Apoptosis and blood–testis barrier during the first spermatogenic wave in the pubertal rat

This research explores the initial assembly of the blood–testis barrier (BTB) during puberty, when a massive physiological apoptosis in the first spermatogenic wave takes place. Fragments of testis from 14‐ to 20‐day‐old rats were studied by conventional transmission electron microscopic techniques. Lanthanum hydroxide was used as an intercellular tracer. Light microscopy was used to confirm apoptotic death when paraffin‐embedded sections were studied by TUNEL analysis. When the seminiferous cords reached the zygotene–pachytene spermatocyte level, they exhibited abundant apoptotic figures, whereas the remaining segments showed sporadic apoptosis. We found a BTB not yet assembled in the cords with zygotene–pachytene spermatocytes and abundant apoptosis. The observed apoptosis frequency diminished drastically when BTB was organized, as confirmed by the use of the tracer. Our conclusion is that the massive apoptosis found in the zygotene–pachytene spermatocytes between days 14 and 20 coincides with an open BTB. The absence of BTB could be one of the factors causing massive apoptosis of zygotene–pachytene spermatocytes, at least within the time span analyzed. The zygotene–pachytene spermatocytes are left exposed in an open environment instead of being isolated in the adluminal compartment to which they are destined. Anat Rec 290:206–214, 2007. © 2007 Wiley‐Liss, Inc.     

Quantitative aspects of water buffalo (Bubalus bubalis) spermatogenesis.

In the buffalo, seminiferous tubules occupy about 82% of the testis. Spermatogenesis can be divided into 6 stages according to characteristic cellular associations in the seminiferous epithelium. A-spermatogonia have a volume of approximately 1,400 microns3 and the highest absolute mitochondrial volume of all spermatogenic cells. B-spermatogonia display cellular, nuclear and mitochondrial volumes of approximately half the values of A-spermatogonia. From preleptotene (approximately 470 microns3) to late diplotene (approximately 2,300 microns3), the volume* of primary spermatocytes increases nearly five-fold; their nuclear volumes increase by 3.5 times within the same period. During zygotene mitochondrial cristae start to dilate. Grouping of mitochondria by a dense intermitochondrial substance is most prominent during pachytene and diplotene. In pachytene the absolute size of the Golgi apparatus more than doubles, indicating a high secretory activity. Through zygotene only rER is encountered; in pachytene and diplotene a tubular sER makes its first appearance. Secondary spermatocytes are found only in stage 4 of the cycle. Due to partial cell necrosis and autolytic events, late maturation phase spermatids display no more than 25% of the size of cap phase spermatids. There is no morphological evidence for an active uptake and digestion of residual bodies by the Sertoli cells. Also, no lipid cycle is present in the buffalo seminiferous epithelium. Morphometric evaluations reveal that 63% of all theoretically possible germ cells disappear from the seminiferous epithelium during spermatogenesis. Heavy cell loss is observed in stage 4 of the cycle in the spermatogonial fraction as well as during the second meiotic division.     

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules

 The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I–III and IX–XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell. Accepted: 27 Oct 1998     

Proliferation and apoptosis of spermatogonia in postpuberal boar (Sus domesticus) testes with spontaneous unilateral and bilateral abdominal cryptorchidism

Cryptorchidism is a frequent male sexual disorder in mammals, which affects the histology of the tunica propria, interstitial tissue, blood vessels, seminiferous epithelium and testis functioning. In this paper, proliferation and apoptosis were examined in the seminiferous epithelium of both testes from unaffected boars and from boars suffering unilateral and bilateral cryptorchidism. In germ cells, proliferation was studied using the immunohistochemical PCNA technique, and apoptosis was analysed by in situ TUNEL labelling. An index was obtained for the proliferation and apoptosis observed in seminiferous tubules. In abdominal testes the epithelium contained few spermatogonia and Sertoli cells. In the testes of unaffected boars, numerous spermatogonia proliferated, whereas in cryptorchid testes such proliferation was lower and the proliferation/apoptosis ratio diminished. In the unaffected group, the TUNEL-positive germ cells were spermatogonia and spermatocytes in different phases of meiosis. In abdominal testes, the TUNEL-positive germ cells were spermatogonia alone. The apoptosis index of both abdominal and scrotal testes was similar. In conclusion, spontaneous cryptorchid testes showed a lower rate of spermatogonia proliferation in the seminiferous epithelium.     

Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle

The male prairie dog (Cynomys ludovicianus) is an annual breeder with complete testicular regression between breeding periods. Knowledge of the seminiferous tubule cycle stages at all phases of the annual cycle is essential for evaluation of testicular effects of endogenous and exogenous hormones. Testis tubule diameter is directly correlated with testicular weight during the annual cycle. Seminiferous tubule stages found during testicular activity start with sperm release and round spermatids in the Golgi stage (I). Then they progress through the cap and acrosome stages (stages II to VI) until elongate spermatids are formed. During these stages preleptotene, leptotene and zygotene cells develop into pachytene cells which mature with the long spermatids (stage VII). Two distinct tubule associations (stages VIII, IX) follow during which the first and second meiotic metaphases occur. These stages are correlated with the middle and late phases of residual lobe retraction and condensation. The last stage (X) has final sperm development and is present with round spermatids that have no Golgi development. During regression changes are initially associated with the seminiferous tubule stages of active testes and end with relocation of Sertoli cell nuclei to a position above the basal layer of spermatogonia. Out of season testes are characterized by few spermatogonial mitoses and absence of viable spermatocytes. In recrudescent testes, Sertoli cell nuclei again become basal, spermatogonia resume mitoses and spermatocytes and spermatids progressively develop. After each cycle of proliferation of germ cells there is sloughing of the most differentiated spermatocytes and spermatids until the final tubule associations of the active testis are present. Anat. Rec. 247:355–367, 1997. © 1997 Wiley-Liss, Inc.     

Etoposide (VP-16) is a potent inducer of micronuclei in male rat meiosis: Spermatid micronucleus test and DNA flow cytometry after etoposide treatment

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids offer different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DMA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating sper-matogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage. © 1994 Wiley-Liss, Inc.     

The blood–testis barrier in the toad (Bufo arenarum hensel): A freeze-fracture and lanthanum tracer study

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.     

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.     

Role of spermatogonia in the stage-synchronization of the seminiferous epithelium in vitamin-A-deficient rats

After 20-day-old rats are placed on a vitamin-A-deficient diet (VAD) for a period of 10 weeks, the seminiferous tubules are found to contain only Sertoli cells and a small number of spermatogonia and spermatocytes. Retinol administration of VAD rats reinitiates spermatogenesis, but a stage-synchronization of the seminiferous epithelium throughout the testis of these rats is observed. In order to determine which cell type is responsible for this synchronization, the germ cell population has been analyzed in whole mounts of seminiferous tubules dissected from the testes of rats submitted to the following treatments. Twenty-day-old rats received a VAD diet for 10 weeks and then were divided into three groups of six rats. In group 1, all animals were sacrificed immediately; in group 2, the rats were injected once with retinol and sacrificed 3 hr later; in group 3, the rats were injected once with retinol, placed on a retinol-containing diet for 7 days and 3 hr, and then sacrificed. Three rats from each group had one testis injected with ³H-thymidine 3 hr (groups 1 and 2) or 7 days and 3 hr (group 3) before sacrifice. Three normal adult rats (approximately 100 days old) served as controls. Labeled and unlabeled germinal cells were mapped and scored in isolated seminiferous tubules. In group 1, type A₁ and type A₀ spermatogonia as well as some preleptotene spermatocytes were present; type A₂ A₃ A₄ In, and B spermatogonia were completely eliminated from the testis. Neither type A₁ mitotic figures nor ³H-thymidine-labeled-type A₁ nuclei were seen. Three hr after retinol injection (group 2), type A₁ mitoses, but no labeled type A₁ nuclei were observed. At 7 days and 3 hr after retinol administration (group 3), type A₄ and In Spermatogonia as well as type A₁ spermatogonia were present. A few residual pachytene spermatocytes were found, and some type A₀ cells were labeled. These results indicate that VAD caused, in addition to an impairment of spermatogenesis at the preleptotene spermatocyte step, a selective momentary arrest of surviving type A₁ spermatogonia at the G₂ phase of their cell cycle. Following administration of vitamin A to VAD rats, these type A₁ cells reinitiated spermatogenesis synchronously and, after several cycless of proliferation and renewai, reconstituted the seminiferous epithelium in a stage-synchronized manner.     

Postnatal formation of the blood-testis barrier in the rat with special reference to the initiation of meiosis

Summary Postnatal formation of the Blood-Testis Barrier (BTB) in the rat was studied by either fixation in hypertonic fixative or employing lanthanum tracer. After 15 days of age, meiosis has reached different stages of spermatogenesis in differnt zones of the seminiferous cords. Only in those parts where germ cells are in the pachytene stage of meiosis do Sertoli cells form an effective barrier or tight compartment. Between 16 and 19 days of age, final formation of the BTB, which is to be found in the adult rat testis, occurs by zygotene and then leptotene stages successively entering the tight compartment. Thus, formation of a BTB by Sertoli cells does not occur synchronously along the length of the seminiferous cord but in accordance with the stage of meiosis of the associated germ cells.     

