小鼠精原细胞移植技术的改进

目的 对现有小鼠精原细胞移植技术进行改进,建立更具操作性,易于推广的新方法。方法 分离含绿色荧光蛋白(GFP)的rC57BL6/tg14 (act-EGFO-Osb Y01)小鼠精原细胞作为供体细胞,应用自制的显微注射器将其从睾丸的精子输出管处,以锥虫蓝作为指示剂直接注入野生型预处理C57BL6小鼠精细管内完成移植。应用荧光显微镜观察移植效果并进行组织学分析。结果 移植后受体小鼠睾丸出现绿色荧光信号,组织学检查发现精细管中荧光信号显著高于周围组织,移植的供体小鼠（GFP）的睾丸细胞在受体小鼠睾丸中形成克隆和形成精子,而预处理的野生型小鼠睾丸精细管未发现。结论 含GFP蛋白的rC57BL6/tg14小鼠精原细胞在受体小鼠体内移植成功,此改进方法在简化精原细胞移植技术的同时可保证移植质量。     

Focal orchitis in undescended testes: discussion of pathogenetic mechanisms of tubular atrophy.

OBJECTIVE: To evaluate seminiferous epithelium lesions in adult cryptorchid testes showing lymphoid infiltrates in seminiferous tubules and interstitium (i.e., focal orchitis). Also, to consider the possible role of this lesion in the etiology of tubular atrophy. METHODS: We performed a histopathologic study of the cryptorchid testes and adjacent epididymides removed from 50 adult men who had not been previously treated for cryptorchidism. The study included morphologic and semiquantitative evaluation of seminiferous tubule pathology (according to germ cell numbers), Sertoli cell morphology, tubular lumen dilation, rete testis pattern (normal, hypoplastic, or cystic), and epididymal pattern (normal or epididymal duct hypoplasia). The study also included immunohistochemical evaluation of immune cell markers. The results were compared with clinical and laboratory findings. RESULTS: Focal lymphoid infiltrates (mainly lymphocytes) in seminiferous tubules and interstitium were found in 22 patients (44%), all of whom had unilateral cryptorchidism. The course of orchitis was asymptomatic, and laboratory data were normal. According to the seminiferous tubule pathology, a variety of histopathologic diagnoses, were made: (1) mixed atrophy consisting of Sertoli cell-only tubules intermingled with tubules showing maturation arrest of spermatogonia (11 testes, 4 of which also showed hyalinized tubules); (2) Sertoli cell-only tubules plus hyalinized tubules (4 testes); (3) Sertoli cell-only tubules (3 testes); (4) intratubular germ cell neoplasia (2 testes, 1 of which also showed hyalinized tubules); (5) complete tubular hyalinization (1 testis); and (6) tubular hyalinization plus some groups of tubules with hypospermatogenesis (all germ cell types were present although in lower numbers, 1 testis). Dysgenetic Sertoli cells, that is, Sertoli cells that had undergone anomalous, incomplete maturation, were observed in all nonhyalinized seminiferous tubules with inflammatory infiltrates. Tubular ectasia was observed in 13 cases. The rete testis was hypoplastic and showed cystic transformation in 18 testes, and the epididymis was hypoplastic in 15 testes. CONCLUSIONS: The causes of these focal inflammatory infiltrates are unknown. It is possible that tubular ectasia and Sertoli cell dysgenesis are involved and that these alterations cause a disruption of the blood-testis barrier and allow antigens to enter the testicular interstitium, giving rise to an autoimmune process.     

