Production of Transgenic Mice by Type-A Spermatogonia-Mediated Gene Transfer

Type-A spermatogonia first appear at between 3-7 d postnatally in mice and are the only immortalized diploid cells that reproduce in adulthood in these animals. In our current study, we explored the feasibility of producing stable transgenic mice using these cells. Enhanced pEGFP-N1 plasmids were suspended in ExGen500 transfection reagent and injected at different angles into the testes of 7-d-old male ICR mice. The resulting type-A spermatogonia-mediated gene transfer （TASMGT） mice were then mated with normal females at different stages of sexual maturity （6, 12, and 24 wk）. The integration and expression of the introduced EGFP gene was evaluated in the F1 transgenic offspring by PCR and Southern blotting analysis. The foreign gene integration rates for a low-dose group （15 μL gene suspension injected into each testis） and a high-dose group （30 μL suspensions injected） at the three stages of female sexual maturity tested were 11.76% （2/17）, 14.29% （3/21）, and 11.11% （2/18）, and 5% （1/20）, 5.56% （1/18）, and 0 （0/17）, respectively. The average integration rates for these two dose groups were 12.5% （7/56） and 3.64% （2/55）, respectively, which was a significant difference （P0.05）. Semi-quantitative RT-PCR analysis further showed that the introduced GFP gene was expressed in 3/9 integration mice. In addition, GFP expression was observed in the sperm cells from the TASMGT mice, and also in the embryos and F2 pups from the F1 generation transgenic mice. Hence, although the foreign gene integration rate for TASMGT is not high and the transgenic offspring show as yet unexplained defects, our results indicate that this method is a potentially feasible and reproducible new approach to creating transgenic mice.

Downregulation of Col lal induces differentiation in mouse spermatogonia

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.

Gene expression dynamics during the gonocyte to spermatogonia transition and spermatogenesis in the domestic yak

Background:Spermatogenesis is a cellular differentiation process that includes three major events:mitosis of spermatogonia,meiosis of spermatocytes and spermiogenesis.Steady-state spermatogenesis relies on functions of spermatogonial stem cells(SSCs).Establishing and maintaining a foundational SSC pool is essential for continued spermatogenesis in mammals.Currently,our knowledge about SSC and spermatogenesis is severely limited in domestic animals.Results:In the present study,we examined transcriptomes of testes from domestic yaks at four different stages(3,5,8 and 24 months of age)and attempted to identify genes that are associated with key developmental events of spermatogenesis.Histological analyses showed that the most advanced germ cells within seminiferous tubules of testes from 3,5,8 and 24 months old yaks were gonocytes,spermatogonia,spermatocytes and elongated spermatids,respectively.RNA-sequencing(RNA-seq)analyses revealed that 11904,4381 and 2459 genes were differentially expressed during the gonocyte to spermatogonia transition,the mitosis to meiosis transition and the meiosis to post-meiosis transition.Further analyses identified a list of candidate genes than may regulate these important cellular processes.CXCR4,a previously identified SSC niche factor in mouse,was one of the up-regulated genes in the 5 months old yak testis.Results of immunohistochemical staining confirmed that CXCR4 was exclusively expressed in gonocytes and a subpopulation of spermatogonia in the yak testis.Conclusions:Together,these findings demonstrated histological changes of postnatal testis development in the domestic yak.During development of spermatogonial lineage,meiotic and haploid germ cells are supported by dynamic transcriptional regulation of gene expression.Our transcriptomic analyses provided a list of candidate genes that potentially play crucial roles in directing the establishment of SSC and spermatogenesis in yak.

