Distribution pattern of testicular sulphydryloxidase immuno-activity in the djungarian hamster (Phodopus sungorus) during photoperiodically induced involution and recrudescence

The distribution pattern of testicular sulphydryloxidase (SOx) immuno-activity was investigated in the djungarian hamster during photoperiodically induced testicular involution and recrudescence. SOx immuno-activity, indicating functional integrity of labelled cells, did not change in pachytene spermatocytes and spermatids as long as these cells were present in the seminiferous epithelium. Its disappearance coincided with the degeneration of spermatocytes in phases IV and V of involution and reappeared during recrudescence, when the first spermatogenic wave had reached the pachytene stage. In tubules at phase VI of involution (showing maximal regression), the apical cytoplasm of Sertoli cells showed immuno-activity. This immuno-activity disappeared during recrudescence prior to the differentiation of pachytene spermatocytes. Changes in SOx immuno-activity resembled those of lactate dehydrogenase-X (LDH-X) in photo-inhibited testes or during puberty, indicating a close functional relationship which still remains to be elucidated. The data suggest that the hamster exposed to different photoperiods can be used as a suitable model to study the relationship between testicular morphology and function in different states of gonadal activity.     

Localisation of RA175 (Cadm1), a cell adhesion molecule of the immunoglobulin superfamily, in the mouse testis, and analysis of male infertility in the RA175-deficient mouse

RA175, a member of the immunoglobulin superfamily, plays an important role in cell adhesion, and RA175 gene-deficient mice (RA175(-/-) ) show oligoastheno-teratozoospermia. To understand the function of RA175, location in the testis and the morphological features of its spermatogenic cells in RA175(-/-) mice were investigated. Immunohistochemical studies revealed that RA175 immunoreactivity was observed on the cell surface of the spermatogenic cells at specific stages. A strong reaction was detected from type A spermatogonia to pachytene spermatocytes at stage IV and from step 6 to step 16 spermatids during spermatogenesis. From pachytene spermatocytes at stage VI to step 4 spermatids, the reaction was not detected by the enzyme-labelled antibody method and was faintly detected by the indirect immunofluorescence method. Abnormal vacuoles in the seminiferous epithelium, showing exfoliation of germ cells, and ultrastructural abnormality of the elongate spermatids were revealed in the RA175(-/-) testes. Other members of the immunoglobulin superfamily such as basigin, nectin-2 and nectin-3, which have an important role in spermatogenesis, were immunohistochemically detected in the RA175(-/-) testis. These observations indicate a unique expression pattern of RA175 in the testis and provide clues regarding the mechanism of male infertility in the testis.     

Neonatally administered diethylstilbestrol retards the development of the blood-testis barrier in the rat

Newborn rats were treated with 10 microg of diethylstilbestrol (DES) on alternate days from the 2nd to the 12th postnatal day, and the testes were sequentially examined up to 105 days of age by light, electron, and confocal laser microscopy. In control rats, spermatozoa and step 19 spermatids were observed in stage VIII seminiferous tubules at 56 days of age. Spermatogenic cells in DES-treated rats differentiated normally from birth until 21 days of age, after which differentiation continued only to the pachytene-spermatocyte stage. From this age onward, spermatogenic cells older than pachytene spermatocytes were not found until 56 days of age. After this point, the cells resumed differentiation and finally became spermatozoa by 91 days of age; that is, 35 days later than control rats. Electron and confocal laser microscopy showed that in the normal rat, the formation of the ectoplasmic specialization between adjoining Sertoli cells was observed as early as 20 days of age. In contrast, the specialization was not formed until 56 days of age in DES-treated rats. Furthermore, the delay in functional maturation of this structure as the blood-testis barrier was confirmed by intercellular tracer experiments. It is clear that neonatal administration of DES delayed the establishment of the blood-testis barrier for 4 weeks. Consequently, during this period, pachytene spermatocytes were exfoliated from the seminiferous epithelium without completion of meiosis.     

