The length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts

Xenografting of immature mammalian testis tissue into mice can accelerate sperm production. To determine whether this shortened time to sperm production is because of reduced length of the spermatogenic cycle, we applied bromodeoxyuridine (BrdU) incorporation to analyze the spermatogenic cycle in porcine and ovine testis xenografts. Small testis fragments from newborn pigs and sheep were ectopically grafted into mice. Once complete spermatogenesis was present in grafted tissue, mice were injected with BrdU and grafts were recovered at different time points thereafter. In porcine grafts, the most advanced germ cells labeled 1 hour, 9 days, 12.3 days, and 18 days after BrdU injection were stage 1 preleptotene/leptotene primary spermatocytes, stage 1 pachytene primary spermatocytes, stage 5 newly-formed round spermatids, and late stage 2 elongating spermatids, respectively. In ovine grafts, the most advanced labeled germ cells at 1 hour, 11 days, and 22 days post-BrdU injection were stage 2 preleptotene/leptotene primary spermatocytes, late stage 1 pachytene primary spermatocytes, and stage 2 elongating spermatids, respectively. These results indicate that each spermatogenic cycle in porcine and ovine xenografts lasts approximately 9 and 11 days, respectively, which is similar to their durations in situ. Therefore, the length of the spermatogenic cycle is conserved in porcine and ovine testis xenografts. This is consistent with earlier reports showing that the cycle length is inherent to the germ cell genotype. The shortened time to sperm production in xenografts therefore appears attributable to accelerated maturation of the testicular somatic compartments. Our results suggest that testis xenografts provide a useful model to study the timing of testicular maturation and spermatogenesis in different mammalian species.     

Immunolocalization assessment of metastasis-associated protein 1 in human and mouse mature testes and its association with spermatogenesis

Aim： To investigate the stage-specific localization of metastasis-associated protein 1 （MTA1） during spermatogenesis in adult human and mouse testis. Methods： The immunolocalization of MTA1 was studied by immunohistochemistry and Western blot analysis. The distribution pattern of MTA1 in mouse testis was confirmed by using quantitative analysis of purified spermatogenic cells. Results： The specificity of polyclonal antibody was confirmed by Western blot analysis. MTA1 was found expressed in the nucleus of germ cells, except elongate spermatids, and in the cytoplasm of Sertoli cells; Leydig cells did not show any specific reactivity. MTA1 possessed different distribution patterns in the two species： in humans, the most intensive staining was found in the nucleus of round spermatids and of primary spermatocytes while in mice, the most intense MTA 1 staining was in the nucleus of leptotene, zygotene and pachytene spermatocytes. In both species the staining exhibited a cyclic pattern. Conclusion： The present communication initially provides new evidence for the potential role of MTA1 in mature testis. In addition, its distinctive expression in germ cells suggests a regulatory role of the peptide during spermatogenesis.     

Acute effects and long-term sequelae of 1,3-dinitrobenzene on male reproduction in the rat. II. Quantitative and qualitative histopathology of the testis

This study determined the quantitative and qualitative histopathologic effects of a single oral dose of 1,3-dinitrobenzene (48 mg/kg) on the rat testis from 1 to 175 days postexposure. The testis was damaged severely by hour 24, as evidenced by increased numbers of regressive seminiferous tubules that exhibited degenerating pachytene spermatocytes, chromatin margination in spermatids, giant cells, deformed spermatid heads, retained spermatids, and reduced numbers of meiotic figures. The major effects during the first 48 hours posttreatment were degeneration or exfoliation of pachytene spermatocytes and round spermatids and the retention of step 19 spermatids. These regressive effects continued until 24 days, after which the tubules either recovered or became atrophic. At the end of the study (175 days), three males were normal, one had regressed testicles, and three males had atrophic tubules (15 to 45%). Several cellular abnormalities were common throughout the period. In addition, the frequency of the stages of spermatogenesis was altered, an indication of a disturbance in the kinetics of spermatogenesis. 1,3-Dinitrobenzene produced profound and specific lesions in the seminiferous tubules, and recovery was slow and incomplete. Atrophic tubules seemed to form if the normal cellular associations were not reestablished within 24 days, possibly due to the inability of Sertoli cells to reorganize the synchrony of germ cell development.     

