Successful intra- and interspecific male germ cell transplantation in the rat

The lumen of the seminiferous tubules has hitherto been regarded as an immunologically privileged site. We report here the birth of young following transplantation of stem spermatogonia from Long-Evans rats to the seminiferous tubules of Sprague-Dawley rats after treatment with the immunosuppressive agent cyclosporin. Follicle-stimulating hormone was also given to stimulate Sertoli cell proliferation, and testosterone to stimulate the recovery of spermatogenesis. Donor germ cells underwent normal spermatogenesis, and progeny were repeatedly produced from the donor germ cells as demonstrated by microsatellite paternity analysis. In addition, donor germ cells from the cryptorchid testes of LacZ mice were also able to colonize the seminiferous tubules of Sprague-Dawley rats using this protocol. Morphologically normal rat and mouse spermatozoa were present in the epididymis and vas deferens of the recipient rats. This highlights the potential for transplantation of male germ cells between different species.     

The making of "transgenic spermatozoa"

The processes of making transgenic animals by microinjecting DNA into the pronucleus of a fertilized oocyte or after the transfection of embryonic stem cells are now well established. However, attempts have also been made, with varying degrees of success, to use spermatozoa as a vector for transgenesis in mammals and other vertebrates during the last decade. A number of different approaches for making transgenic spermatozoa have been developed. These include directly incubating mature, isolated spermatozoa with DNA or pretreating mature, isolated spermatozoa before assisted fertilization. Microinjection procedures have also been established to transfect male germ cells directly in vivo within the seminiferous tubules or to reimplant previously isolated male germ cells submitted to in vitro transfection into a recipient testis. The latter two techniques present the advantage of being able to create transgenic progeny simply by mating with wild-type females, which avoids the possibility of interference or damage as a result of assisted fertilization or the manipulation of embryos. The different aspects of sperm-mediated transgenesis are presented.     

In vivo transfection of testicular germ cells and transgenesis by using the mitochondrially localized jellyfish fluorescent protein gene

We aimed to introduce foreign DNA into spermatogenic cells in the testis by injection of the DNA encoding jellyfish fluorescent proteins, green fluorescent protein (GFP) and yellow fluorescent protein (YFP) into the seminiferous tubules and in vivo electroporation. We obtained fluorescent spermatozoa only when using the gene of the YFP protein fused to a mitochondrial localization signal peptide. Intracytoplasmic injection into oocytes of these spermatozoa gave fluorescent fetuses and pups. Almost all of the individuals produced from fluorescent spermatozoa were transgenic. We confirmed integration of the gene into chromosomes and its transmission into offspring. This is the first report of gene transfer into germ cells and subsequent production of transgenic offspring.     

精子介导转基因动物的制备

近年来 ,通过显微注射DNA至孵育的卵母细胞原核或外源基因转染后的胚胎干细胞进行转基因动物的生产已取得了令人瞩目的成就。在过去的 1 0年中 ,以精子作载体制备转基因哺乳动物或脊椎动物也取得了一些不同程度的进展。这些技术主要包括 :直接将外源DNA与精子共孵育至成熟 ;提取分离的精子DNA或进行预处理至精子发育成熟 ;以及在辅助受精前分离精子细胞等。此外 ,一些显微注射技术 ,如在输精管内进行体内直接转染雄性生殖细胞 ;将体内转染的雄性生殖细胞植入已分离的雄性生殖细胞 ,再显微注射至受体的睾丸 ,这些技术也逐渐成熟起来。研究表明 ,通过体内、体外转染外源DNA的显微操作技术只需将雄性受体与野生型雌性交配就可产生出转基因的后代个体 ,同时也避免了辅助受精和胚胎操作带来的机械损伤 ,因此具有一定的优势。本文综述了精子介导转基因 (SMGT)技术的发展历程、研究现状及前沿进展。     

Restoration of spermatogenesis in infertile mice by Sertoli cell transplantation

The niche is considered to play an important role in stem cell biology. Sertoli cells are the only somatic cells in the seminiferous tubule that closely interact with germ cells to create a favorable environment for spermatogenesis. However, little is known about how Sertoli cells develop to form the male germ line niche. We report here that Sertoli cells recovered and dissociated from testes of donor male mice can be microinjected into recipient testes, form mature seminiferous tubule structures, and support spermatogenesis. Sertoli cells from perinatal donors had a dramatically greater capacity for generating seminiferous tubules than those from adult donors. Furthermore, transplantation of wild-type Sertoli cells into infertile Steel/Steel(dickie) testes created a permissive testicular microenvironment for generating spermatogenesis and spermatozoa. Thus, our results demonstrate that the male germ line stem cell niche can be transferred between animals. In addition, the technique provides a novel tool with which to analyze spermatogenesis and might provide a mechanism for correcting fertility in males suffering from supporting cell defects.     