Possible role of elongated spermatids in control of stage-dependent changes in the diameter of the lumen of the rat seminiferous tubule

Adult male rats were treated with a single dose of 650 mg/kg methoxy acetic acid to deplete the seminiferous tubules specifically of pachytene and later spermatocytes. The effect of this treatment and the subsequent maturation-depletion of later germ cell types on the diameter of the seminiferous tubule and its lumen and the area of the seminiferous epithelium were studied in relation to the stages of the spermatogenic cycle. At 21 days after methoxy acetic acid treatment, the diameter of the tubule and the area of the epithelium were reduced below control values at all stages, consistent with the reduced number of early (stage VIII) or late (all other stages) spermatids. Unexpectedly, diameter of the lumen was also reduced at all stages other than VIII, and especially at stage VII. In controls, lumen diameter at stages VII and VIII was increased by approximately 50% compared with earlier and later stages. In rats treated 21 days previously with methoxy acetic acid no change occurred at stage VII (lacking elongated spermatids) while a normal increase did occur at stage VIII (lacking round but not elongated spermatids). At earlier times after methoxy acetic acid treatment when stage VII tubules were depleted of pachytene spermatocytes alone (3 days) or together with early spermatids (7 days), the diameter of the lumen was not significantly different from the control value. These data suggest that lumen diameter may be regulated by elongated spermatids, especially at stages VII and VIII.     

枸杞多糖对体外培养小鼠睾丸组织形态学影响的研究

本研究是通过对小鼠睾丸组织培养，观察枸杞多糖（LBP）对不同培养条件下的睾丸组织形态学变化的影响。结果显示，32℃条件下培养的睾丸组织结构变化在对照组与LBP实验组之间差异不显著；37℃条件下培养48h时组间差异不显著，但在培养72h时，对照组显示曲精小管上皮细胞结构紊乱，大量细胞脱落和退化，管壁上仅剩精原细胞和支持细胞；在LBP实验组，曲精小管上皮细胞结构明显好于对照组，生精上应仍为复层上皮，管腔内脱落细胞较少。此结果表明，LBP可减弱由于培养温度过高所致的曲精小管结构损伤。     

Cellular regulation of follicle-stimulating hormone (FSH) binding in rat seminiferous tubules

Stage-specific binding of follicle-stimulating hormone (FSH) was measured in rat seminiferous tubules. The binding in single-point assays was over 3-fold higher (P less than 0.05) in stages XIII to I than in stages VI to VII of the epithelial cycle. No difference was found between the equilibrium association constants (Ka) of FSH binding in stages XIV to IV (10 +/- 1.9 X 10(9) 1/mol) and VII to VIII (9.2 +/- 0.6 X 10(9) 1/mol, mean +/- SEM, n = 5). In another experiment, the testes were dosed locally with 3 Gy of 4 MV x-irradiation to selectively lower the number of spermatogonia. After irradiation, FSH binding in staged seminiferous tubule segments was measured when the desired types of spermatogenic cells were reduced in number. Seven days after irradiation when differentiating spermatogonia and preleptotene spermatocytes were reduced in number, FSH binding was decreased in all stages of the cycle, but the cyclic variation remained. Seventeen days after irradiation when intermediate and type B spermatogonia and spermatocytes up to diplotene of stage XIII showed low numbers, FSH binding was decreased in all stages of the cycle and the stage-dependent variation disappeared. At 38 days when pachytene spermatocytes and early spermatids were reduced in number, similar results were found. But at 52 days postirradiation when all spermatids were low in number, FSH binding was slightly elevated compared with days 17 and 38. There were no significant differences in serum FSH or LH levels between irradiated and non-irradiated animals. These findings suggest that all spermatogenic cell types may stimulate FSH binding in the Sertoli cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of levonorgestrel butanoate alone and in combination with testosterone buciclate on spermatogenesis in the bonnet monkey