Specific localization of the basigin protein in human testes from normal adults, normal juveniles, and patients with azoospermia

Basigin is a transmembrane protein belonging to the immunoglobulin superfamily. Specific localization of the protein in normal human testes, from those of a 2-year-old boy to those of a 50-year-old man, and in testes with Sertoli cell only syndrome and germ cell arrest, is reported. Basigin localization was determined using an immunohistochemical technique with an antibody against human basigin. In the normal adult testes, basigin was detected at the periphery of both spermatocytes older than zygotene and round spermatids. In the juvenile testes, it was expressed in accordance with the appearance of pachytene spermatocytes. In this study, pachytene spermatocytes were detected in an 11-year-old boy. Basigin was not expressed in immature testes with germ cells younger than pachytene spermatocytes, namely in testes from boys aged 2-9 years. In testes from adult patients with Sertoli cell only syndrome, basigin was expressed at the periphery of Sertoli cells, but localization was confined to the adluminal compartment of the seminiferous tubule. In testes with germ cell arrest, the protein was expressed on germ cells from pachytene spermatocytes to step 2 spermatids, where present. The results show that in the normal human testes basigin is expressed with the onset of spermatocyte differentiation. Because human basigin is expressed in adult testes with Sertoli cell only syndrome, the protein seems to be synthesized in Sertoli cells and expression continues after these cells dedifferentiate in the seminiferous epithelium.     

Dazl protein expression in adult rat testis is up-regulated at meiosis and not hormonally regulated

The Y-chromosomal DAZ (deleted in azoospermia) gene and the autosomal Dazl (deleted in azoospermia-like) gene are two crucial factors for the achievement and maintenance of spermatogenesis. Whereas Y-chromosomal DAZ is present in certain primates, it is lacking in rodents and other species. We have investigated the expression of Dazl protein during spermatogenesis in the adult rat testis using immunohistochemistry. Dazl immunoreactivity was found predominantly in the cytosol of primary pachytene spermatocytes. A weaker but clearly detectable signal was present in intermediate and B spermatogonia and in early spermatocytes from preleptotene to zygotene. The highest expression patterns were observed between stages IV and VIII during the spermatogenic cycle when spermatocytes prepare for the first meiotic division. Specific staining could also be observed in step 11-19 elongating spermatids in the acrosome region. Treatment for 42 days with a potent GnRH-antagonist abolished gonadotrophin secretion and led to a regressed testis, lacking most of the advanced germ cell types such as spermatids but still bearing spermatogonia and spermatocytes. No difference in staining pattern for Dazl protein was observed in GnRH antagonist-treated rats despite the lack of gonadotrophins and substantial impairment of the spermatogenic process, indicating that Dazl expression is clearly hormone-independent. The localization and level of Dazl expression suggests an important role in the regulation of the first meiotic stages of spermatogenesis. The hormone independent onset of expression points to an autonomous cell-cycle event in which Dazl seems to be essential for the entry into meiosis. The presence of Dazl in the acrosome region of elongating spermatids might reflect an unknown role of Dazl as a morphogenetic factor during spermiogenesis.     

Round spermatids show normal testis-specific H1t but reduced cAMP-responsive element modulator and transition protein 1 expression in men with round-spermatid maturation arrest

During spermiogenesis, histones are replaced by transition proteins, which in turn are replaced by protamines. The TNP1 gene-encoding TP1 (transition protein 1) protein contains a cAMP-responsive element (CRE) that serves as binding site for the CRE modulator (CREM). To gain further insight into the complex regulation of nucleoprotein exchanges in haploid spermatids and its potential role for spermatogenic impairment, we studied the gene expression of testis-specific histone H1t, CREM, and TNP1 in testicular biopsies from men with normal spermatogenesis (n = 20) and with round spermatid maturation arrest (n = 16). During normal spermatogenesis, H1t messenger RNA (mRNA) was present in 86.2%+/-8.7% of pachytene spermatocytes (stages III-V), whereas H1t protein was synthesized in 83.5%+/-13.0% of pachytene spermatocytes (stages III-V) and persisted in 95.2%+/-3.1% of spermatids (steps 1-5). CREM mRNA was detectable in 74.2%+/-9.4% of pachytene spermatocytes (stages IV-V) and in 78.7%+/-10.0% of spermatids (steps 1-3). CREM protein was synthesized in 81.2%+/-14.2% of spermatids (steps 1-3). TNP1 mRNA was present in 80.0%+/-13.5% of spermatids (steps 2-4), whereas TP1 protein was synthesized in 89.7%+/-5.3% of spermatids (steps 3-4). In men with round spermatid maturation arrest, spermatids only develop to step 3 of differentiation. These spermatids were devoid of both CREM and TP1 but did contain H1t. These results indicate that TP1 is likely to be an important parameter in the histone-to-protamine exchange and in the initiation of spermatid elongation. CREM is involved in the regulation of TNP1 gene expression and consequently plays a vital role in the correct differentiation step from round spermatids to mature spermatozoa.     