Primate spermatogonial stem cells colonize mouse testes

In mice, transplantation of spermatogonial stem cells from a fertile male to the seminiferous tubules of an infertile recipient male results in progeny with donor-derived haplotype. Attempts to extend this approach by transplanting human testis cells to mice have led to conflicting claims that no donor germ cells persisted or that human spermatozoa were produced in the recipient. To examine this issue we used the baboon, a primate in which testis cell populations of several ages could be obtained for transplantation, and demonstrate that donor spermatogonial stem cells readily establish germ cell colonies in recipient mice, which exist for periods of at least 6 mo. However, differentiation of germ cells toward the lumen of the tubule and production of spermatozoa did not occur. The presence of baboon spermatogonial stem cells and undifferentiated spermatogonia in mouse seminiferous tubules for long periods after transplantation indicates that antigens, growth factors, and signaling molecules that are necessary for interaction of these cells and the testis environment have been preserved for 100 million years of evolutionary separation. Because germ cell differentiation and spermatogenesis did not occur, the molecules necessary for this process appear to have undergone greater divergence between baboon and mouse.     

Transgenic Stra8-EYFP pigs: a model for developing male germ cell technologies

The male germ line in mammals is composed of self-renewing cells, spermatogonia, the meiotic spermatocytes and spermiogenic spermatids. Identification of these cell stages in vitro has been problematic. Transgenic animals expressing a marker gene with a promoter specific to certain cell stages in the testis would be a useful approach to identifying these cells in a viable state. Towards this end, we have produced transgenic pigs expressing mitochondrial localized enhanced yellow fluorescent protein (EYFP-mito) under control of the germ cell specific Stimulated by Retinoic Acid 8 (Stra8) promoter. Stra8 has been shown to be expressed in pre-meiotic germ cells of mice. Twelve clones harboring the Stra8-EYFP-mito transgene were produced. Analysis by Western blot indicated that expression of the transgene was limited to testicular tissue in the transgenic pigs. Single cells and seminiferous tubules were cultured in vitro and subsequently examined with epifluorescent microscopy. Expression of EYFP was noted in cells cultured for up to 5 days. Both EYFP-mito and STRA8 antibodies were shown to bind and co-localize in seminiferous tubule cells in whole mounts and in histological sections. EYFP-mito in the transgenic pigs co-localized with the endogenous stem cell marker, NANOG. Expression of the Stra8-EYFP transgene in spermatogenic cells indicates that these pigs will be useful by providing labelled cells for use in such technologies such as germ cell transplantation and in vitro spermatogenic studies.     

Real-time observation of transplanted 'green germ cells': proliferation and differentiation of stem cells

To elucidate the mechanism of proliferation and differentiation of testicular germ cells, donor testicular germ cells labeled with enhanced green fluorescent protein (eGFP) were transplanted to recipient seminiferous tubules. The kinetics of colonization as well as of differentiation of the donor cells was followed in the same transplanted tubules (alive) under ultraviolet light. One week after transplantation, clusters of fluorescent cells were randomly spread as dots in the recipient seminiferous tubule, whereas non-homed cells flowed out from the testis to the epididymis. By 4 weeks after transplantation, green germ cells were observed with weak and moderate fluorescence along the recipient seminiferous tubule. By 8 weeks, proliferation and differentiation of the germ cells occurred, resulting in strong fluorescence in the middle part of the seminiferous tubule but in weak and moderate fluorescence at both terminals. The length of the fluorescent positive seminiferous tubule became longer. Detailed histological analyses of the recipient tubules indicated that the portions of the seminiferous tubule in weak, moderate, and strong fluorescence contained the spermatogonia, spermatogonia with spermatocytes, and all types of germ cells including spermatids, respectively. Thus, testicular stem cells colonized first as dots within 1 week, and then proliferated along the basement membrane of the seminiferous tubules followed by differentiation.     

利用雄性生殖细胞建立转基因动物

追溯了用雄性生殖细胞建立转基因动物的发展历程 ,系统阐述了本领域理论和实践的最新进展 ,围绕方法学逐渐改进和完善的过程 ,从利用精子和精原干细胞携带外源DNA两个方向展开 ,分析和评价了DNA转移方法与精子载体法结合、胞浆内单精子注射、蛋白连接的精子介导的基因转移、输精管注射法以及曲细精管显微注射法和精原干细胞移植法 6种实验设计方法。     

The histological changes in the testis, epididymis and prostate gland of the red-bellied tree squirrel, Callosciurus erythraeus, during the growth and reproductive cycle

