血管内皮生长因子和其受体Flt-1在青春期大鼠睾丸、附睾及附睾内精子上的表达研究

目的:通过对血管内皮生长因子(VEGF)及其受体Flt-1在大鼠睾丸、附睾及附睾内精子上表达的研究,探讨其在雄性生殖系统中的作用。方法:免疫组化SP法和免疫荧光法检测20只青春期SD大鼠睾丸、附睾及精子上VEGF和Flt-1蛋白的表达情况。结果:VEGF和Flt-1在大鼠睾丸和附睾组织及精子上均有特征性表达。睾丸内VEGF蛋白表达于生精细胞、精子细胞发育中的顶体、Sertoli和Leydig细胞胞质内;Flt-1只见于精子细胞发育中的顶体及Leydig细胞胞质中。附睾中VEGF表达于各段上皮主细胞胞质内,而Flt-1表达于头、尾段上皮主细胞胞质内,体部免疫染色阴性;两者在附睾上皮亮细胞、晕细胞和基细胞中均为阴性表达。免疫荧光染色显示,VEGF和Flt-1共同定位于附睾内精子头部的顶体,尾部的颈、中和主段。结论:VEGF和Flt-1蛋白在大鼠睾丸、附睾及精子中的特异性表达提示,他们可由不同生精上皮细胞、间质细胞和附睾主细胞产生,可能以自分泌或旁分泌的形式单独或共同作用于睾丸和附睾的生殖细胞或Leydig细胞,直接或间接地影响精子的发生、发育和成熟过程,并与精子的活动和受精能力有关。     

急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70表达的影响

目的:探讨急性热应激对性成熟雄性小鼠睾丸、附睾、输精管中热休克蛋白70(heat shock protein 70,HSP70)表达的影响。方法:将32只8周龄雄性小白鼠随机均分为4组,饲养7d后,进行热应激处理,温度控制在(39±0.5)℃,时间分别为0.5、1和3h。应激后立即采血,分离血清测定谷草转氨酶(GOT)含量。一侧附睾制备精子悬液,用于计算精子密度和顶体畸形率;另一侧附睾、睾丸、输精管用于免疫组化研究。结果:应激后,小鼠体重、睾丸系数、顶体畸形率变化不显著(P>0.05),附睾系数和精子密度有不同程度的下降,GOT含量急剧升高(P<0.01)。随着应激时间的延长,小鼠精子密度呈递减趋势,顶体畸形率呈上升趋势。应激时间最短的0.5h组小鼠体重、睾丸系数、附睾系数的降幅反而最大。免疫组化法观察发现,HSP70在性成熟小鼠睾丸、附睾、输精管中均有表达。正常状态下,HSP70在睾丸组织间质细胞中少量表达,应激后分布于间质细胞核,此外在精母细胞核与精子细胞核中也有大量分布;附睾中HSP70主要分布于主细胞质,基细胞和亮细胞中没有表达,应激后附睾体的纤毛细胞中也发现大量棕色颗粒;输精管中HSP70主要定位在基细胞质,主细胞中不表达。随着应激时间的延长,HSP70在睾丸、附睾中的表达量明显升高,而在输精管中的增幅不明显。结论:急性热应激对性成熟雄性小鼠的生殖系统造成了损伤;HSP70在睾丸、附睾、输精管中的表达与定位具有区域特异性和细胞特异性,提示其可能参与精子的发生与成熟;HSP70在应激状态下表达量大幅上升的作用可能在于保护细胞免受高热损伤。     

