Manipulation of fish germ cell: visualization, cryopreservation and transplantation

Germ-cell transplantation has many applications in biology and animal husbandry, including investigating the complex processes of germ-cell development and differentiation, producing transgenic animals by genetically modifying germline cells, and creating broodstock systems in which a target species can be produced from a surrogate parent. The germ-cell transplantation technique was initially established in chickens using primordial germ cells (PGCs), and was subsequently extended to mice using spermatogonial stem cells. Recently, we developed the first germ-cell transplantation system in lower vertebrates using fish PGCs and spermatogonia. During mammalian germ-cell transplantation, donor spermatogonial stem cells are introduced into the seminiferous tubules of the recipient testes. By contrast, in the fish germ-cell transplantation system, donor cells are microinjected into the peritoneal cavities of newly hatched embryos; this allows the donor germ cells to migrate towards, and subsequently colonize, the recipient genital ridges. The recipient embryos have immature immune systems, so the donor germ cells can survive and even differentiate into mature gametes in their allogeneic gonads, ultimately leading to the production of normal offspring. In addition, implanted spermatogonia can successfully differentiate into sperm and eggs, respectively, in male and female recipients. The results of transplantation studies in fish are improving our understanding of the development of germ-cell systems during vertebrate evolution.     

Biological characteristics of fish germ cells and their application to developmental biotechnology

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.     

Migration and differentiation of gonadal germ cells under cross-sex germline chimeras condition in domestic chickens

A series of experiments was conducted to investigate migration, proliferation and differentiation of gonadal germ cells (GGCs) collected from the gonads of 7-day-old chick embryos under cross-sex germline chimera conditions. The migratory and proliferative abilities of exogenous GGCs were examined by transferring 50 fluorescently labeled GGCs collected from White Leghorn (WL) embryos into the blood of 2-day-old Rhode Island Red (RIR) embryos. No significant difference was observed in the number of fluorescently labeled GGCs in the gonads of recipient embryos among any of the four possible donor and recipient sex combinations. Cross-sex germline chimeras were produced to examine the differentiation of GGCs by transferring 100 GGCs from WL embryos into 2-day-old RIR embryos. Exogenous-GGC-derived progeny were obtained from both male and female recipients, except when female GGCs were transferred into male recipients. The migratory ability of GGCs recovered from the 7-day-old embryonic gonad was not influenced by cross-sex germ cell transfer conditions, whereas the differentiation of the GGCs was affected by the sex combinations of GGCs donors and recipients.     

Male Germ Cell Transplantation

Transplantation of male germ line stem cells from a donor animal to the testes of an infertile recipient was first described in 1994. Donor germ cells colonize the recipient's testis and produce donor-derived sperm, such that the recipient male can distribute the genetic material of the germ cell donor. Germ cell transplantation represents a functional reconstitution assay for male germ line stem cells and as such has vastly increased our ability to study the biology of stem cells in the testis and define phenotypes of infertility. First developed in rodents, the technique has now been used in a number of animal species, including domestic mammals, chicken and fish. There are three major applications for this technology in animals: first, to study fundamental aspects of male germ line stem cell biology and male fertility; second, to preserve the reproductive potential of genetically valuable individuals by male germ cell transplantation within or between species; third, to produce transgenic sperm by genetic manipulation of isolated germ line stem cells and subsequent transplantation. Transgenesis through the male germ line has tremendous potential in species in which embryonic stem cells are not available and somatic cell nuclear transfer has limited success. Therefore, transplantation of male germ cells is a uniquely valuable approach for the study, preservation and manipulation of male fertility in animals.     

Ultrasound Guided Transplantation of Enriched and Cryopreserved Spermatogonial Cell Suspension in Goats

Spermatogonial stem cells transplantation provides a unique approach for studying spermatogenesis. Initially developed in mice, this technique has now been extended in farm animals and provides an alternative means to preserve valuable male germ line and to produce transgenic animals. The aim of this study was to enrich type A spermatogonial cells amongst the isolated cells from goat testis, to cryopreserve these enriched populations of cells and their subsequent transplantation in unrelated recipient goats under ultrasound guidance. The cells were isolated enzymatically and enriched by differential plating and separation on discontinuous percoll gradient. Ultrasound guided injection of trypan blue dye into rete testis resulted in 20–30% filling of the seminiferous tubules. Prior to transplantation, the cells were labelled with a fluorescent dye to trace donor cells in recipient seminiferous tubules after transplantation. The fluorescent‐labelled cells were observed up to 12 weeks after transplantation.     

