Mahoganoid基因在小鼠精子发生中的动态表达及对雄鼠生殖力影响

目的探讨Mahoganoid（Md）基因在小鼠精子第一发生波中的动态表达以及其基因缺失对雄性小鼠生殖力的影响。方法采用荧光实时定量聚合酶链反应（Real-timePCR）相对定量法检测MahoganoidmRNA在7、14、21、28、35、60d龄C3H／HeJ雄鼠睾丸中的表达;对Md基因缺失纯合子（Md^nc）及C3H／HeJ雄鼠精子密度,精子活力,异常精子形态百分率分析,并通过交配实验观察其使正常雌鼠受孕分娩情况。结果MahoganoidmRNA在7d龄C3H／HeJ雄鼠睾丸有低表达,14d表达开始上调,21d上升趋势明显,35d时表达水平为60d91％;Md^nc可以与雌鼠交配,但无幼鼠出生,Md^nc雄鼠精子快速前向运动百分比明显低于C3H／HeJ雄鼠精子（P〈0．01）。结论Mahoganoid基因与精子发生相关,Mahoganoid基因缺失会导致雄性小鼠精子活力下降,影响其生殖能力。     

一种Parkin基因敲除小鼠肾虚证动物模型的建立

目的:构建Parkin基因敲除(knock out, KO)小鼠肾虚证动物模型。方法:本研究设置对照组、Parkin KO组、氢化可的松组,观察并记录小鼠一般情况,完善行为学(旷场实验、轮替实验和新臂探索实验)、检测血清指标(生长激素水平、促肾上腺皮质激素、皮质醇、TSH、T3、T4、雌二醇、睾酮),分析各组小鼠临床表现和相关血清指标与肾虚症是否相符。两组间检测均数的比较采用t检验,多组间比较采用单因素方差分析。结果:Parkin KO组、氢化可的松组表现为拱背蜷缩、反应迟钝、自主活动减少、倦怠、精神萎靡、饮食减少、畏寒喜暖等,与肾虚症候相符,KO组表现更加明显。KO组生长激素水平较对照组明显下降(P0.01)。旷场实验中,KO组直立探索次数、总探索距离、运动速度明显下降(P0.01),氢化可的松组直立探索次数明显下降(P0.01);自发轮替实验中KO组总的进臂次数显著降低(P0.01),氢化可的松组小鼠总的进臂次数也明显降低,KO组下降更加明显;新臂探索实验中,KO组新臂探索次数及总进臂次数明显降低(P0.05),新臂停留时间显著降低(P0.01);上述指标提示KO组、氢化可的松组小鼠对外界事物的自发探索能力、自发活动能力空间学习记忆能力、工作记忆能力显著降低,KO组下降更加明显。与对照组比较,KO组雌鼠雌二醇(E2)水平(P0.05)、E2/T水平(P0.01)、产仔量(P0.01)均显著下降,氢化可的松组雌鼠产仔量明显下降(P0.05),KO组雄鼠血清睾酮(T)水平明显下降(P0.01),血清E2水平(P0.05)、E2/T水平(P0.01)显著升高,提示KO组、氢化可的松组性腺功能降低,生殖能力下降,且KO组下降更加明显。KO组促肾上腺皮质激素、TSH、T3、T4显著降低(P0.01),皮质醇也明显降低(P0.05),氢化可的松组促肾上腺皮质激素、皮质醇、TSH、T4均明显下降(P0.05),KO组下降更明显。与对照组比较,KO组小鼠肾组织Parkin无表达,氢化可的松组Parkin表达呈下降趋势。结论:Parkin基因敲除鼠可作为一种理想的肾虚证动物模型。     

