Spermatogonial Population Research Articles

021. Current progress into testis cell transfer between cattle breeds

Male germline cell transfer has produced offspring in mice (Brinster and Zimmermann 1994). Recently the first livestock animal, a goat, was produced (Honaramooz et al. 2003), while early results in cattle are promising (Oatley et al. 2002; Izadyar et al. 2003). There is an opportunity to develop this technology for the beef industry by transferring male germ line stem cells between breeds to improve the genetics of extensive Australian beef herds. This project is a part of the CSIRO National Research Flagship program that combines expertise and facilities in divisions with complementary expertise at Monash University and the University of Sydney. The environmental constraints of Northern Australia dictate that Brahman type animals show far better survival than Bos taurus cattle, although the carcass value of Brahmans is lower than Bos taurus animals. Artificial insemination is impractical in Northern Australia and thus we aim to develop testis cell transfer technique in cattle to permit Brahman bulls to deliver semen from elite Bos taurus or composite bulls, thereby significantly increasing the growth rate, yield and meat quality of the northern beef herd. Experiments using cattle were performed to determine the applicability of techniques used in the mouse. Initial proof of concept has been achieved that germ cell transfer can result in the donor cells successfully colonizing areas of recipient testis. The viability of isolated testis cells following short term (24 h) culture has been demonstrated through transfer into recipient calves. We have completed >50 male germ cell transfers into recipient calves, using ultrasonographic guided injection into the rete testis. Success of this procedure has been demonstrated by persistence of PKH26 dyed donor cells in the seminiferous tubules of a majority of recipients >2 months after transfer. These recipient male calves have not been depleted of their endogenous spermatogonial populations and we thus expect the efficiency of the procedure to increase as depletion procedures (ongoing) are established. Concurrent with these developments has been research into large scale culture of male germ line stem cells to provide large numbers of stem cells for transplant. Culture of testis cell suspensions has demonstrated survival of enriched testis cells under varying media and culture conditions. Initial passaging of testis cell colonies has revealed mixed cell populations (immunohistochemistry positive for spermatogonia and somatic cells). Further studies will aim to demonstrate that these cultured donor cells are able to undergo spermatogenesis in the recipient animals.

Clonal organization of proliferating spermatogonial stem cells in adult males of two species of non-human primates, Macaca mulatta and Callithrix jacchus.

The present study examines the existence of clonogenic patterns in the proliferation and differentiation of spermatogonial stem cells in two species of non-human primates, the marmoset and the rhesus monkey. We developed a novel approach to detect proliferating spermatogonial clones in whole mounts of seminiferous tubules. Dual fluorescence labeling of bromodeoxyuridine and acrosin in conjunction with confocal microscopy allows the description of the clonogenic and spatial arrangement of proliferating spermatogonia at specific stages of the seminiferous epithelial cycle. Cross-sections of paraffin-embedded tissue were labeled by the same approach. For both monkey species we demonstrate the presence of proliferating spermatogonial clones of variable size at specific stages of the cycle of the seminiferous epithelium. Detailed analysis of the rhesus monkey reveals proliferating Apale spermatogonia at stages VII and IX of the cycle of the seminiferous epithelium, and of proliferating B spermatogonia at stages II, IV, VI, and XII. Proliferating Apale spermatogonia at stages VII and IX of the cycle are organized in pairs or quadruplets. B1 spermatogonia appear as quadruplets or eight-cell clones, and B2 spermatogonia as 8- or 16-cell clones. We conclude that spermatogenesis in the rhesus monkey is initiated by two divisions of duplets or quadruplets of Apale spermatogonia: a first division at stage VII, after which the clones of Apale spermatogonia separate, and a second division at stage IX, which leads to clones of B1 spermatogonia as well as pairs and quadruplets of Apale spermatogonia replenishing the seminiferous epithelium to maintain the original size of the A spermatogonial population.