Birth of normal mice following round spermatid injection without artificial oocyte activation

For fertilization using round spermatid injection (ROSI) in mice, oocytes need to be artificially preactivated because of the lack of oocyte-activating capacity in round spermatids of this species. However, when round spermatids were frozen-thawed before microinjection, 11-71% of injected oocytes developed into 2-cell embryos without any artificial activation. After being transferred into recipient females, 5-27% of these embryos reached term. At least some of the injected oocytes showed intracellular Ca(2+) oscillations, which normally occur after fertilization by mature spermatozoa. Thus, these round spermatids could transmit a sperm-borne oocyte-activating factor, which might have been released from spermatozoa and elongated spermatids in the same suspension by freezing and thawing. This possibility was further supported by activation of intact oocytes following transplantation of the pronuclei from ROSI-generated embryos. Thus, one-step ROSI can be achieved in mice simply by injecting frozen-thawed round spermatids into intact oocytes. Clearly, there is a need for careful interpretation of microinjection experiments when assessing the oocyte-activating capacity of spermatogenic cells, especially when they are derived from frozen-thawed stocks.     

Effects of Intratesticular Injection of 70% Glycerin on Stallions

The removal of endogenous germ cells of recipient stallions is a key step to produce donor germ cell-derived sperm using the germ cell transplantation technique. Six Thoroughbred stallions were divided into a treatment (n = 3) and a control group (n = 3), and 70% glycerin (1, 2, 3-trihydroxypropane, 40 mL per testis) or phosphate-buffered saline, respectively, was locally injected into testes. General semen evaluation, libido, and testicular volume were performed weekly from 3 weeks before to 10 weeks after treatment. The number of round germ cells in the ejaculate was counted using a hemocytometer. The hematoxylin and eosin staining was performed on the cross sections of testicular tissue obtained 11th week of treatment. Plasma testosterone levels in blood collected weekly were measured using a colorimetric competitive enzyme immunoassay kit. The sperm number was significantly lower than that of the control group at 5 and 10 weeks after glycerin injection. No differences in the status of spermatogenesis in the cross sections of seminiferous tubules and testicular volume were found between the two groups. The 70% glycerin-treated stallions had reduced total and progressively motile sperm and exhibited a significantly higher population of round germ cells in the ejaculate. Testosterone levels, testicular volumes, and libido of stallions were not significantly different between the groups. In conclusion, although intratesticular injection of 70% glycerin may have caused disassociation of some germ cells in the seminiferous tubules for several weeks, it did not significantly ablate germ cells in the tubules at 11 week in stallions.     

Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR

1. The present study was carried out to determine whether primordial germ cells isolated from embryonic blood can enter the bloodstream and successfully migrate to the germinal ridges of recipient embryos after transfer to stage X blastoderms, and also whether they can differentiate into blood cells, as is suggested in mice. 2. Primordial germ cells were transfected in vitro by lipofection and then transferred to stage X blastoderms. The introduced GFP gene was efficiently expressed in the gonads of 6-d incubated embryos. 3. Freshly collected primordial germ cells were transferred to stage X blastoderms. The fate of the transferred primordial germ cells was traced by detecting the single nucleotide polymorphism in the D-loop region of the mitochondrial DNA in White Leghorn and Barred Plymouth Rock chickens used in this study. The transferred donor primordial germ cell-derived cells were detected in the gonads, but not in the blood cells, of 17-d incubated embryos by PCR. 4. This procedure for primordial germ cell manipulation could provide a novel method of producing germline chimaeric chickens. 5. In conclusion, our findings indicate that primordial germ cells isolated from embryonic blood can migrate to the germinal ridges of recipient embryos after being transferred to stage X blastoderms. Although these transferred primordial germ cells differentiated into germ cells, no differentiation into blood cells was observed.     