The spermatogenic effects of levonorgestrel butanoate were studied in adult male bonnet monkeys when administered alone and in combination with testosterone buciclate. Levonorgestrel butanoate (0.25, 1.0 and 2.5 mg kg(-1)) given as two injections on days 0 and 60 (groups II, III, IV) resulted in thickening and folding of the basement membrane and disruption of cell associations in groups III and IV (on day 120). In group II, no apparent changes in testicular histology were observed. When these doses of levonorgestrel butanoate were combined with 40 mg of testosterone buciclate (groups V, VI, VII), maximum changes were seen in group VI in which all stages of spermatogenesis were absent on day 120 except for a small number of spermatogonia. The changes caused by lower dose (group V) and higher dose (group VII) of levonorgestrel butanoate were less prominent than in group VI. A significant decrease in the number of dark A (Ad) and B spermatogonia was observed in all groups except for Ad spermatogonia on day 120 in group V, B spermatogonia on day 60 in group IV and B spermatogonia on day 120 in group III. A significant decrease in pachytene spermatocytes was seen on day 120 in groups V only. Early spermatids showed a significant decrease only in groups V and VII on day 120 of treatment. Advanced spermatids were suppressed significantly in group IV on day 60 and in groups IV and V on day 120. These data indicate that levonorgestrel butanoate (1.0 mg kg(-1)) in combination with 40 mg of testosterone buciclate was the most effective treatment in suppressing spermatogenesis. The site of action of this combination regimen is at the level of renewing Ad spermatogonia.     

Regulatory influence of germ cells on sertoli cell function in the pre-pubertal rat after acute irradiation of the testis

While germ cell regulation of Sertoli cells has been extensively explored in adult rats in vivo, in contrast, very little is known about germ cell influence on Sertoli cell function at the time when spermatogenesis begins and develops. In the present study various Sertoli cell parameters (number, testicular androgen binding protein (ABP) and testin, serum inhibin-B and, indirectly, follicle-stimulating hormone (FSH)) were investigated after the exposure of 19-day-old rats to a low dose of 3 Grays of gamma-rays. Differentiated spermatogonia were the primary testicular targets of the gamma-rays, which resulted in progressive maturation depletion, sequentially and reversibly affecting all germ cell classes. Testicular weight declined to a nadir when pachytene spermatocytes and spermatids were depleted from the seminiferous epithelium and complete or near complete recovery of spermatogenesis and testicular weight was observed at the end of the experiment. Blood levels of FSH and ABP were normal during the first 11 days after irradiation, when spermatogonia and early spermatocytes were depleted. While the number of Sertoli cells was not significantly affected by the irradiation, from days 11-66 after gamma-irradiation, ABP production declined and FSH levels increased when pachytene spermatocytes and spermatids were depleted and the recovery of these parameters was only observed when spermatogenesis was fully restored. Comparison of the pattern of change in serum levels of inhibin-B and testicular levels of testin and of germ cell numbers strongly suggest a relationship between the disappearance of spermatocytes and spermatids from the seminiferous epithelium and the decrease in levels of inhibin-B and increase in levels of testin from 7 to 36 days post-irradiation. Levels of testin and inhibin-B were restored before spermatogenesis had totally returned to normal. In conclusion, this in vivo study shows that pre-pubertal Sertoli cell function is under the complex control of various germ cell classes. This control presents clear differences when compared with that previously observed in adult animals and depends on the Sertoli cell parameter of interest, as well as on the germ cell type.     

Effects of subchronic exposure to radio frequency from a conventional cellular telephone on testicular function in adult rats

PURPOSE: We investigated the effects of subchronic exposure to radio frequency emitted from a conventional cellular telephone on the testicular function in adult rats. MATERIALS AND METHODS: A total of 16 male Wistar rats at age 30 days were randomly divided into 2 groups, including experimental and control groups. The experimental group was exposed to radio frequency emitted from a conventional GSM (global system for mobile communications) cellular telephone (1,835 to 1,850 MHz) for 1 hour daily during 11 weeks. Rectal temperature was measured before and after the exposure period. Testicular and epididymal weight, lipid peroxidation levels in these organs, serum total testosterone and the epididymal sperm count were evaluated. Maturation phase spermatid retention at stage IX-X, interstitial infiltration, cellular vacuolation and multinucleate giant cells were among the qualitative testicular histopathological end points analyzed. Each rat had 10 consecutive round seminiferous tubules at stage VII-VIII evaluated for the mean seminiferous tubular diameter measurement, the crude histological count of round spermatids, pachytene spermatocytes and Sertoli's cells with evident nucleoli, and the true histological count (Abercrombie's correction factor) of round spermatids and pachytene spermatocytes. RESULTS: Mean rectal temperature did not alter following exposure. There was no statistical difference between the control and experimental groups in any end points evaluated. CONCLUSIONS: The current study shows that the low intensity pulsed radio frequency emitted by a conventional cellular telephone does not impair testicular function in adult rats.     

