Temporal appearance of seasonal changes in numbers of Sertoli cells, Leydig cells, and germ cells in stallions

The temporal appearance of seasonal changes in numbers of Leydig, Sertoli, and germ cells was evaluated to determine if seasonally increased daily spermatozoan production might be preceded by changes in numbers of either of two somatic testicular cells. A significant increase in numbers of spermatogonia and Sertoli cells preceded the significant increase in number of Leydig cells in the approaching breeding season. Seasonal changes in parenchymal weight and in numbers of Sertoli cells, Leydig cells, and germ cells were maximal in May and June. Numbers of A or B spermatogonia in June were 2.4 to 2.5 times the number present in January. During the same time period, numbers of other germ cells, as well as Leydig cells and Sertoli cells, were increased by 1.5 to 1.9 times. The magnitude of change between January and March (first time period that the change was significant) was greater for A spermatogonia (1.7-fold) than for other cell types (1.3-fold to 1.5-fold). Hence, the need to accommodate more spermatogonial progeny might cause increased testicular size and number of somatic cells, including Sertoli cells. Season did not influence the rate of degeneration between A and B spermatogonia. However, in the breeding season, the conversion of B spermatogonia to primary spermatocytes was reduced. The lack of a seasonal difference in the ratio of primary spermatocytes per Sertoli cell was consistent with a limited capacity of individual Sertoli cells to accommodate primary spermatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH-initiated differentiation of newt spermatogonia to primary spermatocytes in germ-somatic cell reaggregates cultured within a collagen matrix

We previously cultured fragments of newt testes in chemically defined media and showed that mammalian follicle-stimulating hormone (FSH) stimulates proliferation of spermatogonia as well as their differentiation into primary spermatocytes (Ji et al., 1992; Abe and Ji, 1994). Next, we indicated in cultures composed of spermatogonia and somatic cells (mainly Sertoli cells) that FSH stimulates germ cell proliferation via Sertoli cells (Maekawa et al., 1995). However, the spermatogonia did not differentiate into primary spermatocytes, but instead died. In the present study, we embedded large reaggregates of spermatogonia and somatic cells (mainly Sertoli cells) within a collagen matrix and cultured the reaggregates on a filter that floated on chemically defined media containing FSH; in this revised culture system, spermatogonia proliferated and differentiated into primary spermatocytes. The viability and percentage of germ cells differentiating into primary spermatocytes were proportional to the percentage of somatic cells in the culture, indicating that differentiation of spermatogonia into primary spermatocytes is mediated by Sertoli cells.     

Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent).     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Seasonal effects on apoptosis and proliferation of germ cells in the testes of the Chinese soft-shelled turtle, Pelodiscus sinensis

To elucidate the processes involved in the spatial and temporal maturation of spermatogenic cells in the testes of the soft-shelled turtle, Pelodiscus sinensis, we used a histological morphology method, TdT-mediated dUTP nick end-labeling (TUNEL) assay, the proliferating-cell nuclear antigen (PCNA), and electron microscopy. Seminiferous tubules from 100 turtles, normal for size of testes and semen quality, were collected during 10 months of a complete annual cycle (10 turtles/month). The seminiferous epithelium was spermatogenically active through the summer and fall, but quiescent throughout the rest of the year; germ cells progressed through spermatogenesis in a temporal rather than a spatial pattern, resulting in a single spermatogenic event that climaxed with one massive sperm release in November. The TUNEL method detected few apoptotic cells in spermatogenic testis, with much larger numbers during the spermatogenically quiescent phase. Spermatocytes were the most common germ cell types labeled by the TUNEL assay (a few spermatogonia were also labeled). Apoptotic spermatocytes had membrane blebbing and chromatin condensation during the resting phase, but not during active spermatogenesis. We inferred that accelerated apoptosis of spermatogonia and spermatocytes partly accounted for germ cell loss during the nonspermatogenic phase. The PCNA was expressed in nuclei of spermatogonia and primary spermatocytes during the spermatogenically active phase. During the regressive phase, PCNA-positive cells also included spermatogonia and spermatocytes, but the number of positive spermatocytes was less than that during the spermatogenically active phase. We concluded that seasonal variations in spermatogenesis in the soft-shelled turtle were both stage- and process-specific.     

