;Gary D. Smith","doi":"10.2164/jandrol.05062","DOIUrl":null,"url":null,"abstract":"<p>The past 30 years have been marked by unparalleled accomplishments in the medical treatment of malignancy. Prior to advances in chemotherapeutic and radiation treatment, many oncologic conditions had dismal survival rates. Today, medical interventions have success rates that approach complete remission for many malignancies. An inadvertent complication of these therapies, however, has been the high rate of infertility following treatment. Male germinal tissue, like many malignancies, is mitotically active and therefore is particularly susceptible to the toxic effects of both chemotherapy and radiotherapy (Meistrich et al, 1982; Meistrich, 1993). Consequently, posttreatment patients often develop severe oligozoospermia or azoospermia (Wallace et al, 1991). Potential infertility complications can be anticipated, and adult male patients interested in future procreation are counseled to cryopreserve semen before instituting treatment. With present-day capabilities of in vitro fertilization, particularly intracytoplasmic injection, male patients can maintain posttreatment fertility. Pretreatment sperm banking, however, is not a viable option for prepubescent males. These individuals have not yet begun spermatogenesis and thus lack viable spermatozoa. It is estimated that, by the end of the decade, 1 in 250 young men will be childhood cancer survivors (Blatt, 1999). For these patients, infertility has often been an accepted consequence of their life-saving treatment.</p><p>A great deal of interest has recently been shown in testicular autologous transplantation, an intervention that may provide a future therapeutic fertility option for these individuals. Having been successfully demonstrated in rodent models, investigators have now begun to explore the possibility of using testicular autotransplantation to restore fertility in humans. This paper will review the history of spermatogonia transplantation with an emphasis on the clinical pertinence of this field of investigation. Current innovations involving the isolation of spermatogonial stem cells (SSCs) and the present capabilities of in vitro proliferation will additionally be reviewed.</p><p>Spermatogonia are male germinal progenitor cells and are composed of differentiated nonstem and stem cells. Stem cells are characterized by a capacity for self-renewal and an ability to produce differentiating cell lines (Loeffler and CS, 1997; van der Kooy and Weiss, 2000). Spermatogonia are diploid germ cells that originated from primordial germ cells (PGCs). These precursor cells originate from embryonal ectoderm. PGCs migrate to the genital ridge, where they become known as gonocytes. Gonocytes are surrounded by Sertoli precursor cells in what become the seminiferous cords. Tight junctions between adjacent Sertoli cells later become the basis of the blood testis barrier. The gonocytes undergo mitotic division, followed by arrest in the G0 phase of the cell cycle. They are mitotically inactive until after birth, when they become spermatogonia (Clermont and Perey, 1957; de Rooij and van Dissel-Emiliani, 1997).</p><p>Understanding spermatogonial nomenclature, differentiation, and regulation is important for comprehending testicular transplantation. First, distinguishing SSCs from differentiating spermatogonia has been a challenge that has been met with limited success. The term undifferentiated spermatogonia refers to As, Apr, and Aal cell types. Undifferentiated spermatogonia are considered distinct from “differentiating spermatogonia.” The latter group, ordered in succession, consists of A1, A2, A3, A4, Ain, and then B spermatogonia. It has been speculated that only As spermatogonia are stem cells that may divide into 2 identical daughter As spermatogonia or into 2 Apr (paired) daughter cells that are functionally committed to differentiation (Huckins, 1971). Apr cells differentiate into 4, 8, or 16 Aal cells; there is no cell division from Aal to A1; however, there is a transformation while in the G0/G1 phase (de Rooij and van Dissel-Emiliani, 1997). Some controversy surrounds the As-based categorization of spermatogonia, and additional classification and nomenclature schemes exist. However, most concur with the As hypothesis of spermatogonia development (Russell et al, 1990). Throughout the remainder of this review, the As nomenclature will be used.</p><p>Various techniques have been suggested to distinguish As spermatogonia from spermatogonia committed to differentiation. Morphology is inadequate for this purpose. It has been asserted that undifferentiated (As-Aal) spermatogonia can be distinguished from differentiating spermatogonia (A1–4, Ain, B) because the latter cells will be in the G2 or M phase, while undifferentiated cells will not divide synchronously (de Rooij and van Dissel-Emiliani, 1997). Additionally, Apr and further differentiated spermatogonia form cellular bridges, which allow the sharing of gene products and facilitate synchronized development (Weber and Russell, 1987; Braun et al, 1989). SSCs do not have intercellular bridges. However, definitively determining that a cell lacks a bridge is a profound challenge, limited by tissue sectioning. Additionally, the capacity to distinguish As spermatogonia on the basis of the absence of intercellular bridges is limited, since it is known that some gonocytes possess intercellular bridges. There are methods to help identify the spermatogonial stage on the basis of topographical criteria (Huckins, 1971; Oakberg, 1971). However, these methods provide neither an efficient nor an effective means by which to distinguish the As or SSCs from other spermatogonia.</p><p>Lastly, spermatogonial density regulation takes place at the A2 through B spermatogonia (de Rooij and Lok, 1987). This phenomenon is influenced by programmed cell death and ensures that the number of differentiated germ cells does not exceed the organism's need (de Rooij and Grootegoed, 1998). When larger numbers of differentiated spermatogonia are present, degeneration occurs more frequently, thus reducing the cellular population. This apoptosis is similar to events occurring in somatic cells (Conlon and Raff, 1999).</p><p>In 1994, Brinster and Zimmerman published their landmark findings in the field of testicular tissue transplantation. Using a mixed cellular solution obtained from dissociated testicular parenchyma, they infiltrated recipient mouse seminiferous tubules with the donor cells. Among the hallmark findings of this experiment was the discovery that donor spermatogonial cells could interact with the host environment, migrate from the adluminal compartment, and negotiate past Sertoli-Sertoli tight junctions to enter the basal compartment (Griswold, 2000). Brinster and Zimmerman (1994) demonstrated successful donor spermatogenesis from testicular tissue transplanted between different mouse subjects. They used donor testicular tissue harvested from postnatal mice between days 4 and 12 of life. The assumption was that immature mice would have the highest concentration of undifferentiated spermatozoal progenitor cells or gonocytes, thereby providing the largest quantity of viable cells for transplantation. Testicular tissue was mechanically and enzymatically dissociated into a cellular suspension. The suspension was microinjected into mice pretreated with busulfan to eliminate native spermatogenesis. Donor cells came from transgenic mice expressing the LacZ (<i>Escherichia coli</i> B-galactosidase) gene; these cells, when differentiated to the round spermatid phase, stained blue, distinguishing them from the recipient's native sperm cells. The authors identified restored spermatogenesis in the recipient mouse along with colonization and differentiation of the donor tissue (Brinster and Zimmermann, 1994). Brinster and Avarbock in 1994 reported successful spermatogenesis in a mouse allogenic spermatogonial cell transplantation experiment. They found that the donor-derived spermatogonia were responsible for generating offspring; transmission was confirmed by the presence of a donor haplotype in the resulting progeny (Brinster and Avarbock, 1994).