Expression and phosphorylation of mitogen-activated protein kinases during spermatogenesis and epididymal sperm maturation in mice.

The expression and phosphorylation/dephosphorylation of mitogen-activated protein (MAP) kinases during mouse spermatogenesis and epididymal sperm maturation have been investigated by immunoblotting and immunohistochemical staining with commercially available anti-ERK2 and anti-Active MAPK antibodies. Two forms of MAP kinases, p42ERK2 and p44ERK1, were expressed in a similar amount in spermatogenic cells at different stages. ERK1 and ERK2 were phosphorylated (activated) in early spermatogenic cells from primitive spermatogonia to zygotene primary spermatocytes, while only a small quantity of phosphorylated MAP kinases could be detected in pachytene primary spermatocytes and spermatids. MAP kinase activity in primative spermatogonia and preleptotene primary spermatocytes was the highest among spermatogenic cells. ERK1 and ERK2 were also present in epididymal spermatozoa, and their phosphorylation was increased while spermatozoa pass through epididymis and vas deferens for maturation. It would appear that MAP kinase activation may contribute to the mitotic proliferation of primative spermatogonia, an early phase of spermatogenic meiosis, and, later, sperm motility acquirement.     

Spatiotemporal expression patterns of natriuretic peptides in rat testis and epididymis during postnatal development

Natriuretic peptide (NP) family is composed of atrial, brain and C‐type NP (NPPA, NPPB and NPPC). Here, we aimed to investigate NP expression in testis and epididymis during postnatal development. NPPA expression was observed in gonocytes at prepubertal period but in only spermatocytes in pachytene and leptotene/zygotene stage at pubertal period. In prepubertal and pubertal periods, we detected NPPB expression in only Leydig cells. However, NPPC expression was detected in all of the gonocytes and Sertoli cells, spermatocytes and some interstitial cells in prepubertal and pubertal periods. In postpubertal and mature periods, NPPA and NPPB staining were detected in Leydig cells, elongated and round spermatids but not in spermatogonia and spermatocytes. However, we observed NPPC expression in all cells of the seminiferous tubules and Leydig cells in the postpubertal and mature periods. Epididymal epithelium showed intense NPPC expression during postnatal period but weak NPPA and NPPB expression in prepubertal and pubertal periods. The expression of three NPs in the testis significantly increased after puberty. In conclusion, puberty had a significant effect on NP expression in testis. Unlike NPPA and NPPB, expression of NPPC in all cells of the seminiferous tubule suggests that NPPC is effective in each step of spermatogenesis.     

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)     

Spermatogenesis in the immature mouse proceeds faster than in the adult

The first appearance of spermatogenic cell types related to the age of the animal was studied in sections and tubular whole mounts of testes of normal mice (Cpb-N strain) up to 34 days p.p. The first intermediate spermatogonia and leptotene spermatocytes were seen at days 4 and 7 p.p., respectively. It was found that the subsequent types of spermatogenic cells appear earlier than could be expected if spermatogenesis was to proceed at adult speed. [³H]thymidine labelling studies revealed that within a given interval of time, spermatocytes and spermatids in immature mice develop into more advanced cell types than in adults. The labelling studies and the observation that the cellular associations are always identical to those in the adult, indicate that the rate of acceleration in young mice is the same for spermatogonia, spermatocytes and spermatids. The mean duration of the cycle of the seminiferous epithelium during the age interval of 10 to 30 days p.p. is 7.51 pL 0.10 days, compared to 8.61 pL 0.08 in the adult. It increases gradually towards the adult level, reaching the value of 8.45 pL 0.17 days between days 33 and 56 p.p.     

Stage-specific localization of transforming growth factor β1 and β3 and their receptors during spermatogenesis in men

Aim: To investigate the stage-specific localization of transforming growth factor (TGF) β1 and β3 during spermatogenesis in adult human testis, Methods: The localization of TGFβ1 and β3 was investigated by immunohistochemical staining method employing specific polyclonal antibodies. Results: Both TGFβ1 and β3 and their receptors were preponderant in the Leydig cells. TGFβ1 could not be detected in the seminiferous tubules. TGFβ3 and TGFβ-Receptor (R) Ⅰ were mainly seen in the elongated spermatids, while TGFβ-RⅡ in the pachytene spermatocytes and weak in the spermatogonia, spermatids and Sertoli cells. Only TGFβ-RⅡ was detected in the Sertoli cells.TGFβ3, TGFβ-RⅠ and TGFβ-RⅡ showed a staining pattern dependent upon the stages of the seminiferous epithelium cycle. Conclusion: TGFβ isoforms and their receptors are present in the somatic and germ cells of the adult humantestis, suggesting their involvement in the regulation of spermatogenesis.     