Analysis of the gene expression profile of mouse male meiotic germ cells

Wide genome analysis of difference in gene expression between spermatogonial populations from 7-day-old mice and pachytene spermatocytes from 18-day-old mice was performed using Affymetrix gene chips representing ∼12,500 mouse known genes or EST sequences, spanning approximately 1/3rd of the mouse genome. To delineate differences in the profile of gene expression between mitotic and meiotic stages of male germ cell differentiation, expressed genes were grouped in functional clusters. The analysis confirmed the previously described pre-meiotic or meiotic expression for several genes, in particular for those involved in the regulation of the mitotic and meiotic cell cycle, and for those whose transcripts are accumulated during the meiotic stages to be translated later in post-meiotic stages. Differential expression of several additional genes was discovered. In few cases (pro-apoptotic factors Bak, Bad and Bax), data were in conflict with the previously published stage-dependent expression of genes already known to be expressed in male germ cells. Northern blot analysis of selected genes confirmed the results obtained with the microarray chips. Six of these were novel genes specifically expressed in pachytene spermatocytes: a chromatin remodeling factor ( chrac1/ YCL1), a homeobox gene ( hmx1), a novel G-coupled receptor for an unknown ligand ( Gpr19), a glycoprotein of the intestinal epithelium ( mucin 3), a novel RAS activator ( Ranbp9), and the A630056B21Rik gene (predicted to encode a novel zinc finger protein). These studies will help to delineate the global patterns of gene expression characterizing male germ cell differentiation for a better understanding of regulation of spermatogenesis in mammals.

Effect of textile waste water on the spermatogenesis of male albino rats.

Textile waste water released from dyeing and printing industries situated in Sanganer, Jaipur (India), brought about inhibition of spermatogenesis in male rats. Water analysis showed the presence of heavy metals at more than permissible limits. Oral administration of waste water to the rats at the dose level of 26.6 ml kg(-1) body wt. significantly reduced the weights of testes, epididymides and seminal vesicle. Treated animals showed a notable depression of various stages of spermatogenesis. The production of spermatids was inhibited by 70.8% in waste-water-treated rats. The populations of spermatogonia, preleptotene spermatocytes and secondary spermatocytes were decreased by 67.2, 71.1 and 73.2%, respectively. The total number of Sertoli cells was affected after waste water treatment. Reduced sperm count and motility resulted in treated groups. A significant fall in the content of various biochemical parameters of reproductive tissues was observed after water treatment.

51. FSH regulates Sertoli cell and spermatogonial populations in the adult Djungarian hamster

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Use of recombinant human follicle-stimulating hormone in the treatment of male factor infertility

Objective: To evaluate the effects of treatment with recombinant human FSH (r-hFSH) on seminal parameters and seminiferous epithelium in idiopathic patients with oligozoospermia with normal FSH plasma levels. Design: Randomized single-blind study. Setting: Academic setting. Patient(s): Forty-five subjects with idiopathic oligozoospermia (sperm count <10 × 10 6/mL) and normal FSH and inhibin B plasma levels. Intervention(s): Three months of treatment with r-hFSH 50 IU (15 patients) or with r-hFSH 100 IU on alternate days (15 patients) or no treatment (15 patients); bilateral testicular fine-needle aspiration (FNA) performed before and after therapy; FSH and inhibin B plasma levels evaluated during treatment. Main Outcome Measure(s): Seminal parameters; testicular cytological features evaluated by FNA; plasma levels of FSH, LH, T, and inhibin B. Result(s): Treatment with r-hFSH at a dose of 50 IU induced no increase in sperm concentration, while treatment with r-hFSH at a dose of 100 IU induced a significant increase in sperm concentration. In particular, in 11/15 patients a doubling of the pretreatment sperm concentration was observed. No significant increase in sperm parameters was observed in the control group. In both groups of patients treated with r-hFSH, the cytological analysis before treatment showed hypospermatogenesis. An increase in the percentage of spermatogonia and spermatocytes was observed only after the treatment with r-hFSH at a dose of 100 IU. Conclusion(s): The findings of this study demonstrate that r-hFSH at a dose of 100 IU, as previously seen with highly purified FSH, increases the spermatogonial population and sperm production in idiopathic patients with oligozoospermia with normal FSH and inhibin B plasma levels and a cytological picture of hypospermatogenesis.