</p><p>Cryopreservation before transplantation was first described by Avarbock and colleagues (1996). They reported successful transplantation after freezing the donor tissue for up to 156 days. Clouthier and colleagues (1996) published the subsequent landmark investigation in testicular transplantation. In this investigation, rat testicular tissue was introduced into immunodeficient mouse testis. The transgenic rat tissue was identified in the mouse seminiferous tubules, and differentiated rat germinal tissue (including spermatozoa) was recovered from the mouse epididymis (Clouthier et al, 1996). Nagano and colleagues (1998) then demonstrated the capacity to culture spermatogonial cells in vitro, followed by testicular transplantation. Their study found that spermatogonia survived in culture for up to 4 months.</p><p>Further investigations found that the intraluminal transplanted germ cells degenerated and disappeared by 1 month's time. The successfully transplanted spermatogonia localized at the basement membrane and began to show evidence of division by the first week after transplantation. Donor spermatogonia migrated to the basal compartment during the first month, and donor spermatozoa were noted by that time (Parreira et al, 1998; Nagano et al, 1999).</p><p>The limits of spermatogonia transplantation were noted in more distant, xenogeneic transplantation experiments. Although limited colonization did occur with rabbit, monkey, bull, and human transplantation, no spermatozoa or postmeiotic germ cells were found after these transplantations. Schlatt and colleagues (1999a,b) transplanted germinal tissue in primates and found evidence of spermatogonial survival at 4 weeks. When bromodeoxyuridine (BrdU) was introduced into donor tissue before transplantation, immunostaining located cells in the interstitium and seminiferous tubules that were identified with the BrdU label in their nuclei at 4 weeks. Morphologic criteria indicated these were type B or differentiated spermatogonia (Schlatt et al, 1999a). In 2001, Nagano and colleagues demonstrated the transplantation of baboon testicular tissue into nude mice. In this investigation, the authors identified the survival and propagation of the transplanted cells for up to 6 months. They used a rabbit-produced anti-baboon antibody in conjunction with an anti-human antibody to identify the baboon cells. They noted that baboon cells had migrated to the basement membrane of the seminiferous tubules—indicating that mouse Sertoli cells had somehow interacted with the baboon spermatogonia and had allowed passage through the blood testis barrier. Despite this evidence of favorable interactions between the 2 tissue types, the baboon spermatogonia showed no signs of spermatogenesis (Nagano et al, 2001).</p><p>To date, successful donor-derived spermatogenesis has been primarily limited to phylogenetically similar species. In addition to mouse-to-mouse transplants, spermatogenesis has been noted in rat-to-immunodeficient mouse transfers (Clouthier et al, 1996), hamster-to-immunodeficient mouse transfers (Ogawa et al, 1999a), and mouse-to-rat transfers (Ogawa et al, 1999b). It has been theorized that evolutionary distance is primarily responsible for the failure of more distant xenogolous transplantations. Mice and rats are thought to have diverged evolutionarily 10 to 11 million years ago, and hamster and mice are thought to have diverged 16 million years ago (Catzeflis et al, 1993). Transplants with animals separated by greater evolutionary distances have been less successful. This is likely due to failed spermatogonia and Sertoli cell structural association and other functional interactions. Of note, Ogawa and colleagues (1999a) found that hamster spermatogenesis in mice resulted in morphologically defective hamster spermatozoa. This finding suggested that the recipient Sertoli cells (mouse) had influenced the final differentiation of the spermatozoa and that the species differences resulted in the morphologic errors in development. Despite morphologic dissimilarities among the various differentiated germinal tissues, transplantation between different species has uncovered much information in terms of functional similarities. The previously described cooperative interactions between the host testicular environment and the donor germinal tissue underscore these similarities.</p><p>Spermatogonial xenotransplantations using human tissue have resulted in inconsistent findings. Investigators have reported finding the survival of at least some undifferentiated spermatogonia during distant xenotransplantations. Reis et al (2000), however, reported that there was no evidence of donor tissue survival following a human-to-immunodeficient mouse testicular tissue transplantation. These investigators were using the antibody stain proacrosin to attempt to identify successful transplantation. Proacrosin is a marker of differentiated human spermatogonia (primary spermatocytes and spermatids) and would not have detected transplanted cells that had survived or propagated but not differentiated. On the other hand, Sofikitis et al (1999) reported successful spermatogenesis from xenotransplantation of human tissue into rat and mouse recipients. In 2002, Nagano and colleagues reported on the use of anti-baboon testes antibody to identify the survival of human spermatogonia in mouse recipients for up to 6 months posttransplantation. These investigators found no evidence of meiotic activity among the donor tissue. The wide range of outcomes following human-to-mouse testicular tissue transplantation warrants further investigation in this field. On the basis of the findings from other xenotransplantations, it is probable that complete spermatogonial differentiation will not be observed consistently. The large phylogenetic distance between species will likely translate into incompatibilities between the host testicular environment and donor spermatogonia that prohibit complete spermatogenesis.</p><p>Investigations of spermatogonia transplantations have provided great insight into the process of spermatogenesis and different disease states. Ogawa and colleagues (2000) found that c-kit-defective (white spotting W/W<sup>v</sup>) mice demonstrated restored fertility when receiving transplanted spermatogonia from stem cell factor-deficient (Steel Sl/Sl<sup>d</sup>) mice. C-kit, a receptor found on normal differentiated spermatogonia, has been associated with a variety of roles that spermatogonia can play, including situations in which substances such as mitogen (Rossi et al, 1993) and survival factor (Allard et al, 1996; Dirami et al, 1999) are involved.