Evaluation of the effect of selective germ cell depletion on subsequent spermatogenesis and fertility in the rat

Rats were treated with a single high dose of methoxy acetic acid (MAA; 650 mg/kg) specifically to deplete seminiferous tubules of pachytene and later spermatocytes. The impact of this selective depletion on subsequent spermatogenesis, sperm output and fertility was then evaluated at intervals ranging from 3 days to 10 weeks. Cauda epididymal sperm number was reduced progressively beyond 2 weeks post-treatment and reached a nadir at 5-6 weeks (28-34% of control values) before recovering progressively back to control levels at 10 weeks. Sperm motility was reduced significantly at 4-7 weeks post-treatment with a nadir at 6 weeks (35% of control values). Thus, at 5-6 weeks after MAA treatment, motile sperm output was reduced by 82-88%. Despite these changes, there was little evidence for infertility in the majority of treated males during a serial mating trial. Evaluation of seminiferous tubule morphology combined with germ cell counts at stage VII of the spermatogenic cycle confirmed that, initially, MAA induced the specific loss of pachytene and later spermatocytes at all stages other than early to mid stage VII. Maturation depletion of germ cells at later intervals was consistent with the initial effects of MAA, although at 21 days post-treatment a number of unpredicted (? secondary) changes in spermatogenesis were observed. These were (a) a reduction in number of pachytene spermatocytes at late stage VII/early stage VIII, (b) retention of sperm at stages IX-XIV, and (c) increased degeneration of pachytene spermatocytes and round spermatids at stage VII and of secondary spermatocytes at stages XIV-I. Whilst none of these changes was severe, together they probably accounted for the unexpectedly prolonged drop in sperm output. It is concluded that whilst deleterious changes in spermatogenesis may occur secondarily following MAA treatment, for the most part spermatogenesis proceeds normally and fertility is largely maintained despite a massive but transient decrease in sperm output.     

The effect of gossypol acetic acid on the different stages of the spermatogenic cycle in the rat

The reversibility of the effect of gossypol on testicular histology and fertility was studied in rats. Adult males of proven fertility were treated orally with gossypol acetic acid (15 mg/kg) for 9 or 16 weeks (groups 1 and 2, respectively). Another groups of animals (group 3) was given gossypol (15 mg/kg) for 16 weeks and killed 6 weeks after the end of treatment. Control animals (group 4) were given the vehicle only by oral intubation. In the mating studies, although only 33% of the animals in group 1 were infertile, 100% infertility was observed following 16 weeks of gossypol treatment (group 2). All animals in group 3 regained their fertility 6 weeks after cessation of drug treatment. Damage was observed to 15.7% of the seminiferous tubules after 9 weeks of drug treatment, and to 78% after 16 weeks of treatment. Extensive vacuolization, increased numbers of lipid droplets, degeneration of germ cells, loosening of the epithelium, and a significant decrease in the number of pachytene spermatocytes (stages VII-X) and spermatids (steps 7-10 at stages VII-X) were observed after gossypol treatment. There was a decrease in the diameter of only stage VIII seminiferous tubules after 9 weeks of treatment, whereas a reduction was observed in the tubules of all stages after 16 weeks of gossypol treatment. In the recovery phase, the diameter of seminiferous tubules was similar to that of controls, except for tubules at stage VIII. No change in the area of the lumen of the seminiferous tubules and lipid bodies was observed after 9 weeks of drug treatment, but a marked reduction in the area of the lumen (stages II-X) and an increase in lipid bodies (all stages) was observed after 16 weeks of gossypol treatment. Six weeks after cessation of treatment, the area of the lumen and the number of lipid bodies were comparable to values in controls. A reduction in the area of the epithelium was restricted to just a few stages (VIII-XIV) in treated animals at 9 weeks, whereas after 16 weeks the area of the epithelium was decreased in all tubules. In the recovery phase, except for tubules at stage VIII, the area of the seminiferous epithelium was comparable to that in controls.     

