Identification of Leydig cell-specific mRNA transcripts in the adult rat testis.

The adult population of Leydig cells acts to secrete testosterone which is essential for reproductive health and fertility in the adult male. However, other physiological functions of these cells are uncertain, and to address this issue a cell ablation model has been used to identify Leydig cell-specific mRNA transcripts. Ethane dimethane sulphonate (EDS) was synthesised by a novel process and was used to ablate Leydig cells in adult male rats previously treated with butane dimethane sulphonate (busulphan) to delete the germ cell population. Levels of mRNA transcripts were measured in the testis using microarrays 1, 3, 5, 8 and 12 days after EDS injection. During this period, there was a significant change in the levels of 2200 different transcripts with a marked decline in the levels of canonical Leydig cell transcripts, such as Cyp11a1, Cyp17a1 and Insl3. A total of 95 transcripts showed a similar decline in expression after EDS treatment, suggesting that they have a Leydig cell-specific origin. Analysis of selected transcripts confirmed that they were expressed specifically in Leydig cells and showed that most had a late onset of expression during adult Leydig cell development. Apart from transcripts encoding components of the steroidogenic apparatus, the most common predicted function of translated proteins was endogenous and xenotoxicant metabolism. In addition, a number of transcripts encode acute-phase proteins involved in reduction of oxidative stress. Results show that, in addition to androgen secretion, Leydig cells may have a critical role to play in protecting the testis from damage caused by toxicants or stress.

Role of androgen and gonadotrophins in the development and function of the Sertoli cells and Leydig cells: data from mutant and genetically modified mice.

Development and maintenance of the male phenotype and establishment of fertility are all dependent upon the activity of the Sertoli cells and Leydig cells of the testis. This review examines the regulation and function of these cell during fetal and post-natal development. Fetal Leydig cells are sensitive to both luteinising hormone (LH) and adrenocorticotrophic hormone (ACTH) but Leydig cell function appears normal in fetal mice lacking both hormones or their receptors. Post-natally, the Sertoli cells and Leydig cells are reliant upon the pituitary gonadotrophins. Leydig cells are critically dependent on LH but follicle-stimulating hormone (FSH), presumably acting through the Sertoli cell, can also affect Leydig cell function. Testosterone secreted by the Leydig cells acts with FSH to stimulate Sertoli cell activity and spermatogenesis. Study of animals lacking FSH-receptors and androgen-receptors shows that both hormones can act to maintain the meiotic germ cell population but that androgens are critical for completion of meiosis.

Leydig cell re-generation and expression of cell signaling molecules in the germ cell-free testis.

Leydig cells in the rat testis can be specifically ablated with ethane dimethane sulfonate (EDS) and will subsequently re-generate. In this study, we have characterized Leydig cell re-generation and expression of selected cell-signaling molecules in a germ cell-free model of EDS action. This model offers the advantage that re-generation occurs on a stable background without confounding changes from the regressing and repopulating germ cell population. Adult rats were treated with busulfan to remove the germ cell population and Leydig cells were then ablated with EDS. Testicular testosterone levels declined markedly within 24 h of EDS treatment and started to recover after 8 days. After EDS treatment there were marked declines in levels of Leydig cell-specific mRNA transcripts coding for steroidogenic enzymes cytochrome P450 11a1 (Cyp11a1), cytochrome P450 17a1 (Cyp17a1), 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1), 17beta-hydroxysteroid dehydrogenase type 3 (Hsd17b3) and the LH receptor. Levels of all transcripts recovered within 20 days of EDS treatment apart from Hsd17b3, which remained undetectable up to 20 days. Immunohistochemical localization of CYP11A1 during the phase of early Leydig cell re-generation showed that the Leydig cell precursors are spindle-shaped peritubular cells. Studies on factors which may be involved in Leydig cell re-generation showed there were significant but transient increases in platelet-derived growth factor A (Pdgfa), leukemia inhibitory factor (Lif), and neurofilament heavy polypeptide (Nefh) after EDS, while desert hedgehog (Dhh) levels declined sharply but recovered by 3 days. This study shows that the Leydig cell precursors are peritubular cells and that expression of Pdgfa and Lif is increased at the start of the re-generation process when precursor proliferation is likely to be taking place.