</p><p>Stem cell factor, a product of normal Sertoli cells, is the c-kit ligand (Schrans-Stassen et al, 1999). Findings by Ogawa and colleagues demonstrated that infertility resulting from a c-kit defect was a germ cell phenomenon. The testicular microenvironment in these animals, specifically the Sertoli cells, could still facilitate normal spermatogenesis despite never having previously supported differentiated spermatogonia. Additionally, this study showed that mutations affecting the stem cell factor, despite causing infertility, did not affect the differentiating capacity of spermatogonia when placed in a supportive environment.</p><p>The finding that mice W/W<sup>v</sup> could support spermatogenesis has broad implications. The Sertoli cells in these mice had no prior exposure to differentiated spermatogonia. However, they were still capable of supporting spermatogenesis from transplanted germ cells. This discovery provides credence to the use of testicular transplantation to restore fertility in cancer survivors. Select chemotherapies and radiotherapies result in a severe depopulation of germinal tissue in these patients. Like those of the W/W<sup>v</sup> subjects, the testicular microenvironments of these posttreatment patients consist primarily of Sertoli cells. Because Sl/Sl<sup>d</sup> spermatogonia flourished and underwent differentiation in the W/W<sup>v</sup> testicle, it is believed that, after treatment, many cancer survivors could still reinitiate spermatogenesis if viable spermatogonia were reintroduced into their testis.</p><p>Further discoveries regarding spermatogonial transplantation have come from investigations using <i>jsd</i>-mutant mice. Testicular tissue from these infertile mice, transplanted into mice with supportive intratesticular environments, did not result in donor-derived spermatogenesis (Boettger-Tong et al, 2000). This suggested that the <i>jsd</i> mutation prohibited differentiation via a germ cell defect and not from a defect in the supporting intratubular environment.</p><p>The factors determining the timing of spermatogenesis were further clarified by transplantation experiments in the Brinster laboratory. Normal mouse spermatogenesis takes place over about 35 days, while rat spermatogenesis is 52 to 53 days. The xenologous spermatogonial transplantations showed that spermatogenesis of the rat donor germ cells, despite being in a mouse testicular environment, still followed the normal rat timeline. About 52 days transpired before rat spermatozoa were fully developed (Franca et al, 1998). This finding showed that the timing of spermatogenesis is a spermatogonia-controlled event. Furthermore, although supported by the testicular somatic environment, spermatogenesis is not directed by it.</p><p>Testicular transplantation was used by Mahato and colleagues (2001) to investigate the role of estrogen receptor (ER)-alpha gene knockout on infertility. ER-alpha gene knockout mice are known to be infertile due to a failure of spermatogenesis (Eddy et al, 1996). When spermatogonia from ER-alpha knockout mice were transplanted into normal mice, donor-derived spermatozoa resulted. The identities of these spermatozoa were confirmed by the birth of progeny carrying the donor haplotype. This discovery demonstrated that ER-alpha was required for functional spermatogonial differentiation because of its influence on testicular stroma cells, not germinal cells (Mahato et al, 2001).</p><p>The transplantation process was modified as time proceeded to improve outcomes. The changes that facilitated improved host colonization by the donor spermatogonia included the following:</p><p>The most likely clinical application of testicular transplantation will involve prepubertal males facing systemic chemotherapy with sterilizing side effects. These individuals could undergo a pretreatment testicular biopsy. The extracted tissue could be used for SSC isolation and cryopreservation or cultured ex vivo. On completion of treatment with no evidence of recurrence, these patients could undergo an autotransplantation with their own cryopreserved or cultured spermatogonia. The subsequent repopulation of their testes with germinal tissue would result in spermatogenesis and fertility. While the above-described scenario may be technically feasible today or in the near future, there are numerous caveats that must be considered before clinical application.</p><p>Malignant diseases that are blood related, such as leukemia, sarcomas, and lymphomas, should be considered a great risk for reintroduction of malignancy into the patient. On the other hand, nonblood-related malignancies such as Hodgkin lymphoma may not pose a serious risk for patients undergoing autologous transplantations (Aslam et al, 2000). Jahnukainen and colleagues (2001) reported on the transmission of rat T-cell leukemia via testicular transplantation from diseased donors. These findings demonstrate the profound importance of developing accurate ex vivo spermatogonial isolation and quantification assays. The investigators noted that as few as 20 lymphoblastic cells introduced into the recipient testis were capable of transmitting acute leukemia into the healthy hosts (Jahnukainen et al, 2001).</p><p>Additional caution must be exhibited with the application of human xenogeneic transplantation; as a clinical adjunct, it is an unlikely prospect. In the past, porcine retroviruses have infected human kidney cells (Patience et al, 1997). Thus, the possibility of introducing a xenologous viral genotype into human germ line makes the risk of such clinical investigations imposing.</p><p>Among the topics requiring further research is the pretransplant preservation of human spermatogonial cells. Currently, there is no standardized cryopreservation protocol for human spermatogonia. The method employed for previous murine transplantations used dimethylsulphoxide (DMSO) as the cryoprotectant (Avarbock et al, 1996). There have also been reports of using ultrarapid freezing and/or vitrification to cryopreserve testicular tissue and SSCs. However, the lack of investigation in the area of SSC cryobiology is likely indicative of reduced success. It can be anticipated that future research on the cryobiology of SSCs will define optimal combinations of cryoprotectants and rate of temperature change during the freezing and thawing process. This will aid in making the entire process more efficient.</p><p>These caveats, as mentioned, must be systematically and experimentally addressed before SSC isolation, cryopreservation, and transplantation become a clinically feasible technology. For these reasons, continued basic studies of SSC biology are essential and require significant attention, considering the future translation and importance of these technologies in the preservation of reproductive potential in young male cancer survivors.</p>","PeriodicalId":15029,"journal":{"name":"Journal of andrology","volume":"26 4","pages":"442-450"},"PeriodicalIF":0.0000,"publicationDate":"2013-01-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.2164/jandrol.05062","citationCount":"25","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of andrology","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.2164/jandrol.05062","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 25