Acute lymphoblastic leukaemia in rats induces pathomorphological changes in germ cells

Summary.  Concern about the reproductive potential of long-term survivors of acute lymphoblastic leukaemia (ALL) prompted an investigation into the impact of the disease on spermatogenic cells. Using rats as a model, histological, immunocytochemical and electron microscopic analysis was applied to investigate changes in the seminiferous epithelium. In rats transplanted with leukaemic cells at early puberty, degenerate primary spermatocytes and spermatids were prevalent within stage VIII tubules. Electron microscopically, step 8 spermatids showed acrosomal abnormalities and nuclear contour distortion. In the distorted step 9 spermatids, the microtubules of the manchette were abnormally oriented or deficient. Antitubulin antibody staining was reduced in elongating spermatids in the group transplanted with leukaemic cells at early puberty but was not observed in the older leukaemic group. Step 13 spermatids showed extracted chromatin and degenerate step 19 spermatids were occasionally found. Similar but less severe changes were seen in the group of rats transplanted with leukaemic cells at late puberty. We conclude that germinal cell damage induced by ALL is dependent on the developmental maturity of the seminiferous epithelium. The present findings are of particular importance when interpreting the impact of anticancer chemotherapeutics on germinal cells in patients with ALL.     

BCG-induced orchitis: Structural changes during the degeneration of seminiferous tubules of rats and rabbits

Summary The effect of BCG-induced orchitis on the structure of the seminiferous tubules in rats and rabbits was investigated by light and electron microscopy. The formation of cavities between Sertoli cells and the displacement of the cells of the spermatogenic cycle are the earliest changes to be observed. Individual Sertoli cells degenerate and separate from spermatocytes and spermatids. The latter form multinuclear complexes by a broadening of the intercellular bridges. The nuclei of spermatids undergo ring-like chromatin condensation in the rat and swelling in the rabbit. After the loss of spermatocytes and spermatids from the germinal epithelium, the remaining Sertoli cells have a very irregular shape and contain many residual bodies, which are probably derived from previously phagocytosed spermatids. They often contain crystalline inclusions. The nuclei of Sertoli cells show small chromatin condensations. In the rabbit, the tubular wall increases considerably in diameter. In the vicinity of a granuloma in the interstitium caused by BCG inflammatory cells accumulate around the wall of the seminiferous tubules. Although the basal lamina seems to be an obstacle, penetration of macrophages into the tubular lumen could be observed. However, this occurred only after the degeneration of the germinal epithelium.     

Stimulation of Protein Synthesis in Pachytene Primary Spermatocytes from the Human Testis by Pyruvate

A sequential enzymatic incubation in collagenase and trypsin was carried out to yield a suspension of viable single cells from the seminiferous epithelium of adult human testis. The cell suspension predominantly consisted of pachytene primary spermatocytes (15%), round spermatids (32%), and condensing spermatids and residual bodies (21%). Human pachytene spermatocytes were isolated by unit gravity sedimentation using the methods originally developed for murine tissue. The spermatocyte-enriched fraction was 79% pure. When the effect of energy sources on protein synthesis by spermatocytes was examined, the highest rate of protein synthesis with pyruvate was found among four kinds of substrates added at a concentration of 10 mM. As shown with murine spermatocytes, the rate of protein synthesis by the human spermatocytes is probably regulated by pyruvate.     