Sertoli cell and spermatogonial development in pigs

Background:Spermatogenesis is an intricate developmental process during which undifferentiated spermatogonia,containing spermatogonial stem cells(SSCs),undergo self-renewal and differentiation to generate eventually mature spermatozoa.Spermatogenesis occurs in seminiferous tubules within the testis,and the seminiferous tubules harbor Sertoli and germ cells.Sertoli cells are an essential somatic cell type within the microenvironment that support and steer male germ cell development,whereas spermatogonia are the primitive male germ cells at the onset of spermatogenesis.While the developmental progression of Sertoli cells and spermatogonia has been well established in mice,much less is known in other mammalian species including pigs.Results:To acquire knowledge of Sertoli cell and spermatogonial development in pigs,here we collected as many as nine ages of Duroc porcine testes from the neonate to sexual maturity,i.e.,testes from 7-,30-,50-,70-,90-,110-,130-,150-and 210-day-old boars,and performed histological and immunohistochemical analyses on testis sections.We first examined the development of spermatogenic cells and seminiferous tubules in porcine testes.Then,by immunofluorescence staining for marker proteins(AMH,SOX9,DBA,UCHL1,VASA,KIT,Ki67 and/or PCNA),we delved into the proliferative activity and development of Sertoli cells and of spermatogonial subtypes(pro-,undifferentiated and differentiating spermatogonia).Besides,by immunostaining forβ-catenin and ZO-1,we studied the establishment of the blood-testis barrier in porcine testes.Conclusions:In this longitudinal study,we have systematically investigated the elaborate Sertoli cell and spermatogonial developmental patterns in pigs from the neonate to sexual maturity that have so far remained largely unknown.The findings not only extend the knowledge about spermatogenesis and testicular development in pigs,but also lay the theoretical groundwork for porcine breeding and rearing.

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice

Aim： To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. Methods： Mice were surgically rendered cryptorchid, then treated with different doses of 17β- estradiol （E2） s.c. once a day. Mice were killed at sexual maturity （45 days of age）, and histological analysis and inununofluorescence were performed. Serum follicle stimulating hormone （FSH）, estradiol, testosterone and luteinizing hormone （LH） were measured. Results： Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia. Conclusion： E2 has a dose-related mitogenic effect on spermatogonia.

Isolation of Subtype Spermatogonia in Juvenile Rats

The present study was aimed at finding an effective method to isolate and purify the subtype of type A spermatogonial stem cells （SSCs） in juvenile rats. Testes from 9-days-old rats were used to isolate germ cells by using two-step enzymatic digestion. The expression of c-kit in the testes of the rats was immunohistochemically detected. After isolation, cell suspension was enriched further by discontinuous density gradient centrifugation. Then type A1-A4 spermatogonia was isolated from the purified spermatogonia with c-kit as the marker by using fluorescence-activated cell sorting （FACS）. Electron microscopy was used to observe their ultrastructure. Finally, highly purified and viable subtype of SSCs was obtained. Cells separation with discontinuous density gradient centrifugation significantly increased the concentration of c-kit positive cells [（18.65±1.69）% after the centrifugation versus （3.16±0.84）% before the centrifugation, P〈0.01]. Furthermore, the recovery and viability were also high [（65.9±1.24）% and （85.6±1.14）%]. It is concluded that FACS with c-kit as the marker in combination with discontinuous density gradient centrifugation can well enrich type A1-A4 spermatogonia from the testes of 9-days-old rats.

Production of functional sperm from in vitro-cultured premeiotic spermatogonia in a marine fish

In vitro production of functional gametes can revolutionize reproduction by reducing generation intervals and accelerating genetic breeding in aquaculture,especially in fish with relatively long generations.Nevertheless,functional sperm production from in vitro-cultured spermatogonia remains a challenge in most aquaculture fish.In this study,we isolated and characterized premeiotic spermatogonia from marine four-eyed sleepers(Bostrychus sinensis),which are prone to ovotesticular or sterile testicular development,and induced the differentiation of the spermatogonia into flagellated sperm in a three-dimensional(3D)culture system.Artificial insemination indicated that the in vitro-derived sperm were capable of fertilizing mature oocytes to develop into normal larvae.Furthermore,melatonin significantly promoted spermatogonia proliferation and differentiation through the ERK1/2 signaling pathway,and thus increased the efficiency in functional sperm production.The 3D culture system and resulting functional sperm hold great promise for improving the genetic breeding of aquaculture fish.

Ionizing Radiation-Induced RPL23a Reduction Regulates Apoptosis via RPL11-MDM2-p53 Pathway in Mouse Spermatogonia

Objective The expression patterns of ribosomal large subunit protein 23 a(RPL23 a)in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23 a expression and spermatogonia apoptosis upon exposure to X-ray.Methods Male mice and GC-1 cells were irradiated with X-ray,terminal dUTP nick end-labelling(TUNEL)was performed to detect apoptotic spermatogonia in vivo.Apoptotic rate and cell cycle phase of GC-1 cells were analyzed with flow cytometry.Protein interactions were detected by Immunoprecipitation and protein localization as studied by immunofluorescence.Immunoblotting and real-time PCR were applied to analyze to protein and gene expression.Results Ionizing radiation(IR)increased spermatogonia apoptosis,the expression of RPL11,MDM2 and p53,and decreased RPL23 a expression in mice spermatogonia in vivo and in vitro.RPL23 a knockdown weakened the interaction between RPL23 a and RPL11,leading to p53 accumulation.Moreover,knockdown and IR decreased RPL23 a that induces spermatogonia apoptosis via RPL23 a-RPL11-MDM2-p53 pathway in GC-1 cells.Conclusion These results suggested that IR reduced RPL23 a expression,leading to weakened the RPL23 a-RPL11 interactions,which may have activated p53,resulting in spermatogonia apoptosis.These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility.The graphical abstract was available in the web of www.besjournal.com.

