Kanatsu-Shinohara and colleagues continue to reveal the secrets of the rare mammalian spermatogonial stem cells. Their most recent study offers a new approach by applying principles from hematopoietic stem cell resear...Kanatsu-Shinohara and colleagues continue to reveal the secrets of the rare mammalian spermatogonial stem cells. Their most recent study offers a new approach by applying principles from hematopoietic stem cell research to demonstrate that cells which form a cobble- stone-like underlay beneath testicular somatic cells in culture are spermatogonial stem cells. Utilization of mouse models and cell cultures shows how the chemokine, CXCL12, fits into the signalling cascade which governs the fate of these cells and hence is essential to male fertility.展开更多
Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; ho...Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without （untreated control） 3-morpholinosydnonimine （SIN-l）, in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition （MPT）, externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-l, the sperm viability was significantly reduced compared to untreated control （P〈 0.001）. Furthermore, the MPT was induced （P〈 0.01） and increment in DNA oxidation （P 〈 0.01）, DNA fragmentation （P 〈 0.01）, tyrosine nitration （P 〈 0.0001） and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control （P 〈 0.001）, this process was observed in 〈10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.展开更多
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...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。展开更多
Testicular sperm extraction is a common procedure used to find spermatogenic cells in men with nonobstructive azoospermia. The laboratory processing of biopsied testicular tissues needs to be performed meticulously to...Testicular sperm extraction is a common procedure used to find spermatogenic cells in men with nonobstructive azoospermia. The laboratory processing of biopsied testicular tissues needs to be performed meticulously to acquire a high yield of cells. In this study, the effectiveness of mincing the tissues after testicular biopsy was assessed using histological evaluation, as was the possible adverse effect of residual tissue on the migration of spermatogenic cells during density gradient centrifugation. Our results indicate that testicular residual tissue, when laid on the density gradient medium along with the sperm wash, hinders the spermatogenic cells＇ forming a pellet during centrifugation, and therefore impairs the intracytoplasmic sperm injection procedure. Whereas the mean number of recovered cells from the sperm wash medium （SWM） with residual tissue is 39.435 ~ 24.849, it was notably higher （60.189 ~ 28.214 cells） in the SWM without minced tissues. The remaining tissue contained no functional seminiferous tubules or spermatogenic cells in histological sections. In conclusion, the remaining residual tissue after mincing biopsied testicular tissue does not add any functional or cellular contribution to spermatogenic cell retrieval; in fact, it may block the cellular elements in the accompanying cell suspension from migrating through the gradient layers to form a pellet during centrifugation and cause loss of spermatogenic cells.展开更多
Spermatogonial stem cells （SSCs） are a type of adult stem cell found in male mammals.These cells have the capacity for self renewal and are capable of differentiating in the niche of testis.They are also the only ad...Spermatogonial stem cells （SSCs） are a type of adult stem cell found in male mammals.These cells have the capacity for self renewal and are capable of differentiating in the niche of testis.They are also the only adult stem cells in a normal postnatal body that undergo self-renewal throughout life,transferring genetic information to the offspring.Since a technique for transplanting SSCs was first described by Brinster and his colleagues in 1994,more and more researchers have become interested in exploring the possibility of utilizing adult SSCs to generate transgenic animals.In this mini-review,we attempt to summarize the current research progress in the area of spermatogonial stem cells including the source,types and differentiation of the SSCs,and the application on transgenic animals,with a particular focus on the strategy of SSCs delivery including seminiferous tubule injection and spermatogonial stem cell transplantation.展开更多
Col la I （one of the subunit of collagen type I） is a collagen, which belongs to a family of extracellular matrix （ECM） proteins that play an important role in cellular proliferation and differentiation. However, ...Col la I （one of the subunit of collagen type I） is a collagen, which belongs to a family of extracellular matrix （ECM） proteins that play an important role in cellular proliferation and differentiation. However, the role of Col lal in spermatogenesis, especially in the control of proliferation and differentiation of spermatogonial stem cells （SSCs）, remains unknown. In this study, we explored effects of downregulation of Collal on differentiation and proliferation of mouse spermatogonia. Loss-of-function study revealed that Oct4 and Plzf, markers of SSC self-renewal, were significantly decreased, whereas the expression of c-kit and haprin, hallmarks of SSC differentiation, was enhanced after Col la I knockdown. Cell cycle analyses indicated that two-thirds of spermatogonia were arrested in S phase after Collal knockdown. In vivo experiments, DNA injection and electroporation of the testes showed that spermatogonia self-renewal ability was impaired remarkably with the loss-of-function of Collal. Our data suggest that silencing of Collal can suppress spermatogonia self-renewal and promote spermatogonia differentiation.展开更多
文摘Kanatsu-Shinohara and colleagues continue to reveal the secrets of the rare mammalian spermatogonial stem cells. Their most recent study offers a new approach by applying principles from hematopoietic stem cell research to demonstrate that cells which form a cobble- stone-like underlay beneath testicular somatic cells in culture are spermatogonial stem cells. Utilization of mouse models and cell cultures shows how the chemokine, CXCL12, fits into the signalling cascade which governs the fate of these cells and hence is essential to male fertility.
