Effect of high altitude exposure on spermatogenesis and epididymal sperm count in male rats

The present study was designed to determine the effect of exposure to high altitude on spermatogenesis using transillumination technique and sperm count in male rats. In addition, the effect of oral intubation for intragastric administration of vehicle on testicular parameters in adult male rats in a schedule of 42 days was assessed. Male rats were exposed to Cerro de Pasco (Peru) at 4340 m for 3, 7, 14, 21, 28, 35 and 42 days resulting in a modification of the pattern of the seminiferous tubule stages. At day 3, stages I, IV-V, VI, VII and IX-XI were relatively shorter at high altitude than at sea level. At day 7, stages VIII, IX-XI, XII and XIII-XIV were reduced. At day 14, stages VII, VIII and IX-XI were reduced. At day 21 and 28, stages VIII, XII and XIII-XIV were significantly increased at high altitude. At day 35 an increase in stage XIII-XIV was observed. At day 42, stages II-III, IX-XI and XII were significantly increased at high altitude. Epididymal sperm count was significantly reduced at day 7 of exposure to high altitude and maintained low levels with respect to sea level up to 42 days. In conclusion, high altitude exposure affects spermatogenesis, particularly onset of mitosis and spermiation. This in turn affects epididymal sperm count.     

Lepidium meyenii （Maca） reduces spermatogenic damage induced by a single dose of malathion in mice

AIM: To observe the effect of the aqueous extract of hypocotyls of the plant Lepidium meyenii (Maca) on spermatogenic damage induced by the organophosphate insecticide malathion in mice. METHODS: Mice were treated with 80 mg/kg of malathion in the presence or absence of an aqueous extract of Maca, which was orally administered 7, 14 or 21 days after injection of the malathion. Stages of the seminiferous epithelium were assessed by transillumination on days 0, 7, 14 and 21. RESULTS: The administration of Maca increased significantly the length of stage VIII on days 7, 14 and 21 of treatment compared with the controls. An increase in the length of stage IX occurred on day 14 of treatment. Malathion affected spermatogenesis by reducing the lengths of stage IX on day 7, stages VII and IX-XI on day 14 and a recovery of stages IX-XII on day 21. The magnitude of alteration in the length of stage IX produced by malathion was significantly reduced by Maca on days 7 and 14. The length of stage VIII was increased when Maca was administered to mice treated with malathion. Assessment of the relative length of stages of the seminiferous epithelium showed that Maca treatment resulted in rapid recovery of the effect of malathion. CONCLUSION: Maca enhances spermatogenesis following spermatogenic damage caused by the organophosphorous pesticide.     

Attempted protection of spermatogenesis from single doses of gamma-irradiation in the androgen pretreated rat

Spermatogenic stem-cell survival after gamma-irradiation has been investigated in the adult Wistar rat. Single doses of 4.5 and 9 Gy gamma-rays were administered to the testes of rats who received arachis oil (0.1 ml/100 g body weight) or testosterone enanthate (240 micrograms/100 g body weight) subcutaneously three times weekly for 6 weeks prior to radiation and during the week in which the radiations were given. A mean percentage of regenerating seminiferous tubule cross-sections of 32.45% and 7.26% was found in the testes of androgen-pretreated rats at 8 weeks after 4.5 and 9 Gy, respectively. Similar values (33.4% and 6.2%) were obtained in arachis oil-pretreated controls. We therefore conclude that protection of rat spermatogenesis from single doses of gamma-rays cannot be achieved by androgen pretreatment.     

Testicular gametogenic and steroidogenic activities in chlorpyrifos insecticide-treated rats: a correlation study with testicular oxidative stress and role of antioxidant enzyme defence systems in Sprague-Dawley rats

The present works examined an adverse effect of chlorpyrifos insecticide on testes and lipid peroxidation at low doses (5 mg-10 mg kg(-1) body weight) and the role of antioxidant enzymes systems at higher doses (20-30 mg kg(-1) body weight) in albino rats. At low doses, reduction in plasma levels of testosterone and FSH and LH hormones along with the significant shrinkage of seminiferous tubules and gametogenic changes in germ cells were noticed. But these changes were restored with the revival of serum testosterone, FSH and LH along with regression of testis at higher doses. Similarly, level of testicular lipid peroxidation was elevated, whereas levels of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) and steroidogenic enzymes activities (Δ(5) , 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase) were reduced significantly at low doses. But, rat testes showed a significant decrease in lipid peroxidation and concomitant increase in antioxidant enzymes and steroidogenic enzymes activities at higher doses. Results showed that at higher doses of chlorpyrifos treatments, rat testes were shown to trigger their natural defence mechanism which became operative possibly through corrective measure of synthesis of antioxidant defence enzymes and steroidogenic enzymes and pituitary gonadotrophins hormone feedback mechanisms.     