Coordinated expression of UT-A and UT-B urea transporters in rat testis.

The blood-seminiferous tubule barrier is responsible for maintaining the unique microenvironment conducive to spermatogenesis. A key feature of the blood-testis barrier is selective permeability to solutes and water transport, conferred by the Sertoli cells of the seminiferous tubules (SMTs). Movement of fluid into the lumen of the seminiferous tubule is crucial to spermatogenesis. By Northern analysis, we have shown that 4.0-, 3.3-, 2.8-, and ~1.7-kb UT-A mRNA transcripts and a 3.8-kb UT-B mRNA transcript are detected within rat testis. Western analysis revealed the expression of both characterized and novel UT-A and UT-B proteins within the testis. Immunolocalization studies determined that UT-A and UT-B protein expression are coordinated with the developmental stage of the SMT. UT-A proteins were detected in Sertoli cell nuclei at all stages of tubule development and in residual bodies of stage VIII tubules. UT-B protein was expressed on Sertoli cell membranes of stage II-III tubules. Using in vitro perfusion, we determined that a phloretin-inhibitable urea pathway exists across the SMTs of rat testis and conclude that UT-B is likely to participate in this pathway.

The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis (Comet assay) and its relationship to fertilization and embryo development.

BACKGROUND: The integrity of sperm DNA is important for the success of natural or assisted fertilization, as well as normal development of the embryo, fetus and child. ICSI, by bypassing sperm selection mechanisms, increases the risk of transmitting damaged DNA and the significance of this requires investigation. METHODS: DNA damage in sperm from an unselected group of 60 men undergoing IVF treatment was measured by single cell gel electrophoresis (Comet assay) and correlated with semen and treatment cycle parameters. RESULTS: Wide spectra of sperm DNA damage were found both within and between men but no specific subgroups were identified. Semen and treatment cycle parameters were not different in men grouped according to high or low sperm DNA damage. However, regression analysis showed that DNA damage was positively associated with age (29-44 years), abnormal sperm and motility and negatively associated with sperm concentration. In ICSI cycles DNA damage was positively associated with impairment of post-fertilization embryo cleavage. CONCLUSIONS: This study contributes to the evidence of DNA damage within sperm. High loads of DNA damage measured by the Comet assay were predictive of failure of embryo development after ICSI. As it is likely that sperm with DNA damage contributed to successful fertilization and in-vitro development, potential adverse effects remain to be clarified.

Regulated, adenovirus-mediated delivery of tyrosine hydroxylase suppresses growth of estrogen-induced pituitary prolactinomas.

Prolactin-secreting adenomas are one of the most common types of intracranial neoplasm found in humans. The modalities of clinical treatment currently in use include D(2)-dopamine receptor agonists, surgery, and radiotherapy, and the success rates for treatment are good. However, there are prolactinomas that are difficult to treat. As an alternative, we have developed a gene therapy strategy in which the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is overexpressed in the anterior pituitary (AP) gland. Because dopamine is known to have an inhibitory effect on lactotroph growth and prolactin secretion, we developed a system that would enable its local synthesis from freely available precursor amino acids. A dual adenovirus tetracycline-regulatable expression system was generated to control the production of TH. In the absence but not presence of the tetracycline analog doxycycline, TH expression was observed in AP tumor cell lines AtT20, GH3, and MMQ. In both primary AP cell cultures and the AP gland, in situ expression of TH was seen in lactotrophs, somatotrophs, corticotrophs, thyrotrophs, and gonadotrophs in the absence but not presence of doxycycline. The ability of this system to inhibit hyperprolactinemia and pituitary lactotroph hyperplasia was then assessed in a model of estrogen- or estrogen/sulpiride-induced pituitary tumors. In the absence but not presence of doxycycline, a 49% reduction in pituitary growth and 58% reduction in the increase of circulating prolactin levels were observed in estrogen, but not estrogen/sulpiride, treated rats. These results indicate that in situ dopamine enhancement gene therapy can be a useful tool for the treatment of prolactinoma. Dopamine synthesis can be tightly regulated and the therapeutic benefit of the system is only inhibited when local dopamine signaling is impaired.