Clock基因对精子顶体酶的影响

目的研究雄鼠睾丸Clock基因沉默后,小鼠精子顶体酶的活性及功能。方法在ICR小鼠睾丸内注射Clock干扰质粒干扰雄性小鼠睾丸节律基因Clock的表达,研究:(1)进行体外受精,观察精子驱散卵丘颗粒细胞情况及受精率等;(2)检测精子顶体内顶体蛋白酶、透明质酸酶活性及芳香基硫酸酯酶A(ASA)的含量。(3)将精子注射入卵母细胞,观察受精卵及胚胎发育情况。结果睾丸Clock基因沉默后,精子驱散卵丘颗粒细胞时间延长,受精率下降;精子顶体蛋白酶活性下降;透明质酸酶活性和ASA含量无明显变化;干扰组精子对卵母细胞及胚胎发育的影响小于未干扰组(P0.05)。结论干扰睾丸Clock基因表达后,影响精子顶体蛋白酶活性,从而影响雄性小鼠的生殖功能。     

肾阳虚与肾阴虚模型小鼠睾丸生殖相关差异蛋白的比较研究

目的:比较氢化可的松诱导的肾阳虚和肾阴虚模型小鼠睾丸表达的差异蛋白,探讨两种肾虚导致雄性不育共同和特异的物质基础。方法:将30只昆明小鼠随机分为对照组、肾阳虚组和肾阴虚组,对照组:腹腔注射生理盐水0.2 ml,1次/d,连续7 d;肾阳虚组:腹腔注射氢化可的松25 mg/(kg·d),连续10 d;肾阴虚组:腹腔注射氢化可的松50 mg/(kg·d),连续7 d。HE染色分析睾丸组织病理变化,同位素标记相对和绝对定量(iTRAQ)技术及生物信息学方法比较两种肾虚小鼠睾丸表达的差异蛋白。结果:与对照组相比,Sod1为两种肾虚小鼠睾丸表达的生殖相关节点差异蛋白,且在肾阴虚组表达显著高于肾阳虚组(P0.05);两种肾虚小鼠睾丸共同表达的生殖相关节点蛋白有5个,上调的为Rps28,下调的为Rpl11、Rplp2、Svs2和Svs3a(P0.05)。结论:Sod1可能是区别两种肾虚不育小鼠的关键物质基础之一,Rps28、Rpl11、Rplp2、Svs2和Svs3a则可能是两种肾虚不育小鼠共同的重要的物质基础。     

蛋白酶体REGγ对雄性小鼠生精功能的影响

目的研究蛋白酶体REGγ对雄性小鼠生精功能的影响。方法利用免疫蛋白印迹检测小鼠睾丸中REGγ的表达;通过组合酶消化法分离精原干细胞,流式细胞仪检测精原干细胞中c-kit及α6-integrin的表达;通过小鼠精子分析仪检测REGγ基因敲除雄鼠的精子浓度和精子活力;进行小鼠合笼实验检测正常雌鼠和REGγ基因敲除雄鼠合笼后的生育力。结果 REGγ在小鼠睾丸中高表达;应用组合酶消化法得到了纯度70%的精原干细胞;REGγ基因敲除雄鼠精原干细胞的c-kit及α6-integrin表达量均显著低于野生型组(P0.05);REGγ基因敲除型雄鼠的平均精子浓度(37.1×106/ml)和精子活力(54%)均显著低于野生型雄鼠(75.4×106/ml、74%)(P0.05);REGγ基因敲除型雄鼠与野生型雌鼠合笼后的产仔数均低于野生型雄鼠(P0.05)。结论蛋白酶体REGγ参与调节雄鼠的生精功能。     