Experimental allergic orchitis in mice

Summary Experimental allergic orchids was induced in (C57BL/6J × A/J)F₁ mice by two injections of syngeneic testicular homogenate emulsified with adjuvants immediately followed by intravenous injection of pertussis vaccine, at a 2 week interval.Histologically, in the initial stage there was occasional focal degeneration and desquamation of both spermatogonia and Sertoli cells within limited parts of the seminiferous tubules, in the peripheral region of the testis. No inflammatory change was present. In some cases, however, inflammatory reaction in the rete testis and focal lymphocytic infiltration in the interstitium were also observed. Subsequently, marked infiltration of lymphocytes, monocytes, and polymorphs were found not only in the testes, but also in rete testis and epididymis. In later stages the inflammatory reaction gradually subsided, but the testes became atrophic due to progression of spermatogenic arrest. Many tubules were lined only with monolayers of Sertoli cells, surrounded by hyperplastic Leydig cells in the interstitium. At 5 months after the 2nd immunization, there was still variable depression of spermatogenesis and hyperplasia of Leydig cells with scattered fibrous scars in the seminiferous tubules, although good regeneration of germ cells appeared in some tubules.Immunological studies revealed that lymphocytes obtained from mice bearing developed orchitis showed a significantly enhanced response in the mixed culture with syngeneic testicular cells, and suggest that cellular immunity plays an important role in the induction of experimental allergic orchitis in mice.     

F4/80-positive cells rapidly accumulate around tubuli recti and rete testis between 3 and 4 weeks of age in the mouse: an immunohistochemical study

PROBLEM: Previous studies demonstrated that F4/80 antigen (murine macrophage-specific antigen)-positive cells in testes of normal adult mice accumulate particularly in the interstitium adjacent to the tubuli recti and rete testis (i.e., the central region). However, it remains unknown whether this accumulation is a congenital or acquired phenomenon. METHOD OF STUDY: The distribution of F4/80-positive cells on frozen sections of testes obtained from various aged mice was immunohistochemically examined to determine when the positive cells specifically accumulate in the central region. RESULTS: F4/80-positive cells were homogeneously distributed throughout the testicular interstitium with no specific accumulation until 2 weeks of age. However, at 3 weeks of age, the density of positive cells in the central region became slightly, but significantly, higher than that in the interstitium between the seminiferous tubules. Between 3 and 4 weeks of age, the cell density in the central region increased rapidly, the density at 4 weeks of age reaching the level of the mature testes of 8-week-old mice. CONCLUSION: These results demonstrate that the specific accumulation of F4/80-positive cells in the central region is an acquired phenomenon, which starts and ends before puberty.     

Histologic changes in the mouse testis after treatment with gossypol tetra-acetic acid

The effect of oral administrations (20 or 40 mg/kg body weight/day, for 21 days) of gossypol tetraacetic acid on the testis of the Parkes strain mouse was investigated. Gossypol treatment did not affect the body weight or testicular weight, but caused a significant depression in the weight of the seminal vesicle. Histologically, the testes in mice treated with gossypol possessed regressed seminiferous tubules showing the exfoliation of germ cells, the occurrence of giant cells, a disorganization of the germinal epithelium, the degeneration of germinal elements, intraepithelial vacuolation and dislocation of the Sertoli cells into the luminal portion. However, the effect of gossypol was not uniform, and normal features were also observed in the majority of the tubules in the testes of the gossypol-treated mice. When quantitatively analysed, the frequency of regressed seminiferous tubules was significantly higher in the testes in the treated mice than the controls. The results suggest that the gossypol treatment induces non-uniform regressive changes in the seminiferous tubules in the mouse testis.     

Histological and morphometric analyses of seasonal testicular variations in the Jungle Crow (<Emphasis Type="Italic">Corvus macrorhynchos</Emphasis>)