Transgenerational effects of preconception paternal contamination with (55)Fe.

The conjecture that germline mutations induced by radiation exposure before conception may predispose subsequent offspring to cancer remains contentious. Previous experimental studies have shown that preconception paternal irradiation with (239)Pu induces perturbations in the hemopoietic systems of offspring and influences sensitivity to a secondary carcinogen. In the present study, male DBA2 mice were injected intravenously with the Auger electron emitter (55)Fe (4 kBq g(-1)) 18 or 84 days before mating with normal females. Comet analysis showed an increased incidence of DNA strand breaks in sperm from contaminated animals after 84 days, but not after 18 days, indicating spermatogonial rather than spermatid damage. Offspring were either assayed for changes in bone marrow stem cells and committed progenitors or challenged with the chemical carcinogen methyl nitrosourea (MNU, 50 mg/kg) at 10 weeks of age and monitored for the onset of malignancy. Offspring from irradiated fathers had normal peripheral blood profiles, although the stem cell population was amplified in offspring arising from those exposed to (55)Fe at 84 days before conception. Exposure to MNU significantly increased the incidence of lympho-hemopoietic malignancies in offspring from the 84-day group, but not in those from the 18-day group. These findings support the hypothesis that aberrations that are potentially leukemogenic may be transmitted to offspring after radiation damage to the paternal germline.

Increased levels of comet-detected spermatozoa DNA damage following in vivo isotopic- or X-irradiation of spermatogonia.

To investigate whether DNA damage arising in spermatogenic germ cells can be detected in resultant sperm, we have irradiated murine testis and collected spermatozoa from the vas deferens 45 days later. These cells were derived from spermatogonia present at the time of irradiation. Two forms of irradiation were used, external X-rays (4Gy) and internal auger electrons from contamination of the male mouse with the isotope Indium-114m (1.85MBq), which was localised in the testis. Both forms of irradiation produced a profound fall in vas deferens sperm count and testis weight, Indium-114m being more effective. Using the neutral Comet assay for double strand break detection, significant increases in sperm comet tail length and moment were observed. The levels of damage were similar for both treatments. Care had to be taken during the assay to distinguish between sperm and somatic cells as the proportion of the latter increased after irradiation. We conclude that the comet assay can detect DNA damage in spermatozoa after the in vivo exposure of male germ cells to a known testicular genotoxic agent. The assay may be useful for the assessment of sperm DNA damage (double stranded) associated with male infertility and post-fertilization developmental abnormalities in the offspring.

The locomotion of dairy cows on floor surfaces with different frictional properties.

The locomotion of dairy cows was evaluated on floors with a smooth epoxy resin surface or with a surface-applied bauxite aggregate of mean diameter 0.5, 1.2, or 2.5 mm (coefficients of static friction, mu 0.35, 0.42, 0.49, and 0.74, respectively). Locomotion was recorded as cows walked to receive a food reward. Cows on the floor with least friction walked rapidly (0.85 m/s), with frequent, short steps. At the start of the supporting phase the upper limbs were more vertical. Joint arcs during this phase were reduced. Cows on 0.5-mm aggregate also walked rapidly (0.84 m/s); they had the least vertical limb angles and long steps but held the hoof more vertical, probably to offset any increased slip risk. On floors with larger aggregate, cows decreased speed and step frequency but maintained long steps, keeping their upper forelimbs more vertical to reduce the supporting limb phase. It is concluded that on a low friction floor (mu < 0.4), cows walk quickly with frequent, short steps. As mu increases to 0.5, step length increases and the number of steps decreases to maintain speed at increased friction, producing an optimal coefficient of friction between 0.4 and 0.5. Further increases in mu may increase the hanging limb phase at the expense of the supporting limb phase, to reduce friction, while maintaining a long stride to expedite arrival at the reward.