Elimination of male germ cells in transgenic mice by the diphtheria toxin A chain gene directed by the histone H1t promoter.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Evidence for a Stimulatory Role of Follicle-Stimulating Hormone on the Spermatogonial Population in Adult Males

Abstract Objective: To evaluate the effects of treatment with FSH on seminal indices and on the seminiferous epithelium of oligozoospermic subjects with normal FSH plasma levels. Design: Placebo-controlled, double-blind randomized study. Setting: Academic setting. Patient(s): Ninety subjects with idiopathic oligozoospermia (sperm count of 6 /mL) and normal plasma levels of FSH. Intervention(s): Three months of treatment with FSH (60 patients) or placebo (30 patients); bilateral testicular fine-needle aspiration. Main Outcome Measure(s): Seminal indices; testicular cytologic features; plasma levels of FSH, LH, and testosterone; and ultrasonographic testicular examination. Result(s): According to seminal indices, patients treated with FSH and placebo were classified as nonresponders or as responders (as determined by at least a doubling of sperm count). No placebo-treated patients responded to treatment. Among FSH-treated patients, 20 responded to hormonal treatment and 40 did not. The results of pretreatment cytologic examination of testicular specimens from patients who did not respond to FSH treatment were consistent with hypospermatogenesis associated with maturational disturbances at the spermatid level. In contrast, patients who responded to treatment with FSH had isolated hypospermatogenesis without maturational disturbances. After FSH therapy, we detected an increase of spermatogonia and spermatocyte population in both the responder and nonresponder subjects. This increase was associated with an activation of spermatogenic and spermiogenic processes and with a rise in ejaculated sperm concentration only when isolated hypospermatogenesis was present (responder patients). Conclusion(s): The findings of this study demonstrate that FSH treatment increases the spermatogonial population in men. This treatment may be appropriate for oligozoospermic subjects who have normal FSH plasma levels and a testicular evaluation characterized by hypospermatogenesis without maturational disturbances.

Evidence for a Stimulatory Role of Follicle-Stimulating Hormone on the Spermatogonial Population in Adult Males

Evidence for a Stimulatory Role of Follicle-Stimulating Hormone on the Spermatogonial Population in Adult Males

HISTOLOGIA TESTICULAR HUMANA COMPARADA, ADULTO JOVEN Y SENIL

In general, sperm production decreases with advanced age in men. Therefore the present work compares the histology and cellularity of aging human testis with that of a young adult. Gonads from three patients 69 years old and testis from a young subject (aged 25 years) were examined after histological standard techniques (PAS-Haematoxyline). Histological, morphometric and cell counting analyses of testicular sections revealed that the seminiferous tubules of senile patients were of smaller diameter, with lower seminiferous epithelium, incomplete germ cell line and vacuolization of the epithelium. Sertoli cells showed a market quantitative decrease, similar to the spermatogonial and primary spermatocyte population. It is possible to postulated that in the senile individues there is a programmed damage of seminiferous epithelial cells that may result in lowered sperm production