实验性精索静脉曲张对青春期大鼠睾丸、附睾中CRES蛋白表达的影响

目的:研究青春期大鼠实验性左侧精索静脉曲张(ELV)对睾丸和附睾中胱蛋白酶抑制剂相关的附睾精子发生(CRES)蛋白表达的影响,探索精索静脉曲张导致不育的机制。方法:建立青春期雄性SD大鼠ELV模型,采用免疫组化及蛋白印迹方法检测CRES蛋白在ELV 2周组(n=8)、4周组(n=8)及其相应的对照组(n均=5)大鼠睾丸和附睾头、体、尾中的表达变化。结果:免疫组化显示,CRES蛋白在ELV实验组和对照组大鼠睾丸和附睾中均有表达。在睾丸中,CRES蛋白主要定位于圆形和长形精子细胞的胞质、精子的顶体以及残余体中,其表达与生精周期密切相关,Ⅰ~Ⅲ和Ⅸ~ⅩⅣ期表达最强,Ⅶ~Ⅷ期表达次之,Ⅳ~Ⅵ期表达减弱。在附睾中,CRES蛋白主要表达于从附睾起始段到尾部的上皮主细胞的胞质中,且以体、尾部表达最强,头部次之,管腔分泌物也呈阳性表达。实验组比对照组CRES蛋白表达增强。W estern印迹显示:实验组和对照组大鼠睾丸和附睾在相对分子质量(Mr)为19 000和14 000处均可见CRES蛋白条带,其中以Mr19 000处表达更为明显。实验组CRES蛋白的表达比对照组明显增强。对免疫组化和W estern印迹结果进行灰度值和积分吸光度值(IA)测定,并经统计学分析显示:ELV 2周组和4周组比其相对应的对照组表达增强且差异有显著性(P<0.05或P<0.01),而ELV各组间未见CRES蛋白表达有明显差异(P>0.05)。结论:CRES蛋白在大鼠睾丸和附睾中均有表达,睾丸中表达呈现生精周期特异性和细胞特异性,附睾中表达呈现附睾上皮区段和细胞特异性;CRES蛋白在青春期ELV大鼠睾丸和附睾中的表达比对照组明显增强。这些结果提示CRES蛋白在精子发生和精子成熟过程中可能起着重要的作用,并且可能与ELV引起的不育或生育力低下有关。     

Ccdc70基因在小鼠睾丸精子发生过程中的表达特征

目的:探讨Ccdc70(Coiled-coil domain containing 70)基因在小鼠睾丸中的表达特征并分析其在生精过程中的潜在功能。方法:经表达谱芯片筛选出小鼠睾丸特异性基因Ccdc70,通过RT-PCR、real-time PCR、Western印迹及免疫组化检测Ccdc70基因在成年小鼠睾丸中表达特征,并对该蛋白做相关的生物信息学分析。结果:RT-PCR、real-time PCR和Western印迹结果表明Ccdc70基因在小鼠睾丸中高表达,在附睾中低表达;免疫组化结果表明Ccdc70蛋白在睾丸中主要表达于小鼠精母细胞和圆形精子胞质,在附睾中主要表达在附睾管上皮细胞。生物信息学分析显示该蛋白存在一个CCDC结构域,并在哺乳动物进化过程中高度保守。结论:Ccdc70为小鼠睾丸高表达基因,且主要表达于精母细胞、圆形精子及附睾管上皮细胞,提示其可能参与调控小鼠精子发生过程及附睾精子的进一步成熟。     

细胞外囊泡：与精子成熟、运动以及受精相关的生理功能

细胞外囊泡是一种细胞间通讯工具,在精子成熟、运动以及受精过程中发挥重要作用。精子通过附睾的过程中,附睾小体为精子提供蛋白质、脂质和遗传物质,促进精子成熟,也可在表观遗传水平影响精子和后代表型。射精后,前列腺小体与精子融合,精子质膜组成以及精子内容物改变,不仅可以维持精子质膜稳定性,也增强了精子活力。子宫小体可增强精子获能、增加顶体反应水平。输卵管小体除了参与精子获能、运动中,还可以给予精子非编码RNA,进而影响胚胎发育。尚且不足的是,有关细胞外囊泡与精子的研究多为动物实验,其在人类的适用性仍有待进一步研究。     

小鼠Lyzls的表达特征及人LYZL6蛋白的抗菌特性研究

C类溶菌酶基因（Lyzls）属于溶菌酶基因家族,高表达于小鼠睾丸和附睾。该家族基因Lyzl4和Spaca3被报道在小鼠精卵融合和受精过程中发挥作用。然而其他两种小鼠C类溶菌酶基因Lyzll他HLyzl6的功能尚不清楚。本研究分析了小鼠C类溶菌酶基因的组织表达、表达与雄激素的关联以及重组人LYZL6蛋白（rLYZL6）在免疫中可能发挥的作用。通过RT-PCR、Westernblots、免疫组织化学和免疫荧光的方法分析了Lyz＆的表达,细菌集落形成测定实验分析了重组人LYZL6蛋白的抗菌活性。小鼠lyzls主要在睾丸和附睾中表达,受发育调控及雄激素或睾丸因子调控。免疫检测LYzL6蛋白定位于小鼠睾丸的初级精母细胞、圆形精子和成熟精子的顶体后及中段。重组人LYZL6蛋白呈现抗菌活性。我们推测Lyzls可能在精子线粒体的功能中发挥作用,LYZL6蛋白在雄性生殖道免疫中发挥作用。     

