Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes

It was recently demonstrated that rat spermatogenesis can occur in the seminiferous tubules of an immunodeficient recipient mouse after transplantation of testis cells from a donor rat. In the present study, hamster donor testis cells were transplanted to mice to determine whether xenogeneic spermatogenesis would result. The hamster diverged at least 16 million years ago from the mouse and produces spermatozoa that are larger than, and have a shape distinctly different from, those of the mouse. In four separate experiments with a total of 13 recipient mice, hamster spermatogenesis was identified in the testes of each mouse. Approximately 6% of the tubules examined demonstrated xenogeneic spermatogenesis. In addition, cryopreserved hamster testis cells generated spermatogenesis in recipients. However, abnormalities were noted in hamster spermatids and acrosomes in seminiferous tubules of recipient mice. Hamster spermatozoa were also found in the epididymis of recipient animals, but these spermatozoa generally lacked acrosomes, and heads and tails were separated. Thus, defects in spermiogenesis occur in hamster spermatogenesis in the mouse, which may reflect a limited ability of endogenous mouse Sertoli cells to support fully the larger and evolutionarily distant hamster germ cell. The generation of spermatogenesis from frozen hamster cells now adds this species to the mouse and rat, in which spermatogonial stem cells also can be cryopreserved. This finding has immediate application to valuable animals of many species, because the cells could be stored until suitable recipients are identified or culture techniques devised to expand the stem cell population.     

Development of germ cell transplants in mice

Development of spermatogonial transplants was studied by using 5- to 6-wk-old histocompatible mice as cell donors and sterile (W-locus) mice as recipients. Groups of animals transplanted with germ cell suspensions were killed at 10 min, 9 h, 24 h, 1 wk, 1 mo, 2 mo, and 3 mo along with age-matched "start" and "end" W-locus controls. Weight of testes increased significantly at 24 h through 3 mo after germ cell transplantation, suggesting that the infused cells quickly stimulated organ function. Small clones of young spermatocytes were evident at 1 mo and sperm at 2 mo. The percentage of tubular profiles containing active spermatogenesis originating from spermatogonia increased with time (0.8% at 1 mo, 8.9% at 2 mo, and 28.2% at 3 mo). Most transplanted germ cells were eliminated from the seminiferous epithelium through phagocytosis by Sertoli cells that occurred primarily before 1 wk, although some pachytene cells were able to proceed through meiosis by 1 wk. A variety of abnormal features are described that characterize developing spermatogenesis in the transplanted testis. Spermatogenesis improved quantitatively and qualitatively with time although released sperm were frequently engulfed by intratubular macrophages and Sertoli cells. A quantitative analysis of spermatogenesis from transplanted germ cells will serve as a basis for improving spermatogonial transplant efficiency.     

Differential responses of the heart and vasculature to chronic blood pressure reduction in essential hypertension

In the current study, localization of D-aspartic acid (D-Asp) in rat testis was studied by immunohistochemical and biochemical techniques. Immunohistochemical staining of this tissue using specific polyclonal antibody to D-Asp revealed D-Asp immunoreactivity (IR) in the cytoplasm of germ cells, especially around the region rich in elongate spermatids, the most mature of the germ cells. Weak IR was also noted in cytoplasm of spermatocytes and round spermatids; however, it was negligible in interstitial cells and Sertoli cells. The intensity of immunostaining in each seminiferous tubule differed according to its distinct germ cell composition. In testis of young rats, seminiferous tubules lack elongated spermatids, and D-Asp was found to be localized in spermatocytes, the most mature population of germ cells at that age. We used various toxicants to destroy specific testicular cell populations and to confirm the localization of D-Asp in rat testis. Administration of ethane dimethane sulfonate induced a selective destruction of all Leydig cells in this tissue. This resulted in a significant decrease in the D-Asp level, which was probably due to a drop in testosterone brought about by this treatment, and this was followed by a modulation of spermatogenesis. Three days after treatment with methoxyacetic acid (MAA), many seminiferous tubules were found to lack or to have severe depletions of pachytene spermatocytes, but not of elongate spermatids. This caused reductions in protein content and in the total amount of L-Asp, but not that of D-Asp. Twenty days after treatment with MAA, the depleted population of germ cells progressed through the spermatogenic cycle from pachytene spermatocytes to elongate spermatids. At this time, the level of D-Asp decreased significantly, as did that of L-Asp and protein, consistent with D-Asp localization in elongate spermatids. This decrease in the D-Asp level was also seen with immunostaining.     