scRNA-seq reveals that origin recognition complex subunit 6 regulates mouse spermatogonial cell proliferation and apoptosis via activation of Wnt/β-catenin signaling

The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis.The single-cell RNA sequencing(scRNA-seq)analysis of the testis was performed to identify genes upregulated in spermatogonia.Using scRNAseq analysis,we identified the spermatogonia upregulated gene origin recognition complex subunit 6(Orc6),which is involved in DNA replication and cell cycle regulation;its protein expression in the human and mouse testis was detected by western blot and immunofluorescence.To explore the potential function of Orc6 in spermatogonia,the C18-4 cell line was transfected with control or Orc6 siRNA.Subsequently,5-ethynyl-2-deoxyuridine(EdU)and terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assays,flow cytometry,and western blot were used to evaluate its effects on proliferation and apoptosis.It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells.Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated(Wnt)/β-catenin signaling.Western blot revealed that the expression ofβ-catenin protein and its phosphorylation(Ser675)were significantly decreased when silencing the expression of ORC6.Our findings indicated that Orc6 was upregulated in spermatogonia,whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.

Ginsenosides stimulated the proliferation of mouse spermatogonia involving activation of protein kinase C

The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0～10 μg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10-8 to 10-7 mol/L and the PKC inhibitor H7 inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H7. These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.

PLZF^posc-KIT^pos-delineated A1-A4-differentiating spermatogonia by subset and stage detection upon Bouin fixation

While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2–A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.

Mechanistic target of rapamycin kinase(Mtor)is required for spermatogonial proliferation and differentiation in mice

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility.However,the exact mechanisms underlying the behavior of spermatogonia,including spermatogonial stem cell(SSC)self-renewal and spermatogonial proliferation and differentiation,are not fully understood.Recent studies demonstrated that the mTOR complex 1(mTORC1)signaling pathway plays a crucial role in spermatogonial development,but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined.In this study,we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia.The Mtor knockout(KO)mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age.These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre.Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice,suggesting that spermatogonial differentiation was inhibited.Spermatogonial proliferation was also impaired in Mtor KO mice,leading to a diminished spermatogonial pool and total germ cell population.Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.

Successful transplantation of cryopreserved spermatogonia in Sebastes schlegelii:A simple and suitable alternative approach for conservation of viviparous fish

Black rockfish(Sebastes schlegelii)is one of the most important marine economic viviparous fishes.Recently,germplasm degradation and genetic diversity reduction have occurred due to overfishing and long-term artificial breeding.Germ cell transplantation combined with cryopreservation may be an alternative way to protect genetic resources.However,in viviparous fish that undertake fertilization and embryo development in vivo,transplantation is more difficult than in oviparous fish,including selection of transplantation stage,isolation of germ stem cells,and preparation of sterile recipients.This seriously restricts the development of viviparous transplantation.Therefore,in this study,we aimed to explore a transplantation method suitable for these species.Donor cells were isolated from cryopreserved whole testes of 300–400​g male Sebastes schlegelii in May,labeled by PKH26,and intra-peritoneally transplanted into allogeneic larvae at 5–10 days post-birth.Subsequently,the development of donor-derived cells in recipients were continuously detected by fluorescence labeling,histology,microsatellite markers,and fecundity tests.The results showed that donors were rich in spermatogonia(75%)and recipients maintained a high survival rate after transplantation,with a rate of>20%at sexual maturity.Further,donor-derived cells successfully migrated(100%),colonized,and incorporated into the developing recipient gonad(93.33%).Finally,transplanted recipients could normally develop and differentiate into male and female individuals,with donor-derived gametes found in 65.38%of mature recipients.In the present study,we first establish a simple and suitable transplantation method for Sebastes schlegelii using immature males and specific larvae,which will serve as a promising tool in the protection of germplasm resources for this transplantation-restricted marine viviparous species.