人源类溶菌酶蛋白4在受精过程中的作用及酶学性质研究

目的:对人源类溶菌酶蛋白4(LYZL4)在受精过程中的作用和重组蛋白的酶学性质进行研究,从而揭示LYZL4的生理功能。方法:细胞免疫荧光法检测LYZL4在精子细胞上的亚细胞定位,RT-PCR分析精子表面LYZL4的来源,通过精子穿透试验分析LYZL4在受精过程中的作用。构建真核重组表达载体p PIC9K-LYZL4,转化毕赤酵母GS115菌株后,甲醇诱导表达;甲壳素亲和层析和凝胶过滤层析从发酵上清中纯化重组LYZL4蛋白(rLYZL4),通过Western印迹进行验证。ELISA测定rLYZL4的透明质酸结合能力,分光光度法测定rLYZL4的胞壁质酶活性、透明质酸酶活性和自由基清除能力。结果:LYZL4免疫荧光定位于成熟精子头部的顶体膜上,RTPCR检测表明精子表面的LYZL4蛋白来源于睾丸和附睾的分泌,兔抗LYZL4多克隆抗体可明显抑制精卵结合。毕赤酵母表达系统成功表达了rLYZL4,酶活性分析显示rLYZL4无胞壁质酶和透明质酸酶活性,但能够结合透明质酸,并具有较强的自由基清除能力。结论:LYZL4由睾丸和附睾分泌后定位于成熟精子头部的顶体膜上,可在精卵结合过程中发挥作用,其还能够结合透明质酸并清除自由基,提示LYZL4可能作为一种多功能分子在精子保护及精卵结合过程中发挥作用。     

小鼠BPI体内外抗溶脲脲原体生长的实验研究

目的:通过体内外实验,检测小鼠杀菌渗透性增强蛋白(bactericidal/permeability-increasing protein,BPI)是否对溶脲脲原体(Ureaplasma urealyticum,UU)的生长有抑制作用。方法:UU与Bac-to-Bac系统真核表达的BPI共孵育0.5~2h后培养16~20h,通过颜色变化单位(CCU)观察BPI体外抗UU生长的效果;膀胱注射UU,逆行感染小鼠生殖道,通过观察睾丸和附睾BPI的mRNA和BPI蛋白表达的变化来检测BPI体内抗UU的效果。结果:UU与BPI孵育0.5h,在蛋白量40μg时,BPI可抑制UU生长;随孵育蛋白量的增加,BPI对UU抑制效应增加。UU与BPI孵育2h,BPI对UU的抑制效应无明显变化。UU逆行感染成功的小鼠睾丸和附睾的BPI在基因和蛋白水平均显著提高(P0.01)。结论:小鼠BPI在体内外均对溶脲脲原体的生长有抑制作用。     

精子蛋白Ropporin的研究进展

Ropporin一直以来被认为是精子细胞特异性蛋白,在睾丸组织中特异性表达,并参与精子的运动、获能、顶体反应等生理活动。然而,新的研究表明Ropporin在正常的非睾丸组织也有微弱表达,而且作为一种新的肿瘤睾丸抗原(CT抗原)在恶性血液病的肿瘤细胞中高表达。Ropporin在哺乳动物中的表达具有高度保守性,说明它对生命的重要性。本文就Ropporin的特性、表达与分布、生理学功能及临床研究的新进展进行了综述。     