Male germ cell transplantation: present achievements and future prospects

Germ cells are unique, since their surviving descendants can undergo meiosis and differentiate into gametes, which transmit genetic material from one generation to another. We now know that male germ cells, whether they be primordial germ cells in gonadal ridges, gonocytes, or stem spermatogonia, are transplantable. The donor cells can be transferred by direct microinjection into the seminiferous tubules, rete testis or efferent ducts, depending on the recipient species. Following transplantation, the donor cells undergo spermatogenesis in the host's seminiferous tubules in rats and mice, and have even sired offspring in mice. Interspecific germ cell transfer is possible if the recipient's immune system is defective; nude or SCID mice can even produce rat spermatozoa. However, the major obstacle restricting widespread use of this new technology is its extremely low success rate. This article discusses some ideas for improving the success rate of the transfer technique, and considers several potential applications.     

Histopathology of the seminiferous tubules in mice injected with syngeneic testicular germ cells alone

Previously, a model of murine experimental autoimmune orchitis was produced by active immunization with viable syngeneic testicular germ cells without resorting to any adjuvants. The histological mode of the spermatogenic disturbance of this autoimmunity was investigated in A/J mice. A significant spermatogenic disturbance was consistently induced after the appearance of inflammatory cell responses around the tubuli recti. It first appeared seminiferous epithelium adjacent to the tubuli recti, then spread to the peripheral epithelium. The histopathology of the seminiferous tubules in the early phase ranged from partial degeneration and depletion of all kinds of germ cells to complete loss of germ cells other than some remaining spermatogonia, while both Sertoli cells and the basal lamina of the tubules appeared intact. In the late phase, depletion of Sertoli cells, disorganization of the seminiferous tubular wall or filling with many round-shaped degenerating germ cells, appearance of malformed spermatids with signet ring nuclei, depletion of immature germ cells with remaining elongated spermatids, or complete loss of the seminiferous epithelium were observed in addition to the early histopathological features.     

LDL subclass phenotypes and the insulin resistance syndrome in women

An experiment was conducted to examine the appearance of the seminiferous tubule 20 days after a single exposure of the testes of rams to a scrotal temperature of about 42 degrees C for 45 min. Ten of the animals were surgically hypophysectomized and five were simultaneously heated; these rams were treated twice a day with ovine pituitary extract to avoid modifications in the negative feedback from the testes to the pituitary and consequent changes in gonadotrophin secretion. Six intact rams (three heated and three unheated) were also studied. The pituitary extract significantly increased the testis weight and spermatogonial multiplications from A1 spermatogonia onwards. Twenty days after the heat treatment, testis weight was significantly reduced by heating; both tubular and intertubular tissues were affected. The total length of seminiferous tubules per testis was not modified, whereas the mean seminiferous tubule diameter was significantly reduced after heating. The total number of Sertoli cells per testis was not significantly modified, while their mean cross-sectional nuclear area was significantly reduced by heat treatment. A decrease in the number of all germ cells except A0 spermatogonia, from A1 spermatogonia onwards, was observed. The number of round spermatids decreased by 95 and 90%, slightly more than the diplotene primary spermatocytes (76 and 77%) and elongated spermatids (79 and 85%) in hypophysectomized pituitary extract-treated and intact rams, respectively. Round and elongated spermatids would be derived from germ cells that were respectively leptotene and young pachytene primary spermatocytes at the time of heating, whereas diplotene primary spermatocytes would have been type B spermatogonia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.     