Apoptosis related gene products in differentiated and tumorigenic rat Leydig cells and following regression induced by the cytotoxin ethane dimethanesulphonate.

Androgen secreting Leydig cells in the adult are differentiated with a very low turnover, however, Leydig cell tumours can arise spontaneously or after treatment with toxins. This study in the rat investigated whether changes in components of programmed cell death could be involved. In contrast to their absence in differentiated Leydig cells, antiapoptotic Bcl-2 and proapoptotic Bax were expressed in tumours. Bak and Bcl-xl were found in both tumour and normal Leydig cells. Apoptosis was induced in subcutaneous implants of Leydig cell tumour by ethane dimethanesulphonate (EDS) which is known to kill differentiated Leydig cells. The marked regression of the tumour following EDS treatment was transient and re-growth occurred between 6 and 14 days later. Tumour regression and growth was associated with a similar weight pattern in the seminal vesicles caused by changes in serum testosterone. During tumour regression, clusterin and Bax proteins were elevated but Bak, Bcl-xl and Bcl-2 were unchanged. Fas-R, Fas-L and Bax were upregulated after tumour regression had taken place. These data show that Leydig cell tumours possess many of the apoptosis related gene products and can die by apoptosis, however, regulation is clearly different in differentiated and mitotic Leydig cells.

Leptin acts on metabolism in a photoperiod-dependent manner, but has no effect on reproductive function in the seasonally breeding Siberian hamster (Phodopus sungorus).

Leptin may play a role in appetite regulation and metabolism, but its reproductive role is less clear. In photoperiodic Siberian hamsters, seasonal changes in fatness, leptin gene expression, and metabolism occur synchronously with activation or suppression of reproduction, analogous to puberty. Here, we test the hypothesis that seasonal changes in leptin secretion mediate the photoperiodic regulation of reproduction. Mature male and ovariectomized estrogen-treated female Siberian hamsters were kept in long (LD; 16 h of light, 8 h of darkness) or short days (SD; 8 h of light, 16 h of darkness) for 8 weeks, and recombinant murine leptin (15 microg/day) was infused for 2 weeks via osmotic minipumps. SD hamsters exhibited significant weight and fat losses, reduced serum leptin and food intake, and suppressed pituitary LH concentration. Leptin did not suppress food intake over the 2-week treatment on either photoperiod, but significantly reduced fat reserves in SD hamsters. Leptin had no significant effect on pituitary LH concentrations in either sex or photoperiod or on testicular size and testosterone concentrations in males. These results suggest hamsters are more responsive to leptin on SD than on LD and that effects on food intake and fat loss can be dissociated in this species. Our data suggest that leptin does not mediate photoperiodic reproductive changes.

Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis.

Urea movement across plasma membranes is modulated by specialized transporter proteins that are products of two genes, termed UT-A and UT-B. These proteins play key roles in the urinary concentrating mechanism and fluid homeostasis. We have isolated and characterized a 1.4-kb cDNA from testes encoding a new isoform (UT-A5) belonging to the UT-A transporter family. For comparison, we also isolated a 2. 0-kb cDNA from mouse kidney inner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNA has a putative open reading frame encoding a 323-amino acid protein, making UT-A5 the smallest UT-A family member in terms of molecular size. Its putative topology is of particular interest, because it calls into question earlier models of UT-A transporter structure. Expression of UT-A5 cRNA in Xenopus oocytes mediates phloretin-inhibitable urea uptake and does not translocate water. The distribution of UT-A5 mRNA is restricted to the peritubular myoid cells forming the outermost layer of the seminiferous tubules within the testes and is not detected in kidney. UT-A5 mRNA levels are coordinated with the stage of testes development and increase 15 days postpartum, commensurate with the start of seminiferous tubule fluid movement.

The locomotion of dairy cows on concrete floors that are dry, wet, or covered with a slurry of excreta.