Lactate inhibits germ cell apoptosis in the human testis

Dysregulation of male germ cell apoptosis has been associated with the pathogenesis of male infertility. Therefore, factors involved in the regulation of germ cell death are being actively investigated. Here, we studied the effects of lactate on human male germ cell death, using as a model a testis tissue culture in which physiological contacts are maintained between the germ cells and the supportive somatic Sertoli cells. Apoptosis of spermatocytes, spermatids and a few spermatogonia was induced by culturing segments of seminiferous tubules under serum-free conditions. This germ cell death was inhibited effectively and dose-dependently by lactate, indicating that it plays a crucial role in controlling cell death cascades of male germ cells. Interestingly, the anti-apoptotic role of lactate was not associated with changes in testicular adenine nucleotide (ATP, ADP and AMP) levels. In the seminiferous tubules, the final site of the death-suppressing action of lactate appeared to be downstream along the cell death pathway activated by the Fas receptor of the germ cells. In conclusion, testicular cell death was effectively regulated by lactate, which may be regarded as a potential compound for optimizing in-vitro methods involving male germ cells for assisted reproduction.     

Spermatogenesis in the vasectomized monkey: Quantitative analysis

The seminiferous epithelium in mature vasectomized Macaca fascicularis was examined quantitatively to assess spermatogenesis. Monkeys were bilaterally vasectomized and controls were bilaterally sham operated. At postoperative periods of 10 and 18 months, groups of monkeys were castrated and their testes prepared for morphologic analysis. Diameters were measured in 100 cross sections of seminiferous tubules from each animal. Numbers of spermatogonia (Ad and Ap), preleptotene spermatocytes, pachytene spermatocytes, and step 7 spermatids, relative to Sertoli cell nucleoli, were counted in stage VII tubules. Tubule diameter and germ cell numbers per Sertoli cell nucleoli were not altered by vasectomy. Our study demonstrates quantitatively that spermatogenesis in the monkey is not inhibited up to 18 months following vasectomy.     

低剂量电离辐射诱导小鼠睾丸生精细胞凋亡及P53基因和蛋白表达

目的:研究低剂量电离辐射对小鼠睾丸生精细胞凋亡及 P53 基因和蛋白表达的影响。方法:应用密度梯度离心法分离不同种类生精细胞, 流式细胞术检测其细胞凋亡, 免疫组化法观察生精细胞P53蛋白表达, 原位杂交法观察其 P53 mRNA水平。结果:0.025-0.2 Gy X射线全身照射后, 生精细胞凋亡具有明显的细胞种类规律性。在较低剂量照射(0.025和0.05 Gy)时, 以精原细胞凋亡为主, 随照射剂量增加(0.075-0.2 Gy)逐渐累及精母细胞, 并且前者凋亡率明显高于后者, 很少累及精子细胞和精子。P53蛋白表达主要见于精原细胞和精母细胞, 并且前者阳性率高于后者, 随照射剂量增加, 其阳性率逐渐升高, 而精子细胞和精子阳性率较低; P53 mRNA表达在较低剂量照射(0.025 Gy)时, 主要以精母细胞和精子细胞为主, 随剂量增加(0.05-0.2 Gy)逐渐累及精原细胞。精原细胞和精母细胞 P53 mRNA表达呈明显的剂量依赖性关系, 但精子细胞表现不明显。结论:低剂量电离辐射可选择性诱导小鼠睾丸生精细胞凋亡, 具有明显的剂量和时程效应关系。提示, 这种选择性诱导凋亡调控机制可能与 P53 基因和蛋白表达相关联。     

Detection of germ cell mutagenicity of trophosphamide by the spermatid micronucleus test in the rat

The effects of the antineoplastic drug trophosphamide (TP) onmale rat germ cells were examined with the spermatid micronucleustest (SMNT). We used the microdissection technique in orderto isolate stage I of the seminiferous epithelial cycle, wherethe cells that have just passed the meiotic divisions can befound. Micronuclei (MN) were scored at different time-pointsafter TP treatment at dose levels of 25 and 50 mg/kg. An inductionof MN was detected in cells exposed at preleptotene (18 and19 days) and late pachytene (3 days), as well as at the diplotene-diakinesisstage (1 day). The dose-response for MN induction was linearat all time intervals studied, except for 18 days time point.The highest frequency of MN (5.20 ± 0.57/1000 spermatids)could be found with the lower TP dose at 18 days, correspondingto exposed preleptotene spermatocytes and reflecting S-dependentclastogenicity. While a significant increase in MN could onlybe detected in exposed preleptotene spermatocytes with the lowerTP dose, the higher dose level also induced MN significantlyin late pachytene and the diplotene-diakinesis stage. DNA flowcytometry at 18 days showed cytotoxicity of TP to exposed primaryspermatocytes at pachytene, but no cytotoxicity to the preleptotenesypermatocytes that exhibited a significant MN induction. Theresults show that the SMNT using the stage-I-specific examinationof the rat seminiferous epithelium can detect the germ cellmutagenicity of TP and gives further evidence of the usefulnessof this technique in the testing of chemicals for genotoxiceffects in male germ cells. ¹To whom correspondence should be addressed     