Effect of alcoholic extract of Lepidium meyenii （Maca） on testicular function in male rats

Aim: To evaluate the effect of the alcoholic extract of Lepidium meyenii (Maca) on the spermatogenesisin male rats. Methods: In Holtzman rats, Maca alcoholic extract (5 %) was given by oral route at doses of 48 mg/day or 96 mg/day for 7 days, 14 days and 21 days. Testicular function was assessed by measurements of lengths of different stages of seminiferous epithelia and by epididymal sperm count. Results: Ethanolic extract of Maca increased the length of stages IX-XI of seminiferous epithelium at treatment day 7, day 14 and day 21. Progression of spermatogenesis was evident only after day 21 when lengths of stages XII-XIV of seminiferous epithelium were increased; at day 7 and day 14, no important change in spermatogenesis was observed. Epididymal sperm count was increased with 48 mg/day at all times. With 96 mg/day an increase in sperm count was observed at day 7, but it was reduced at day 14 and day 21 of treatment. Serum testosterone levels were not affected. Conclusion: The alcoholic extract of Maca activates onset ant progression of spermatogenesis at 48 mg/day or 96 mg/day in rats.     

Effects of Boesenbergia rotunda （L.） Mansf. on sexual behaviour of male rats

Aim： To study the effects of Boesenbergia rotunda （Krachai） on sexual behaviour in male albino rats. Methods： Thirty-two male Wistar rats were equally divided into four groups： experimental groups were gavaged with the ethanolic extract of the rhizome of B. rotunda at doses of 60, 120 and 240 mg/kg and a control group received distilled water, for 60 days. Sexual behaviour, reproductive organs, diameter of seminiferous tubule, epididymal sperm density, and androgenic hormones were evaluated. Results： Within 30-min observation, there was no significant difference of courtship behaviour, mount frequency （MF）, intromission frequency （IF）, mount latency （ML）, intromission latency （IL）, copulatory efficiency or intercopulatory interval in male rats. In three 10-min intervals over a 30-min period, courtship behaviour and MF during the first 10-min were significantly higher than those in the second and third i0-min observation in all groups, whereas IF had no significant difference. All doses of B. rotunda extract significantly increased the relative testicular weight and the diameter of the seminiferous tubules. The dose of 60 mg/kg also significantly increased the relative weight of the seminal vesicle. Nevertheless, the sperm density, serum testosterone and androstenedione levels were not affected by the B. rotunda extract. Conclusion： B. rotunda does not affect sexual behaviour nor serum androgenic levels.     

Local regulation of Leydig cells by the seminiferous tubules. Effect of short-term cryptorchidism

Unilateral cryptorchism was induced in adult rats for 24 h, and its effect on testicular morphology and intratesticular testosterone concentration after hCG-stimulation were studied. In seminiferous, tubules from abdominal testes an increased number of degenerating germ cells was noted in stages XIV-III of the spermatogenic cycle and Sertoli cells contained an increased amount of lipid droplets in stages XIV-VIII. However, germ cells and Sertoli cells from tubules at other stages of the cycle appeared unaffected. In scrotal testes the size of peritubular Leydig cells varied in phase with the spermatogenic cycle. The largest cells were found adjacent to stage VII-VIII and the smallest adjacent to stage XI-XII. In abdominal testes no stage-dependent variation in the size of peritubular Leydig cells was seen. Perivascular Leydig cells were of equal size in abdominal and scrotal testes. The testicular testosterone concentration following stimulation with a low dose of hCG was significantly lower in abdominal testes. It is suggested that the seminiferous tubules locally modulate Leydig cell function and that the stage specific stimulatory influence from stage VII-VIII is rapidly lost during experimental cryptorchidism.     

