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.     

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.     

Changes in spermatogenesis and endocrine function in the ram testis due to irradiation and active immunization against luteinizing hormone-releasing hormone

Spermatogonial stem cell transplantation is a technique that has potential in livestock to enhance genetic gain and generate transgenic offspring through the male germ line. A means for depletion of endogenous germ cells in a recipient's seminiferous tubules is necessary for this technology to be applied. The objectives of this study were to evaluate several methods for depletion of endogenous germ cells in the testes of adult rams and to evaluate ultrasound-guided injections into the rete testes as a means for infusing a suspension into the seminiferous tubules. Sixteen adult rams were randomly divided into 4 treatment groups (n = 4 per group). Treatments consisted of active immunization against LHRH (IMM), localized testicular irradiation (IR), LHRH immunization + irradiation (IMM+IR), and untreated control. Serial bleedings were conducted pretreatment and monthly after treatment for 4 mo, at which time all rams were castrated. Both IMM and IMM+IR rams received exogenous gonadotropin in the form of Perganol weekly for 8 wk before castration to bypass the immunization. All rams also received an ultrasound-guided injection of PBS containing 0.4% trypan blue into the rete testis of one testicle before castration. Rams receiving IMM and IMM+IR treatments had higher (P < 0.05) average percentages of seminiferous tubule cross sections with depleted germ cells compared with controls. Serum testosterone was decreased (P < 0.05) in IMM and IMM+IR rams 1 mo after treatment and throughout the remainder of the study compared with controls and IR rams, which were not different from each other. Serum inhibin concentration was unchanged in all rams following treatment indicating that Sertoli cell function was unaltered. A greater (P < 0.05) average percentage of the total testicular area could be filled with the trypan blue solution by rete testis injection in IMM and IMM+IR rams. These data demonstrate the depletion of endogenous germ cells in adult ram testes without alteration of Sertoli cell viability and function that have potential as methods for preparing recipient animals for germ cell transplantation.     

Cell-cycle-dependent Colonization of Mouse Spermatogonial Stem Cells After Transplantation into Seminiferous Tubules

Spermatogonial stem cells (SSCs) migrate to the niche upon introduction into the seminiferous tubules of the testis of infertile animals. However, only 5–10% of the transplanted cells colonize recipient testes. In this study, we analyzed the impact of cell cycle on spermatogonial transplantation. We used fluorescent ubiquitination-based cell cycle indicator transgenic mice to examine the influence of cell cycle on SSC activity of mouse germline stem (GS) cells, a population of cultured spermatogonia enriched for SSCs. GS cells in the G1 phase are more efficient than those in the S/G2-M phase in colonizing the seminiferous tubules of adult mice. Cells in the G1 phase not only showed higher expression levels of GFRA1, a component of the GDNF self-renewal factor receptor, but also adhered more efficiently to laminin-coated plates. Furthermore, this cell cycle-dependency was not observed when cells were transplanted into immature pup recipients, which do not have the blood-testis barrier (BTB) between Sertoli cells, suggesting that cells in the G1 phase may passage through the BTB more readily than cells in the S/G2-M phase. Thus cell cycle status is an important factor in regulating SSC migration to the niche.     

Leydig cell hyperplasia in an ENU-induced mutant mouse with germ cell depletion

Repro22 is an N-ethyl-N-nitrosourea (ENU)-induced mutation in mice showing depletion of both male and female germ cells. In the present study, we investigated the male phenotypes of the mutant mouse at the adult stage. The repro22/repro22 homozygous mice showed reduced body weights as well as markedly reduced testis weights. Histological examination of the testes at 4 and 10 months of age showed no germ cells in the seminiferous tubules of the affected testis while a number of Sertoli cells were observed in the tubules. In addition to the germ cell depletion, the testes of the affected mouse contained expanded intertubular spaces that were filled by Leydig cell-like interstitial cells. These interstitial cells were confirmed to be Leydig cells by immunohistochmical staining using anti-3beta-HSD antibody. The estimated number of Leydig cells in the affected testes at 10 months of age increased approximately 2 fold compared with those of normal testes. Furthermore, the plasma testosterone levels of the affected mice at 10 months of age were significantly higher than those of the normal mice. These findings indicated that the repro22/repro22 mouse developed hyperplasia of Leydig cells that was presumably caused by the absence of germ cells in the seminiferous tubules.     