Follicle-stimulating hormone is indispensable for the last spermatogonial mitosis preceding meiosis initiation in newts (Cynops pyrrhogaster).

We previously reported that mammalian FSH induced differentiation of secondary spermatogonia into primary spermatocytes in organ culture of newt testicular fragments, whereas in medium lacking FSH primary spermatocytes never appeared. Here, we investigated why spermatogonia fail to form primary spermatocytes in the absence of FSH. Spermatogonia maintained proliferative activity and viability at about half the level of those cultured in the presence of FSH, progressed into the seventh generation, but became moribund during the G2/M phase. Thus, the eighth generation of spermatogonia never appeared, suggesting that cell death is the chief reason why primary spermatocytes fail to form in the absence of FSH. The presence of Dmc1, a molecular marker for the spermatocyte stage, confirmed our microscopic observations that spermatogonia differentiated into primary spermatocytes in the presence of FSH. Thus, FSH is indispensable for the completion of the last spermatogonial mitosis, a prerequisite for the conversion of germ cells from mitosis to meiosis. Because prolactin induced apoptosis in spermatogonia during the seventh generation, we propose that a checkpoint exists for the initiation of meiosis in the seventh generation whereby spermatogonia enter meiosis when the concentration ratio of FSH to prolactin is high but fail to do so when the ratio is low.     

Human recombinant stem cell factor promotes spermatogonial proliferation,but not meiosis initiation in organ culture of newt testis fragments

We previously showed that mammalian FSH stimulates the proliferation of newt spermatogonia and induces their differentiation into primary spermatocytes in vitro. In the current study, to examine a possibility that stem cell factor (SCF) is involved in the proliferation of newt spermatogonia and/or their differentiation into primary spermatocytes, human recombinant SCF (rhSCF) was added to organ culture of testicular fragments. rhSCF was found to stimulate the spermatogonial proliferation and the spermatogonia progressed to the seventh generation that is the penultimate stage before primary spermatocyte stage. However, the spermatogonia did not differentiate into primary spermatocytes, but instead died of apoptosis. These results indicate that rhSCF promotes the proliferation of newt spermatogonia, but not the initiation of meiosis.     

Chromosome aberrations induced by monomeric acrylamide in bone marrow and germ cells of mice

Acrylamide induces chromatid exchanges and breaks with considerable frequency in spermatogonia of mice with long-term administration (3 weeks), though not, remarkably, with short-term administration (1–2 weeks). At 12 and 24 h after single injections with 50, 100 and 150 mg/kg acrylamide, evaluation of the cytogenetic effect is difficult in the spermatogonia because of an extreme reduction of mitotic cells. Aneuploid and polyploid cells increase with ti,e after treatment in both marrow and spermatogonial cells, while the aberration frequency shows no increase in marrow after both oral-administration and injection. Evidently the spermatogonia are thus rather more sensitive to acrylamide than marrow cells. On the other hand, the SCE frequency is at the control level in treated subjects in marrow and spermatogonia. Acrylamide induces chain quadrivalents, ring quadrivalents, fragments and univalents which are particularly evident in primary spermatocytes in both oral administration and injection, though it is questionable whether these structural changes deal with spermatogonia, or otherwise with the S-phase primary spermatocytes. There is a possibility that the aberrant cells thus produced can develop into spermatozoa carrying a certain type of reciprocal translocation which leads to semi-sterile progeny. In relation to the above problem detailed investigations into this type of rearrangement in primary spermatocytes are needed.     