Histological Characterization of Defective Spermatogenesis in Mice Lacking the Basigin Gene

Basigin is a transmembrane protein belonging to the immunoglobulin superfamily. In the light of the fact that knockout mice lacking the basigin gene (Bsg) are azoospermic, the phenotype in the male reproductive system was extensively examined in this study. Spermatogenesis in Bsg (-/-) mice was found to be disrupted, and arrested at the metaphase of the first meiotic division. A few germ cells differentiated into young spermatids, but they were exfoliated. The lumens of the male reproductive system were filled with round degenerated cells. Using the TUNEL method and electron microscopy, some of the degenerated cells in the testis and epididymal head were shown to be apoptotic. Crystalloids of fine tubules and unusual ectoplasmic specializations were also observed in the Sertoli cells of Bsg (-/-) mice. These specializations displayed unusual 'circular' structures. Furthermore, unusual ectoplasmic specializations covering the spermatocytes rather than the mature spermatids were found. These structures were formed as a result of the lack of mature spermatids in the Bsg (-/-) testis. Results from analyses of azoospermia in the Bsg (-/-) mice suggest that basigin, through the interactions between germ cells and Sertoli cells, is an essential factor in the growth and/or survival of spermatids.     

Formation of male and female sex cords in gonadal development of C57BL/6 mouse

The sex cords of male and female fetal C57BL/6 mice were studied by light and transmission electron microscopy to elucidate the origins of Sertoli cells (male) and follicle cells (female) in detail. In the testes of fetal mice from day 12 to day 14 post coitum (p.c.), PAS-positive substances were detected exclusively throughout the cytoplasm of Sertoli cells. On day 12 p.c., pre-Sertoli cells, identified by PAS-reaction, formed irregular and cord-like arrangements around germ cells. The arrangements were not associated with the coelomic epithelium. Thus, it is suggested that Sertoli cells are originated from the mesonephric tissues. In the ovaries, germ cell cords were still not observed at a stage immediately following the gonadal sex differentiation in male. On about day 15 p.c., connective tissues including many capillaries penetrated into the ovaries, resulting in obvious formation of germ cell cord-like arrangements. At the same time, the coelomic epithelium-derived cells (CEd cells) as well as the mesonephros-derived cells (Md cells) invaded the adjacent arrangements. It is concluded that the follicle cells are originated most from Md cells and some from CEd cells.     

Microinsemination with first-wave round spermatids from immature male mice

In several mammalian species, including mice, round spermatids have been used to produce normal offspring by means of microinsemination techniques. In this study, we examined whether mouse round spermatids retrieved from immature testes undergoing the first wave of spermatogenesis had acquired fertilizing ability comparable to cells from mature adults. Microinsemination with round spermatids was performed by direct injection into preactivated oocytes, as previously reported. About 60-85% of the successfully injected oocytes developed to the morula/blastocyst stage after 72 h in culture, irrespective of the age of the males (17-25 days old). After embryo transfer, normal pups were obtained from all age groups, including the day-17 group, the stage at which the first round spermatids appeared. A high correlation (r=0.90) was found between the birth rate and male age (P<0.01, Spearman rank correlation), indicating that the efficiency of producing offspring was dependent on the age of the donor males. Imprinted genes (H19, Igf2, Meg3, and Igf2r) were expressed from the correct parental alleles (maternal, paternal, maternal, and maternal, respectively) in all (n=12) day-9.5 fetuses derived from day-20 spermatids. These results clearly indicate that at least some first-wave spermatogenic cells have a normal haploid genome with the correct paternal imprint and are capable of supporting full-term embryo development, as do mature spermatozoa from adults. The use of male germ cells from immature animals may save time in the production of inbred/congenic strains and rescue male-factor infertility of early onset.     

Effect of the strain combination of the donor and recipient on the production efficiency of W‐bearing sperm in mixed‐sex germline chimeric chickens

Effect of the strain combination of the donor and recipient on production efficiency of W‐bearing sperm in mixed‐sex chimeric testes was analyzed. The combinations of the donors and recipients were White Leghorn (WL) and Rhode Island Red (RIR), and vice versa. Generated mixed‐sex chimeras that had the male phenotype at sexual maturity were classified into four groups: (1) a female WL donor and a male RIR recipient; (2) a male WL donor and a female RIR recipient; (3) a female RIR donor and a male WL recipient; (4) a male RIR donor and a female WL recipient. The mean number of W‐bearing sperm detected by in situ hybridization among 10 000 sperm observed were 147, 165, 30 and 45 in groups 1, 2, 3 and 4, respectively. The numbers in groups 1 and 2 were both significantly higher than those of groups 3 and 4 (P < 0.05). The combination of a WL donor and a RIR recipient produced W‐bearing sperm more efficiently than the reverse combination.     