Six dairy cows were trained to individually walk down a concrete aisle for a food reward. Their locomotion was then examined in a switchback experiment as the floor surface of the aisle was changed from dry to wetted concrete or concrete covered by shallow (5 cm) or deep (12.5 cm) slurry from cattle excreta. The static and dynamic frictional coefficients were measured by a tribometer, but did not give a clear indication of the risk of slipping. Cow locomotion was measured over the second half of the aisle, and limb angles recorded as the cow passed a video camera. Wetting the floor did not affect the walking or stepping rate, but it reduced the arc made by the joints of the hindlimb during the supporting phase. Slurry caused the cows to keep their legs more vertical at the end of the support phase, probably to aid lifting the limb out of the slurry. It also caused the cows to place their forelimbs down less vertically at the start of the support phase, probably because of the reduced risk of slip in the slurry. When the floor was covered with either the deep or, to a lesser extent, the shallow slurry, the cows' walking and stepping rates were reduced, and on the floor covered with deep slurry their step length was increased. Therefore slurry reduces the cow's walking speed and alters limb angles during the support phase, producing a different walking pattern from cows on dry or wetted concrete.

Testicular and sperm DNA damage after treatment with fludarabine for chronic lymphocytic leukaemia.

This study investigated whether chemotherapy using fludarabine (FLU) caused testicular damage and if cytotoxicity could be detected as sperm DNA damage in the single cell Comet assay. A patient with chronic lymphocytic leukaemia requesting preservation of fertility was treated with seven monthly cycles of fludarabine (45.8 mg total dose per cycle). Testicular assessments, serum follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone measurements, semen analysis and sperm Comet assays were carried out at presentation (pre-FLU therapy), after 1 and 7 months of FLU treatment, and finally at 11 months after completion of chemotherapy. We found that testicular damage occurred within a month, as indicated by reduced testicular volume, oligozoospermia, elevated FSH and LH, and lower testosterone concentrations. Spermatozoa with a large range of DNA damage were detected in the samples from both the control and treated men. DNA damage in the spermatozoa was marked by 7 months of FLU treatment. The high levels of sperm DNA damage seen during and possibly persisting after treatment suggests that caution should be exercised if the ejaculates from these men are used for in-vitro fertility treatment. Further experiments are needed to assess the biological significance of these DNA changes; it may, however, be prudent at present to be cautious when counselling these patients.

Adenovirus-mediated herpes simplex virus type-1 thymidine kinase gene therapy suppresses oestrogen-induced pituitary prolactinomas.

We tested the hypothesis that gene transfer using recombinant adenovirus vectors (RAds) expressing herpes simplex virus type 1 thymidine kinase (HSV1-TK) might offer an alternative therapeutic approach for the treatment of pituitary prolactinomas that do not respond to classical treatment strategies. HSV1-TK converts the prodrug ganciclovir (GCV) to GCV monophosphate, which is in turn further phosphorylated by cellular kinases to GCV triphosphate, which is toxic to proliferating cells. One attractive feature of this system is the bystander effect, whereby untransduced cells are also killed. Our results show that RAd/HSV1-TK in the presence of GCV is nontoxic for the normal anterior pituitary (AP) gland in vitro, but causes cell death in the pituitary tumor cell lines GH3, a PRL/GH-secreting cell line, and AtT20, a corticotrophic cell line. We have used sulpiride- and oestrogen-induced lactotroph hyperplasia within the rat AP gland as an in vivo animal model. Intrapituitary infection of rats bearing oestrogen-induced lactotroph hyperplasia, with RAd/ HSV1-TK and subsequent treatment with GCV, decreases plasma PRL levels and reduces the mass of the pituitary gland. More so, there were no deleterious effects on circulating levels of other AP hormones, suggesting that the treatment was nontoxic to the AP gland in situ. In summary, our results show that suicide gene therapy using the HSV1-TK transgene could be further developed as a useful treatment to complement current therapies for prolactinomas.

Administration of exogenous testosterone in the adult rat and its effects on reproductive organs, sex hormones and body-weight.