Influence of stress on gonadotrophin induced testicular recrudescence in the lizard Mabuya Carinata

Administration (ip) of FSH (10 IU/0.1 ml distilled water (dw)/lizard/alternate days/30 days) to adult male lizards, Mabuya carinata, during the early recrudescence phase of the reproductive cycle caused activation of spermatogenic and steroidogenic activity of the testis, as shown by a significant increase in mean number of spermatogonia, primary spermatocytes and spermatids, and serum levels of testosterone, as compared to initial controls. In addition, there were abundant spermatozoa in the lumen of the seminiferous tubules. Interestingly, administration of a similar dosage of FSH to lizards exposed to stressors (handling, chasing, and noise randomly applied, five times a day for 30 days) resulted in a significant increase in mean number of spermatogonia and primary spermatocytes over initial control values, whereas the number of secondary spermatocytes and spermatids and serum levels of testosterone did not significantly differ from those of initial controls, and were significantly lower than FSH treated normal lizards. Further, spermatozoa were infrequently found in the seminiferous tubules of these lizards. Treatment controls (receiving 0.1 ml dw/lizard/alternate days for 30 days) did not show significant variation in mean number of spermatogonia, spermatocytes and spermatids, and serum levels of testosterone from initial controls. Another group of lizards was exposed to stressors and did not receive FSH. These lizards showed a significant decrease in mean number of secondary spermatocytes compared to treatment controls and all other parameters did not significantly differ from those of both control groups. The results reveal that gonadotrophin-induced spermatogonial proliferation occurs under stressful conditions, whereas progress of spermatogenesis beyond primary spermatocyte stage is impaired due to inhibition (under stress) of gonadotrophin induced steroidogenic activity in M. carinata.     

Immunolocalization of proliferating cell nuclear antigen (PCNA) in cynomolgus monkey (Macaca fascicularis) testes during postnatal development

The age-related distribution of proliferating cell nuclear antigen (PCNA) in the testes of cynomolgus monkeys (Macaca fascicularis) during postnatal development was detected using light-microscopic immunohistochemistry. In neonatal testes, some PCNA-positive spermatogonia, Sertoli cells, peritubular cells, and Leydig cells were detected. In early infantile testes, only a few of these cell types were positive. In late infantile testes, the numbers of positive cells were greater than in the earlier developmental stages. In pubertal testes, the numbers of positive spermatogonia, spermatocytes, Sertoli cells, peritubular cells, and Leydig cells were considerably higher. In adult testes, a larger percentage of spermatogonia and spermatocytes was positive, and peritubular cells and Leydig cells were occasionally positive; secondary spermatocytes, spermatids, and Sertoli cells were not positive. We concluded that immunolocalization of PCNA can serve as a tool for studying proliferation status in developing testes of cynomolgus monkeys. A relatively low proliferative activity in early infantile testes and a remarkable increase of proliferative activity in pubertal testes correlate with the fluctuations of steroidogenic functions during postnatal development in cynomolgus monkeys.     

Murine male germ cell apoptosis induced by busulfan treatment correlates with loss of c-kit-expression in a Fas/FasL- and p53-independent manner

Male germ cell apoptosis has been extensively explored in rodents. In contrast, very little is known about the susceptibility of developing germ cells to apoptosis in response to busulfan treatment. Spontaneous apoptosis of germ cells is rarely observed in the adult mouse testis, but under the experimental conditions described here, busulfan-treated mice exhibited a marked increase in apoptosis and a decrease in testis weight. TdT-mediated dUTP-X nicked end labeling analysis indicates that at one week following busulfan treatment, apoptosis was confined mainly to spermatogonia, with lesser effects on spermatocytes. The percentage of apoptosis-positive tubules and the apoptotic cell index increased in a time-dependent manner. An immediate effect was observed in spermatogonia within one week of treatment, and in the following week, secondary effects were observed in spermatocytes. RT-PCR analysis showed that expression of the spermatogonia-specific markers c-kit and Stra 8 was reduced but that Gli I gene expression remained constant, which is indicative of primary apoptosis of differentiating type A spermatogonia. Three and four weeks after busulfan treatment, RAD51 and FasL expression decreased to nearly undetectable levels, indicating that meiotic spermatocytes and post-meiotic cells, respectively, were lost. The period of germ cell depletion did not coincide with increased p53 or Fas/FasL expression in the busulfan-treated testis, although p110Rb phosphorylation and PCNA expression were inhibited. These data suggest that increased depletion of male germ cells in the busulfan-treated mouse is mediated by loss of c-kit/SCF signaling but not by p53- or Fas/FasL-dependent mechanisms. Spermatogonial stem cells may be protected from cell death by modulating cell cycle signaling such that E2F-dependent protein expression, which is critical for G1 phase progression, is inhibited.     