A histological and morphometric study was conducted to examine the seasonal testicular variations in the Jungle Crow (Corvus macrorhynchos) of the Kanto area, Japan, from January to July. The paired testes mass, diameter and number of germ cells of the seminiferous tubules, and proportion of seminiferous tubule area and interstitium were examined. Hematoxylin and eosin-stained testis sections and ImageJ Software were used. Paired testes weight was found to increase by 55-fold from January to late March–early May, thereafter declining by 18-fold by June–July. Seminiferous tubule diameter increased fivefold from January to late March–early May, followed a fourfold decrease in June–July. The increase in testes weight correlated well with the increase in the diameter of the seminiferous tubule. In January, the seminiferous tubules constituted 56% of the testicular tissue and the interstitium 44%. During late March–early May, there was very little testicular interstitium (7.9%), and the seminiferous tubules were significantly enlarged (P < 0.05) (92%); this was followed by a gradual increase in the interstitial regression of testes. In January, the seminiferous epithelium contained a single layer of spermatogonia and Sertoli cells. The number of spermatogonia, primary and secondary spermatocytes, spermatids, and maturing spermatozoa were significantly increased (P < 0.05) in late March–early May, followed by regression from mid May. Our results indicate that the Jungle Crow has a non-breeding season in January, a pre-breeding season during February–mid March, a main breeding season during late March–early May, a transition period during mid May–late May, and a post-breeding season beginning in June.     

Germ cell transfer into rat, bovine, monkey and human testes.

Germ cell transplantation is a potentially valuable technique offering oncological patients gonadal protection by reinitiating spermatogenesis from stem cells which were reinfused into the seminiferous tubules. In order to achieve an intratubular germ cell transfer, intratubular microinjection, efferent duct injections and rete testis injections were applied on dissected testes of four different species: rat, bull, monkey and man. Ultrasound-guided intratesticular rete testis injection was the best and least invasive injection technique with maximal infusion efficiency for larger testes. Deep infiltration of seminiferous tubules was only achieved in immature or partially regressed testes. This technique was applied in vivo on two cynomolgus monkeys. In the first monkey a deep infusion of injected cells and dye into the lumen of the seminiferous tubules was achieved. In the second, transplanted germ cells were present in the seminiferous epithelium 4 weeks after the transfer. These cells were morphologically identified as B-spermatogonia and located at the base of the seminiferous epithelium. In summary, this paper describes a promising approach for germ cell infusion into large testes. The application of this technique is the first successful attempt of a germ cell transfer in a primate.     

Three-dimensional organization of testicular interstitial tissue and lymphatic space in the rat

Rat testes fixed by vascular perfusion were examined by scanning and transmission electron microscopy. This revealed a complex organization of the interstitial tissue and an extensive “testicular lymphatic space” composed of continuous “peritubular lymphatic sinusoids.” No cellular connections exist between the interstitium and the tubules. Each sinusoid completely surrounds an individual seminiferous tubule, and adjacent sinusoids communicate freely through fenestrae in the interstitium. Thus, material must enter the lymph to gain access to the tubules. The sinusoids are delimited by separate endothelial cell layers over the tubules and interstitium. The layer over the interstitium is discontinuous in specific areas according to the geometry of the tubule packing. Interstitial tissue located in the flattened biconcave interstices between two adjacent tubules is consistently covered with endothelial cells, while that located in the open, triangular interstices between three or more tubules consistently lacks an endothelium. Most of the Leydig cells are located in the “open interstitium” and are directly exposed to the lymph. Physiological implications of the “testicular lymphatic space” and additional features of the interstitium are discussed.     

Androgen insensitivity syndrome: an immunohistochemical, ultrastructural, and morphometric study

OBJECTIVE: To evaluate the morphometric, immunohistochemical, and ultrastructural lesions of the testes in prepubertal and adult patients with androgen insensitivity syndrome. METHODS: We examined the testicular biopsy using immunohistochemistry for vimentin, smooth muscle actin, and collagen IV antigens. Quantification of seminiferous tubules and testicular interstitium was performed in prepubertal and adult patients with androgen insensitivity syndrome and results were compared with normal testes from both infants and adults. RESULTS: The adult testes presented nodular and diffuse lesions that consisted of Sertoli-cell-only seminiferous tubules. Two types of Sertoli cells could be distinguished, namely, immature vimentin-positive Sertoli cells and nearly mature Sertoli cells. In the nodules, the lamina propria was thin and contained a scant number of actin-positive peritubular cells. Leydig cells were hyperplastic. The prepubertal patients showed only diffuse lesions characterized by Sertoli cell hyperplasia, decreased germ cell numbers, and a discontinuous immunoreaction to collagen IV. CONCLUSIONS: The testicular lesions in androgen insensitivity syndrome are probably caused by primary alterations that begin during gestation. These lesions become progressively more pronounced at puberty, when the nodular lesion pattern (adenomas) is completely developed.     

Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model

The side population phenotype is associated with the Hoechst dye efflux activity of the Abcg2 transporter and identifies hematopoietic stem cells (HSCs) in the bone marrow. This association suggests the direct use of Abcg2 expression to identify adult stem cells in various other organs. We have generated a lineage tracing mouse model based on an allele that coexpresses both Abcg2 and a CreERT2 expression cassette. By crossing these mice with lox-STOP-lox reporter lines (LacZ or YFP), cells that express Abcg2 and their progeny were identified following treatment with tamoxifen (Tam). In the liver and kidney, in which mature cells express Abcg2, reporter gene expression verified the expected physiologic expression pattern of the recombinant allele. Long-term marking of HSCs was seen in multiple peripheral blood lineages from adult mice, demonstrating that Abcg2(+) bone marrow HSCs contribute to steady-state hematopoiesis. Stem cell tracing patterns were seen in the small intestine and in seminiferous tubules in the testis 20 months after Tam treatment, proving that stem cells from these organs express Abcg2. Interstitial cells from skeletal and cardiac muscle were labeled, and some cells were costained with endothelial markers, raising the possibility that these cells may function in the repair response to muscle injury. Altogether, these studies prove that Abcg2 is a stem cell marker for blood, small intestine, testicular germ cells, and possibly for injured skeletal and/or cardiac muscle and provide a new model for studying stem cell activity that does not require transplant-based assays.     

Congenital Leydig cell hyperplasia

The testes and epididymes collected at autopsy from 21 newborns showed apparent Leydig cell hyperplasia which was studied by light and electron microscopy. Twelve newborns were the sons of diabetic mothers, two had undergone rhesus isoimmunization, two were twins of a non-diabetic mother, three had Beckwitz-Widemann's syndrome, and two had leprechaunism. In the first two groups the placentas were also collected and studied. All the testes showed normal seminiferous tubules and diffuse Leydig cell hyperplasia in the testicular interstitium. In addition one son of a diabetic mother and another with Beckwitz-Widemann's syndrome presented multiple Leydig cell nodules in the mediastinum testis and epididymis. The number of Leydig cells per unit area of the testis was calculated on histological sections stained with the peroxidase-anti-peroxidase method for the detection of testosterone. These numbers varied from 1.4 to 3.2 times those found in age-matched controls, except for the two testes with nodular hyperplasia in which the increase in Leydig cells was even greater. The differential diagnosis between Leydig cell hyperplasia, ectopic adrenal cells and leydig cell tumour is discussed. It is proposed that the cause of congenital Leydig cell hyperplasia might be related to placental secretion of human chorionic gonadotrophin.     

Early testicular changes after vasectomy and vasovasostomy in Lewis rats

The testes of Lewis rats were studied at intervals from 2 weeks to 3 months after bilateral vasectomy, vasectomy followed 1 month later by vasovasostomy, or sham operations. Aims were to determine the nature of early alterations after vasectomy, and to determine whether vasovasostomy after 1 month would result in reversal of vasectomy-induced changes. Approximately one-fourth of the testes in the vasectomy and vasovasostomy groups displayed histological changes, which consisted mainly of depletion of germ cells. The extent of the depletion varied greatly in different seminiferous tubules. In testes altered in this way, no abnormal infiltrations of lymphocytes, macrophages, or other cells were observed in the seminiferous epithelium or in the interstitium. The rete testis and straight tubules were normal in testes with altered seminiferous epithelium. A few testes in the vasectomy and vasovasostomy groups had necrotic centers. The results suggest that depletion of germ cells occurred as a result of shedding from the seminiferous epithelium into the lumen of the tubules. A cellular immune response, such as occurs in experimental allergic orchitis in other species, did not appear to be responsible for the observed loss of germ cells. This suggests a possible role for humoral antibody in this model, since there is an association between testicular changes and serum antisperm antibodies at longer intervals after vasectomy. Testicular alterations were not reversed by performance of a vasovasostomy 1 month after vasectomy.     