微波辐射对雄性小鼠生殖系统的影响

目的观察微波辐射对雄性小鼠生殖系统的影响,并初步探讨其作用机制。方法将48只雄性昆明小鼠随机分成微波辐射低、中、高强度组(微波辐射功率密度分别为5、10、15mW/cm~2强度)和对照组,对小鼠辐射1h/d,连续30d。微波辐射结束后进行小鼠性行为能力的观察扑捉潜伏期(capture incubation period,CIP)、扑捉次数(capture times,CT)、精子相对计数、精子畸形数、超氧化物歧化酶(SOD)和丙二醛(MDA)等指标的检测。结果 15mW/cm~2强度辐射后15d小鼠的扑捉潜伏期显著延长、扑捉次数显著减少(P0.05),10mW/cm~2强度辐射后30d小鼠的扑捉潜伏期显著延长、扑捉次数显著减少(P0.05);精子相对计数明显减少(P0.05);精子畸形率明显增加(P0.05);血清和睾丸组织中SOD的活性显著降低(P0.05);血清和睾丸组织中MDA的含量显著增加(P0.05);5mW/cm~2强度辐射组上述各项指标与对照组相比均未见明显变化。结论低功率微波辐射可对雄性小鼠生殖系统产生影响,其作用机制可能与生殖细胞的氧化损伤有关。     

醋酸铅对雄性小鼠生殖功能的毒性作用

目的　探讨醋酸铅对雄性小鼠生殖功能的毒性作用。　方法　不同浓度醋酸铅 ( 1 .5、6及 2 4mg/kg)腹腔注射 4周龄雄性小鼠 ,共 1 0次。 50d后与正常雌鼠合笼交配 ( 1∶2 ) ,观察雌鼠受孕率、胚胎总数、异常胚胎数 (率 )、胎鼠重量 ;测定睾丸指数、附睾精子数量、精子活动率和精子畸形率。　结果　对照组、醋酸铅各组合笼 2 1d,雌鼠受孕率和胚胎总数无显著差异 (P >0 .0 5)。醋酸铅 2 4mg/kg组异常胚胎数 (率 )显著高于对照组和 1 .5mg/kg组 (P <0 .0 5) ,胎鼠重量和雄鼠睾丸指数显著低于对照组 (P <0 .0 5)。其他实验组异常胚胎率、胎鼠重量和雄鼠睾丸指数与对照组比较无显著性差异。各实验组附睾精子数显著低于对照组 (P <0 .0 5,P <0 .0 0 5) ,附睾精子畸形率显著高于对照组 (P<0 .0 5,P <0 .0 0 5) ,中、高浓度醋酸铅组附睾精子活动率显著低于对照组 (P <0 .0 5)。　结论　醋酸铅 2 4mg/kg处理雄鼠对睾丸、精子数量和质量、合笼雌鼠胚胎产生影响。 1 .5及 6mg/kg只影响精子数量和质量而不影响生殖功能和子代。合笼雌鼠异常胚胎率增高和胎鼠重量下降可能与醋酸铅引起精子质量下降有关     

淫羊藿苷与睾酮治疗亚急性衰老雄性大鼠的实验研究

目的观察淫羊藿苷及睾酮对D-半乳糖所致亚急性衰老大鼠血清SOD活性、T、E2含量,睾丸组织P16蛋白表达与细胞凋亡的影响。方法随机将40只SD成年雄性大鼠分为正常对照组、模型组、淫羊藿组、睾酮组。检测各组大鼠血清SOD活性、T、E2含量,HE染色观察睾丸组织变化,SP法观察睾丸组织P16蛋白表达情况,TUNEL法检测睾丸生殖细胞凋亡情况。结果D-半乳糖致亚急性衰老大鼠血清SOD活性、T含量下降,与正常组比较差异显著(P<0．01,P<0．01);各组E2变化无统计学意义。睾丸组织出现退行性变化,睾丸生殖细胞P16阳性细胞百分率(PI)和凋亡指数增加,较正常组差异显著(P<0．01);淫羊藿组和睾酮组SOD活性增加,T水平升高,生殖细胞凋亡指数下降,较模型组差异显著,睾丸组织的退行性变化明显改善。淫羊藿组生殖细胞P16阳性细胞百分率较模型组差异显著(P<0．01),而睾酮组变化无显著意义。结论与睾酮相比,淫羊藿不仅可以提高亚急性衰老雄性大鼠血清SOD活性和雄激素水平,减少生殖细胞凋亡,改善睾丸组织的退行性变化,还可通过抑制生殖细胞衰老基因P16蛋白表达这一途径延缓性腺衰老。     