Abstract

The past 30 years have been marked by unparalleled accomplishments in the medical treatment of malignancy. Prior to advances in chemotherapeutic and radiation treatment, many oncologic conditions had dismal survival rates. Today, medical interventions have success rates that approach complete remission for many malignancies. An inadvertent complication of these therapies, however, has been the high rate of infertility following treatment. Male germinal tissue, like many malignancies, is mitotically active and therefore is particularly susceptible to the toxic effects of both chemotherapy and radiotherapy (Meistrich et al, 1982; Meistrich, 1993). Consequently, posttreatment patients often develop severe oligozoospermia or azoospermia (Wallace et al, 1991). Potential infertility complications can be anticipated, and adult male patients interested in future procreation are counseled to cryopreserve semen before instituting treatment. With present-day capabilities of in vitro fertilization, particularly intracytoplasmic injection, male patients can maintain posttreatment fertility. Pretreatment sperm banking, however, is not a viable option for prepubescent males. These individuals have not yet begun spermatogenesis and thus lack viable spermatozoa. It is estimated that, by the end of the decade, 1 in 250 young men will be childhood cancer survivors (Blatt, 1999). For these patients, infertility has often been an accepted consequence of their life-saving treatment.

A great deal of interest has recently been shown in testicular autologous transplantation, an intervention that may provide a future therapeutic fertility option for these individuals. Having been successfully demonstrated in rodent models, investigators have now begun to explore the possibility of using testicular autotransplantation to restore fertility in humans. This paper will review the history of spermatogonia transplantation with an emphasis on the clinical pertinence of this field of investigation. Current innovations involving the isolation of spermatogonial stem cells (SSCs) and the present capabilities of in vitro proliferation will additionally be reviewed.

Spermatogonia are male germinal progenitor cells and are composed of differentiated nonstem and stem cells. Stem cells are characterized by a capacity for self-renewal and an ability to produce differentiating cell lines (Loeffler and CS, 1997; van der Kooy and Weiss, 2000). Spermatogonia are diploid germ cells that originated from primordial germ cells (PGCs). These precursor cells originate from embryonal ectoderm. PGCs migrate to the genital ridge, where they become known as gonocytes. Gonocytes are surrounded by Sertoli precursor cells in what become the seminiferous cords. Tight junctions between adjacent Sertoli cells later become the basis of the blood testis barrier. The gonocytes undergo mitotic division, followed by arrest in the G0 phase of the cell cycle. They are mitotically inactive until after birth, when they become spermatogonia (Clermont and Perey, 1957; de Rooij and van Dissel-Emiliani, 1997).

Understanding spermatogonial nomenclature, differentiation, and regulation is important for comprehending testicular transplantation. First, distinguishing SSCs from differentiating spermatogonia has been a challenge that has been met with limited success. The term undifferentiated spermatogonia refers to As, Apr, and Aal cell types. Undifferentiated spermatogonia are considered distinct from “differentiating spermatogonia.” The latter group, ordered in succession, consists of A1, A2, A3, A4, Ain, and then B spermatogonia. It has been speculated that only As spermatogonia are stem cells that may divide into 2 identical daughter As spermatogonia or into 2 Apr (paired) daughter cells that are functionally committed to differentiation (Huckins, 1971). Apr cells differentiate into 4, 8, or 16 Aal cells; there is no cell division from Aal to A1; however, there is a transformation while in the G0/G1 phase (de Rooij and van Dissel-Emiliani, 1997). Some controversy surrounds the As-based categorization of spermatogonia, and additional classification and nomenclature schemes exist. However, most concur with the As hypothesis of spermatogonia development (Russell et al, 1990). Throughout the remainder of this review, the As nomenclature will be used.

Various techniques have been suggested to distinguish As spermatogonia from spermatogonia committed to differentiation. Morphology is inadequate for this purpose. It has been asserted that undifferentiated (As-Aal) spermatogonia can be distinguished from differentiating spermatogonia (A1–4, Ain, B) because the latter cells will be in the G2 or M phase, while undifferentiated cells will not divide synchronously (de Rooij and van Dissel-Emiliani, 1997). Additionally, Apr and further differentiated spermatogonia form cellular bridges, which allow the sharing of gene products and facilitate synchronized development (Weber and Russell, 1987; Braun et al, 1989). SSCs do not have intercellular bridges. However, definitively determining that a cell lacks a bridge is a profound challenge, limited by tissue sectioning. Additionally, the capacity to distinguish As spermatogonia on the basis of the absence of intercellular bridges is limited, since it is known that some gonocytes possess intercellular bridges. There are methods to help identify the spermatogonial stage on the basis of topographical criteria (Huckins, 1971; Oakberg, 1971). However, these methods provide neither an efficient nor an effective means by which to distinguish the As or SSCs from other spermatogonia.

Lastly, spermatogonial density regulation takes place at the A2 through B spermatogonia (de Rooij and Lok, 1987). This phenomenon is influenced by programmed cell death and ensures that the number of differentiated germ cells does not exceed the organism's need (de Rooij and Grootegoed, 1998). When larger numbers of differentiated spermatogonia are present, degeneration occurs more frequently, thus reducing the cellular population. This apoptosis is similar to events occurring in somatic cells (Conlon and Raff, 1999).

