The A-type cyclins and the meiotic cell cycle in mammalian male germ cells

There are two mammalian A-type cyclins, cyclin Al and A2. While cyclin A1 is limited to male germ cells, cyclin A2 is widely expressed. Cyclin A2 promotes both Gl/S and G2/M transitions in somatic cells and cyclin A2-deficient mice are early embryonic lethal. We have shown that cyclin Al is essential for passage of spermatocytes into meiosis I (MI) by generating mice null for the cyclin A1 gene Ccna1. Both Ccna1(-/-) males and females were healthy but the males were sterile because of a cell cycle arrest before MI. This arrest was associated with desynapsis abnormalities, low M-phase promoting factor activity, and apoptosis. We have now determined that human cyclin A1 is expressed in similar stages of spermatogenesis and are exploring its role in human male infertility and whether it may be a novel target for new approaches for male contraception.     

Reproductive stem cell research and its application to urology

Abstract:   Germ cells are defined by their innate potential to transmit genetic information to the next generation through fertilization. Males produce numerous sperm for long periods to maximize chances of fertilization. Key to the continuous production of large numbers of sperm are germline stem cells and their immediate daughter cells, functioning as transit amplifying cells. Recently, it has become possible to expand germline stem cells of rodents in vitro . In addition, multipotent stem cells, which are functionally the same as embryonic stem cells, have been established from neonatal mouse testes. These stem cells derived from the testis should contribute to biological research and technologies. On the other hand, the nature of human spermatogenesis is largely unknown due to the lack of an appropriate experimental system. However, the prevailing testicular sperm extraction procedure unraveled hitherto unknown facets of human spermatogenesis. The establishment of a culturing method for human spermatogonial stem cells in hopefully the near future would be a great benefit for achieving further insight into human spermatogenesis and should lead to more sophisticated diagnostic and therapeutic clinical measures for male infertility.     

脐带间充质干细胞增强无精子症模型睾丸特异性基因表达

在生殖男科领域,探索人脐带间充质干细胞（HUCMSCs）对无精子症的治疗作用。HUCMSCs具有潜在的免疫抑制功能,并能够分泌多种细胞因子和生长因子,因此具有潜在的临床应用价值。作为探索,我们移植HUCMSCs进入无精子症小鼠睾丸间质,检测是否能够促进精子发生过程。从不同来源的脐带中分离HUCMSCs,移植进入白消安处理的小鼠睾丸间质中,采用注射生理盐水和HEK293细胞作为对照,对侧睾丸不注射。三周之后,RT-PCR检测10个生殖细胞特异性表达的基因,并检N3个特异性蛋白的表达。结果表明,注射人脐带间充质干细胞之后的表达明显高于对照组,证实生殖细胞特异性基因的表达上调,从而表明HUCMSCs对睾丸生精功能的恢复具有促进作用,为治疗无精子症探索一条新的途径。     

A matter of death and life: the significance of germ cell death during spermatogenesis

The significance of cell death occurring during spermatogenesis is a subject of interest because of its potential medical importance. Unfortunately, the field has been difficult for andrologists to penetrate, in part because of the difficulties of studying germ cells in vitro and the complexity of designing suitable models in which to dissect the molecular signalling pathways involved in control of germ cell apoptosis. As a result, the reasons for these deaths remain unclear despite considerable investigative effort. As developments which have occurred over the last few years in understanding of apoptosis can shed light on this important topic, this review focuses on what is currently known about germ cell apoptosis and outlines the emerging picture of what might be the causes and biological role of germ cell deaths in spermatogenesis.     