人源类溶菌酶蛋白4的多克隆抗体制备及其表达分析

目的:以原核表达的人源类溶菌酶蛋白4(LYZL4)为免疫原制备多克隆抗体,检测LYZL4在睾丸组织中的定位,为阐明其生理功能提供依据。方法:将LYZL4编码基因克隆于原核表达载体pET32a,IPTG诱导重组LYZL4(r LYZL4)表达。分别使用Ni-NTA树脂和甲壳素亲和层析纯化rLYZL4,采用双层琼脂平板扩散法检测杀菌活性。以Ni-NTA树脂纯化产物为免疫原制备兔抗rLYZL4多克隆抗体,通过ELISA测定抗体效价,Western印迹检测抗体特异性后,检测LYZL4蛋白在人体各组织的分布及其在精子和精浆中存在情况,免疫组化确认LYZL4在人睾丸组织中的定位。结果:原核表达系统可有效表达rLYZL4,其对溶壁微球菌及大肠杆菌无杀灭活性。制备的兔抗rLYZL4多克隆抗体具有很高的效价和特异性,在睾丸、附睾及人精子蛋白提取物中可检测到LYZL4存在,在生精小管中,LYZL4定位于圆形精子细胞及长形精子细胞的顶体上。结论:本研究成功以原核表达的rLYZL4制备了该蛋白的多克隆抗体,确认LYZL4在睾丸和附睾有表达,并定位于精子顶体上,提示其可能与顶体结构或功能相关。     

Migration and ultrastructural localization of the c-kit receptor protein in spermatogenic cells and spermatozoa of the mouse

PURPOSE: The c-kit receptor is a proto-oncogene important in germ cell migration and maturation and has also been demonstrated on the acrosomal region of mature sperm. The purpose of the present study was to examine the ultrastructural location of the c-kit receptor in mouse testis and sperm. MATERIALS AND METHODS: Testis and sperm from mature male mice were examined for the c-kit receptor utilizing electron microscopy and Western blot analysis techniques. Thin sections of mouse testis and sperm were stained with immunogold-labeled anti-c-kit antibodies. The protein from these testes and sperm was also utilized for Western blot analysis. RESULTS: The c-kit protein was localized within the mouse testes to the type A spermatogonia, the round spermatids, and the mature testicular spermatozoa. The c-kit receptor was noted to migrate from the lumen of the acrosomal vesicles in the early spermatids to the plasma membrane of the late spermatids. It was also noted in the acrosomal region of the testicular spermatozoa, as well as the sperm from the epididymis. Sperm undergoing the acrosome reaction demonstrated association of the c-kit receptor with the plasma membrane of the acrosome, but not on the acrosomal membrane itself. Western blot analysis demonstrated protein bands of 150 kDa in testis and intact sperm. CONCLUSIONS: The present study confirms the presence of the c-kit receptor in mouse testis and sperm. It also demonstrates that this receptor is localized to the region of the developing acrosome.     

Decreased expression of AKAP4 and TyrPho proteins in testis,epididymis, and spermatozoa with low sexual performance of mice induced by modified CUMS

The molecular mechanism of chronic stress especially reduced motility, a major cause of male infertility, has not been proved. It is known that A-kinase anchor protein 4 (AKAP4) and tyrosine-phosphorylated (TyrPho) proteins are involved in progressive motility. This study aimed to investigate the effect of chronic unpredictable mild stress (CUMS) on sexual behaviours, sperm quality, and expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa. Sixteen male mice were divided into control and CUMS groups (n = 8/group). Animals were induced by a stressor from twelve stressors for 36 days. Sexual behaviours, corticosterone and testosterone, sperm parameters, and histopathology were observed. The expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa were examined. Results showed that CUMS significantly increased corticosterone while serum testosterone level was decreased. Sexual behaviours and sperm parameter quality were significantly decreased. CUMS mice showed vacuolisation and pyknotic cells in seminiferous epithelium and less sperm mass was observed within epididymal lumen. CUMS decreased expressions of AKAP4 and TyrPho proteins in testis, epididymis, and spermatozoa. In conclusion, the decreased expression of AKAP4 and TyrPho proteins may be a mechanism associated with low semen qualities particularly decrease of sperm motility in CUMS.     