大鼠非特异性睾丸炎模型的建立及生精上皮的变化

目的:探讨大鼠生精上皮在非特异性炎症状态下的变化。方法:20只成年W istar雄性大鼠随机分为2组(对照组和实验组),对照组腹腔注射生理盐水(1 m l/kg),实验组注射细菌内毒素(LPS),剂量为1 mg/kg,两组均隔1 d注射1次,距第1次注射10 d后取材。HE染色观察睾丸组织的病理改变,免疫组化方法探讨生精上皮内增殖细胞核抗原(PCNA)、α连接素的变化。结果:实验组睾丸组织有明显炎性改变;生精上皮内PCNA表达量显著降低:每个生精小管(仅精原细胞着色的小管)阳性细胞个数为(59±5)个,比对照组明显减少(P<0.01),而这种小管占总管数的比例显著升高(P<0.01),为0.673±0.054;实验组大鼠睾丸内α连接素表达量亦减少(P<0.01),阳性反应颗粒平均光密度为0.150±0.014。结论:睾丸炎症时精原细胞增殖和生精上皮内细胞间粘附力度均减弱,这可能是睾丸炎导致男性不育的原因之一。     

Quantitative （stereological） study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats

Aim: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate(TU) treatment. Methods: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. Results: In response to TU treatment, the numbers of non-type B spermatogonia, type B sperrnatogonia and late elongated spermatids per testis were reduced to 51%, 66% and 14% of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51%-65% of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0% of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. Conclusion: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gonadotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular test-osterone levels. (Asian J Androl 2004 Dec; 6: 291-297)     

The selective removal of pachytene spermatocytes using methoxy acetic acid as an approach to the study in vivo of paracrine interactions in the testis

Testicular weight and morphology, serum gonadotropins, intratesticular levels of testosterone and ABP levels in testicular interstitial fluid were studied in adult rats at intervals of 1 to 70 days after a single oral dose of 650 mg/kg methoxy acetic acid. At 3 days, this treatment resulted in the selective loss or depletion of pachytene and later spermatocytes from seminiferous tubules at all stages other than VIII to XI of the spermatogenic cycle. At later times this lesion was expressed as an absence mainly of round (14 days) or elongated (21 days) spermatids from the majority of seminiferous tubules. Other than these changes, spermatogenesis did not appear to be affected by treatment and was qualitatively normal in all tubules at 70 days after treatment. As deduced from cell counts at 3 days posttreatment, the initial action of methoxy acetic acid was restricted to late zygotene spermatocytes (stage XII) and pachytene spermatocytes at all stages other than early- to mid-stage VII. Levels of FSH in serum and those of ABP in testicular interstitial fluid indicated that Sertoli cell function was altered in rats treated with methoxy acetic acid. Both were increased at 1 to 3 days posttreatment, returned to normal at 7 to 14 days but were increased again at 21 days before finally returning to control levels at 28 days. In contrast, the levels of testosterone in serum, isolated seminiferous tubules and testicular interstitial fluid were unaffected by treatment, as also were the serum levels of LH. The two periods of increase in FSH and ABP levels coincided with the times of greatest decrease (approximately 20%) in testicular weight, and may be related either to the type of germ cell missing from the affected tubules and/or to the stage of the cycle of the affected (or unaffected) tubules. These data suggest that chemicals such as methoxy acetic acid may prove useful in the study of paracrine interactions in vivo.     

Apoptosis and molecular pathways in the seminiferous epithelium of aged and photoinhibited Syrian hamsters (Mesocricetus auratus)