诱导小鼠骨髓间充质干细胞向雄性生殖细胞的定向分化

目的探讨小鼠骨髓间充质干细胞是否能够在体外被诱导发生向雄性生殖细胞方向的分化。方法从雄性小鼠骨髓中分离能够长期贴壁生长的细胞,并鉴定其是否为间充质干细胞。对分离的细胞进行生殖细胞特异性报告基因标记(stra-8-GFP)。采用视黄酸诱导标记的细胞发生向生殖细胞方向的分化。通过观察报告基因表达和生殖细胞相关基因mRNA表达情况确定是否发生了分化。结果从小鼠骨髓中分离到的贴壁生长的细胞表达间充质干细胞的表面标志CD90、CD44、CD105和Sca-1;细胞在体外可以被诱导分化为成骨、成软骨及成脂肪细胞。报告基因标记的间充质干细胞在被视黄酸诱导2d后开始表达绿色荧光蛋白和生殖细胞相关基因Mvh、Fragilis和Stella的mRNA。未经视黄酸诱导的细胞不表达绿色荧光蛋白和生殖细胞相关基因。结论小鼠骨髓间充质干细胞在体外可以被视黄酸诱导发生向雄性生殖细胞方向的分化。     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

High dose chemotherapy and stem cell support in the treatment of germ cell cancer

PURPOSE: The current status of high dose chemotherapy with autologous stem cell support in patients with germ cell cancer is reviewed. MATERIALS AND METHODS: Advanced germ cell cancer can be cured in most patients using chemotherapy with or without surgery. A small fraction of patients fail to achieve a marker remission, have residual viable carcinoma at post-chemotherapy surgery or have relapse after remission. Phase II trials suggest that autologous stem cell support is more active than standard dose chemotherapy in patients with relapse. A comprehensive literature review, focusing on trials published in the last decade, is followed by a discussion of current trials and recommendations for the use of autologous stem cell support in germ cell cancer. RESULTS: In early trials about 15% of patients with multiple relapsed and refractory disease had durable remission with high dose carboplatin and etoposide. Most regimens now add high dose cyclophosphamide or ifosfamide to carboplatin and etoposide. Together with the use of autologous stem cell support in less heavily-pretreated patients, these regimens have produced durable remissions in 40% to 50% of patients. Multivariate analyses led to the identification of prognostic factors at diagnosis and predictive factors during therapy which were associated with a low rate of durable remission. Ongoing randomized trials of autologous stem cell support early in relapse or as part of initial therapy are designed to study and validate further these prognostic factors. CONCLUSIONS: For patients with poor risk presenting features, the role of autologous stem cell support has not been proven and awaits the results of an ongoing United States intergroup trial. Patients with residual cancer at post-chemotherapy surgery may have a substantial risk of relapse despite additional cycles of the same drugs used to achieve marker remission. For select patients in this category alternatives to additional cycles of the original chemotherapy may include established second line regimens or autologous stem cell support. The role of autologous stem cell support for germ cell tumor in relapse may be challenged by the future discovery of new agents for these diseases.     

Evaluation of the effects of cryopreservation on viability,proliferation and colony formation of human spermatogonial stem cells in vitro culture

Proliferation of spermatogonial stem cells (SSCs) in vitro system is very important. It can enhance SSCs numbers for success of transplantation and treatment of infertility in cancer patients. In this study, testicular cells that obtained from azoospermia patients (n = 8) by enzymatic digestion were cryopreserved at the beginning and after 2 weeks of culture. Then, frozen‐thawed SSCs were co‐cultured on fresh Sertoli cells (experimental group 1), and frozen‐thawed Sertoli cells (experimental group 2) for another 3 weeks. In control group, fresh SSCs were co‐cultured on fresh Sertoli cells. Viability rate after enzymatic digestion was 93.4%±5.0. Frozen‐thawed testicular cells after 2 weeks of culture had a significantly (P < 0.05) higher percentage of living cells compared to frozen‐thawed testicular cells at the beginning of culture (59.2 ± 7.05 and 46.3 ± 8.40 respectively). The number of colonies in the experimental group 1 was significantly higher than experimental group 2 (19.6 ± 2.8 and 8.33 ± 1.5, respectively, P < 0.05). The diameter of the colonies in the experimental group 1 was significantly higher than control and experimental group 2 (P < 0.05) after 3 weeks of culture (269.7 ± 52.1, 204.34 ± 24.1 and 112.52 ± 23.5 μm, respectively). Cryopreservation technique will raise the possibility of banking SSCs for men who have a cancer‐related illness and waiting for radiotherapy and/or chemotherapy.     