COMMENTS

Retinoblastoma family proteins pRb, p107 and p130 are differentially expressed in the rat testis. They function in specific cell types during testicular development and spermatogenesis, participating in the control of proliferation, differentiation, and survival. Their expression levels and phosphorylation status are modulated during germ cell cycle progression and apoptosis. Hyperphosphorylated states and elevated levels of p107 are correlated with cell cycle progression, whereas hypophosphorylated states and reduced levels are associated with suppression of proliferation and apoptosis in germ cells and Leydig cells. These proteins may also serve as markers of cell cycle status of germ cells during spermatogenesis.     

Proliferation of spermatogonia and Sertoli cells in maturing mice

Summary Spermatogonial proliferation was studied in mice from day 13 p.p. when the seminiferous epithelium is incomplete, until week 12 p. p. when a steady state at adult levels has been attained. Counts of undifferentiated, A ₁ and intermediate spermatogonia and primary spermatocytes in stages IV and IX of the cycle of the seminiferous epithelium were made in whole mounted seminiferous tubules. Sertoli cell proliferation was studied in a separate series from 6 to 14 days p.p. employing the ³H-thymidine labeling index.It appeared that 1. Sertoli cell proliferation stops at day 12 whereafter the cells obtain their adult appearance; 2. The numbers of stem cell spermatogonia and the production of differentiating A ₁ spermatogonia increase almost twofold between day 13 and week 12; 3. The efficiency of the divisions of the differentiating A ₁-B spermatogonia is similar to that in the adult throughout this period; 4. At all ages studied, the cell counts revealed an almost constant numerical relationship between Sertoli cells and germ cells, which suggests a function of Sertoli cells in the regulation of spermatogonial proliferation.     

Effects of testosterone enanthate on the structure of the male reproductive tract of the rat

The histology and fine structure of the testis, epididymis and sex accessory glands were studied in young adult male rats administered testosterone enanthate, 120 μg/100 g body weight, three times weekly for 4, 8, or 12 weeks. The weights of the testis and epididymis decreased, and animals treated for 11 weeks were infertile. Alterations were found in the seminiferous tubules of all rats treated for 8 or 12 weeks, including the presence of many degenerating germ cells and a-large decrease or absence of late spermatids. Study of different stages of the cycle of the seminiferous epithelium showed that the greatest number of degenerating germ cells, step 7 spermatids and pachytene primary spermatocytes, occurred at stages VII-VIII of the cycle. Some normal appearing spermatogonia, primary spermatocytes and early spermatids remained in most seminiferous tubules. Sertoli cells contained many lipid droplets and lysosome-like bodies, and degenerating cells were surrounded by Ser-toli cell cytoplasm. The Leydig cells of treated animals were greatly reduced in size. Sperm progressively disappeared from the lumen of the middle segment and proximal part of the terminal segment of the epididymis after treatment for 8 or 12 weeks. Changes in the middle segment also included the appearance of intraepithelial cavities containing debris, and the presence within the epithelium of phagocytic cells that resembled leukocytes. The lumen of the proximal part of the terminal segment was often collapsed, while in the distal part of the terminal segment, the lumen was filled with cellular debris and degenerating sperm. Organelles of the principal cells of the epididymal epithelium appeared to be qualitatively unaltered. The weight of the sex accessory glands remained close to normal, and the presence of normal ultrastructural features suggested that production of secretions continued.     

Apurinic/apyrimidinic endonuclease immunoreactivity in germ cells of experimental varicocele-induced rat testes

Increased germ cell apoptosis is related to oxidative DNA damage; therefore, we investigated whether there was a significant change in apurinic/apyrimidinic endonuclease (APE) in varicoceles. Experimental varicoceles were created by partial ligation of the left renal vein of adult male Sprague-Dawley rats, which were sacrificed at 1, 3 and 6 weeks after varicocele creation. Testicular tissues were sampled for TUNEL, Western blotting and immunohistochemistry. There was a significant increase in apoptotic germ cells in the ipsilateral testes 6 weeks after varicocele creation. Increased activation of p53, Bax and cleaved caspase-3 in the left testes was also noted. APE increased activation until 3 weeks after varicocele creation, and then decreased at 6 weeks after varicocele surgery. The spermatocytes were immunostained for both 8-hydroxy-2′-deoxyguanosine and APE, but the spermatogonia revealed only APE immunopositivity in the defective tubules. These results suggest that repression of APE is an underlying mechanism of augmented p53-dependent apoptosis in varicocele-induced rat testes and that remaining APE in the spermatogonia plays a decisive role in regaining testicular spermatogenic function after varicocelectomy.