In 1994, Brinster and Zimmerman published their landmark findings in the field of testicular tissue transplantation. Using a mixed cellular solution obtained from dissociated testicular parenchyma, they infiltrated recipient mouse seminiferous tubules with the donor cells. Among the hallmark findings of this experiment was the discovery that donor spermatogonial cells could interact with the host environment, migrate from the adluminal compartment, and negotiate past Sertoli-Sertoli tight junctions to enter the basal compartment (Griswold, 2000). Brinster and Zimmerman (1994) demonstrated successful donor spermatogenesis from testicular tissue transplanted between different mouse subjects. They used donor testicular tissue harvested from postnatal mice between days 4 and 12 of life. The assumption was that immature mice would have the highest concentration of undifferentiated spermatozoal progenitor cells or gonocytes, thereby providing the largest quantity of viable cells for transplantation. Testicular tissue was mechanically and enzymatically dissociated into a cellular suspension. The suspension was microinjected into mice pretreated with busulfan to eliminate native spermatogenesis. Donor cells came from transgenic mice expressing the LacZ (Escherichia coli B-galactosidase) gene; these cells, when differentiated to the round spermatid phase, stained blue, distinguishing them from the recipient's native sperm cells. The authors identified restored spermatogenesis in the recipient mouse along with colonization and differentiation of the donor tissue (Brinster and Zimmermann, 1994). Brinster and Avarbock in 1994 reported successful spermatogenesis in a mouse allogenic spermatogonial cell transplantation experiment. They found that the donor-derived spermatogonia were responsible for generating offspring; transmission was confirmed by the presence of a donor haplotype in the resulting progeny (Brinster and Avarbock, 1994).

Cryopreservation before transplantation was first described by Avarbock and colleagues (1996). They reported successful transplantation after freezing the donor tissue for up to 156 days. Clouthier and colleagues (1996) published the subsequent landmark investigation in testicular transplantation. In this investigation, rat testicular tissue was introduced into immunodeficient mouse testis. The transgenic rat tissue was identified in the mouse seminiferous tubules, and differentiated rat germinal tissue (including spermatozoa) was recovered from the mouse epididymis (Clouthier et al, 1996). Nagano and colleagues (1998) then demonstrated the capacity to culture spermatogonial cells in vitro, followed by testicular transplantation. Their study found that spermatogonia survived in culture for up to 4 months.

Further investigations found that the intraluminal transplanted germ cells degenerated and disappeared by 1 month's time. The successfully transplanted spermatogonia localized at the basement membrane and began to show evidence of division by the first week after transplantation. Donor spermatogonia migrated to the basal compartment during the first month, and donor spermatozoa were noted by that time (Parreira et al, 1998; Nagano et al, 1999).

The limits of spermatogonia transplantation were noted in more distant, xenogeneic transplantation experiments. Although limited colonization did occur with rabbit, monkey, bull, and human transplantation, no spermatozoa or postmeiotic germ cells were found after these transplantations. Schlatt and colleagues (1999a,b) transplanted germinal tissue in primates and found evidence of spermatogonial survival at 4 weeks. When bromodeoxyuridine (BrdU) was introduced into donor tissue before transplantation, immunostaining located cells in the interstitium and seminiferous tubules that were identified with the BrdU label in their nuclei at 4 weeks. Morphologic criteria indicated these were type B or differentiated spermatogonia (Schlatt et al, 1999a). In 2001, Nagano and colleagues demonstrated the transplantation of baboon testicular tissue into nude mice. In this investigation, the authors identified the survival and propagation of the transplanted cells for up to 6 months. They used a rabbit-produced anti-baboon antibody in conjunction with an anti-human antibody to identify the baboon cells. They noted that baboon cells had migrated to the basement membrane of the seminiferous tubules—indicating that mouse Sertoli cells had somehow interacted with the baboon spermatogonia and had allowed passage through the blood testis barrier. Despite this evidence of favorable interactions between the 2 tissue types, the baboon spermatogonia showed no signs of spermatogenesis (Nagano et al, 2001).

To date, successful donor-derived spermatogenesis has been primarily limited to phylogenetically similar species. In addition to mouse-to-mouse transplants, spermatogenesis has been noted in rat-to-immunodeficient mouse transfers (Clouthier et al, 1996), hamster-to-immunodeficient mouse transfers (Ogawa et al, 1999a), and mouse-to-rat transfers (Ogawa et al, 1999b). It has been theorized that evolutionary distance is primarily responsible for the failure of more distant xenogolous transplantations. Mice and rats are thought to have diverged evolutionarily 10 to 11 million years ago, and hamster and mice are thought to have diverged 16 million years ago (Catzeflis et al, 1993). Transplants with animals separated by greater evolutionary distances have been less successful. This is likely due to failed spermatogonia and Sertoli cell structural association and other functional interactions. Of note, Ogawa and colleagues (1999a) found that hamster spermatogenesis in mice resulted in morphologically defective hamster spermatozoa. This finding suggested that the recipient Sertoli cells (mouse) had influenced the final differentiation of the spermatozoa and that the species differences resulted in the morphologic errors in development. Despite morphologic dissimilarities among the various differentiated germinal tissues, transplantation between different species has uncovered much information in terms of functional similarities. The previously described cooperative interactions between the host testicular environment and the donor germinal tissue underscore these similarities.

Spermatogonial xenotransplantations using human tissue have resulted in inconsistent findings. Investigators have reported finding the survival of at least some undifferentiated spermatogonia during distant xenotransplantations. Reis et al (2000), however, reported that there was no evidence of donor tissue survival following a human-to-immunodeficient mouse testicular tissue transplantation. These investigators were using the antibody stain proacrosin to attempt to identify successful transplantation. Proacrosin is a marker of differentiated human spermatogonia (primary spermatocytes and spermatids) and would not have detected transplanted cells that had survived or propagated but not differentiated. On the other hand, Sofikitis et al (1999) reported successful spermatogenesis from xenotransplantation of human tissue into rat and mouse recipients. In 2002, Nagano and colleagues reported on the use of anti-baboon testes antibody to identify the survival of human spermatogonia in mouse recipients for up to 6 months posttransplantation. These investigators found no evidence of meiotic activity among the donor tissue. The wide range of outcomes following human-to-mouse testicular tissue transplantation warrants further investigation in this field. On the basis of the findings from other xenotransplantations, it is probable that complete spermatogonial differentiation will not be observed consistently. The large phylogenetic distance between species will likely translate into incompatibilities between the host testicular environment and donor spermatogonia that prohibit complete spermatogenesis.

Investigations of spermatogonia transplantations have provided great insight into the process of spermatogenesis and different disease states. Ogawa and colleagues (2000) found that c-kit-defective (white spotting W/Wv) mice demonstrated restored fertility when receiving transplanted spermatogonia from stem cell factor-deficient (Steel Sl/Sld) mice. C-kit, a receptor found on normal differentiated spermatogonia, has been associated with a variety of roles that spermatogonia can play, including situations in which substances such as mitogen (Rossi et al, 1993) and survival factor (Allard et al, 1996; Dirami et al, 1999) are involved.