Extracellular quality control in the epididymis

The epididymal lumen represents a unique extracellular environment because of the active sperm maturation process that takes place within its confines. Although much focus has been placed on the interaction of epididymal secretory proteins with spermatozoa in the lumen, very little is known regarding how the complex epididymal milieu as a whole is maintained, including mechanisms to prevent or control proteins that may not stay in their native folded state following secretion. Because some misfolded proteins can form cytotoxic aggregate structures known as amyloid, it is likely that control/surveillance mechanisms exist within the epididymis to protect against this process and allow sperm maturation to occur. To study protein aggregation and to identify extracellular quality control mechanisms in the epididymis, we used the cystatin family of cysteine protease inhibitors, including cystatin-related epididymal spermatogenic and cystatin C as molecular models because both proteins have inherent properties to aggregate and form amyloid. In this chapter, we present a brief summary of protein aggregation by the amyloid pathway based on what is known from other organ systems and describe quality control mechanisms that exist intracellularly to control protein misfolding and aggregation. We then present a summary of our studies of cystatinrelated epididymal spermatogenic （CRES） oligomerization within the epididymal lumen, including studies suggesting that transglutaminase cross-linking may be one mechanism of extracellular quality control within the epididymis. （Asian J Androl 2007 July; 9： 500-507）     

Origin of the luminal fluid proteins of the rat epididymis

Using a combined microperfusion and high resolution gel electrophoresis technique, the origin of the epididymal fluid proteins of the rat has been investigated. Some proteins originate from the testis, others are secreted by the epididymis or are released by spermatozoa. Of particular interest is a 32 000 dalton protein found to be actively secreted by the caput epithelium in situ and concenrated in the lumen. The cauda epididymidis contained the highest concentration of this protein. Radioactive labelling of the sperm surface proteins revealed that this protein was present on the surface of the mature cauda but not on the immature caput or corpus sperm, suggesting its acquisition by the sperm surface during epididymal transit. Another sperm surface protein of interest (MW 40 000) is present only on the plasma membrane of the cauda but not on that of the caput or corpus sperm. Since this protein was not identified in the epididymal perfusates or luminal fluids, its presence may result from some modification events taking place in the sperm membrane during maturation.     

Orchestration of occludins, claudins, catenins and cadherins as players involved in maintenance of the blood-epididymal barrier in animals and humans

Although spermatozoa are formed during spermatogenesis in the testis, testicular spermatozoa are immature and cannot swim or fertilize. These critical spermatozoal functions are acquired in the epididymis where a specific luminal environment is created by the blood-epididymal barrier; proteins secreted by epididymal principal cells bind to maturing spermatozoa and regulate the maturational process of the spermatozoa. In the epididymis, epithelial cell-cell interactions are mediated by adhering junctions, necessary for cell adhesion, and by tight junctions, which form the blood-epididymal barrier. The regulation of these cellular junctions is thought to represent a key determinant in the process of sperm maturation within the epididymis. Tight junctions between adjacent principal cells permit the formation of a specific microenvironment in the lumen of the epididymis that is essential for sperm maturation. Although we have made significant progress in understanding epididymal function and the blood-epididymal barrier, using animal models, there is limited information on the human epididymis. If we are to understand the normal and pathological conditions attributable to human epididymal function, we must clearly establish the physiological, cellular and molecular regulation of the human epididymis, develop tools to characterize these functions and develop clinical strategies that will use epididymal functions to improve treatment of infertility. （Asian J Androl 2007 July; 9： 463- 475）     

Mouse sperm acquire a new structure on the apical hook during epididymal maturation

Spermatozoa emerging from the testis undergo a maturation process in the epididymis during which they change morphologically, biochemically and physiologically to gain motility and the ability to fertilize ova. We examined mouse epididymal sperm with immunostaining and transmission electron microscopy (EM) and identified a previously unknown structure on the apical hook. The structure has a coiled configuration around 11 nm in thickness and is present at the tip of each corner of the triangular-rod shaped perforatorium. Surveying sperm isolated from various regions of the epididymis indicated that mouse sperm acquire the hook rim (HR) structure during its passage through the proximal two-thirds of the caput epididymidis. The structure withstands vigorous sonication and harsh chemical treatments and remains intact after the acrosome reaction. Its location and sturdiness suggest a function in protecting the apical hook from mechanical wear during fertilization. Our EM images of epididymal sperm also revealed additional novel structures as well as lateral asymmetry of the sperm head, indicating that mouse sperm head has a structure more complex than previously recognized.     