杜仲补天素胶囊对雄性小鼠生育力的影响

目的:探讨杜仲补天素胶囊对雄性小鼠生育力的影响。方法:SPF级4周龄雄性昆明种小鼠(体重14～16 g)随机分为对照组,生精胶囊组[0.8 g/(kg·d)],杜仲补天素胶囊低[0.694 g/(kg·d)]、高剂量[1.388 g/(kg·d)]组,每组12只。雄鼠灌胃给药1周后将小鼠尾根部负重系重约2 g的铁片(体质量5%～10%)进行适应性游泳练习,给药2周后记录小鼠力竭游泳时间,力竭判断标准按照Dawson法。灌胃给药3周,给药2周后雌雄2∶1合笼1周,合笼期间雄鼠继续给药,合笼结束后雄鼠取材检测小鼠精子数量和活力,计算各组脏器系数,HE染色观察睾丸、附睾形态学变化,ELISA法检测各组小鼠血清E2、LH、FSH、T水平,免疫组化法检测各组小鼠睾丸雄激素受体(AR)表达情况,检测血清中谷草转氨酶(AST),谷丙转氨酶(ALT),肌酐(Cr)、尿素氮(BUN)水平。合笼1周后处死雌鼠,统计受孕情况。结果:与对照组受孕率54%相比,杜仲补天素胶囊低剂量组(受孕率70%)、高剂量组(受孕率75%)均能提高雌鼠受孕率。与对照组小鼠负重游泳时间[(173±17) s]相比,杜仲补天素胶囊低剂量组能够显著增加小鼠负重游泳时间[(394±51) s,P<0.01],高剂量组[(266±42) s]虽也有提高,但无显著差异;与对照组精原细胞数量(25.7±5.3)、精母细胞数量(92.5±10.7)、成熟精子数量(481±56)相比,杜仲补天素胶囊低剂量组雄性小鼠原细胞数量(77.8±5)、精母细胞数量(132.4±8.9)、成熟精子数量(734±67)显著提高(P<0.01);杜仲补天素胶囊低、高剂量组均能显著性提高睾丸AR的表达水平(P<0.01);与对照组相比,杜仲补天素胶囊低、高剂量组血清T、FSH、LH、E2水平均无显著性差异(P>0.05),血清ALT水平无显著性差异(P>0.05);与对照组相比,杜仲补天素胶囊高剂量组AST水平显著性增加[(44.2±11) U/L vs(30.5±13.7) U/L,P<0.05],Cr、BUN水平有增加但无显著性差异(P>0.05)。结论:杜仲补天素胶囊对雄性小鼠具有一定的促生育作用,为杜仲补天素胶囊的临床应用提供实验基础。     

苯甲酸雌二醇诱导不育小鼠睾丸中生精细胞增殖紊乱的研究

目的:探讨外源性雌激素苯甲酸雌二醇(EB)对诱导雄性不育小鼠中生精细胞增殖紊乱的影响。方法:60只雄性昆明小鼠随机分为3组,对照组肌肉注射150μl玉米油,EB处理组注射浓度分别为5、10 mg/kg的EB,隔天1次,持续4周。实验结束后取睾丸称其质量并计算睾丸指数;睾丸、附睾尾常规石蜡切片,HE染色;附睾尾制备精子悬液进行精子计数;免疫组化检测睾丸PCNA表达;qRT-PCR分析睾丸细胞周期蛋白A1、细胞周期蛋白B1、VASA、p53的表达变化;Western印迹检测p53及磷酸化p53蛋白表达。结果:与对照组相比,EB处理组小鼠睾丸指数显著下降[(0.56±0.09)%vs(0.43±0.08)%、(0.38±0.10)%,P0.05],精子悬液镜下观察未见精子,HE染色附睾管内未见精子;生精小管中精原细胞、初级精母细胞及支持细胞数量显著下降(P0.05)。免疫组化结果显示,与对照组相比,EB处理组生精小管中细胞PCNA阳性表达细胞数量显著下降(P0.05)。qRTPCR结果表明EB处理组PCNA、细胞周期蛋白A1、细胞周期蛋白B1、VASA mRNA表达与对照组相比显著下降(P0.05);p53表达随EB剂量升高而显著升高(P0.05)。Western印迹结果显示,与对照组相比,EB处理组p53及磷酸化p53蛋白表达水平显著升高,且存在剂量依赖效应,10 mg/kg组显著高于5 mg/kg组(P0.05)。结论:EB以剂量依赖的方式下调细胞周期相关因子表达抑制生精细胞的增殖,是导致雄性不育小鼠睾丸中生精细胞增殖紊乱的重要原因。     