The present experiment was performed to observe the effects produced by high dose of a testosterone ester on the reproductive organ and body weight changes in the adult rat, and to correlate these effects with the serum hormone changes. The present study has used the benzoate ester of testosterone (Testosterone benzoate, TB) in the adult male rat (300-350 g). The aim was to co-relate the reproductive organ and body-weight changes with changes in the serum hormone levels following the administration of the ester. TB was injected i.p. for five (5) consecutive days at a dose of 100 mg/kg body-weight. The control rats were injected with vehicle (arachis oil) at the same dose. The rats were killed on the 6th, 12th, 18th, 24th and 36th days. Controls for only the 6th and 36th days were kept. Reproductive organ weight, body-weight and testosterone (T) levels in serum and testis together with serum FSH and serum LH levels were observed. The testes weights remained similar (p < 0.05) to those in the control rats until the 18th days and were reduced on the 36th day. The epididymis weights were not changed until the 36th days, while the androgen-dependent seminal vesicle and ventral prostate weights were increased (< 0.05) compared to those in the control rats. The body-weights remained unchanged at the 6th day but were significantly (p < 0.05) decreased on the 36th day. The serum testosterone (ST) concentrations were highly raised on the 6th day, came at the control level on the 18th day and were significantly decreased (< 0.05) on the 36th day. The testicular testosterone (TT) content remained significantly lower (p < 0.05) from the 6th to the 36th days post-injection. The serum LH and FSH levels also remained significantly lower (p < 0.05) throughout the treatment period. It appears that the elevated serum T levels exerted dual effect in the adult rats, namely, enhanced growth of the androgen-dependent organs and an inhibition over the hypothalamo-pituitary-testicular axis. Inhibition of the said axis was evident by the lower levels of the serum LH and FSH; probably due to this, the TT-content remained all through lower, and perhaps this low TT-content for the long period had led to the low testis weights (p < 0.05) on the 36th day. This experiment therefore, demonstrates the effects of exogenous androgen administration in the adult male rat physiology.

Leydig cell apoptosis after the administration of ethane dimethanesulfonate to the adult male rat is a Fas-mediated process.

Leydig cells undergo apoptosis in response to the cytotoxin ethane dimethanesulfonate (EDS), with numbers declining at 12-18 h and maximal apoptosis at 24 h postinjection. The Bcl-2 family members, Bcl-2, Bcl-xl, and Bax, appear not to be involved in this process. To further investigate this phenomena, a single dose of EDS was administered to adult rats to induce the killing of Leydig cells. The interstitial cells were examined up to 3 days after EDS administration by Western blot analysis for the Bcl-2 family members (Bak and Bcl-w). Western blotting showed that Bak expression in the interstitial cell preparations was unchanged after EDS, and immunohistochemistry showed that it was not up-regulated in Leydig cells in response to EDS. Bcl-w expression in the Leydig cells and interstitial cell preparations was unchanged until 48 h when it became undetectable, suggesting that Leydig cell-associated Bcl-w is not involved in initiating apoptosis. We also investigated the role of the Fas system in Leydig cell apoptosis. Both Fas receptor and Fas ligand protein levels increased after EDS, peaking at 12-18 h and declining thereafter. Fas receptor and ligand were shown by immunohistochemistry to be present in Leydig cells, and after EDS all Leydig cells became strongly positive for both proteins. The intensity of staining increased in the early stages of apoptosis and decreased as the nuclear morphology became more fragmented. These data suggest that Bcl-2 family members are not involved in Leydig cell apoptosis after EDS administration. However, up-regulation of the Fas system does occur, implicating activation of Fas receptor in the induction of Leydig cell apoptosis.

Effects of endocrine-disrupting contaminants on amphibian oogenesis: methoxychlor inhibits progesterone-induced maturation of Xenopus laevis oocytes in vitro.