磁性活性细胞分选技术在男性不育研究中的应用

磁性活性细胞分选法(MACS)根据细胞表面特异的标记物,在分子水平对目的细胞进行有效分选,具有简易、快速、灵活、特异性高的特点,在临床方面有着广泛的应用。MACS也为男性不育提供了一个新的研究平台,将MACS用于精液质量优化与生殖细胞分离是男性不育研究的一个新思路。本文简要介绍了MACS的基本原理,综述了MACS在精子优选、冷冻保存、精原干细胞及生精细胞分离等男性不育研究方面的应用现状和临床应用前景。     

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.     

Prenatal development of murine gonads with special reference to germ cell differentiation: a morphological and immunohistochemical study

The prenatal differentiation of male and female gonads of the mouse was investigated both morphologically and immunohistochemically. Sexual dimorphism could be detected as early as 12 days post-coitum (dpc) by the appearance of the primary elements of the tunica albuginea and positive immunoreactivity for anti-Muellerian hormone in the Sertoli cells of the male gonad. Male germ cells passed two waves of mitotic activity, a first wave between 12 and 14 dpc, which is followed by apoptosis of the old germ cell generation, and a second wave between 17 and 20 dpc. Oct-4 was expressed as a juxtanuclear ring in the cytoplasm of germ cells up to 17 dpc. Subsequently, it was down-regulated and completely disappeared in 20 dpc full-term fetuses. By contrast, M2A antigen revealed only a weak immunoreaction in some germ cells of 14 dpc gonads, but exhibited strong signals in all germ cells of 20 dpc full-term fetuses. Therefore, we postulate that, in the mouse, prenatal germ cells represent two populations: the first is immunopositive for Oct-4 and disappeared in full-term fetuses, whereas the second appeared in 14 dpc and is immunopositive for M2A antigen.     

人羊水间充质干细胞的分离鉴定及其对抗Thy-1肾炎的治疗作用

探讨羊水间充质干细胞(amniotic fluid mesenchymal stem cells,AF-MSCs)对抗Thy-1肾炎模型的治疗作用。 方法采用差异性贴壁与机械性分离法从人孕中期羊水中分离出羊水间充质干细胞,流式细胞术鉴定其表面标志物,成脂、成骨诱导分化检测其分化能力。利用尾静脉注射抗Thy-1抗体建立SD大鼠抗Thy-1系膜增生性肾炎模型;将第5代羊水间充质干细胞通过尾静脉注射治疗抗Thy-1系膜增生性肾炎大鼠,检测大鼠尿蛋白的变化,PAS染色观察大鼠肾脏病理改变。 结果羊水间充质干细胞顺利分离,流式结果显示其表达间充质干细胞标志物(CD29、CD44、CD73、CD90、CD105)和胚胎干细胞标志物(SSEA-4)而不表达造血干细胞标志物(CD34、CD45、CD133),且在适当条件下能够被诱导分化为脂肪细胞和骨细胞。羊水间充质干细胞治疗后,与模型组相比,治疗组在第7、12天时24 h尿蛋白明显下降(P<0.01),且肾脏病理结果表明系膜细胞增殖减轻,系膜区细胞外基质积聚减少。 结论成功的从人孕中期羊水中分离出羊水间充质干细胞,羊水间充质干细胞治疗系膜增生性肾小球肾炎有效。     