Development of renin expression in the mouse kidney

During metanephric kidney development, renin is expressed in the walls of larger intrarenal arteries, but is restricted to the terminal part of the preglomerular arterioles in the adult kidney. Our study describes the three-dimensional development of renin expression in mouse kidneys during fetal and postnatal life. Renin immunoreactivity first appeared at day 14 of development in the cells expressing alpha-smooth muscle actin (alphaSMA) in the arcuate arteries. Before adulthood, the branching of the arcuate arterial tree increased exponentially and renin expression shifted from proximal to distal parts of the tree. Renin expression at branching points or in the cones of growing vessels was not seen. Instead, renin expression appeared after vessel walls and branches were already established, disappeared a few days later, and remained only in the juxtaglomerular regions of afferent arterioles. In these arterioles, coexpression of renin and alphaSMA disappeared gradually, with the terminal cells expressing only renin. At all stages of kidney development, renin expression among comparable vessel segments was heterogeneous. Renin expression remained stable after it reached the terminal parts of afferent arterioles.     

WNK家族基因在小鼠肾脏表达的特性分析

目的检测WNK1和WNK4基因在小鼠肾脏组织中的表达位点。方法RT-PCR方法扩增WNK1和WNK4基因片段作为探针,在小鼠多组织膜上进行Northern印迹杂交。将上述片段克隆入pGEM-T载体,测序证实后,体外转录RNA探针并在小鼠肾脏石蜡组织切片上进行原位杂交。结果WNK1基因广泛表达在小鼠各组织中,在肾脏有9.5kb强杂交信号。WNK4基因主要表达在肾脏组织中,有4.4kb杂交信号。原位杂交显示WNK1基因仅表达在肾脏远曲小管中,而WNK4除表达在肾脏远曲小管外,还表达在髓质集合管上。结论WNK1和WNK4基因均在肾脏中有表达,WNK4基因在肾脏中表达比WNK1基因更广泛。     

Systematic review of mouse kidney transplantation

A mouse model of kidney transplantation was first described in 1973 by Skoskiewicz et al. Although the mouse model is technically difficult, it is attractive for several reasons: the mouse genome has been characterized and in many aspects is similar to man and there is a greater diversity of experimental reagents and techniques available for mouse studies than other experimental models. We reviewed the literature on all studies of mouse kidney transplantation to report the donor and recipient strain combinations that have been investigated and the resultant survival and histological outcomes. Some models of kidney transplantation have used the transplanted kidney as a life‐supporting organ, however, in many studies the recipient mouse's native kidney has been left in situ. Several different combinations of inbred mouse strains have been reported, with varying degrees of injury, survival or tolerance because of haplotype differences. This model has been exceptionally useful as an investigational tool to understand multiple aspects of transplantation including acute rejection, cellular and humoral rejection mechanisms and their treatment. Furthermore, this model has been used to investigate disease mechanisms beyond transplant rejection including intrinsic renal disease and infection‐associated pathology.     