PLZF的克隆及其对犏牛未分化精原细胞的增殖作用

【目的】犏牛作为牦牛与黄牛的种间杂交产物,具有优良的生产性能,但其杂种优势的进一步应用却受限于犏牛雄性不育。通过克隆犏牛PLZF,明确其在犏牛和牦牛睾丸组织和未分化精原细胞中的差异表达,并进一步揭示过表达该基因对犏牛未分化精原细胞活性的影响。为阐明犏牛生精停滞的作用机制提供理论基础。【方法】以24月龄公麦洼牦牛和F1代公犏牛为实验动物,通过RT-PCR法克隆得到了犏牛PLZF的CDS序列,并进行了生物信息学分析;通过RT-qPCR法分析PLZF在犏牛和牦牛睾丸组织中的差异表达;采用同源重组的方法构建了PLZF的表达载体,并利用RT-qPCR检测了PLZF过表达效率及其下游靶基因的表达;通过PDT、CCK-8、EdU和免疫荧光检测了过表达PLZF对犏牛未分化精原细胞增殖活性的影响。【结果】克隆获得了犏牛PLZF的CDS区,并通过生物信息学分析发现该基因编码的蛋白序列不包含跨膜结构域和信号肽序列,其三级结构以α螺旋和无规卷曲为主。系统进化树分析表明犏牛PLZF与黄牛PLZF的亲缘关系更近。三级结构预测发现,虽然犏牛、牦牛和黄牛的PLZF蛋白三级结构高度相似,但牦牛PLZF蛋白在531—540位氨基酸处与犏牛和黄牛有较大差异。RT-qPCR发现,PLZF在犏牛睾丸组织和未分化精原细胞中的表达均显著低于牦牛(P<0.05),而在犏牛未分化精原细胞中过表达PLZF后,该基因的表达上调了13.8倍(P<0.01),且能显著增加犏牛未分化精原细胞的增殖活性(P<0.05),表明PLZF表达下调影响了犏牛未分化精原细胞增殖活性。此外,过表达PLZF后,犏牛未分化精原细胞中与增殖相关的基因(Etv5、Bcl6b、Pcna和c-fos)全部显著上调(P<0.05),与分化相关的基因(Stra8、Kit、Dmrt1和Sohlh2)全部显著下调(P<0.05),表明PLZF通过上调增殖相关基因、下调分化相关基因的表达促进犏牛未分化精原细胞增殖。【结论】PLZF在犏牛未分化精原细胞中表达异常降低了犏牛未分化精原细胞增殖活性,导致其数量减少,影响了犏牛的精子发生。本试验为进一步阐明犏牛生精停滞的作用机制提供了理论基础,并为解决犏牛雄性不育问题提供了新的思路。

三氯异氰尿酸通过诱导氧化应激和铁死亡抑制精原细胞增殖

目的探索三氯异氰尿酸(TCCA)对小鼠睾丸精原细胞GC-1氧化应激和DNA甲基化的影响。方法以TCCA 0(细胞对照),97,194和387μmol·L^(-1)处理GC-1细胞24 h;CCK-8法检测细胞存活率;Hoechst 33342染色检测各组细胞形态和凋亡率;流式法检测细胞周期;实时荧光定量PCR(RT-qPCR)检测细胞凋亡相关基因Bax和Fas、氧化应激相关基因线粒体超氧化物歧化酶(SOD2)、铁死亡相关基因谷胱甘肽过氧化酶4(GPX4)、核因子红细胞2相关因子2(Nrf2)、溶质载体家族7成员11(SLC7A11)、二氢乳清酸脱氢酶(DHODH)、DNA甲基化转移酶3A(DNMT3A)和DNA甲基转移酶3L(DNMT3L)mRNA表达水平;Griess法测定GC-1细胞一氧化氮(NO)含量,比色法测定丙二醛(MDA)水平;DCFH-DA荧光探针法测定GC-1细胞活性氧(ROS)水平,DNTB比色法测定还原型谷胱甘肽(GSH)含量,WST-8法测定还原型辅酶Ⅱ(NADPH)含量。结果与细胞对照组相比,TCCA 194和387μmol·L^(-1)组细胞存活率降低(P<0.01),出现细胞核固缩及核碎裂,细胞凋亡率显著增加(P<0.01),细胞被阻滞在G2/M期(P<0.05);TCCA 387μmol·L^(-1)组细胞NO和MDA含量增加(P<0.01),ROS水平升高(P<0.01),GSH和NADPH含量降低(P<0.01),SOD2,GPX4,Nrf2,SLC7A11和DHODH mRNA表达减少(P<0.05,P<0.01);Bax,Fas,DNMT3L和DNMT3A mRNA表达增加(P<0.05,P<0.01)。结论TCCA可降低GC-1细胞存活率,抑制细胞增殖,引起细胞凋亡。其机制可能与其破坏抗氧化系统、增强氧化应激、诱导铁死亡及干扰GC-1细胞甲基化有关。