Localization of Hsp60 and Grp78 in the human testis, epididymis and mature spermatozoa

Molecular chaperones of the heat shock proteins (HSP) family are important in numerous cellular processes. In this study, the expression of Hsp60 and Grp78 proteins was investigated in the male reproductive tract. The cellular distribution of Hsp60 and Grp78 proteins was analysed in the human testis and epididymis by immunohistochemical approaches. DNA microarray technology was used to analyse HSP60 and GRP78 gene expression along human epididymis. The cellular localization of these chaperone proteins in ejaculated spermatozoa was investigated by indirect immunofluorescence and by Western blot following sperm sub-cellular fractionation. In the human testis, Hsp60 was detected in spermatogonia, whereas a strong Grp78 staining was observed in spermatocytes and round spermatids. Grp78 protein was also observed in the epididymal epithelium, whereas no Hsp60 staining was observed in this organ by immunohistochemistry. The presence of both Hsp60 and Grp78 RNA in human epididymis was confirmed by microarrays. In ejaculated spermatozoa, Hsp60 was localized in the mid-piece, whereas Grp78 was detected in the neck region. These results indicate that in addition to being expressed in human testis spermatogenic cells, both Hsp60 and Grp78 proteins persist in ejaculated spermatozoa. These findings are in agreement with the involvement of Hsp60 and Grp78 during spermatogenesis and in sperm functions such as fertilization.     

ConA-binding proteins of the sperm surface are conserved through evolution and in sperm maturation

Summary.  Lectin-binding glycoconjugates present on the surface of spermatozoa are believed to play a crucial role in sperm maturation, capacitation, acrosome reaction, or sperm-egg interaction. We have studied ConA-binding surface proteins on spermatozoa from different mammalian species. First, ConA-binding proteins were isolated from boar spermatozoa by affinity chromatography. ConA-binding ability was confirmed by Enzyme-linked Lectin assay (ELLA). Monoclonal (MAb436/10) and polyclonal antibodies were raised against chromatography fractions containing purified ConA-binding proteins of boar spermatozoa. MAb436/10 (IgG_2a) recognizes a 40 kD ConA-binding antigen. Indirect immunofluorescence on fixed and unfixed boar spermatozoa with MAb436/10 indicated a plasma membrane localization of antigen 436/10 in the acrosomal macrodomain. Interspecies cross-reactivity with MAb436/10 was found by whole cell ELISA and immunocytochemistry. MAb436/10 cross-reacted with human, horse, guinea-pig, bull, and ram spermatozoa in both assays. Expression of ConA-binding antigen 436/10 on guinea pig sperm surface was detectable during spermiogenesis and in early stages of sperm maturation. Change of regionalization of the antigen did not occur during the epididymal passage. ConA-binding antigen 436/10 was also detectable in testis and caudal segments of the epididymis. These findings suggest that ConA-binding proteins located in the acrosomal region are highly conserved through evolution as well as in sperm maturation indicating an important role for the physiology of spermatozoa.     

The Effect of Epididymal and Testicular Fluids on the Fertilising Capacity of Testicular and Epididymal Spermatozoa

It is possible that the fertilising capacity of spermatozoa in the epididymis is influenced by the epididymal secretion. We have studied this problem by obtaining spermatozoa before entry into the epididymis and after passage through it, incubating both types of spermatozoa in fluids from the rete testis and cauda epididymidis and then checking their fertilising capacity. While spermatozoa from the rete testis were infertile, rete testis fluid did not decrease the fertilising capacity of epididymal sperm from the cauda epididymidis. Fluid from the cauda epididymidis did not promote the fertilising capacity of testicular spermatozoa. These results are discussed in the light of the current understanding of epididymal physiology.     

Forward motility protein (FMP): localization in hamster epididymal spermatozoa

The presence of FMP was investigated by immunocytochemistry in spermatozoa from the hamster caput and cauda epididymis. Spermatozoa from the caput showed no staining whereas spermatozoa pre-incubated with FMP were stained on the acrosome. Pre-treatment of the same sperm with epididymal plasma induced staining on the principal piece of the flagellum. Spermatozoa from the cauda were stained without previous incubation both on the acrosome and on the principal piece of the flagellum. These results suggest that the action of FMP, which prevents head-to-head agglutination of motile spermatozoa and allows acquisition of forward motility, may be due to at least two proteins. The first localizes to the acrosome during epididymal transit (anti-sticking factor), whilst the second localizes to the principal piece of the flagellum (forward motility initiation factor).