There is currently little evidence of pollution-induced endocrine dysfunction in amphibia, in spite of widespread concern over global declines in this ecologically diverse group. Data regarding the potential effects of endocrine-disrupting contaminants (EDCs) on reproductive function in amphibia are particularly lacking. We hypothesized that estrogenic EDCs may disrupt progesterone-induced oocyte maturation in the adult amphibian ovary, and tested this with an in vitro germinal vesicle breakdown assay using defolliculated oocytes from the African clawed frog, Xenopus laevis. While a variety of natural and synthetic estrogens and xenoestrogens were inactive in this system, the proestrogenic pesticide methoxychlor was a surprisingly potent inhibitor of progesterone-induced oocyte maturation (median inhibitive concentration, 72 nM). This inhibitory activity was specific to methoxychlor, rather than to its estrogenic contaminants or metabolites, and was not antagonized by the estrogen receptor antagonist ICI 182,780, suggesting that this activity is not estrogenic per se. The inhibitory activity of methoxychlor was dose dependent, reversible, and early acting. However, washout was unable to reverse the effect of short methoxychlor exposure, and methoxychlor did not competitively displace [3H]progesterone from a specific binding site in the oocyte plasma membrane. Therefore, methoxychlor may exert its action not directly at the site of progesterone action, but downstream on early events in maturational signaling, although the precise mechanism of action is unclear. The activity of methoxychlor in this system indicates that xenobiotics may exert endocrine-disrupting effects through interference with progestin-regulated processes and through mechanisms other than receptor antagonism.

Apoptosis in the rat spermatogenic epithelium following androgen withdrawal: changes in apoptosis-related genes.

Programmed cell death is an important regulatory event in spermatogenesis. However, the molecular events governing apoptosis have not been characterized. Using the Leydig cell-specific toxin ethane dimethanesulfonate (EDS) to withdraw androgen support, we have investigated the relationship between apoptosis and apoptosis-related genes. Adult male Sprague-Dawley rats were injected (i.p.) with 100 mg/kg EDS and killed at times of androgen depletion 2, 5, and 8 days postinjection. A 24-fold increase in the apoptotic index 8 days after EDS administration was demonstrated in tissue sections by in situ end-labeling of fragmented DNA. Leydig cell death and androgen withdrawal were confirmed by the absence of 3beta-hydroxysteroid dehydrogenase in testes from animals treated with EDS for 2 days. After androgen withdrawal, there were no significant changes in the levels of clusterin, Bcl-xl, Bak, and Bad. However, the expression of Bcl-2 and Bax was up-regulated at 8 days after EDS administration. The induction of Bax at this time suggests that it may play a role in germ cell apoptosis following androgen withdrawal. The concomitant elevation in Bcl-2 expression may represent a survival mechanism for the remaining germ cells. There was also a decline in the expression of Fas-L and Fas-R in the pachytene spermatocytes and spermatids. Fas-R was also present in Sertoli cells, although Fas-L staining was minimal. As the colocalization of Fas-L and Fas-R correlates with the germ cell types that die in response to androgen withdrawal, the potential exists for apoptosis in the rat spermatogenic epithelium to be regulated by the Fas pathway.

Mutation studies in lacI transgenic mice after exposure to radiation or cyclophosphamide.

We have used the Big Blue lacI transgenic mouse reporter system to investigate mutation induction in the testes, spleen and liver after exposure to an internally incorporated radionuclide, 114mIn, whole body irradiation with 60Co gamma-rays and systemically administered cyclophosphamide. Spontaneous mutation frequencies were 6-17x10(-6). No statistically significant mutation induction was observed in testes or spleen at 35 days after exposure to any test agent, although mutation frequencies tended to be increased (by approximately 1.5-fold) after exposure to 1 Gy gamma-rays. However, liver mutation frequencies were doubled after treatment with 100 mg/kg cyclophosphamide and were elevated by approximately 2.5-fold after systemic administration of 114mIn and 4.5-fold after 1 Gy 60Co gamma-rays. When data from all organs were pooled, mutation frequency was doubled after exposure to 1 Gy gamma-rays, but no other significant increases were observed. These findings support the hypothesis that the lacI transgenic mouse may be relatively inefficient at detecting mutations induced by exposure to ionizing radiation or other agents which produce a spectrum of deletion sizes, including those which are larger than the lacI transgene.