Alteration of Sertoli cell differentiation in the presence of carcinoma in situ in human testes

PURPOSE: We investigated Sertoli cells in testicular biopsies with carcinoma in situ (CIS) in respect to cytokeratin expression to elucidate the status of Sertoli cell differentiation adjacent to CIS in human testes. Cytokeratin 18 intermediate filaments indicate a state of undifferentiation usually observed in Sertoli cells of prepubertal testes. MATERIALS AND METHODS: 29 testicular biopsies presenting CIS were investigated by means of immunohistochemistry, using a polyclonal antibody against placental-alkaline phosphatase to detect CIS cells and a monoclonal antibody against human cytokeratin 18 to show expression of cytokeratin 18 intermediate filaments in Sertoli cells. RESULTS: All tubules bearing CIS showed positive cytokeratin expression of Sertoli cells if tubules were devoid of normal germ cells. However, a total of 13 specimen revealed CIS cells together with normal germ cells. In the presence of CIS cells together with round or elongated spermatids, adjacent Sertoli cells did not express cytokeratin immunoreactivity. In the case of combined presence of CIS and spermatogonia and primary spermatocytes, Sertoli cells could be found either immunopositive or immunonegative, and were positive in tubules with CIS and spermatogonia only. CONCLUSIONS: Sertoli cells associated with CIS cells undergo a process of dedifferentiation, seen by the re-expression of cytokeratin intermediate filaments. We suggest that this dedifferentiation results in a loss of Sertoli cell function and leads to a cessation of spermatogenic activity.     

A novel stage-specific differentiation antigen is expressed on mouse testicular germ cells during early meiotic prophase

A murine cell surface antigen exhibiting stage-specific expression during spermatogenesis was detected with two monoclonal antibodies (mAbs), designated BC7 and CA12. In mouse testis, these mAbs recognized a small population of cells located near the periphery of seminiferous tubules at stages XII and I-VI, and these spermatogenic cells were identified as zygotene and early pachytene spermatocytes. Expression of the antigens was transient and was not detected in germ cells at more advanced stages of spermatogenesis such as late pachytene spermatocytes and round spermatids. Immunoprecipitation and immunoblotting studies showed that both mAbs CA12 and BC7 reacted with the same antigenic molecule, which had an estimated molecular mass of 95 kDa. CA12/BC7 antigen, detected in plasma membrane fraction, was a glycoprotein with sialic acid residues and had affinity with WGA lectin. Furthermore, intraperitoneal injection of mAb BC7 caused an apparent spermatogenic disturbance in prepubertal mice. These results suggested that CA12/BC7 antigen, a novel cell surface glycoprotein, is an essential molecule that plays an important role during early meiotic prophase of spermatogenesis.     

RNA synthesis in spermatocytes and spermatids and preservation of meiotic RNA during spermiogenesis in the mouse

The rate of RNA synthesis at different stages of spermatogenesis in the mouse, and the preservation of RNA from the diploid to the haploid phase of spermatogenesis were studied in homogeneous germ cell fractions separated by velocity sedimentation at unit gravity. The uridine pool expansion method was used for determining the rate of RNA synthesis: seminiferous tubules were labelled in culture with increasing concentrations of [3H]-uridine and the incorporated radioactivity was estimated in cell fractions separated by velocity sedimentation. The results indicate that before nuclear elongation, round spermatids (steps 1 to 8 of spermiogenesis) synthesize RNA at the same rate per DNA content as middle-late pachytene spermatocytes. The preservation of RNA molecules synthesized in meiosis was investigated by labelling pachytene spermatocytes with T3H]uridine in vivo and collecting samples of germ cells at definite stages of spermatogenesis at various time intervals thereafter. The results show that a considerable proportion the RNA synthesized during the pachytene stage is preserved through spermatid development until late spermiogenesis.     

A randomized, controlled trial of intravenous clodronate in patients with metastatic bone disease and pain

The present study analyses cell loss and proliferation which account for the decrease in the number of germ cell populations in the senile male Octodon degus. This is a good model to study ageing in wild animals, since it has recently been incorporated as a laboratory animal but still has a high degree of genetic heterogeneity, thus representing a situation found in natural systems. The cell loss from pachytene spermatocytes to round spermatids is estimated by cell counts in the cross section of seminiferous tubules. DNA testicular synthesis is measured by scintillation counting and the index of labelling of spermatogonia by radioautography of testes comparing sexually mature young animals and senile animals. Other determinations in both groups are testis weight, thickness of the albuginea and tubular wall, daily sperm production, percentage of depleted seminiferous tubules and nuclear cell diameters of germ cells. The results suggest a decrease in the number of cell population in the senile animals resulting from an increase in physiological cell loss coupled with a decreased proliferative spermatogonial activity. There is also a decreased yield of meiosis in terms of round spermatid production. Lowered testosterone levels both in plasma and testicular parenchymal fluid are found in senile animals. All these senescent changes reflect an altered remodelling activity of the seminiferous epithelium and presumably also of Leydig cells.     