Induction of chromosomal aberrations in male mouse germ cells by uranyl fluoride containing enriched uranium

Cytogenetic damage induced by a wide range of concentrations of uranyl fluoride injected into mouse testes was evaluated by determining the frequencies of chromosomal aberrations in spermatogonia and primary spermatocytes. Breaks, gaps and polyploids were observed in spermatogonia. The frequencies of the significant type of aberration, breaks, were induced according to the injected doses of uranyl fluoride. Primary spermatocytes were examined for fragments, univalents and multivalents. The multivalents observed in this study resulted either from chromatid interchanges or from reciprocal translocations. The reciprocal translocations were induced in spermatogonia and recorded in primary spermatocytes. For primary spermatocytes the incidence of aberrant cells largely depended on the administered dose. Sampling time after treatment could affect the frequencies of chromosomal aberrations in male mouse germ cells.     

SPERMATOGONIAL STEM CELL RENEWAL IN THE MOUSE: II AFTER CELL LOSS

The repair of the mouse seminiferous epithelium after cell loss has been studied in seminiferous tubules mounted in toto . Cell loss was inflicted by injection of Myleran in a dose of 10 mg/kg body weight. In stages 7–8, in which we mainly counted, the numbers of A_isolated (A_is), A_paired (A_pr), A_aligned (A_al) and A₁ spermatogonia and resting primary spermatocytes decreased after injection. After about 24 days normal numbers of A₁ spermatogonia were found again. Thereafter a substantial overshoot in the number of A₁ spermatogonia was found.
While normally most of the A_pr and A_al cells differentiate into A₁ spermatogonia in stages 3 and 4 and do not divide until stage 9, during repair they pass through one more division during stages 6 and 7. Normally, during these stages divisions of these spermatogonia are rare. Owing to this extra division the transformation of A_pr and A_al into A₁ spermatogonia is delayed from stage 3 or 4 to stage 8, i.e. still before stage 9, in which A₁ spermatogonia divide. From 16 days after the injection onwards the extra division takes place less generally and more and more cells transform into A₁ spermatogonia at the normal time.     

Appearance of cell surface auto- and isoantigens during spermatogenesis in the rabbit

The appearance of spermatogenic cell surface auto- and isoantigens can be precisely determined by utilizing techniques that separate spermatogenic cells. Using cytotoxic (complement dependent) auto- and iso- rabbit antirabbit whole semen sera, specific spermatogenic auto- and isoantigens were first detected following the maturation of spermatogonia into primary pachytene spermatocytes. The antisera employed were cytotoxic (complement dependent) for pachytene diplotene and primary spermatocytes and spermatids but not for type A, intermediate, or type B spermatogonia. Furthermore, Sertoli cells, endothelial cells, Leydig cells, and erythrocytes were not lysed by the antisera. These observations support the concept of a blood-testis barrier. Only after migration of spermatogonia to the luminal side of the barrier can autoantigenic molecules be synthesized and/or inserted into the plasma membrane of spermatogenic cells. Thus, the appearance of surface autoantigens offers a model system to study the synthesis of specific molecules which are inserted into the plasma membrane at a precise time during development.     