Aging and short photoperiod exposure induce germ cell apoptosis in the Syrian hamster; however, the specific germ cells affected and the molecular pathways triggered have not been elucidated. We analyzed germ cell apoptosis and the expression of the Fas/Fas-L system, Bcl-2 family, and p53 in aged and photoinhibited hamsters and compared with those young maintained in natural photoperiod. Aging increased apoptosis in spermatogonia and spermatocytes, but in photoinhibited hamster testes only an increase in apoptotic spermatocytes was observed. Apoptosis was higher in aged hamsters in stages I-IV, V-VI and VII-VIII. Aging increased apoptosis of spermatogonia in stages I-IV and V-VI. Apoptotic pachytene spermatocytes were significantly higher in stages I-IV, V-VI, and VII-VIII in aging. Apoptotic preleptotene and pachytene spermatocytes were higher in aging, but no differences were observed in leptotene-zygotene. Fas-L was expressed by Sertoli cells, of young, aged, and photoinhibited hamsters. Bcl-x(L) was strongly expressed in germ cells on young hamsters and slightly in aging and after short photoperiod exposure. Spermatocytes of photoinhibited hamsters were intensively stained with Fas, Bax, Bcl-xs/L, and p53. In conclusion, aging increases apoptosis in spermatogonia and spermatocytes, depending on the stage of the seminiferous epithelium cycle, whereas after a short photoperiod exposure only an increase in apoptotic spermatocytes is observed. The results suggest that Fas, Bcl-x(L), Bax, and p53 participate in germ cell apoptosis induction after short photoperiod exposure, whereas only Bcl-x(L) is involved in aging.     

Quantitative morphology of the testicular tubular epithelium in the water buffalo (Bubalus bubalis).

Ultrastructural features and morphometric evaluations of water buffalo seminiferous epithelium are reported for the 6 phases of the spermatogenic cycle. The relative Sertoli cell volume varies between 30% (phase 4) and 39% (phase 8), the calculated volume of a Sertoli cell between 7118 microns3 and 8968 microns3 (phase 4). Smooth ER is the organelle that exhibits the most prominent changes in Sertoli cells during the spermatogenic cycle: it occupies about 6% in phase 3 and 21% in phase 4. All spermatogenic cells of the same clone present cytoplasmic bridges among them. From preleptotene (about 470 microns3) to late diplotene (about 2300 microns3) the volume of a primary spermatocyte increases nearly 5-fold; their nuclear volumes increase 3.5-fold in the same period. Secondary spermatocytes are found only in phase 4 of the cycle. Due to partial cell necrosis and autolysis late maturation phase spermatids display not more than 25% of the size of early cap phase spermatids. 63% of all numerically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Particularly heavy cell loss is observed in phase 4 and involves the spermatogonial fraction as well as cells during the second meiotic division.     

Quantitative analysis of spermatogenic DNA synthesis in the rat using a monoclonal anti-5-bromodeoxyuridine antibody

Summary. The utility of the 5-bromodeoxy-uridine (BrdUrd) labelling technique for the quantitative analysis of spermatogenic deoxyribonucleic acid (DNA) synthesis was investigated in the rat. Rat testicles were labelled by a single intraperitoneal injection of 100 mg kg⁻¹ of BrdUrd. The testicles were removed 1 h after injection, fixed in Bouin's fluid and embedded in paraffin. BrdUrd-labelled cells were detected by immunohistochemical staining using a monoclonal anti-BrdUrd antibody. The number of BrdUrd-labelled tubules per total number of tubules (percent L.T.), the number of BrdUrd-labelled cells per total number of tubules (tubular ratio) and the number of BrdUrd-labelled cells per number of Sertoli cells (Sertoli cell ratio in BrdUrd-labelled cells) were calculated as indices of spermatogenic DNA synthesis during each stage of the seminiferous epithelial wave. BrdUrd labelling was found exclusively in the nuclei of spermatogonia and in preleptotene spermatocytes in the seminiferous epithelium. The percent L.T. was generally greater than 50%, except in stages VI, VII and XIV, and the tubular as well as Sertoli cell ratios in BrdUrd-labelled cells was greater than 2.0 and 0.15, respectively, in stages I, II-III, V, VIII, X, and XII. The tubular ratio and Sertoli cell ratio in BrdUrd-labelled cells along the seminiferous epithelial wave had two distinct peaks. The distribution of the tubular ratio using the BrdUrd-labelling technique correlated well with the distribution previously established by measuring tritiated thymidine uptake per tubule. Thus, the BrdUrd labelling technique, which is more efficient than the tritiated thymidine labelling technique, can be used to quantitatively evaluate spermatogenic DNA synthesis.