Comparative pharmacokinetics of testosterone enanthate and testosterone cyclohexanecarboxylate as assessed by serum and salivary testosterone levels in normal men

The pharmacokinetics of 2 testosterone esters, testosterone enanthate and testosterone cyclohexanecarboxylate, were compared in a single blind crossover study in healthy young men. Their effects on serum and salivary levels of testosterone, as well as on the serum levels of LH, FSH and prolactin were measured after the injection of doses equivalent to 140 mg free testosterone. Both preparations yielded supraphysiological testosterone levels in serum and saliva as early as 2 h following injection, reaching peak levels 4 to 5 times above basal between 8 and 24 h. LH and FSH levels were suppressed as long as serum testosterone levels were elevated. Nine days after injecting testosterone enanthate and 7 days after giving testosterone cyclohexanecarboxylate, serum and salivary levels of testosterone had returned to basal. The longer activity of testosterone enanthate was also evidenced from more extended suppression of gonadotrophin levels. Although neither preparation is ideal because of the initial supraphysiological peaks, testosterone enanthate appears preferable for clinical use because of its slightly longer duration of action.     

The effect of selective destruction and regeneration of rat Leydig cells on the intratesticular distribution of testosterone and morphology of the seminiferous epithelium

This study was designed to explore the relationship between the intratesticular distribution of testosterone and spermatogenesis by completely destroying the Leydig cells of mature male rats with injection of a single i.p. dose of ethane dimethanesulphonate. After such treatment, testosterone levels in serum, testicular interstitial fluid, seminiferous tubules, and whole testis declined significantly 6 to 24 hours after injection and fell below assay detection limits between 3 and 7 days. At 3 and 7 days, serum LH and FSH levels rose significantly and remained elevated up to 4 and 6 weeks, respectively, in comparison with vehicle-treated controls. Leydig cells disappeared from the interstitium by day 3, but between 2 and 4 weeks postinjection a new generation of fetal-like Leydig cells repopulated the testicular interstitium and, during weeks 6 to 10, were transformed into, or replaced by, Leydig cells with an adult type of morphology. Histologic examination of the seminiferous tubules showed progressive disruption of spermatogenesis between 3 and 14 days post-ethane dimethanesulphonate. The first histologic sign of spermatogenic damage was noted at day 3, with the occurrence of stage-specific degenerating pachytene primary spermatocytes at stages VII to VIII of the spermatogenic cycle. On day 7, these cells and degenerating round, or step 19, spermatids often were observed during stages VII to XI, although qualitatively normal spermatogenesis also was seen in these and all other stages of the cycle. Maximum impairment of spermatogenesis occurred 2 weeks post-ethane dimethane sulphonate, at which time the tubules commonly lacked one or more germ cell generations or, alternatively, showed accumulation of lipid inclusions, extracellular spaces, and variable numbers of degenerating germ cells. Following repopulation of the testis by Leydig cells during weeks 3 and 4, spermatogenesis recovered. By 10 weeks after treatment, qualitatively normal spermatogenesis was seen in the great majority of seminiferous tubules, although a few tubules still remained in which the germ cell complement was severely reduced, and contained only Sertoli cells and spermatogonia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of green fluorescent protein under beta-actin promoter in living spermatogenic cells of the mouse: stage-specific regulation by FSH

Spermatogenic cells from a mouse strain expressing enhanced green fluorescent protein (EGFP) under chicken beta-actin promoter were studied under living conditions to analyse stage- and cell-specific expression and hormonal regulation of the transgene. The isolated seminiferous tubules were examined by transillumination and the live cell squashes by phase contrast and fluorescence microscopy. FSH effects were measured in whole seminiferous tubules comparing stages I-VI, VII-VIII and IX-XII of the cycle. Beta-actin was highly expressed in spermatogonia, but almost no expression was found at early meiosis (leptotene spermatocytes). A gradual increase in translation of beta-actin was found during later stages of meiosis and early spermiogenesis, with a maximum in elongating spermatids. FSH increased the translation of beta-actin after 4 h and 24 h of incubation at stages I-VI, after 24 h at stages VII-VIII but not at stages IX-XII of the cycle. The results support the view that beta-actin plays a role in the nuclear elongation of spermatids and that its expression is regulated by FSH in a stage-specific fashion. Techniques used in this study give us new insight to study temporal and hormonal regulation of gene products in living spermatogenic cells.     