Expression of Ia in mouse kidney

Expression of Ia antigens in mouse kidney was studied by absorption of diluted anti-Ia sera with crude suspensions of kidney issue. Specific absorption of anti-Ia activity was seen for all Ia specificities tested: Ia.1,2, Ia.3, Ia.4,5,12, Ia.7, Ia.8, Ia.9, Ia.15, and Ia.16. Certain polyspecific antisera (against the I-Ak products Ia.1,2,3,15) were more difficult to absorb than oligo-specific antisera against other Ia specificities (e.g., Ia.7 and Ia.9). This observation may indicate that polyspecific sera are less absorbable by limited numbers of antigenic sites because of steric hindrance, although differences in the extent of antigen expression in kidney have not been excluded. Ia absorption could be demonstrated either in microcytotoxicity or in 51Cr release assays. Both mechanically disrupted and enzyme-disrupted kidney tissue suspensions absorbed Ia specifically, although the former method was used routinely. As estimated from the efficiency of absorption, the amount of Ia in kidney was small, about 2 to 5% of that in spleen. One kidney was equivalent in absorptive capacity to about 3 X 10(6) splenocytes, and to greater than 3 X 10(6) buffy coat cells. Comparisons of the rates of absorption indicated that the amount of Ia in kidney was less than the amount of an H-2K or D alloantigen. Ia was expressed in kidney in an immunogenic form, since animals immunized repeatedly with I region-incompatible kidney tissue produced anti-Ia antibodies. Thus, Ia antigens are expressed in and are immunogenic in mouse kidney and can be studied by conventional serological techniques.     

Chronic rejection of mouse kidney allografts

BACKGROUND: Chronic renal allograft rejection is the leading cause of late graft failure. However, its pathogenesis has not been defined. METHODS: To explore the pathogenesis of chronic rejection, we studied a mouse model of kidney transplantation and examined the effects of altering the expression of donor major histocompatibility complex (MHC) antigens on the development of chronic rejection. RESULTS: We found that long-surviving mouse kidney allografts develop pathological abnormalities that resemble chronic rejection in humans. Furthermore, the absence of MHC class I or class II antigens did not prevent the loss of graft function nor alter the pathological characteristics of chronic rejection. Expression of transforming growth factor-beta (TGF-beta), a pleiotropic cytokine suggested to play a role in chronic rejection, was markedly enhanced in control allografts compared with isografts. However, TGF-beta up-regulation was significantly blunted in MHC-deficient grafts. Nonetheless, these differences in TGF-beta expression did not affect the character of chronic rejection, including intrarenal accumulation of collagens. CONCLUSIONS: Reduced expression of either class I or II direct allorecognition pathways is insufficient to prevent the development of chronic rejection, despite a reduction in the levels of TGF-beta expressed in the allograft. This suggests that the severity of chronic rejection is independent of the level of MHC disparity between donor and recipient and the level of TGF-beta expression within the allograft.     

小鼠移植肾再次移植动物模型的建立

目的探讨小鼠移植肾再次移植动物模型建立方法。方法将首次移植供体小鼠左侧供肾肾静脉(RV)同首次移植受体小鼠肾下下腔静脉(IVC)端侧连续吻合,首次移植供体小鼠左侧供肾肾动脉(RA)连带小段首次移植供体小鼠腹主动脉(AO)同首次移植受体小鼠AO端侧间断吻合,首次移植供体小鼠左侧供肾输尿管拖入并固定在首次移植受体小鼠膀胱顶后壁完成小鼠首次肾移植术。首次移植术后2~4周,将首次移植受体小鼠体内移植肾脏RV连带部分首次移植受体IVC同移植肾再次移植受体小鼠IVC端侧连续吻合,移植肾脏RA连带小段首次移植供体和受体小鼠AO同移植肾脏再次移植受体小鼠AO端侧间断吻合,将再次移植肾输尿管拖入并固定在再次移植受体小鼠膀胱顶后壁完成小鼠移植肾再次肾移植术。首次移植和移植肾再次移植术中均切除受体双侧自体肾脏。记录手术时间,随访移植肾再次移植受体存活,监测再次移植肾功能和病理。结果移植肾再次移植供体手术时间为(50±10)min,受体手术时间为(55±5)min。共完成8例小鼠移植肾再次移植术。2例同系,6例非同系。第1例尝试性非同系移植肾再次移植受体存活11 d。后续5例非同系移植肾再次移植受体中1例存活21 d,其余4例均存活到术后70 d获取标本。2例同系移植肾再次移植受体均存活到术后30 d获取标本。8例移植肾再次移植受体在获取标本时或非预期死亡前血清肌酐均<0.2 mg/dl。苏木精-伊红(HE)染色提示同系移植肾再次移植术后30 d移植肾未见病理性改变。结论本文描述了建立小鼠移植肾再次移植动物模型的方法,为开展移植免疫相关研究提供了新手段。     