The male reproductive glands of the red-bellied tree squirrel, Callosciurus erythraeus, in the infantile, and prepubertal males, as well as sexually functional, degenerating and redeveloping adults were studied histologically. In the infant, testes are characterized with solid seminiferous tubules filled with primordial germ cells and Sertoli cells. Interstitial cells are sparse. The prostate is composed of condensed cell cords grouped into lobules dispersed with interlobular tissues rich in fibroblasts. In the epididymis the highly convoluted tubule is lined with a simple cuboidal or columnar epithelium and thin smooth musculature without. In the prepubertal male, germ cells are engaged actively in mitosis. Primary spermatocytes are readily recognized. Leydig cells appear in groups in the interstitial tissue. In the prostate, cell cords become highly branched and collecting tubules make their appearance. The tubules in the epididymis are enlarged in diameter but their peripheral musculature becomes thinner. In functional males, meiosis is active and bundles of spermatozoa are scattered along the central lumen. Leydig cells have their cytoplasm highly enriched. The prostate is in the secretory phase. The tubule in the epididymis is filled with sperm. In the degenerating adult, meiosis is interrupted and necrotic germ cells are detached from germinal epithelium. In the prostate, secretory and collecting ducts are eventually reduced to condensed lobules separated by interlobular fibrous tissue. The tubule in the epididymis often fills with necrotic germ cells but no sperm. In the redeveloping adult, the histology of the testes, prostate and epididymis is similar to that of the prepubertal male. However, there is more fibrous tissue in the interlobular septa in the prostate gland and thick musculature at the periphery of the tubule in the epididymis.     

Germ cell transplantation in goats

Transplantation of spermatogonial stem cells provides a unique approach for the study of spermatogenesis and manipulation of the male germ line. This technique may also offer an alternative to the currently inefficient methods of producing transgenic domestic animals. We have recently established the technique of spermatogonial transplantation, originally developed in laboratory rodents, in pigs, and this study was aimed to extend the technique to the goat. Isolated donor testis cells were infused into the seminiferous tubules of anesthetized recipient goats through an ultrasonographically-guided catheter inserted into the rete testis. Donor cells were obtained by enzymatic digestion of freshly collected testes from immature goats (either from the recipients' contralateral testis or from unrelated donors). Prior to transplantation, testis cells were labeled with a fluorescent marker to allow identification after transplantation. Recipient testes were examined for the presence and localization of labeled donor cells at 3-week intervals up to 12 weeks after transplantation. Labeled donor cells were found in the seminiferous tubules of all testes, comprising 10-35% of the examined tubules. Histological examination of the recipient testes did not reveal evident tissue damage, except for limited fibrotic changes at the site of needle insertion. Likewise there were no detectable local or systemic signs of immunologic reactions to the transplantations. These results indicate that germ cell transplantation is technically feasible in immature male goats and that donor-derived cells are retained in the recipient testis for at least three months and through puberty. This study represents the first report of germ cell transplantation in goats.     

Spermatogonial stem cell transplantation, cryopreservation and culture.

Testis cells of a fertile male mouse can be transplanted to the seminiferous tubules of an infertile male, where the donor spermatogonial stem cells will establish spermatogenesis and produce spermatozoa that transmit the donor haplotype to progeny. In addition, stem cells can be cryopreserved for long periods, thereby making male germ lines immortal. Recently, mouse testis cells have been cultured for longer than 3 months and, following transplantation, produced spermatogenesis. These techniques are likely to be applicable to many species, since rat testis cells can be cryopreserved and generate spermatogenesis in the seminiferous tubules of immunodeficient mice.     

Plasmid DNA adsorbed to pH-sensitive liposomes efficiently transforms the target cells

We have previously reported that plasmid DNA entrapped in the pH-sensitive immunoliposomes effectively transforms the target cells (Proc. Natl. Acad. Sci. USA, in press). In the present study, we demonstrate that DNA adsorbed on the same liposome also transforms the target cells. The transformation activity is antibody dependent, as liposomes containing no targeting antibody had reduced activity. The activity could be significantly inhibited by excess non-specific DNA (salmon sperm). Since some DNA are likely adsorbed to the liposomes during the entrapment process, the activity of the entrapped DNA is partially accounted for by the adsorbed DNA. The possibility of developing a simple DNA-mediated transfection protocol using liposome adsorbed DNA is discussed.     