Stem cell factor, a product of normal Sertoli cells, is the c-kit ligand (Schrans-Stassen et al, 1999). Findings by Ogawa and colleagues demonstrated that infertility resulting from a c-kit defect was a germ cell phenomenon. The testicular microenvironment in these animals, specifically the Sertoli cells, could still facilitate normal spermatogenesis despite never having previously supported differentiated spermatogonia. Additionally, this study showed that mutations affecting the stem cell factor, despite causing infertility, did not affect the differentiating capacity of spermatogonia when placed in a supportive environment.

The finding that mice W/Wv could support spermatogenesis has broad implications. The Sertoli cells in these mice had no prior exposure to differentiated spermatogonia. However, they were still capable of supporting spermatogenesis from transplanted germ cells. This discovery provides credence to the use of testicular transplantation to restore fertility in cancer survivors. Select chemotherapies and radiotherapies result in a severe depopulation of germinal tissue in these patients. Like those of the W/Wv subjects, the testicular microenvironments of these posttreatment patients consist primarily of Sertoli cells. Because Sl/Sld spermatogonia flourished and underwent differentiation in the W/Wv testicle, it is believed that, after treatment, many cancer survivors could still reinitiate spermatogenesis if viable spermatogonia were reintroduced into their testis.

Further discoveries regarding spermatogonial transplantation have come from investigations using jsd-mutant mice. Testicular tissue from these infertile mice, transplanted into mice with supportive intratesticular environments, did not result in donor-derived spermatogenesis (Boettger-Tong et al, 2000). This suggested that the jsd mutation prohibited differentiation via a germ cell defect and not from a defect in the supporting intratubular environment.

The factors determining the timing of spermatogenesis were further clarified by transplantation experiments in the Brinster laboratory. Normal mouse spermatogenesis takes place over about 35 days, while rat spermatogenesis is 52 to 53 days. The xenologous spermatogonial transplantations showed that spermatogenesis of the rat donor germ cells, despite being in a mouse testicular environment, still followed the normal rat timeline. About 52 days transpired before rat spermatozoa were fully developed (Franca et al, 1998). This finding showed that the timing of spermatogenesis is a spermatogonia-controlled event. Furthermore, although supported by the testicular somatic environment, spermatogenesis is not directed by it.

Testicular transplantation was used by Mahato and colleagues (2001) to investigate the role of estrogen receptor (ER)-alpha gene knockout on infertility. ER-alpha gene knockout mice are known to be infertile due to a failure of spermatogenesis (Eddy et al, 1996). When spermatogonia from ER-alpha knockout mice were transplanted into normal mice, donor-derived spermatozoa resulted. The identities of these spermatozoa were confirmed by the birth of progeny carrying the donor haplotype. This discovery demonstrated that ER-alpha was required for functional spermatogonial differentiation because of its influence on testicular stroma cells, not germinal cells (Mahato et al, 2001).

The transplantation process was modified as time proceeded to improve outcomes. The changes that facilitated improved host colonization by the donor spermatogonia included the following:

The most likely clinical application of testicular transplantation will involve prepubertal males facing systemic chemotherapy with sterilizing side effects. These individuals could undergo a pretreatment testicular biopsy. The extracted tissue could be used for SSC isolation and cryopreservation or cultured ex vivo. On completion of treatment with no evidence of recurrence, these patients could undergo an autotransplantation with their own cryopreserved or cultured spermatogonia. The subsequent repopulation of their testes with germinal tissue would result in spermatogenesis and fertility. While the above-described scenario may be technically feasible today or in the near future, there are numerous caveats that must be considered before clinical application.

Malignant diseases that are blood related, such as leukemia, sarcomas, and lymphomas, should be considered a great risk for reintroduction of malignancy into the patient. On the other hand, nonblood-related malignancies such as Hodgkin lymphoma may not pose a serious risk for patients undergoing autologous transplantations (Aslam et al, 2000). Jahnukainen and colleagues (2001) reported on the transmission of rat T-cell leukemia via testicular transplantation from diseased donors. These findings demonstrate the profound importance of developing accurate ex vivo spermatogonial isolation and quantification assays. The investigators noted that as few as 20 lymphoblastic cells introduced into the recipient testis were capable of transmitting acute leukemia into the healthy hosts (Jahnukainen et al, 2001).

Additional caution must be exhibited with the application of human xenogeneic transplantation; as a clinical adjunct, it is an unlikely prospect. In the past, porcine retroviruses have infected human kidney cells (Patience et al, 1997). Thus, the possibility of introducing a xenologous viral genotype into human germ line makes the risk of such clinical investigations imposing.

Among the topics requiring further research is the pretransplant preservation of human spermatogonial cells. Currently, there is no standardized cryopreservation protocol for human spermatogonia. The method employed for previous murine transplantations used dimethylsulphoxide (DMSO) as the cryoprotectant (Avarbock et al, 1996). There have also been reports of using ultrarapid freezing and/or vitrification to cryopreserve testicular tissue and SSCs. However, the lack of investigation in the area of SSC cryobiology is likely indicative of reduced success. It can be anticipated that future research on the cryobiology of SSCs will define optimal combinations of cryoprotectants and rate of temperature change during the freezing and thawing process. This will aid in making the entire process more efficient.

These caveats, as mentioned, must be systematically and experimentally addressed before SSC isolation, cryopreservation, and transplantation become a clinically feasible technology. For these reasons, continued basic studies of SSC biology are essential and require significant attention, considering the future translation and importance of these technologies in the preservation of reproductive potential in young male cancer survivors.