文摘Peroxynitrite is a highly reactive nitrogen species and a potent inducer of apoptosis and necrosis in somatic cells. Peroxynitrite-induced nitrosative stress has emerged as a major cause of impaired sperm function; however, its ability to trigger cell death has not been described in human spermatozoa. The objective here was to characterize biochemical and morphological features of cell death induced by peroxynitrite-mediated nitrosative stress in human spermatozoa. For this, spermatozoa were incubated with and without （untreated control） 3-morpholinosydnonimine （SIN-l）, in order to generate peroxynitrite. Sperm viability, mitochondrial permeability transition （MPT）, externalization of phosphatidylserine, DNA oxidation and fragmentation, caspase activation, tyrosine nitration, and sperm ultrastructure were analyzed. The results showed that at 24 h of incubation with SIN-l, the sperm viability was significantly reduced compared to untreated control （P〈 0.001）. Furthermore, the MPT was induced （P〈 0.01） and increment in DNA oxidation （P 〈 0.01）, DNA fragmentation （P 〈 0.01）, tyrosine nitration （P 〈 0.0001） and ultrastructural damage were observed when compared to untreated control. Caspase activation was not evidenced, and although phosphatidylserine externalization increased compared to untreated control （P 〈 0.001）, this process was observed in 〈10% of the cells and the gradual loss of viability was not characterized by an important increase in this parameter. In conclusion, peroxynitrite-mediated nitrosative stress induces the regulated variant of cell death known as MPT-driven necrosis in human spermatozoa. This study provides a new insight into the pathophysiology of nitrosative stress in human spermatozoa and opens up a new focus for developing specific therapeutic strategies to better preserve sperm viability or to avoid cell death.
文摘目的: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。
文摘Testicular sperm extraction is a common procedure used to find spermatogenic cells in men with nonobstructive azoospermia. The laboratory processing of biopsied testicular tissues needs to be performed meticulously to acquire a high yield of cells. In this study, the effectiveness of mincing the tissues after testicular biopsy was assessed using histological evaluation, as was the possible adverse effect of residual tissue on the migration of spermatogenic cells during density gradient centrifugation. Our results indicate that testicular residual tissue, when laid on the density gradient medium along with the sperm wash, hinders the spermatogenic cells＇ forming a pellet during centrifugation, and therefore impairs the intracytoplasmic sperm injection procedure. Whereas the mean number of recovered cells from the sperm wash medium （SWM） with residual tissue is 39.435 ~ 24.849, it was notably higher （60.189 ~ 28.214 cells） in the SWM without minced tissues. The remaining tissue contained no functional seminiferous tubules or spermatogenic cells in histological sections. In conclusion, the remaining residual tissue after mincing biopsied testicular tissue does not add any functional or cellular contribution to spermatogenic cell retrieval; in fact, it may block the cellular elements in the accompanying cell suspension from migrating through the gradient layers to form a pellet during centrifugation and cause loss of spermatogenic cells.
基金supported by a grant from the Major Science and Technology Project of New Variety Breeding of Genetically Modified Organisms,China(2009ZX08008-004B)the National High-Tech R&D Program of China (863 Program,2008AA10Z140)the National Natural Science Foundation of China(30571339)
文摘Spermatogonial stem cells （SSCs） are a type of adult stem cell found in male mammals.These cells have the capacity for self renewal and are capable of differentiating in the niche of testis.They are also the only adult stem cells in a normal postnatal body that undergo self-renewal throughout life,transferring genetic information to the offspring.Since a technique for transplanting SSCs was first described by Brinster and his colleagues in 1994,more and more researchers have become interested in exploring the possibility of utilizing adult SSCs to generate transgenic animals.In this mini-review,we attempt to summarize the current research progress in the area of spermatogonial stem cells including the source,types and differentiation of the SSCs,and the application on transgenic animals,with a particular focus on the strategy of SSCs delivery including seminiferous tubule injection and spermatogonial stem cell transplantation.
文摘Col la I （one of the subunit of collagen type I） is a collagen, which belongs to a family of extracellular matrix （ECM） proteins that play an important role in cellular proliferation and differentiation. However, the role of Col lal in spermatogenesis, especially in the control of proliferation and differentiation of spermatogonial stem cells （SSCs）, remains unknown. In this study, we explored effects of downregulation of Collal on differentiation and proliferation of mouse spermatogonia. Loss-of-function study revealed that Oct4 and Plzf, markers of SSC self-renewal, were significantly decreased, whereas the expression of c-kit and haprin, hallmarks of SSC differentiation, was enhanced after Col la I knockdown. Cell cycle analyses indicated that two-thirds of spermatogonia were arrested in S phase after Collal knockdown. In vivo experiments, DNA injection and electroporation of the testes showed that spermatogonia self-renewal ability was impaired remarkably with the loss-of-function of Collal. Our data suggest that silencing of Collal can suppress spermatogonia self-renewal and promote spermatogonia differentiation.