Restoration of fertility in infertile mice by transplantation of cryopreserved male germline stem cells

BACKGROUND: The development of a spermatogonial transplantation technique has provided new possibilities for the treatment of male infertility. Previous studies have shown that spermatogonial stem cells could reinitiate spermatogenesis after cryopreservation and reintroduction into the seminiferous tubules of infertile recipient males, and this raised the possibility of banking frozen stem cells for male infertility treatment. It remains unknown, however, whether germ cells from freeze-thawed stem cells are fertile, leaving the possibility that the procedure compromises the integrity of the stem cells. METHODS AND RESULTS: Dissociated mouse testis cells were cryopreserved and transplanted into infertile recipient testes. The freeze-thawed testis cell populations contained higher concentrations of stem cells than fresh testis cell populations. Offspring were obtained from freeze-thawed stem cells transplanted into infertile males, and fertility restoration was more efficient in immature (5-10 days old) than in mature (6-12 weeks old) recipients. However, offspring were also obtained from infertile adult recipients using in-vitro microinsemination. CONCLUSIONS: This first successful application of frozen stem cell technology in the production of offspring by spermatogonial transplantation suggests the superiority of immature recipients for clinical applications. Thus, the combination of cryopreservation and transplantation of stem cells is a promising approach to overcome male infertility.     

Androgen aromatization in cryptorchid mouse testis

Estrogens play an important role in germ cell development. Therefore, we have studied expression patterns of aromatase that converts testosterone into estrogens in 2 recombinant inbred mouse strains that differ in efficiency of spermatogenesis. In order to show whether germ cells are a target for estrogens, estrogen receptors (ER)alpha and beta were localized as well. Adult male CBA and KE mice were made unilaterally cryptorchid to determine alterations in testicular steroidogenesis and spermatogenesis. Differences between control and cryptorchid testes have been studied with respect to (1) cellular sites of aromatase, the enzyme responsible for estrogen formation, (2) the presence of ERalpha and ERbeta in various types of testicular cells, and (3) steroidogenic activity in the testes. Additionally, unilaterally control testes of cryptorchid mice were compared with bilaterally descended testes. Histological or hormonal differences were not found between control testes of cryptorchid and untreated mice. In cryptorchid testes from both strains, degeneration of germ cells was observed as well as a decrease in size of the seminiferous tubules, whereas the amount of interstitial tissue increased, especially in testes of CBA mice. Using immunohistochemistry, aromatase was localized in Leydig cells and germ cells in both control and cryptorchid testes. Sertoli cells were immunopositive in control testes only. In cryptorchid testes of KE mice, aromatase was strongly expressed in spermatids, that were still present in a few tubules. Other cell types in tubules were negative for aromatase. In both control and cryptorchid testes of both mouse strains, ERalpha were present in Leydig cells only, whereas ERbeta were found in Leydig cells and in germ cells in early stages of maturation. In homogenates of testes of CBA control mice, testosterone levels were 3-fold higher than in those of control KE mice, whereas the difference in estradiol levels between both strains was small. Cryptorchidism resulted in decreased testosterone levels and increased estradiol levels. The results of the present study show functional alterations due to cryptorchidism in both mouse strains. Strong aromatase expression in germ cells in control and cryptorchid testes indicates an additional source of estrogens in the testis besides the interstitial tissue and the relevance of estrogen in spermatogenesis.     