Mitochondrial respiratory function during rat thymocyte induced apoptosis

Different doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.1, 1, 5, and 10 μg/kg body wt) were administered ip to 21-day-old male Sprague–Dawley rats. Control animals received the same volume of the vehicle (acetone:corn oil, 1:19). Body weight and daily food intake were recorded during the 90-day time course of the study. Random samples of five rats were sacrificed at 34, 49, 62, and 90 days of age. Epidermal growth factor receptor (EGFR) in whole testis was measured, as were the activities of c-Src kinase, protein tyrosine kinase (PTK), mitogen-activated protein 2 kinase (MAP2K also termed as Erk2), protein kinase A (PKA), and protein kinase C (PKC). Testicular tissue from 90-day-old rats was evaluated for histopathology, and sperm numbers in whole testis were counted to estimate daily sperm production. The motility of sperm in the vas deferens and caudal segments of the epididymis of 90-day-old rats was measured by computer assisted sperm analysis (CASA) and the function of the sperm was tested by assessment of acrosome reactions. A dose of 10 μg/kg resulted in testicular atrophy and histopathologic examination revealed a decrease in the diameter of the seminiferous tubules. Sertoli cell nuclei were clearly seen, but the spermatogonial population was totally absent. Lower doses of TCDD did not affect testicular histology, but doses as low as 1 μg/kg significantly decreased testicular sperm numbers and affected some sperm functions (motility parameters and acrosome reactions) in 90-day-old rats. Significant decreases in EGFR were found in 34-day-old rats and this effect on EGFR was sustained until the end of the experiment (90 days). Although TCDD significantly increased c-Src kinase activity in immature and mature rats, opposite effects of TCDD on activities of PTK, PKA, and PKC were found in 34-day-old rats vs 49-, 62-, and 90-day-old rats. When 10 μg TCDD/kg was administered to 21-day-old rat, 24-h after c-Src kinase inhibitor geldanamycin, there was no testicular atrophy and no change in the daily sperm production was found. These findings provide evidence for involvement of Src kinase signaling and EGFR in the mechanism by which TCDD disrupts testicular development and subsequently affects testis function.

Treatment of Rats during Pubertal Development with 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Alters Both Signaling Kinase Activities and Epidermal Growth Factor Receptor Binding in the Testis and the Motility and Acrosomal Reaction of Sperm

Different doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (0.1, 1, 5, and 10 μg/kg body wt) were administered ip to 21-day-old male Sprague–Dawley rats. Control animals received the same volume of the vehicle (acetone:corn oil, 1:19). Body weight and daily food intake were recorded during the 90-day time course of the study. Random samples of five rats were sacrificed at 34, 49, 62, and 90 days of age. Epidermal growth factor receptor (EGFR) in whole testis was measured, as were the activities of c-Src kinase, protein tyrosine kinase (PTK), mitogen-activated protein 2 kinase (MAP2K also termed as Erk2), protein kinase A (PKA), and protein kinase C (PKC). Testicular tissue from 90-day-old rats was evaluated for histopathology, and sperm numbers in whole testis were counted to estimate daily sperm production. The motility of sperm in the vas deferens and caudal segments of the epididymis of 90-day-old rats was measured by computer assisted sperm analysis (CASA) and the function of the sperm was tested by assessment of acrosome reactions. A dose of 10 μg/kg resulted in testicular atrophy and histopathologic examination revealed a decrease in the diameter of the seminiferous tubules. Sertoli cell nuclei were clearly seen, but the spermatogonial population was totally absent. Lower doses of TCDD did not affect testicular histology, but doses as low as 1 μg/kg significantly decreased testicular sperm numbers and affected some sperm functions (motility parameters and acrosome reactions) in 90-day-old rats. Significant decreases in EGFR were found in 34-day-old rats and this effect on EGFR was sustained until the end of the experiment (90 days). Although TCDD significantly increased c-Src kinase activity in immature and mature rats, opposite effects of TCDD on activities of PTK, PKA, and PKC were found in 34-day-old rats vs 49-, 62-, and 90-day-old rats. When 10 μg TCDD/kg was administered to 21-day-old rat, 24-h after c-Src kinase inhibitor geldanamycin, there was no testicular atrophy and no change in the daily sperm production was found. These findings provide evidence for involvement of Src kinase signaling and EGFR in the mechanism by which TCDD disrupts testicular development and subsequently affects testis function.

Radioprotective effect of zinc aspartate on mouse spermatogenesis: a flow cytometric evaluation

The radioprotective effect of zinc aspartate on spermatogonial cells of whole-body irradiated mice was studied using flow cytometry. Adult male Swiss albino mice were treated with 30 mg/kg body weight of zinc aspartate 30 min before exposure to 0, 0.5, 1, 2 and 3 Gy of γ-radiation. The animals were killed 7 to 70 days postirradiation and the relative percentages of different germ cells were analyzed by flow cytometry. A significant increase ( p<0.002, 0.0001, 0.005 and 0.008 for 0.5, 1, 2 and 3 Gy, respectively) in the relative percentage of spermatogonial (2C) population was observed in mice treated with zinc aspartate before exposure to different doses of γ-radiation, compared to the irradiated controls on day 35 posttreatment. Also mean of each radiation dose of all the intervals studied showed a significant ( p<0.03) increase in the relative percentage of spermatogonia. Despite the increase in the relative percentage of spermatogonia, the relative percentage of tetraploid cells (4C) remained higher in the zinc aspartate treated mice, compared to the irradiated controls. However, there was no change in the haploid populations viz. round (1C) and elongated (HC) spermatids of the zinc aspartate pretreated animals compared to irradiated controls. These data suggests that zinc aspartate pretreatment protects spermatogonia and tetraploid cells from radiation-induced cell killing.