In-vitro contractility of human seminiferous tubules in response to testosterone,dihydrotestosterone and estradiol

Summary The effects of steroids (testosterone, dihydrotestosterone and estradiol) on human seminiferous tubules in vitro were ascertained by recording the intratubular pressure with a servonull micropressure measuring device. We describe here the first response of the human seminiferous tubule to steroids. Testosterone and dihydrotestosterone had a biphasic effect on tubular contractility. Higher doses of both testosterone and dihydrotestosterone induced contractions of the seminiferous tubules whereas lower doses of these compounds induced relaxation. Estradiol (10^-9 M to 10^-6 M) induced relaxation of the seminiferous tubules in a dose-dependent manner. The results from these experiments suggested that steroids may be involved in the control of contraction of the human seminiferous tubule and may regulate the movement of spermatozoa from the tests.     

Flow cytometric DNA analysis of defined stages of rat seminiferous epithelial cycle during in vitro differentiation

The stages of the rat seminiferous epithelial cycle have been isolated for flow cytometric analysis of DNA and for culture, using transillumination-assisted microdissection. Precise stages have been identified by phase contrast microscopy of live cell squashes from adjacent segments. Each stage of the cycle showed a characteristic flow cytometric pattern with haploid (1C), diploid (2C) and tetraploid (4C) peaks. Stages I to VIII of the cycle showed an additional hypofluorescent (0.25-0.70C) peak due to a reduced dye-binding capacity of maturation phase-spermatids at steps 15 through 19. The appearance of the hypofluorescent haploid peak coincided with the second nucleoprotein transition at step 15 of spermiogenesis and the homogeneous condensation of the chromatin seen in electron microscopy. As a concomitant of the formation of disulphide bonds during epididymal maturation, the fluorescence intensity decreased further to reach a relative value of 0.07C in the cauda epididymidis. The constant 1C peak was raised by round and elongating spermatids (steps 1-14), 2C by spermatogonia, secondary spermatocytes and Sertoli cells, and the 4C peak by primary spermatocytes and spermatogonia at G2 or M phase of the mitotic cycle. The proportion of each peak accurately reflected the relative proportion of cells in most stages of the cycle when compared with morphometric measurements of histologic preparations. DNA flow cytometry is a suitable method for quantitative evaluation of cultured seminiferous tubule segment DNA. Although the relative yield of the meiotic reductive divisions in vitro is comparable with that observed in vivo, steps 9 and 15 of spermiogenesis involving nucleoprotein transitions and spermiation itself did not occur under the present culture conditions.     

Suppression of pituitary and testicular function in rats by a transplanted adrenocortical carcinoma

Snell adrenocortical carcinoma was transplanted into immature 4-week-old male rats, and the animals were sacrificed 3 weeks afterward for study of adenohypophyseal and testicular function. The weight of the tumour was 11pL5 g; plasma corticosterone levels were elevated and plasma progesterone levels were massively increased compared to those in rats with no tumours. The weights of the adrenals, testes and androgen-dependent accessory reproductive glands were significantly decreased, as was the diameter of seminiferous tubules. The concentrations of testosterone, LH and FSH in the plasma were significantly reduced, whereas the levels of Prl and oestradiol were not affected. We suggest that steroid products of the transplanted tumour suppressed release of gonadotrophins from the pituitary, leading to a severe reduction of testosterone synthesis in the testes.     

Intratesticular factors and testosterone secretion: The effect of treatment with ethane dimethanesulphonate (EDS) and the induction of seminiferous tubule damage