Incipient germ cell tumor in Sertoli-cell-only syndrome testis, accompanied with retroperitoneal teratocarcinoma and widespread metastases

Incipient germ cell tumor in Sertoli-cell-only syndrome testis was examined in an autopsy case of retroperitoneal teratocarcinoma with widespread metastases. Although both testes of a 28-year-old man had clinically been small and free from tumor mass to palpation, histopathological examinations revealed a malignancy in the right testis with the appearance of Sertoli-cell-only syndrome. The left testis showed solely the histology of Sertoli-cell-only syndrome. The testicular malignancy consisted of undifferentiated, atypical germ cells mainly confined within approximately one-tenth of seminiferous tubules, and only one small cartilage nodule. Some tubules showed intratubular growth pattern suggestive of seminoma. A few syncytiotrophoblast-like giant cells occurred in the tubules. These findings seem to furnish substantial evidence to the concept that atypical germ cells are the origin of testicular germ cell tumors of different types.     

Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells

Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. We report here in vitro generation of a germ cell line (SSC1) from the pluripotent teratocarcinoma cells by a novel promoter-based sequential selection strategy and show that the SSC1 cell line form mature seminiferous tubule structures, and support spermatogenesis after transplantation into recipient testes. To select differentiated germ cell population, we generated a fusion construct (Stra8-EGFP) harbouring the 1.4 kb promoter region of germ line specific gene Stra8 and coding region of enhanced green fluorescence protein. This region was sufficient to direct gene expression to the germinal stem cells in testis of transgenic mice. The purified cells expressed the known molecular markers of spermatogonia Rbm, cyclin A2, Tex18, Stra8 and Dazl and the beta1- and alpha6-integrins characteristic of the stem cell fraction. This cell line undergoes meiosis and can develop into sperm when transplanted into germ cell depleted testicular tubules. Sperm were viable and functional, as shown by fertilization after intra-cytoplasmic injection into mouse oocytes. This approach provides the basis that is essential for studying the development and differentiation of male germ line stem cell, as well as for developing new approaches to reproductive engineering and infertility treatment.     

Germline niche transplantation restores fertility in infertile mice

BACKGROUND: Stem cells interact closely with their microenvironment or niche, and abnormalities in niche compromise the self-renewing tissue. In testis, for example, Sertoli cells interact with germ cells, and defects in Sertoli cells compromises spermatogenesis, leading to male infertility. However, it has not been possible to restore spermatogenesis from endogenous stem cells in infertile testis with environmental defects. METHODS AND RESULTS: When healthy Sertoli cells from infertile white spotting (W) mouse were transplanted into the seminiferous tubules of infertile Steel (Sl) mouse testis that had defective Sertoli cells, spermatogenesis occurred from Sl stem cells in the recipient testis. On average, 1.1% of the recipient tubules showed spermatogenesis. Furthermore, in a microinsemination experiment with germ cells that developed in the testis, we obtained four normal offspring from 114 successfully injected oocytes. CONCLUSIONS: This study demonstrates that defects in male germline microenvironment can be corrected by Sertoli cell transplantation. Although further improvements are required to enhance the low efficiency of spermatogenesis, the ability to correct environmental defect by niche transplantation has important implications in developing new strategies for treating incurable disorders in self-renewing tissues.     

Germ cell transplantation: a review and progress report on ICSI from spermatozoa generated in xenogeneic testes

Results from the transplantation of donor male germ cells into xenogeneic recipient seminiferous tubules indicate that donor spermatogonia are capable of differentiating to form spermatozoa morphologically characteristic of the donor species. Germ cell transplantation procedures combined with developments in freezing, culturing or enriching germ cell populations have applications of paramount importance in medicine, basic sciences and animal reproduction. Additionally, these techniques can serve as an alternative approach for gonadal protection and fertility preservation in patients with cancer. This article is a chronological critical review of the technological advances that followed the initial successful transplantation of mouse germ cells into recipient mice. Furthermore, the factors responsible for the immunological privilege properties of the testis and the parameters influencing the potential of mammalian germ cells to undergo mitosis and meiosis within a xenogeneic testis are described. Finally, the role of human germ cell transplantation procedures in the therapeutic management of non-obstructive azoospermia is discussed.