犊牛睾丸支持细胞的分离与冷冻保存

以新生犊牛睾丸为实验对象,应用组合酶法进行支持细胞分离培养,并研究了冷冻保存后支持细胞的生长特性。结果表明:在细胞分离时,消化睾丸组织,分离曲细精管法所获得的细胞悬液中的有效细胞数高于组织剪碎法。支持细胞体外培养,4 h后开始贴壁,3~4 d铺满培养皿底壁,传代后细胞生长较快,2 d即可增殖一代。HE染色,胞质染色较淡,而细胞核染色较深,呈圆形或椭圆形位于细胞质中央或偏位,核仁明显。采用10%FBS+10%DMSO的DMEM液做冷冻液,对细胞进行冷冻保存时,支持细胞的复苏率在65%以上。解冻后的支持细胞体外培养,4h开始有细胞贴壁,24h后大部分细胞贴壁,3~4d铺满培养皿底壁。     

Persistence of undifferentiated spermatogonia in aged Japanese Black cattle

Aging is a major risk factor for spermatogenesis deterioration. However, the influence of age on spermatogenic stem cells and their progenitors in bulls is largely unknown. Here, we report age-related changes in undifferentiated and differentiating spermatogonia in Japanese Black cattle with nearly constant sperm output, by using spermatogonial markers. The numbers of differentiating spermatogonia and more differentiated spermatogenic cells were significantly decreased in aged bovine testes compared with those in young testes. In contrast, the number of undifferentiated spermatogonia was maintained, and their proliferative activity did not differ significantly between young and aged bovine testes. Although severe calcification was only observed to a small extent in aged testes, fewer Sertoli cells and interstitial fibrosis were observed in noncalcified testicular regions. These results suggest that, even in old bulls with nearly constant sperm output, testicular spermatogenic activity declined whereas undifferentiated spermatogonia numbers were maintained. Thus, we propose that undifferentiated spermatogonia may be resistant to age-related changes in bovine testes. Because undifferentiated spermatogonia may contain stem cell activity, our findings highlight the potential utility of undifferentiated spermatogonia as an agricultural resource to produce spermatozoa beyond the natural bovine lifetime through transplantation and in vitro spermatogenesis in future animal production.     

Pathophysiology of small testes in beef bulls: relationship between scrotal circumference, histopathologic features of testes and epididymides, seminal characteristics, and endocrine profiles

Tissue sections from testes and epididymides obtained from 17 young beef bulls with scrotal circumference (SC) between 27 and 40.5 cm were studied to determine whether small testes were a manifestation of lesions or a result of less, but otherwise normal, seminiferous epithelium. The SC correlated negatively with the estimates of germinal epithelial loss and positively with seminiferous epithelial area. Four bulls with SC less than 30 cm had severe lesions in their testes. Hypoplastic tubules were characterized by Sertoli's cells only with no evidence of germinal cells. Loss of germinal cells, leaving vacuolated epithelium and atrophy, were observed in degenerated tubules. Hyperplasia of Leydig's cells was observed in the vicinity of Sertoli's cell-only tubules, resulting either from degeneration or hypoplasia, and atrophy of Leydig's cells was associated with tubules devoid of Sertoli's cells. These findings indicated that Sertoli's cells may produce a factor(s) required for maintenance and regulation of Leydig's cell function. Epididymal epithelium, especially in the head, had regressed in bulls with hypoplastic and degenerative changes in their testes. Decreased sperm concentration and motility and an increased frequency of morphologic defects were observed in the 4 bulls with testicular lesions and regressed epididymal epithelium. Blood plasma profiles of cortisol, follicle-stimulating hormone, luteinizing hormone, and testosterone were determined in the 4 bulls with SC less than 30 cm and 10 of the 13 bulls with SC greater than 30 cm. There were no statistically significant (P greater than 0.1) differences in the responses to exogenous gonadotropin-releasing hormone or base-line patterns of blood plasma follicle-stimulating hormone and luteinizing hormone between the 2 groups. However, in the bulls with SC less than 30 cm, the mean concentration of testosterone was lower, whether spontaneous (P less than 0.05) or exogenous gonadotropin-releasing hormone induced (P less than 0.1). The fact that these bulls were not deficient in gonadotropins indicated that Leydig's cell function was impaired by local factors, either the factors that caused the tubular damage or those consequent to the tubular damage.     

Immunohistochemical study of osteopontin in boar testis

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.     

Characterization and in vitro culture of male germ cells from developing bovine testis

The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro.     

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.     

Germ cell weakness as a cause of testicular hypoplasia in bulls.

Sporadic cases of testicular hypoplasia were earlier found in bulls of the Swedish Red and White breed. An accumulation of cases have occurred since 1970 in sons of 2 outstanding progenytested bull sires, 2 F and 27 U, which had a common father, 545 B. The history and clinical examination of affected bulls varied. Some had azoospermia and very small testes at a young age, while others could be normal in all respect when they were young but had a short reproductive life and had to be culled at about 3 years of age. Most of the affected bulls were between these 2 extremes. The histologic examination showed principally different degrees of testicular degeneration. There were always some germ cells left in all affected seminiferous tubules indicating that there was not a lack of germ cells causing the hypoplasia. Germ cell weakness is obviously a hereditary condition. The sires 545 B, 2 F and 27 U had a relatively low fertility. In their pedigree were several bulls known to have had a low fertility. No sons of 2 F and only a few sons of 27 U were used for A.I. services and at present only few cases of testicular hypoplasia are seen.     