Ectopic Hermaphroditic Gonad in the Cranial Cavity of Japanese Medaka

Abstract

Diseases were monitored for 1 year in a cohort of approximately 560 Japanese medakas Oryzias latipes kept in a laboratory at Tulane University. The fish were maintained as part of a breeding colony for embryological studies. Eighty-six fish exhibiting disease or injury during the year were examined histopathologically. Two adult Japanese medakas (one male and one female) had ectopic hermaphroditic gonads in the cranial cavity. The ectopic gonads were bordered by meninges and cranium. The ectopic gonads in both cases consisted mostly of spermatocytes and spermatids with numerous islands of primary germ cells and a few ovarian follicles. An ectopic gonad in the cranial cavity has not been previously reported in vertebrates.     

Embryo production by intracytoplasmic injection of sperm retrieved from Meishan neonatal testicular tissue cryopreserved and grafted into nude mice

Testicular xenografting, combined with cryopreservation can assist conservation of the genetic diversity of indigenous pigs by salvaging germ cells from their neonatal testes. Using Meishan male piglets as an example, we examined whether testicular tissue would acquire the ability to produce sperm after cryopreservation and grafting into nude mice (MS group). For comparison, testicular tissue from neonatal Western crossbreed male piglets was used (WC group). Sixty days after xenografting (day 0 = grafting), MS grafts had already developed seminiferous tubules containing sperm, whereas in the WC grafts, sperm first appeared on day 120. The proportion of tubules containing spermatids and sperm was higher in the MS group than in the WC group between days 90 and 120. Moreover, in vitro‐matured porcine oocytes injected with a single sperm obtained from the MS group on day 180 developed to the blastocyst stage. The blastocyst formation rate after injection of the xenogeneic sperm was 14.6%, whereas the ratio in the absence of such injection (attributable to parthenogenesis) was 6.7%. Thus, cryopreserved Meishan testicular tissue acquired spermatogenic activity in host mice 60 days earlier than Western crossbreed tissue. Such xenogeneic sperm are likely capable of generating blastocysts in vitro.     

Possible Role of ZPAC,Zygote-specific Proteasome Assembly Chaperone,During Spermatogenesis in the Mouse

In the mammalian testis, the ubiquitin-proteasome system plays important roles in the process that promotes the formation of mature sperm. We recently identified zygote-specific proteasome assembly phaperone (ZPAC), which is specifically expressed in the mouse gonads and zygote. ZPAC mediates a unique proteasome assembly pathway in the zygote, but the expression profile and function of ZPAC in the testis is not fully understood. In this study, we investigated the possible role of ZPAC during mouse spermatogenesis. First, we analyzed the expression of ZPAC and 20S proteasome subunit α4/PSMA7 in the adult mouse testis. ZPAC and α4 were expressed in spermatogonia, spermatocytes, and round spermatids. In elongating spermatids, ZPAC was expressed until step 10, whereas expression of α4 persisted until step 12. We then examined the expression profile of ZPAC and α4 in a mouse model of experimental unilateral cryptorchidism. Consistent with appearance of morphologically impaired germ cells following cryptorchidism, the ZPAC protein level was significantly decreased at 4 days post induction of experimental cryptorchidism (D4) compared with the intact testis, although the amount of α4 protein persisted at least until D10. Moreover, intense ZPAC staining was co-localized with staining of annexin V, an early indicator of apoptosis in mammalian cells, in germ cells of cryptorchid testis, but ZPAC was also expressed in germ cells showing no detectable expression of annexin V. These results suggest that ZPAC plays a role during spermatogenesis and raises the possibility that 20S proteasome mediated by ZPAC may be involved in the regulation of germ cell survival during spermatogenesis.     

Synthesis, processing, and subcellular localization of mouse ADAM3 during spermatogenesis and epididymal sperm transport

To elucidate synthesis, processing, and subcellular localization of mouse ADAM3 (cyritestin) during spermatogenesis and epididymal sperm transport, we carried out immunoblotting and immunohistochemical analysis of testicular germ cells, and epididymal and vas deferens sperm, using affinity-purified anti-ADAM3 antibody. ADAM3 was initially synthesized as a 110-kDa precursor in round spermatids, and the precursor was then processed into a 42-kDa mature protein during the sperm transport into and/or once in the epididymis. The mature ADAM3 was localized on the anterior part of capacitated sperm heads and was rapidly removed from the head region during the calcium ionophore A23187-induced acrosome reaction. These results demonstrate that the mature form of ADAM3 is involved in the binding of sperm to the egg zona pellucida, not in the membrane fusion between sperm and egg.     