精原干细胞的分离、储存和移植
过去30年来,恶性肿瘤的医学治疗取得了无与伦比的成就。在化疗和放射治疗取得进展之前,许多肿瘤疾病的生存率都很低。今天,医疗干预的成功率接近完全缓解许多恶性肿瘤。然而,这些疗法的一个无意的并发症是治疗后不孕症的高发生率。与许多恶性肿瘤一样,男性生发组织具有有丝分裂活性,因此特别容易受到化疗和放疗的毒性作用(mestrich等人,1982;Meistrich, 1993)。因此,治疗后患者往往会出现严重的少精症或无精症(Wallace et al ., 1991)。潜在的不育并发症是可以预见的,建议对未来生育感兴趣的成年男性患者在进行治疗前冷冻保存精液。随着目前体外受精的能力,特别是胞浆内注射,男性患者可以维持治疗后的生育能力。然而,预处理精子库对于青春期前的男性来说并不是一个可行的选择。这些个体尚未开始精子发生,因此缺乏可存活的精子。据估计,到本十年结束时,每250名年轻男子中将有1人是儿童癌症幸存者(Blatt, 1999年)。对于这些患者来说,不孕症通常是他们接受的挽救生命的治疗的结果。近年来,人们对睾丸自体移植表现出极大的兴趣,这种干预可能为这些个体提供未来治疗生育的选择。在啮齿类动物模型中成功证明了这一点后,研究人员现在开始探索使用睾丸自体移植来恢复人类生育能力的可能性。本文将回顾精原细胞移植的历史,重点介绍这一研究领域的临床意义。目前的创新涉及精原干细胞(SSCs)的分离和目前的体外增殖能力也将进行审查。精原细胞是男性生发祖细胞,由分化的非干细胞和干细胞组成。干细胞的特点是具有自我更新的能力和产生分化细胞系的能力(Loeffler和CS, 1997;van der Kooy and Weiss, 2000)。精原细胞是起源于原始生殖细胞的二倍体生殖细胞。这些前体细胞起源于胚胎外胚层。PGCs迁移到生殖嵴,在那里它们被称为性腺细胞。卵母细胞被支持前体细胞包围,形成精索。邻近的支持细胞之间的紧密连接后来成为血睾丸屏障的基础。性腺细胞经历有丝分裂,随后在细胞周期的G0期停止。它们在出生后成为精原细胞(Clermont and Perey, 1957;de Rooij and van Dissel-Emiliani, 1997)。了解精原细胞的命名、分化和调控对理解睾丸移植具有重要意义。首先,区分ssc和分化精原细胞一直是一个挑战,但取得的成功有限。未分化精原细胞是指As、Apr和Aal细胞类型。未分化精原细胞被认为不同于“分化精原细胞”。后一组,按顺序排列,包括A1, A2, A3, A4, Ain,然后B精原细胞。据推测,只有As精原细胞是干细胞,可以分裂成2个相同的子As精原细胞或2个Apr(配对)子细胞,并在功能上致力于分化(Huckins, 1971)。Apr细胞分化为4个、8个或16个Aal细胞;从Aal到A1没有细胞分裂;然而,在G0/G1阶段有一个转变(de Rooij和van Dissel-Emiliani, 1997)。围绕着基于a的精原细胞分类存在一些争议,并且存在其他分类和命名方案。然而,大多数人同意精原细胞发育的As假说(Russell et al, 1990)。在本综述的其余部分中,将使用As命名法。人们提出了各种技术来区分As精原细胞和分化精原细胞。形态学不足以达到这个目的。有人认为,未分化(As-Aal)精原细胞可以与分化精原细胞区分(A1-4, Ain, B),因为后者的细胞将处于G2或M期,而未分化的细胞不会同步分裂(de Rooij and van Dissel-Emiliani, 1997)。 在移植前将溴脱氧尿苷(BrdU)引入供体组织,免疫染色发现间质和精小管中的细胞在4周时细胞核中有BrdU标记。形态学标准表明这些是B型或分化精原细胞(Schlatt et al ., 1999a)。2001年,长野和他的同事演示了将狒狒睾丸组织移植到裸鼠体内。在这项研究中,作者发现移植细胞的存活和繁殖长达6个月。他们使用了一种兔子产生的抗狒狒抗体和一种抗人类抗体来识别狒狒细胞。他们注意到狒狒细胞已经迁移到精管的基底膜上,这表明小鼠的支持细胞以某种方式与狒狒的精原细胞相互作用,并允许通过血睾丸屏障。尽管有证据表明这两种组织类型之间存在有利的相互作用,狒狒的精原细胞却没有显示出精子发生的迹象(Nagano et al, 2001)。迄今为止,成功的供体精子发生主要局限于系统发育相似的物种。除了小鼠到小鼠的移植外,在大鼠到免疫缺陷小鼠的移植(Clouthier等人,1996年)、仓鼠到免疫缺陷小鼠的移植(Ogawa等人,1999a)和小鼠到大鼠的移植(Ogawa等人,1999b)中也发现了精子发生。从理论上讲,进化距离是导致更远的异种器官移植失败的主要原因。小鼠和大鼠被认为在1000万到1100万年前进化分化,仓鼠和小鼠被认为在1600万年前进化分化(Catzeflis et al, 1993)。动物之间进化距离越远,移植就越不成功。这可能是由于精原细胞和支持细胞结构关联和其他功能相互作用失败所致。值得注意的是,Ogawa及其同事(1999a)发现仓鼠精子在小鼠体内发生会导致仓鼠精子在形态上存在缺陷。这一发现表明,受体支持细胞(小鼠)影响了精子的最终分化,物种差异导致了发育过程中的形态错误。尽管各种分化的生发组织在形态上存在差异,但不同物种之间的移植在功能相似性方面揭示了许多信息。先前描述的宿主睾丸环境和供体生发组织之间的合作相互作用强调了这些相似性。使用人类组织的精原异种移植导致了不一致的结果。研究人员报告发现,在远距离异种移植中,至少有一些未分化的精原细胞存活下来。然而,Reis等人(2000)报告说,没有证据表明,在人-免疫缺陷小鼠睾丸组织移植后,供体组织存活。这些研究人员正在使用抗体染色原克rossin,试图确定成功的移植。Proacrosin是已分化的人精原细胞(原代精母细胞和精母细胞)的标记物,无法检测到存活或繁殖但未分化的移植细胞。另一方面,Sofikitis等人(1999)报道了人类组织异种移植在大鼠和小鼠受体中成功发生精子。