Differentiation of Secondary Spermatogonia to Primary Spermatocytes by Mammalian Follicle-Stimulating Hormone in Organ Culture of Testes Fragments from the Newt, Cynops pyrrhogaster

In order to elucidate essential factors responsible for the initiation and promotion of spermatogenesis, we developed an organ culture system with a chemically defined medium. When newt testes fragments, consisting of somatic cells and germ cells almost exclusively secondary spermatogonia, were cultured in control medium for three weeks, most of the testicular cysts still contained only secondary spermatogonia. On the other hand, in the medium supplemented with various kinds of hormones and vitamins primary spermatocytes (zygotene-pachytene) appeared in about 60% of the cysts by the second week. Selective removal of specific hormones and vitamins revealed that follicle-stimulating hormone (FSH) alone was indispensable and sufficient for the differentiation of secondary spermatogonia to primary spermatocytes. Neither the addition of luteinizing hormone (LH) nor androgens (testosterone and 5α-dihydrotestosterone) to the control medium stimulated differentiation. Consistent with these findings was the fact that radioreceptor assays revealed high affinity specific binding sites for FSH but none for LH. Since our ultrastructural studies revealed a major loss of contact between spermatogonia and Sertoli cells following exposure to FSH, we suggest that FSH triggers differentiation of spermatogonia by acting on Sertoli cells which in turn act on spermatogonia.     

Temporal expression of membrane antigens during mouse spermatogenesis.

The temporal expression of cell surface antigens during mammalian spermatogenesis has been investigated using isolated populations of mouse germ cells. Spermatogenic cells at advanced stages of differentiation, including pachytene primary spermatocytes, round spermatids, and residual bodies of Regaud and mature spermatozoa, contain common antigenic membrane components which are not detected before the pachytene stage of the first meiotic prophase. These surface constituents are not detected on isolated populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, or leptotene and zygotene primary spermatocytes. These results have been demonstrated by immunofluorescence microscopy, by complement-mediated cytotoxicity, and by quantitative measurements of immunoglobulin (Ig) receptors on the plasma membrane of all cell populations examined. The cell surface antigens detected on germ cells are not found on mouse thymocytes, erythrocytes, or peripheral blood lymphocytes as determined by immunofluorescence and by cytotoxicity assays. Furthermore, absorption of antisera with kidney and liver tissue does not reduce the reactivity of the antibody preparations with spermatogenic cells, indicating that these antigenic determinants are specific to germ cells. This represents the first direct evidence for the ordered temporal appearance of plasma membrane antigens specific to particular classes of mouse spermatogenic cells. It appears that at late meiotic prophase, coincident with the production of pachytene primary spermatocytes, a variety of new components are inserted into the surface membranes of developing germ cells. The further identification and biochemical characterization of these constituents should facilitate an understanding of mammalian spermatogenesis at the molecular level.     

Protection from radiation-induced male germ cell loss by sphingosine-1-phosphate

Male germ cells are susceptible to radiation-induced injury, and infertility is a common problem after total-body irradiation. Here we investigated, first, the effects of irradiation on germ cells in mouse testis and, second, the role of sphingosine-1-phosphate (S1P) treatment in radiation-induced male germ cell loss. Irradiation of mouse testes mainly damaged the early developmental stages of spermatogonia. The damage was seen by means of DNA flow cytometry 21 days after irradiation as decreasing numbers of spermatocytes and spermatids with increasing amounts of ionizing radiation (0.1-2.0 Gy). Intratesticular injections of S1P given 1-2 h before irradiation (0.5 Gy) did not protect against short-term germ cell loss as measured by in situ end labeling of DNA fragmentation 16 h after irradiation. However, after 21 days, in the S1P-treated testes, the numbers of primary spermatocytes and spermatogonia at G2 (4C peak as measured by flow cytometry) were higher at all stages of spermatogenesis compared with vehicle-treated testes, indicating protection of early spermatogonia by S1P, whereas the spermatid (1C) populations were similar. In conclusion, S1P appears to protect partially (16%-47%) testicular germ cells against radiation-induced cell death. This warrants further studies aimed at development of therapeutic agents capable of blocking sphingomyelin-induced pathways of germ cell loss.