Sodium-phosphate transport in the kidney and intestine of the hypophosphatemic mouse

X-linked hypophosphatemic vitamin D-resistant rickets is the most common inherited form of vitamin D-resistant rickets in man. The current studies were designed to characterize the defect in the sodium (Na⁺)-phosphate transporter in the (Hyp) mouse model. The slope of initial rate of phosphate uptake was significantly decreased in the kidney but not in intestinal brush border membranes of the (Hyp) mice compared with genetically matched controls. Phosphate uptake by the basolateral membranes of the intestine and kidney was similar in the (Hyp) and control mice. Kinetic analysis of phosphate uptake by renal brush border membranes showed aV _max of 0.32±0.06 and 1.6±0.1 nmol/mg protein per 15 s (P<0.01) andK _m of 0.07±0.06 and 0.39±0.05 mM in (Hyp) and control mice respectively (P<0.05).V _max andK _max of jejunal uptake of phosphate were similar in (Hyp) and control mice. To confirm these findings, we expressed the Na⁺-phosphate transporter inXenopus laevis oocytes. Na⁺-dependent phosphate uptake in the oocytes was expressed 6 days after renal and intestinal poly(A)⁺ RNA injection, however, uptake values were significantly lower in oocytes injected with renal poly(A)⁺ RNA from the (Hyp) mice compared with controls (P<0.01). No differences were noted in phosphate uptake by oocytes injected with poly(A)⁺ RNA from the jejunum of the (Hyp) or control mice. These studies suggest that the defect in the (Hyp) mice is localized to the kidney and is secondary to diminished activity and/or function of the Na⁺-phosphate transporter.     

Morphogenesis during mouse embryonic kidney explant culture

BACKGROUND: Renal organogenesis is routinely studied using cultured murine embryonic kidneys, but the application of this model has not yet been subjected to rigorous standards. METHODS: We measured ex vivo growth and morphogenesis of day 13 murine kidneys and evaluated the importance of culture conditions and biological variables. RESULTS: Kidney size was measured in two dimensions as planar surface area and was shown to correlate highly with volume (R2 = 0.60, P < 0.005). The final surface area of kidneys was directly dependent on the initial starting size (R2 = 0.61, P < 0.05), suggesting that the final surface area is not a valid outcome measurement unless starting size is equal among treatments. Relative growth rate, defined as (final surface area - initial surface area)/initial surface area, was a good measure of growth and independent of size and anatomical position (P> 0.05). Significant differences in size and growth rates were observed among litters (P < 0.05), implying that kidneys from a given litter must be randomized to avoid confounding results. Planar surface area of each explant increased in proportion to ureteric bud branching (R2 = 0.6854, P < 0.05). In a comparison of a variety of base media and supplements, kidney explants were observed to grow best in Dulbecco's modified Eagle's medium (DMEM)/F12 with 5% fetal bovine serum and to sustain growth for up to 96 hours, despite decreased proliferation and increased apoptosis at this time point. CONCLUSIONS: These results represent an important step in establishing standardized procedures for the use of cultured embryonic kidneys and will improve our ability to apply the model to better understand kidney morphogenesis.