Susceptibility of alpine ibex to conjunctivitis caused by inoculation of a sheep-strain of Mycoplasma conjunctivae

The genetic expression of insulin-like growth factor II (IGF-II) mRNA was studied in healthy adult testes and in hypoplastic testes of polled Murciano-Granadina goats by means of in situ hybridization. A positive reaction in spermatogonia, pachytene spermatocytes and a few peritubular myoid cells was observed using the ovine antisense oligonucleotide in healthy testes. The hypoplastic testes displayed a loss of germinal epithelium and a slight thickening of the basement membranes. A limited number of immature germinal cells displayed a lesser hybridization reaction, while the expression of IGF-II mRNA observed in the peritubular myoid cells was similar to that seen in healthy testes. In hypoplastic testes, IGF-II mRNA expression within germinal cells decreased with increasing hypoplasia within the seminiferous epithelium and there was no hybridization within the tubules in cases of severely disrupted spermatogenesis. These results suggest that testicular hypoplasia is associated with changes in the expression of IGF-II mRNA.     

Flow cytometric analysis of the effects of 50 Hz magnetic fields on mouse spermatogenesis

The cellular effects of an extremely-low-frequency (ELF) magnetic field on mouse spermatogenesis were assessed by DNA flow cytometry and serum testosterone. Seven week old male ICR mice were exposed to a 50 Hz magnetic field the strength of which was 1.0 m Tesla. Seven mice per treatment group were exposed for 13, 26, 39 or 52 days. For each experimental point, an equal number of mice per sham-treated group were used as a control and were exposed only to the background field below 1 mu Tesla in the same room as the treatment group. In the control mice, the testis cellular DNA content distribution by flow cytometory was characterized by four quantifiable populations; round spermatids (1C), spermatogonia and other diploid cells (2C), spermatogonial cells synthesizing DNA (S-phase) and primary spermatocytes (4C). In animals exposed for 26 days the number of cells in the 4C and the 4C:2C ratio was significantly lower, and the 1C:4C ratio (meiotic transformation) was significantly higher than the corresponding control groups. In animals exposed for 52 days the cell population in 1C and the 1C:2C ratio (total germ-cell transformation) was significantly higher, and the cell population in 2C was significantly lower than the corresponding control groups. The concentration of serum testosterone in animals exposed for 13 days was significantly higher than in the corresponding control group. These changes suggest that long-term exposure to an ELF magnetic field had a possible effect on the proliferation and differentiation of spermatogonia.     

A race-specific insertion of transposable element IS801 in Pseudomonas syringae pv. phaseolicola

To detect free zinc ions in the rat testes four rats were transcardially perfused with Na2S, and the seminiferous tubules from two other rats were incubated in Na2S. Sections from the two sources were autometallographically (AMG) developed, whereby zinc sulphide crystal lattices created in the tissue by the sulphide treatment were silver enhanced. Light microscopical analysis showed zinc ions in primary spermatogonia until the zygotene primary spermatocytes (stage I), in late pachytene spermatocytes (stages XII and XIII), and in late spermatids from step 15 to step 19 (stages I-VIII). The highest intensity of AMG grains was detected in the residual bodies and tails of step 19 spermatids. Grains were occasionally found in the cytoplasm of Leydig cells. Sections from animals treated with the chelator diethyldithiocarbamate prior to sulphide treatment showed a complete lack of AMG staining. At ultrastructural levels the AMG grains were found in smooth-surfaced endoplasmic reticulum of all spermatogonial stages, and in the acrosome, midpiece, and tail of late spermatids. The presence of zinc ions in preleptotene spermatocytes and cytoplasmic lobes of late spermatids suggests a specific role of free zinc at the onset of meiosis and at spermiation.     