Evidence for a Stimulatory Role of Follicle-Stimulating Hormone on the Spermatogonial Population in Adult Males

Objective: To evaluate the effects of treatment with FSH on seminal indices and on the seminiferous epithelium of oligozoospermic subjects with normal FSH plasma levels. Design: Placebo-controlled, double-blind randomized study. Setting: Academic setting. Patient(s): Ninety subjects with idiopathic oligozoospermia (sperm count of <10 × 10 6/mL) and normal plasma levels of FSH. Intervention(s): Three months of treatment with FSH (60 patients) or placebo (30 patients); bilateral testicular fine-needle aspiration. Main Outcome Measure(s): Seminal indices; testicular cytologic features; plasma levels of FSH, LH, and testosterone; and ultrasonographic testicular examination. Result(s): According to seminal indices, patients treated with FSH and placebo were classified as nonresponders or as responders (as determined by at least a doubling of sperm count). No placebo-treated patients responded to treatment. Among FSH-treated patients, 20 responded to hormonal treatment and 40 did not. The results of pretreatment cytologic examination of testicular specimens from patients who did not respond to FSH treatment were consistent with hypospermatogenesis associated with maturational disturbances at the spermatid level. In contrast, patients who responded to treatment with FSH had isolated hypospermatogenesis without maturational disturbances. After FSH therapy, we detected an increase of spermatogonia and spermatocyte population in both the responder and nonresponder subjects. This increase was associated with an activation of spermatogenic and spermiogenic processes and with a rise in ejaculated sperm concentration only when isolated hypospermatogenesis was present (responder patients). Conclusion(s): The findings of this study demonstrate that FSH treatment increases the spermatogonial population in men. This treatment may be appropriate for oligozoospermic subjects who have normal FSH plasma levels and a testicular evaluation characterized by hypospermatogenesis without maturational disturbances.

Endocrine control of germ cell proliferation in the primate testis. What do we really know?

The present chapter reviews current knowledge concerning hormonal regulation of gametogenesis in the primate testis. LH/testosterone and FSH are the prime regulators of primate spermatogenesis. Although either hormone is capable of stimulating all phases of the spermatogenic process including the formation of sperm, the combination of both hormones is necessary in most instances to achieve quantitatively normal germ cell numbers. Sertoli cell proliferation can also be induced by either hormone in juvenile monkeys. Evidence for differential effects of testosterone and FSH on gametogenesis, however, is lacking and a synergistic effect is observed when they are combined. Receptors for androgens and FSH occur exclusively on testicular somatic cells and, hence, the trophic effects of these hormones on germ cell numbers are indirect ones. Interestingly, both hormones seem to have a common target, the spermatogonial population but it is unknown how such an indirect albeit highly specific effect is mediated. Whether the trophic hormone action influences germ cell numbers via increased proliferation or decreased cell death or both remains to be seen. There is evidence to suggest that the local androgen requirements for primate spermatogenesis might be comparatively high.

Preparation of spermatogonia, spermatocytes, and round spermatids for analysis of gene expression using fluorescence-activated cell sorting.

A new method is described for the purification of spermatogenic cell populations from mouse testis. Through use of this method, it is possible to purify leptotene, zygotene, and pachytene primary spermatocytes as well as round spermatids from adult mouse testis. In addition, spermatogonial populations can be purified from mice at 9 days postpartum. The leptotene and zygotene primary spermatocytes that can be prepared by this method are impossible to separate successfully by the unit gravity method. The cells were used to prepare RNA for reverse transcriptase-polymerase chain reactions.