Production of germ-line chimeras by the transfer of cryopreserved gonadal germ cells collected from 7- and 9-day-old chick embryos

Gonadal germ cells (GGC) were collected from the gonads of 7‐ or 9‐day‐old White Leghorn chick embryos and suspended in freezing medium containing 10% dimethylsulfoxide (DMSO). The cell suspension was frozen at ?1°C/min. until the temperature reached ?80°C. Then, the cells were immersed in liquid nitrogen at ?196°C and stored for 3–4 months. Approximately 50 frozen/thawed GGC were injected into the dorsal aorta of each 2‐day‐old Rhode Island Red (RIR) embryo, from which blood was drawn before germ‐cell injection. The injected embryos were incubated until they hatched and the chicks were raised until sexually mature. On reaching sexual maturity, a progeny test was performed by mating recipient chicks with normal RIR of the opposite sex. Progenies were obtained from male germ cell recipients that were injected with germ cells collected from 7‐ and 9‐day‐old embryos. The results demonstrated that frozen/thawed GGC collected from 7‐ or 9‐day‐old fertilized eggs can be used to produce male germ‐line chimeras.     

Changes in intracellular and cell surface localization of Le(x) epitope during germ cell differentiation in fetal mice.

Changes in the intracellular and cell surface localization of Lewis x (Le(x)) determinants in germ cells during fetal development in mice were examined by light and electron microscopy. Light microscopically, in undifferentiated gonads on day 12 post coitum (p.c.), the anti-Le(x) monoclonal antibody (MAb) was specifically bound to the plasma membranes and to the cytoplasmic granule-like structures of germ cells. In the testes on day 13 p.c., most of the germ cells were enclosed within the testicular cords and showed an MAb-positive reaction which was restricted mainly to the cytoplasmic granule-like structures. The reaction on the plasma membranes almost disappeared. On the other hand, the ectopic germ cells still showed a positive reaction on their plasma membranes. In the ovaries on day 13 p.c., the germ cells also exhibited positive reactions both on the plasma membranes and on the granule-like structures. Immunoelectron microscopic observations agreed well with these light microscopic observations in such a way that both the plasma membranes and the "small dense bodies" (SDB) were positive in undifferentiated gonads on day 12 p.c. In the germ cells organized into the testicular cords, the reaction to anti-Le(x) MAb became restricted to the SDB. These results may indicate that such intracellular changes in Le(x) determinants during germ cell differentiation are associated with the enclosure of germ cells within the testicular cords.     

雄性关中奶山羊胎儿生殖细胞增殖分化的研究

旨在阐明山羊胚性腺的发生规律,这对于山羊的繁殖育种、丰富胚胎学有关研究内容都具有重要意义.该研究对22枚从39日胎龄胎儿至初生奶山羊性腺进行石蜡组织切片,用组织学和免疫组化、免疫荧光方法对山羊胎儿性腺发育的组织学特点,山羊胎儿性腺发育过程中VASA及细胞增殖核抗原(PCNA)的表达规律进行了系统研究.结果表明:奶山羊性腺在39～47日胎龄时已出现精索和白膜,已发生性别分化,并可分辨出生殖细胞、支持细胞及间质细胞,细胞增殖率较高.在大约50日胎龄前生殖细胞可称作性原细胞,细胞为球形、核质比高.在51～59日胎龄,生殖细胞发生剧烈变化:体积增大、核质比降低.此后逐渐由性原细胞发育为前精原细胞,并且在此期间表达VASA的生殖细胞增多.50～90日胎龄,生殖细胞增殖率最高,生殖细胞所占比例也达到峰值.在90日胎龄后生殖细胞增殖率下降,比例下降,间质细胞大量退化.结果表明,山羊胎儿的生殖细胞在50日胎龄后由性原细胞发育为前精原细胞,生殖细胞在50～90日胎龄的增殖率最高,VASA可作为雄性山羊胎儿前精原细胞的标记.