The distribution pattern of cytokeratin and vimentin immunoreactivity in testicular biopsies of infertile men

Testicular biopsies of infertile patients are often characterized by a mixed atrophy, in which different types of spermatogenic lesions are found in adjacent tubules. In order to evaluate a possible involvement of the state of differentiation of the Sertoli cells, the distribution pattern of cytokeratin and vimentin intermediate filaments within the seminiferous epithelium of 228 biopsy specimens with normal spermatogenesis (n = 10), mixed atrophy (n = 206) or Sertoli Cell Only Syndrome (n = 12) were investigated by means of immunohistochemical techniques. Sertoli cells were regularly found to show vimentin expression in tubules with normal spermatogenesis as well as in tubules with any kind of spermatogenic impairment including SCO. Cytokeratin expression as a marker showing lack of differentiation was common in Sertoli cells of tubules with arrest of spermatogenesis at the level of spermatogonia, and was occasionally associated with arrest at the level of primary spermatocytes or with SCO. Ultrastructural examination of tubules with spermatogonial arrest revealed Sertoli cells with features of typical fetal or prepubertal Sertoli cells, such as round to ovoid nuclei without indentations, stacks of rough ER and spot desmosomes. These data suggest that spermatogenic arrest at the level of spermatogonia might be due to functional impairment of the associated Sertoli cells, which have maintained or regained an undifferentiated state and are not able to initiate or trigger the process of spermatogonial differentiation.     

Classification of several types of maturational arrest of spermatogonia according to Sertoli cell morphology: an approach to aetiology

Bilateral testicular biopsies and clinical histories from 34 adult men with maturational arrest of spermatogonia were examined. According to the morphology of Sertoli cell nuclei, five testicular types of spermatogonial maturational arrest were established. In type I lesion, Sertoli cells resembled the immature Sertoli cells of infant testes. These cells had a round, regularly outlined, dark nucleus with a small nucleolus. The seminiferous tubules showed no apparent lumen and a poorly developed lamina propria lacking in elastic fibres. This lesion was found in patients exhibiting a eunuchoid phenotype, with small tests and low serum levels of gonadotrophins and testosterone (hypogonadotrophic hypogonadism). Type II lesion showed morphologically normal, mature, adult Sertoli cells which had a pale, irregularly outlined nucleus, many often triangle-shaped, with a large, centrally located nucleolus. The seminiferous tubules were reduced in diameter and showed a few spermatocytes and spermatids. This lesion was found in patients with varicocoele, epididymitis, testicular trauma or idiopathic infertility. Serum FSH levels were normal or increased while LH and testosterone levels were normal. In type III lesion, Sertoli cells resembled the involuting Sertoli cells found in the testes of aging men, and displayed very infolded nuclei, with abundant dense chromatin patches and a large nucleolus. The seminiferous tubules showed a slightly dilated lumen and a normal tubular wall. The most relevant clinical findings in patients with this lesion were alcoholism, varicocoele, falciform cell anaemia, epididymitis and germ cell tumour. Serum follicle stimulating hormone (FSH) levels were normal or increased while luteinizing hormone (LH) and testosterone levels were normal. Type IV lesion Sertoli cells presented with a de-differentiated appearance. These cells had a small, round euchromatic nucleus with a small nucleolus and vacuolated cytoplasm. The seminiferous tubules were devoid of lumen or ectatic, and the tubular wall was thick and contained abundant elastic fibres. This lesion was characteristic of patients who underwent hormonal treatment because of prostatic carcinoma or sex change. Type V lesion showed abnormally differentiated, probably dysgenetic, Sertoli cells which had a round to ovoid regularly outlined nucleus, with small heterochromatin granules, and the number of these cells was increased. The seminiferous tubules had a central lumen, or were ectatic with vacuolated Sertoli cells, and the amount of elastic fibres was decreased. The most relevant clinical finding in patients with this lesion was orchidopexy. Serum FSH and LH levels were normal or slightly increased. These findings indicate that spermatogonial maturational arrest is associated with a characteristic Sertoli cell morphology that can be easily identified. This morphology may shed light on the aetiology of the disorder, and be useful for establishing the prognosis and bases for treatment in subfertile patients.