2002年,Nagano和他的同事报道了使用抗狒狒睾丸抗体鉴定人类精原细胞在小鼠受体移植后存活长达6个月的情况。这些研究人员在供体组织中没有发现减数分裂活性的证据。人-小鼠睾丸组织移植的广泛结果值得在这一领域进一步研究。根据其他异种移植的发现,很可能不一致地观察到完全的精原细胞分化。物种之间较大的系统发育距离可能会导致宿主睾丸环境与供体精原细胞之间的不相容,从而禁止完整的精子发生。对精原细胞移植的研究为了解精子发生的过程和不同的疾病状态提供了很大的帮助。Ogawa及其同事(2000)发现,当接受来自干细胞因子缺乏(Steel Sl/Sld)小鼠的精原细胞移植后,c-kit缺陷(白色斑点W/Wv)小鼠表现出恢复的生育能力。C-kit是在正常分化的精原细胞中发现的一种受体,它与精原细胞的多种作用有关,包括有丝分裂原(Rossi et al ., 1993)和生存因子(Allard et al ., 1996;Dirami et al, 1999)。 干细胞因子是正常支持细胞的产物,是c-kit配体(schranss - stassen et al ., 1999)。Ogawa及其同事的研究结果表明,c-kit缺陷导致的不孕症是一种生殖细胞现象。这些动物的睾丸微环境,特别是Sertoli细胞,尽管以前从未支持过分化的精原细胞,但仍然可以促进正常的精子发生。此外,本研究表明,影响干细胞因子的突变尽管会导致不育,但在支持环境中并不影响精原细胞的分化能力。小鼠W/Wv可能支持精子发生的发现具有广泛的意义。这些小鼠的支持细胞没有事先暴露于分化的精原细胞。然而,它们仍然能够从移植的生殖细胞中支持精子发生。这一发现为使用睾丸移植来恢复癌症幸存者的生育能力提供了依据。选择性化疗和放疗导致这些患者的生发组织严重减少。与W/Wv受试者一样,这些治疗后患者的睾丸微环境主要由支持细胞组成。由于Sl/Sld精原细胞在W/Wv睾丸中发育旺盛并分化,因此人们认为,在治疗后,如果将有活力的精原细胞重新引入他们的睾丸,许多癌症幸存者仍然可以重新启动精子发生。关于精原移植的进一步发现来自对jsd突变小鼠的研究。将这些不育小鼠的睾丸组织移植到具有支持性睾丸内环境的小鼠中,没有导致供体来源的精子发生(Boettger-Tong et al, 2000)。这表明jsd突变通过生殖细胞缺陷阻止分化,而不是通过支持小管内环境的缺陷。布伦斯特实验室的移植实验进一步阐明了决定精子发生时间的因素。正常小鼠的精子发生时间约为35天,而大鼠的精子发生时间为52至53天。结果表明,大鼠供体生殖细胞尽管处于小鼠睾丸环境中,其精子发生仍遵循正常大鼠时间线。大鼠精子发育完全大约需要52天(Franca et al ., 1998)。这一发现表明精子发生的时间是一个精原细胞控制的事件。此外,尽管睾丸的体细胞环境支持精子的发生,但精子的发生并不是由它来指导的。Mahato及其同事(2001)利用睾丸移植研究了雌激素受体(ER) α基因敲除在不育中的作用。已知er - α基因敲除小鼠由于精子发生失败而不育(Eddy等,1996)。将er - α敲除小鼠的精原细胞移植到正常小鼠体内,产生供体来源的精子。携带供体单倍型的后代的出生证实了这些精子的身份。这一发现表明,er - α是功能性精原细胞分化所必需的,因为它影响睾丸基质细胞,而不是生发细胞(Mahato et al, 2001)。移植过程随着时间的推移而改变,以改善结果。促进供体精原细胞宿主定植的变化包括以下方面:睾丸移植最有可能的临床应用将涉及青春期前的男性,他们面临着全身化疗,并且有不育的副作用。这些个体可以进行预处理睾丸活检。提取的组织可用于SSC分离、低温保存或离体培养。在治疗结束且无复发迹象时,这些患者可以用自己冷冻保存或培养的精原细胞进行自体移植。随后用生发组织重新繁殖他们的睾丸将导致精子发生和生育。虽然上述情况在今天或不久的将来在技术上是可行的,但在临床应用之前必须考虑许多注意事项。与血液相关的恶性疾病,如白血病、肉瘤和淋巴瘤,应被认为是恶性肿瘤再次传入患者的巨大风险。另一方面,非血液相关的恶性肿瘤,如霍奇金淋巴瘤,可能不会对接受自体移植的患者构成严重风险(Aslam等,2000)。Jahnukainen及其同事(2001)报道了通过患病供体的睾丸移植传播大鼠t细胞白血病。这些发现证明了发展准确的离体精子分离和定量分析的深远重要性。 研究人员注意到,传入受体睾丸的淋巴母细胞只有20个,能够将急性白血病传播到健康宿主(Jahnukainen et al ., 2001)。在应用人类异种移植时必须格外谨慎;作为临床辅助疗法,它的前景不太可能。过去,猪逆转录病毒曾感染过人类肾细胞(Patience et al, 1997)。因此,将异种病毒基因型引入人类生殖系的可能性使这种临床研究的风险增加。需要进一步研究的课题之一是人精原细胞的移植前保存。目前,人类精原细胞还没有标准化的冷冻保存方案。以前的小鼠移植方法使用二甲基亚砜(DMSO)作为冷冻保护剂(Avarbock et al ., 1996)。也有报道使用超快速冷冻和/或玻璃化冷冻保存睾丸组织和ssc。然而,在SSC低温生物学领域缺乏研究可能表明成功率降低。可以预见,未来对SSCs的低温生物学研究将确定冷冻保护剂的最佳组合以及冻融过程中温度变化的速率。这将有助于使整个过程更有效率。如上所述,在SSC分离、冷冻保存和移植成为临床可行的技术之前,必须系统地和实验地解决这些问题。由于这些原因,考虑到这些技术在保存年轻男性癌症幸存者生殖潜力方面的未来翻译和重要性,继续进行SSC生物学的基础研究是必不可少的,需要得到极大的关注。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of andrology
Journal of andrology 医学-男科学
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