Expression pattern of asialoglycoprotein receptor in human testis

During acute or chronic hepatitis B virus (HBV) infection, the virus can invade the male reproductive system, pass through the blood–testis barrier and integrate into the germ line, resulting in abnormal spermatozoa. However, the pathway remains unclear. The asialoglycoprotein receptor (ASGR), a potential receptor for HBV, is mainly distributed in hepatocytes. We have examined the distribution of ASGR in human testis and found it in the seminiferous tubules and interstitial region but its enrichment in human testis is much lower than that in liver. By multiple immunoenzyme histochemistry staining, ASGR was precisely co-localized with vimentin (Sertoli cell marker) but not proliferating cell nuclear antigen (spermatogonial cell marker) in testis tissue. ASGR was expressed in human Leydig cells, stromal cells in the seminiferous tubules and Sertoli cells but seldom in spermatogonial cells. Therefore, ASGR could provide HBV with access to the luminal compartment of human testis. The mechanism by which HBV invades germ cells remains unknown.     

Radioautographic demonstration of the methods of incorporation of tritiated leucine into germ cells of the testis incubated in a modified Rose chamber

The influence of the blood-testis barrier on the synthetic capabilities of male germ cells in the mouse as investigated by quantitative radioautography in aggregates of seminiferous tubules incubated in a modified Rose chamber, in the presence of tritiated leucine. In the seminiferous tubule, a distribution gradient of radioactivity could be seen : radioactivity decreased from the periphery towards the lumen. Labelling intensity was approximately equivalent in type B spermatogonia and pachytene primary spermatocytes but was only one third as heavy in spermatids (steps 4-5). These results which confirm prior observations carried out in both animal and germ cells isolated from the seminiferous epithelium by differential sedimentation velocity indicate that the blood-testis barrier has no significant effect on the capacity for synthesis peculiar to each germ-cell population.     

Regulation of proliferation and differentiation in spermatogonial stem cells: the role of c-kit and its ligand SCF

To study self-renewal and differentiation of spermatogonial stem cells, we have transplanted undifferentiated testicular germ cells of the GFP transgenic mice into seminiferous tubules of mutant mice with male sterility, such as those dysfunctioned at Steel (Sl) locus encoding the c-kit ligand or Dominant white spotting (W) locus encoding the receptor c-kit. In the seminiferous tubules of Sl/Sl(d) or Sl(17H)/Sl(17H) mice, transplanted donor germ cells proliferated and formed colonies of undifferentiated c-kit (-) spermatogonia, but were unable to differentiate further. However, these undifferentiated but proliferating spermatogonia, retransplanted into Sl (+) seminiferous tubules of W mutant, resumed differentiation, indicating that the transplanted donor germ cells contained spermatogonial stem cells and that stimulation of c-kit receptor by its ligand was necessary for maintenance of differentiated type A spermatogonia but not for proliferation of undifferentiated type A spermatogonia. Furthermore, we have demonstrated that their transplantation efficiency in the seminiferous tubules of Sl(17H)/Sl(17H) mice depended upon the stem cell niche on the basement membrane of the recipient seminiferous tubules and was increased by elimination of the endogenous spermatogonia of mutant mice from the niche by treating them with busulfan.     

Spontaneous degeneration of germ cells in normal rat testis: assessment of cell types and frequency during the spermatogenic cycle.

The occurrence of degenerating germ cells in the cycle of the seminiferous epithelium was measured in testicular tissues from eight normal adult rats. Testes were perfusion fixed, embedded in epoxy resin and, after sectioning a total of 180 randomly selected blocks at 1 microns, stained sections were examined by light microscopy; all cross-sectioned seminiferous tubules were categorized into one of 14 stages of the spermatogenic cycle. The number of degenerating cells per tubule was recorded in 2103 tubules. Degenerating germ cells were not detected at stages II-VI, and only rarely at stage VII (n = 366 tubules) in which one primary spermatocyte and one step 19 spermatid degenerated. All other stages exhibited a greater incidence of degenerative germ cells, particularly at stage XIV where, on average, the frequency of degenerating cells per round seminiferous tubule was about 40 times greater than at stage VII. The results indicated that, in the normal adult rat testis, the germ cells are least at risk of degeneration as they pass through stage VII.     

Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.     

Expression of L-histidine decarboxylase in mouse male germ cells

Histamine synthesis in male reproductive tissues remains largely unknown. The interaction between stem cell factor and its receptor, c-Kit, has been found to be essential for the maturation of male germ cells and peripheral mast cells. Based on this analogy, we investigated the expression of histidine decarboxylase (HDC), the rate-limiting enzyme of histamine synthesis, in mouse male germ cells. Immunohistochemical analyses revealed that HDC is localized in the acrosomes of spermatids and spermatozoa. In the testis, epididymis, and spermatozoa, a significant amount of histamine and HDC activity were detected. W/W(V) mice, known to lack most of their germ cells in the seminiferous tubules, were found to lack HDC protein expression as well as HDC activity in the testis. An in vitro acrosome reaction induced by a calcium ionophore, caused the release of histamine from epididymal spermatozoa. Our observations indicate that histamine is produced in and released from the acrosomes.