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.     

Influence of a leptin deficiency on testicular morphology, germ cell apoptosis, and expression levels of apoptosis-related genes in the mouse

Leptin-deficient (ob/ob) male mice are morbidly obese and exhibit impaired reproductive function. The objective of this study was to assess the effect of a leptin deficiency on testicular morphology, germ cell development, apoptotic activity within germ cells, and expression levels of apoptosis-related genes in the testis. Sixteen week-old ob/ob male mice (n = 8) and controls (n = 8) were killed, and their reproductive organs were weighed. Testes were processed for either histomorphological analysis (hematoxylin and eosin [H&E] staining), germ cell apoptosis assessment (deoxy-UTP-digoxigenin nick end labeling [TUNEL] method), or apoptosis-related gene expression analysis (microarray). Cross sections of the testes of leptin-deficient animals showed reduced seminiferous tubule area, fewer pachytene spermatocytes, and fewer tubules exhibiting elongated spermatids/mature spermatozoa. Condensation of germ cell nuclei and Sertoli cell vacuolization were evident in the testes of some ob/ob animals. Overall there was an elevation of apoptotic activity in the germ cells of ob/ob mice, particularly within the pachytene spermatocytes. With microarray technology, we identified 9 proapoptosis-related genes that were expressed at a significantly higher level in the testes of ob/ob mice than in the testes of the controls. Among these were members of the tumor necrosis factor receptor super family 1A and 5 (TNFR1 and 5) and peptidoglycan recognition proteins (associated with the extrinsic apoptotic pathway), and granzymes A and B, growth arrest and DNA damage inducible 45 gamma, sphingosine phosphate lyase 1, and caspase 9 (associated with the intrinsic apoptotic pathway). The results of the current study show that a leptin deficiency in mice is associated with impaired spermatogenesis, increased germ cell apoptosis, and up-regulated expression of proapoptotic genes within the testes.     

A possible direct action of oxytocin on spermatogenesis and steroidogenesis in pre‐pubertal mouse

The aim of this study was to evaluate the effects of in vivo and in vitro treatments of oxytocin (OT) on the testis of pre‐pubertal mice. The OT treatment produced significant changes in the spermatogenic and steroidogenic activity by increasing expression of OT‐receptor in the testis of pre‐pubertal mice. Treatment with OT showed increased proliferation of germ cells as indicated by increased number of spermatocytes and round spermatids. Dose‐dependent increase in expression of PCNA, Bcl‐2 and AR proteins was observed in the testis of OT‐treated mice as compared with the control and further supports the role of OT in germ cell proliferation and survival. The pre‐pubertal mice treated with increasing dose of OT showed significant increase in testosterone synthesis due to dose‐dependent stimulatory effects on 3β‐HSD activity and increased expression of STAR, LH‐receptor (LH‐R) and gonadotrophin‐releasing hormone receptor (GnRH‐R) proteins in the testis. The in vitro study has confirmed in vivo finding showing direct action of OT on testicular steroidogenesis. Thus, OT stimulates testicular spermatogenesis and steroidogenesis by directly acting on testis in mice.     

β-肌动蛋白在大鼠精子发生过程中的特殊表达

目的:寻找大鼠精子发生相关蛋白,研究β-肌动蛋白在大鼠睾丸组织的表达和分布。方法:用牛血清白蛋白梯度沉降(STAPUT)法从9日龄雄性SD大鼠睾丸中分离出A型精原细胞,从成年雄性大鼠睾丸中分离出粗线期精母细胞、圆形精子细胞;分别提取这3种细胞的总蛋白,进行双向电泳;对所得到的双向电泳图谱用Im ageM aster2D E lite图像分析软件分析,找出差异蛋白,对挑选出的差异蛋白做质谱分析。进一步用β-肌动蛋白抗体做免疫组化的睾丸组织定位研究。结果:在双向电泳图谱中,β-肌动蛋白在A型精原细胞、粗线期精母细胞中表达量较高,在圆形精子细胞中则表达量极少。免疫组化研究发现:A型精原细胞、粗线期精母细胞有阳性颗粒反应,圆形精子细胞则无阳性颗粒反应;在接近成熟的精子细胞中,呈现极强的阳性颗粒反应,并且越接近排放期的精子细胞,阳性反应越强。在接近成熟的精子头部,阳性颗粒反应最强。β-肌动蛋白主要在精原细胞和精母细胞的细胞质中表达;在接近成熟的精子细胞中主要表达在细胞核。结论:β-肌动蛋白在精子发生过程中有明显的阶段差异表达,推测其对精子发生起重要调节作用。     

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)