一种在体标记小鼠精原细胞新生蛋白合成的方法

目的 哺乳动物精子发生是一种由一系列生精细胞组成的连续细胞增殖和分化并最终产生精子的过程。然而,生精细胞的蛋白稳态分析仍缺乏有效手段。本研究的目的是探索一种在体标记并检测生精细胞新生蛋白合成的方法。方法 利用氨基酰tRNAs结构类似物——嘌呤霉素(Puromycin,Puro)以65 mg/kg小鼠体质量的剂量腹腔注射小鼠,1.5 h后,处死小鼠,分离两侧睾丸,其中一侧睾丸用于总蛋白提取,另一侧睾丸用于石蜡切片制备,随后利用Puro特异性抗体分别通过Western blot和免疫荧光检测样品中Puro的含量及定位。结果 Western blot结果显示Puro能够标记总体新生蛋白合成情况;免疫荧光共定位显示Puro在小鼠睾丸中特异性标记的是精原细胞。结论 Puro能够用于小鼠精原细胞新生蛋白质的在体标记,并可通过相应免疫学技术定量分析新生蛋白合成水平。

长白猪精原细胞的分离和纯化

目的 分离和纯化猪的精原细胞 ,从而对与人类具有高同源性的猪的精子发生进行研究。 方法 酶消化法制备 2月龄未成熟长白猪睾丸组织的单细胞悬液 ,以 2 %～ 4 %牛血清白蛋白 (BSA)连续梯度作为分离介质 ,采用重力沉降法并结合细胞贴壁培养的方法分离精原细胞。 结果 重力沉降法分离细胞后所获精原细胞纯度为 91% ,进一步经过贴壁培养纯化后 ,精原细胞纯度达到 94 2 %。 结论 该方法方便、快捷 ,分离效果好 ,能满足在分子水平研究精原细胞的需要。

体内精原干细胞转染法建立转基因小鼠

将人Bcl-2 cDNA与小鼠乳清酸蛋白(WAP)5’上游调控序列融合后,与脂质体按一定比例混合,再加入适量的台盼蓝制成转染液,注入到小鼠睾丸中的曲细精管中,转染精原干细胞以探讨建立转基因小鼠的可行性。共注射了3只公鼠,4天后将公鼠与发情母鼠合笼交配,共生仔鼠20只。检测结果表明,有3只呈PCR阳性,Southern blot检测,阳性鼠2只,1只公鼠,1只母鼠,其中,公鼠意外死亡;Western blot证实,1只母鼠的乳腺组织表达了Bcl-2蛋白,其F1代的16只小鼠中,有7只呈PCR阳性。证实了体内精原干细胞转染建立转基因动物的可行性。

中草药远志对实验性小鼠雄性生殖细胞遗传物质损伤的保护作用

目的 :观察中草药远志对小鼠雄性生殖细胞遗传物质损伤的保护作用。方法 :采用小鼠精原细胞姐妹染色单体互换实验。结果 :铅能诱发小鼠精原细胞姐妹染色单体互换 ,而在腹腔注射铅的同时给予远志 ,可使铅诱发的小鼠精原细胞姐妹染色单体互换频率明显降低。结论 :远志对雄性生殖细胞遗传物质具有保护作用 。

新生小鼠精原细胞分离和纯化的实验研究

目的:分离和纯化7～8d新生雄性小鼠精原细胞,为深入研究生精机理及其影响因素提供细胞来源和技术支持。方法:采用组合酶消化法制备7～8d雄性小鼠的生精细胞悬液;Percoll密度梯度离心法分离精原细胞,贴壁培养法进一步纯化精原细胞;滴片进行碱性磷酸酶染色;取同时间段雄性小鼠睾丸组织冰冻切片进行碱性磷酸酶染色。结果:精原细胞主要分布于45%～55%梯度间的Percoll中,经贴壁培养后纯度达75.2%。结论:用上述方法成功地分离和纯化了小鼠精原细胞。