Transduction and Transplantation of Spermatogonia into the Testis of Ram Lambs through the Extra-testicular Rete

Spermatogonial transplantation will provide a new way to study spermatogenesis in domestic animals, disseminate male genetics and produce transgenic animals, if efficiency can be improved. We evaluated a 'surgical' method for transplanting donor cells into testes of ram lambs, where the head of the epididymis is reflected, and a catheter introduced into the extra-testicular rete testis. We also tested transduction of ram spermatogonia with a lentiviral (LV) vector as a means to identify permanent colonization, and introduce genes into donor cells. Eight ram lambs, 11- to 13-week olds, were the recipients: in five, spermatogonia were injected into one testis, and the contralateral testis was an un-manipulated control: in two, spermatogonia were injected into one testis and the contralateral was sham-injected: in one, both testes were injected. Six lambs received spermatogonia labelled with a cell-tracking dye and these were collected 1 or 2 weeks after transplantation; three lambs received spermatogonia transduced with a LV vector driving the expression of enhanced Green Fluorescence Protein and these were collected after 2 months. Donor cells were detected by immunohistochemistry in tubules of seven of nine recipient testes. Approximately 22% of tubule cross-sections contained donor cells immediately after transplantation, and 0.2% contained virally transduced cells 2 months after transplantation. The onset of spermatogenesis was delayed, and there were lesions in both injected and sham-injected testes. Despite the effects of the surgery, elongated spermatids were present in one recipient testis 2 months after surgery. The results suggest that, after modifying the surgical and transduction techniques, this approach will be a means to produce good colonization by donor spermatogonia in sheep testes.     

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.     

Morphological study of metaphase-specific apoptosis in MRL mouse testis

Apoptosis of male germ cells is a complex phenomenon in many animal species. Understanding its mechanisms could be useful in the diagnosis and therapy of male infertility. To examine the differences of distribution of apoptosis among mouse strains, the terminal transferase-mediated nick end labelling (TUNEL) method was employed. In the testes of MRL mice, many TUNEL-positive cells were identified at the metaphases of meiotic spermatocytes. Morphometrical analysis revealed that metaphase-specific apoptosis occurred at the region between secondary spermatocytes and step 1 spermatids in stage XII seminiferous tubules. In the investigation of the developing first-wave of seminiferous tubules, there were some metaphases showing apoptotic morphology prior to becoming secondary spermatocytes. Details of the apoptotic structure revealed by electron microscopy showed that cellular arrest occurred after the beginning of the M phase of the cell cycle. These results suggested that metaphase-specific apoptosis in the testis of the MRL mouse strain took place at least at the first meiotic division, perhaps showing the spindle assembly checkpoint of the cell cycle.     

Growth and Development of Testes in Domesticated and Hybrid(wild × domesticated)Yak Bulls

Testicular tissue was studied in domesticated yak and in wild yak × domesticated yak(F1 and F2)bulls at 6,12,18 and 24 months of age by stereology for quautitative histology and by comparative studies on the lactate dehydrogenase(LDH)isoenzyme spectrum, LDH activity and the percentage content of LDH isoenzymes in testes. The results indicated that all three types of yak were similar in both the characteristics of ultrastructure of testicular tissue and the degree of germ cell development. The process of spermatogenesis was initiated and sperm were produced for the first time at the age of 12months in all three yak types. All yak types exhibited similar age-related, increases in weight of testes,volume density of both the seminiferous tubules and seminiferous epithelium and height of seminiferous epithelium but all these indexes were slightly affected by season. The number of LDH isoenzyme bands after electrophoresis varied with age as follows: four bands were present at 6 months of age, five bands at 12 and 18 months and six bands at 24 months of age. The sixth band was LDH -x ,which appeared between the LDH4 and LDH5 band. These results indicated that yak bulls reach sexual maturity at 24months of age and that puberty is not affected by infusion of wild strains.     

Immunolocalization of steroidogenic enzymes P450scc, 3betaHSD, P450c17, and P450arom in Göttingen miniature pig testes

The objective of this study was to investigate immunolocalization of steroidogenic enzymes in G?ttingen miniature (GM) pig testes. Testes of 6 adult GM pigs were obtained in September 1996 (n=2), February (n=2) and June (n=2), 1997. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Histologically, all types of spermatogenic cells including mature-phase spermatozoa in seminiferous tubules were observed in all testes throughout the year. Moreover, P450scc, 3betaHSD, P450c17and P450arom were identified in Leydig cells but not in Sertoli cells of all testes. These results suggested that adult GM pig testes have the ability to produce germ cells throughout the year, and the synthesis of progestin, androgen and estrogen occurs in the Leydig cells of GM pig testes.     

RADIOGRAPHIC CHANGES IN TESTES OF BULLS INFECTED WITH BESNOITIOSIS

Ten beef bulls infected with Besnoitiosis were examined radiographically and pathologically. Eight of these were slaughtered soon after diagnosis; two were left for a six-month observation period. The radiographic findings were focal and branching-tree calcification of the testes. Histopathologic examination proved these calcifications to be necrotic sections of the seminiferous tubules. Radiography of the testes as a means of evaluating the fertility status of the infected bull is recommended.