The role of seminiferous tubule dysfunction in regulating the levels of a factor (or factors) in testicular interstitial fluid (IF) which stimulates Leydig cell testosterone secretion in vitro, was assessed by injecting rats with the Leydig cell toxin, EDS. Within 72 h of treatment EDS destroyed the Leydig cells and concomitantly reduced IF testosterone to undetectable levels. This was associated with nearly a 2-fold increase (P less than 0.001) in levels of the IF-factor(s) as judged by the enhancement of hCG-stimulated testosterone production (= IF bioactivity). By 3 weeks, and thereafter up to 10 weeks post-EDS, Leydig cells regenerated within the testis, and testosterone levels returned to control values, but IF-bioactivity remained significantly increased. The latter was associated with seminiferous tubule dysfunction as indicated initially by testicular morphology, raised serum levels of FSH and reduced testicular weight. For animals with normal testosterone levels, there was a significant negative correlation (r = -0.57, N = 46; P less than 0.001) between testicular weight and IF bioactivity. A similar increase in IF bioactivity in the presence of normal testosterone levels was observed in rats in which patchy severe seminiferous tubule damage had been induced by short-term cryptorchidism. It is concluded that, in addition to testosterone, seminiferous tubule function may dictate the intratesticular levels of the testosterone-stimulating factor(s) in IF.     

Morphometric studies on the testes of rats treated neonatally with oestrogen and subsequently with gonadotrophins and testosterone

The experiments involved male rats, which were given a single subcutaneous dose of 1 mg stilboestrol on the first day of life. Beginning on day 28, subgroups of the rats received either gonadotrophins or testosterone for 39 days. The weight of the testes, serum luteinizing hormone and testosterone levels were determined while sections of the testes were subjected to morphological analysis and morphometric measurements, based on computerized techniques. The results demonstrated that a single dose of oestrogen caused a reduction in the cross-sectional area of the seminiferous tubules and a reduction in the thickness of the seminiferous epithelium, accompanied by inhibition of spermatogenesis. The number of and area occupied by Leydig cells, as well as the size of their cell nuclei, were also diminished, and the levels of serum testosterone decreased by 73%. All the experimental animals manifested significantly increased serum luteinizing hormone levels. Stimulation with gonadotrophins markedly increased the number of Leydig cells, their size and the size of their cell nuclei. This was associated with significantly increased levels of serum testosterone. Under these conditions, the cross-sectional area of the seminiferous tubules and the thickness of seminiferous epithelium remained less than those in the untreated controls. Following stimulation with testosterone the pattern of the seminiferous tubules resembled that noted after stimulation with gonadotrophins; the number of Leydig cells was markedly reduced but the size of both the cells themselves and of their nuclei approached normal values.     

Basal and FSH-stimulated steady state levels of SGP-2, alpha 2-macroglobulin, and testibumin in culture media of rat seminiferous tubules at defined stages of the epithelial cycle.

Production of several proteins by rat Sertoli cells is dependent on the stage of the cycle of the seminiferous epithelium. The authors have determined steady state levels and follicle-stimulating hormone responsiveness of three Sertoli cell products in culture media of rat seminiferous tubule segments at different stages of the epithelial cycle: SGP-2 (sulfated glycoprotein-2), alpha 2-macroglobulin, and testibumin. Basal SGP-2 levels were twofold higher in stages VII through VIII compared with stages XIII to I to VI (P less than 0.05). Highest basal alpha 2-macroglobulin levels were found in stages II through VIII; this was about 35% greater than in stages XIII through I of the cycle (P less than 0.05). Basal testibumin levels were twofold higher in stages II through VI compared with stages IX through XII of the cycle. Follicle-stimulating hormone had no effect on SGP-2, but by contrast it (50 mg/L) increased the level of alpha 2-macroglobulin significantly (P less than 0.05) in stages XIII through I. Follicle-stimulating hormone treatment (10 mg/L) elevated testibumin levels at each stage-pool by about 40% (P less than 0.05). The current results using staged tubular segments in vitro demonstrate cyclic basal steady-state levels of the three proteins along the seminiferous tubules and follicle-stimulating hormone regulation of alpha 2-macroglobulin and testibumin.     