hBMP-2基因改良修饰骨髓间充质干细胞和人脐血间充质干细胞研究

目的研究hBMP-2基因改良修饰骨髓和人脐血间充质干细胞的方法,并比较修饰结果。 方法联合应用密度梯度离心法和贴壁培养法分离、培养骨髓和人脐血间充质干细胞,通过高效非脂质体试剂X-treme GENE介导重组hBMP-2质粒转染骨髓和人脐血间充质干细胞,倒置荧光显微镜检测荧光强度并计算转染效率,qPCR检测hBMP-2及软骨连接蛋白在两种细胞中的表达,免疫组化检测细胞中Ⅱ型胶原的变化。 结果成功分离、培养出骨髓和人脐血间充质干细胞,两种细胞具有不同的生长特性。高效非脂质体试剂介导的重组hBMP-2质粒成功转染骨髓和人脐血间充质干细胞,转染骨髓间充质干细胞效率[（18.44±5.94）%]低于人脐血间充质干细胞[（27.74±7.59）%],且差异有统计学意义（t=3.027,P<0.05）。转染后的两种细胞均检测到hBMP-2、软骨连接蛋白及Ⅱ型胶原的表达。 结论hBMP-2基因可以有效改良修饰骨髓和人脐血间充质干细胞,且能促进其向软骨细胞方向分化。     

皮肤扩张后表皮干细胞异位现象初步研究

目的 了解皮肤软组织扩张术后表皮干细胞(ESC)的分化和分布情况,初步探讨扩张皮肤组织的相关生长机制. 方法 取15例行Ⅱ期头、颈部皮肤扩张术患者扩张后(平均注水期45d)皮肤标本及正常皮肤标本,按取材部位分为:(1)头部近扩张器中心组:取与扩张器中轴线垂直距离为3cm处的扩张后头皮;(2)头部扩张器侧壁组:取与扩张器中轴线垂直距离为5～7 cm的扩张后头皮;(3)颈部扩张皮肤组;(4)未扩张头皮对照组;(5)未扩张颈部皮肤对照组.各组皮肤标本行HE染色观察组织结构,行免疫组织化学染色观察细胞角蛋白19(CK19)阳性细胞的分化及分布特征. 结果 与2个末扩张对照组比较,HE染色可见各扩张组表皮层凹凸不平且相对增厚,皱褶明显,细胞层次增多;细胞呈密集分布,以靠近基底层最为显著,但排列欠整齐,极性过度不明显.免疫组织化学染色显示,各扩张组基底层CK19阳性细胞的连续性基本存在,基底层个别部位阳性细胞明显增多,呈复层排列;基底层之外亦有少量成团或散在分布的CK19阳性细胞.2个未扩张对照组未见上述现象. 结论 J 皮肤软组织扩张后,ESC 在修复过程中增殖和分化加强,并出现异化分布.     

The platelet-rich plasma and mesenchymal stem cell milieu: A review of therapeutic effects on bone healing

Platelet-rich plasma is autologous plasma that contains concentrated platelets compared to whole blood. It is relatively inexpensive to produce, can be easily isolated from whole blood, and can be administered while the patient is in the operating room. Further, because platelet-rich plasma is an autologous therapy, there is minimal risk for adverse reactions to the patient. Platelet-rich plasma has been used to promote bone regeneration due to its abundance of concentrated growth factors that are essential to wound healing. In this review, we summarize the methods for producing platelet-rich plasma and the history of its use in bone regeneration. We also summarize the growth factor profiles derived from platelet-rich plasma, with emphasis on those factors that play a direct role in promoting bone repair within the local fracture environment. In addition, we discuss the potential advantages of combining platelet-rich plasma with mesenchymal stem cells, a multipotent cell type often obtained from bone marrow or fat, to improve craniofacial and long bone regeneration. We detail what is currently known about how platelet-rich plasma influences mesenchymal stem cells in vitro, and then highlight the clinical outcomes of administering platelet-rich plasma and mesenchymal stem cells as a combination therapy to promote bone regeneration in vivo.