Effects of quercetin on rat testis aerobic glycolysis.

Lactate production by testicular fragments and isolated germinal cells at various stages of spermatogenesis was studied in aerobic and anerobic conditions. Several ATPase inhibitors were used to determine the role of ATPase activities in the control of aerobic lactate production. Aerobic glycolysis reached a high level in spermatogonia plus Sertoli cell and in primary spermatocyte populations. The activity was twice that found in early spermatids. Neither Na+-K+ ATPase nor mitochondrial F1 ATPase seemed to participate directly in the control of aerobic glycolysis. The uncoupling of oxidative phosphorylation revealed the potential role of F1 ATPase in providing ADP and P(i) for the glycolytic pathway. Lactate production was inhibited by quercetin in all the experimental conditions tested. Quercetin (100 microM) halted lactate production by the Sertoli cell plus spermatogonia population and by isolated primary spermatocytes. In spermatids, quercetin inhibited aerobic glycolysis only by 40%, even at higher concentrations. Only during the first meiotic prophase did quercetin inhibit the activity of a cytosolic Ca(2+)-Mg2+ ATPase. This ATPase was also inhibited by erythro-9-[3-3(hydroxynonyl)]adenine (EHNA), suggesting that a cytoplasmic dynein could be involved in the control of glycolysis in Sertoli cells, spermatogonia, and early primary spermatocytes.

Evolution and ultrastructure of the bovine spermatogonia precursor cell line

The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells.

Stem cell kinetics in rat testis after irreversible injury induced by 2,5-hexanedione.

Stem cells provide a continuous supply of committed progenitor cells for the process of spermatogenesis. In rodents, stem cells have been identified as single, undifferentiated type A spermatogonia. The rate of stem cell division has not been definitively determined because of difficulty in locating stem cells among a normal compliment of germ cells. The testicular toxicant 2,5-hexanedione (2,5-HD) induces irreversible testicular atrophy with only Sertoli cells and spermatogonia remaining after injury. Stem cell kinetics could be assessed in this toxicant model because of the absence of most mature germ cells. It is also not known if 2,5-HD-exposed rats possess an active stem spermatogonia population. Charles River CD rats were exposed to 1% 2,5-HD in drinking water for 5 wk. At 7 or 35 wk following toxicant exposure, rats were exposed to bromodeoxycytidine continuously via Alzet mini-pumps for 1-28 days. Serial cross sections of testis were used to identify single stem spermatogonia and to determine whether the cells were positive or negative for bromodeoxyuridine incorporation. We obtained a continuous labeling index for stem cells from rats 7 and 35 wk after 2,5-HD exposure and found that stem cells had a cell cycle time of approximately 8-14 days at both time points after toxicant exposure. In conclusion, we have developed a method for the assessment of stem cell kinetics and verified the presence of an actively dividing stem cell population in irreversibly injured testes.

Differential Effects of Fractionated X Irradiation on Mouse Spermatogonial Stem Cells

The response of spermatogonial stem cells to fractionated X irradiation was studied in the various stages of the spermatogenic cycle of the CBA mouse. Fractionated doses of 2 + 2, 1 + 3, and 3 + 1 Gy with a 24-h interval between the doses were compared with a single dose of 4 Gy. The numbers of undifferentiated spermatogonia present 10 days after (the second) irradiation were taken as a measure of stem cell survival. Twenty-four hours after the first irradiation a sensitization was observed that was found to be stage-dependent. The greatest sensitization occurred in that part of the spermatogonial stem cell population that was in stages X-I during the first irradiation, i.e., the part that is stimulated to proliferate or actively proliferating at that time. In stages that were quiescent during the first irradiation (VI-VII), fractionation did not influence the response. Therefore, only the spermatogonial stem cells that are initially radioresistant become sensitized 24 h after irradiation. When two unequal doses (3 + 1 Gy or 1 + 3 Gy) are given, damage correlates with the size of the second dose, indicating that priming doses of 1 and 3 Gy are both capable of inducing the sensitizing effect.