Regulation of interstitial cell function by seminiferous tubules in intact and cryptorchid rats

The effects of experimental cryptorchidism on seminiferous tubule secretions and interstitial cell testosterone production were studied in vitro. Spent media obtained from incubations of seminiferous tubules (SMST) from cryptorchid rats caused a significant increase in testosterone production when added to interstitial cells isolated from intact rats. The previously noticed inhibitory activity of the SMST from stages VIII–XI of the sperma-togenic epithelial cycle gradually disappeared after the induction of experimental cryptorchidism. SMST obtained from both sham-operated or cryptorchid rats stimulated basal testosterone production when added to interstitial cells from cryptrochid rats. SMST from rats had been cryptorchid for 7, 14 and 28 days stimulated testosterone production when added to interstitial cells prepared from intact animals. Seminiferous tubules from cryptorchid rats therefore appear to be the source of a heat stable, trypsin-resistant factor with an apparent molecular weight of between 5000 and 10 000 daltons which stimulates testosterone production when added to interstitial cells in vitro. Its activity could not be blocked by an LRH antagonist. This factor enhances both basal and LH-stimulated secretion of testosterone in contrast to the inhibitory activity which involves only a partial blockade of LH-dependent steroidogenesis.     

Classification of plastic-embedded rat seminiferous epithelium prior to electron microscopy

Systematic electron microscopic studies of the changes which take place during the cycle of the rat seminiferous epithelium require that each of the 14 stages of the cycle are identified in the light microscope in plastic-embedded material allowing each stage to be trimmed out separately before the electron microscopic study. The classification of the 14 stages has originally been based on the morphological characteristics seen in paraffin-embedded tissue after PAS-staining. PAS-staining is less effective in plastic-embedded material, and this may be a main reason why electron microscopic findings of the seminiferous epithelium relatively seldom are related to the stages of the cycle. In the present paper a method is described by which the 14 stages of the cycle can be easily identified in the light microscope in plastic-embedded tissue. Some of the stages could be subdivided and a total of 23 stages were distinguished. This constitutes a safe method allowing accurate classification prior to electron microscopic examination of the seminiferous epithelium.     

The Anti-Reproductive Effects of Long Term Oral Administration of Cadmium on the Adult Male Rat

The toxic effects of prolonged oral administration of cadmium on gametogenic and endocrine function of testes of adult rats were investigated. The experimental animals received daily, for 3, 6, 12 or 15 months, pellets containing 8.8 mg or 88 mg of cadmium chloride per kg body weight.
The rats treated with the higher doses of cadmium for 12 and 15 months showed a marked reduction in absolute weight of testes accompanied by histological signs of impairment of seminiferous tubules. There were no necrotic changes but a number of cross-sections of seminiferous tubules were deprived of spermatocytes and spermatids, reaching about 50 per cent of the tubules in the rats treated with higher doses of cadmium. The appearance of histological changes in rats treated for 12 and 15 months correlated with cumulation step of cadmium in testes and with alterations in serum concentration of LH but not of testosterone. Therefore, we suppose that under these experimental conditions the impairment of seminiferous tubules was induced by the direct influence of cadmium on germinal epithelium beginning the moment when cadmium reaches an effectively toxic concentration in testis.     

Heterogeneity of sperm density profiles following 16-week therapy with continuous infusion of high-dose LHRH analog plus testosterone

LHRH agonist analogs have been investigated as potential male contraceptives. It has been shown that the LHRH agonistic analog [D-Trp6,Pro9-NEt] LHRH (LHRHA) given to men in single doses up to 500 micrograms daily for up to 20 weeks with the coadministration of testosterone enanthate produces reversible oligozoospermia. Individual responses to the treatment, however, were variable. In this study, we gave the same analog to eight normal male volunteers as a continuous infusion of 500 micrograms daily for 16 weeks. Testosterone enanthate, 100 mg, was given by injection every second week. Six of the subjects became oligozoospermic but the other two retained sperm counts that were greater than 20 million/ml, although their treatment continued for 20 weeks. The reasons for this variability of response are not clear. Serum immunoreactive LH values increased during the infusion period whereas testosterone declined. FSH values fell during treatment in all subjects except the two non-responders. The acute pituitary response to LHRHA during the treatment or shortly thereafter (48 h) was completely abolished, and bioactive LH values were suppressed totally. FSH, LH, testosterone and sperm counts returned to normal in all subjects following discontinuation of LHRHA infusion. Continuous infusion of 500 micrograms of LHRHA daily for 16 weeks with 100 mg of testosterone enanthate every 2 weeks induced desensitization of the pituitary, loss of LH bioactivity, and decreases of FSH and testosterone. This mode of administration, however, did not improve sperm density results obtained earlier by single daily injections of the analog. Heterogeneity of sperm density profiles still persists for reasons that are not yet clear.