类雄激素受体在尖唇散白蚁繁殖蚁和工蚁卵子发生中的免疫细胞化学表达

为探讨类雄激素受体 (androgen receptor-like,AR-like)在白蚁卵子发生过程中的作用,采用免疫细胞化学方法对尖唇散白蚁Reticulitermes aculabialis雌性繁殖蚁和工蚁卵子发生中的类雄激素受体进行了定位检测。结果发现：繁殖蚁和工蚁卵巢中均有类雄激素受体免疫阳性反应。类雄激素受体在末龄若虫和成虫卵母细胞的分化期和生长期定位于卵母细胞质,而在成虫的卵黄形成期定位于滤泡细胞;在工蚁卵巢中,类雄激素受体定位于分化期和生长期的卵母细胞质,无卵黄形成期。结果提示类雄激素受体在白蚁的卵子发生过程中有重要作用： 工蚁的卵巢发育受抑制,其卵子发生相当于末龄若虫水平,但根据群体的变化又可以发育为补充繁殖蚁,有发育的潜能。     

尖唇散白蚁工蚁和繁殖蚁卵子发生比较

为探讨白蚁工蚁品级性腺不育的原因,采用组织学观察与测量对尖唇散白蚁Reticulitermes aculabialis Tsai et Hwang的工蚁和繁殖蚁的卵子发生各阶段进行比较和分析。结果表明,成熟工蚁的卵子发生与繁殖蚁相比,仅有卵母细胞的分化期和生长期,没有卵黄形成期,其分化期和生长期的卵母细胞大小与繁殖蚁这2个时期的卵母细胞大小呈显著性差异;生长期时,工蚁的卵母细胞和滤泡细胞之间出现较大间隙并且呈现萎缩退化状态;工蚁卵子发生相当于雌性繁殖蚁末龄若虫水平。该结果将为工蚁不能生殖提供组织学上的证据,同时又表明了工蚁具有潜在生殖能力的结构基础。     

类雄激素受体在尖唇散白蚁繁殖蚁和工蚁精子发生中的免疫细胞化学定位

为了比较和探讨类雄激素受体 (androgen receptor-like,AR-like)在白蚁生殖品级和非生殖品级精子发生过程中的作用,运用免疫细胞化学方法对尖唇散白蚁Reticulitermes aculabialis繁殖蚁和工蚁精子发生中的AR-like的定位进行了研究。结果显示：在繁殖蚁和工蚁精子发生过程中都有AR-like免疫阳性细胞的分布,均分布于初级精母细胞的细胞质和细胞核中;与繁殖蚁相比,AR-like在工蚁中的表达较弱。结果提示,AR-like的表达与精子发生过程中初级精母细胞的减数分裂有关,雄激素及其受体通过调控精母细胞的第一次减数分裂来影响白蚁精子的发生。虽然工蚁的精巢发育受抑制,但其精子发生和维持具有与繁殖蚁相同的激素调节机制,能形成精子。本研究为工蚁性腺退化不育而根据群体的变化又可以发育为补充繁殖蚁这一特殊的生理功能提供了组织学依据。     

尖唇散白蚁兵蚁和工蚁卵巢和卵子形态特征的比较

兵蚁和工蚁是白蚁中的非生殖品级,兵蚁是由工蚁分化产生。为了探讨兵蚁品级性腺不育的原因以及兵蚁和工蚁性腺发育的差异,采用组织学染色观察与测量方法对尖唇散白蚁Reticulitermes aculabialis Tsai et Hwang的兵蚁和工蚁的卵子发生各阶段进行比较和分析。结果显示:两者性腺发育大小呈极显著性差异(P<0.01),兵蚁与工蚁卵巢横切面面积之比约为1∶7;兵蚁的卵子发生与工蚁相比,仅有卵母细胞的分化期,没有生长期;兵蚁的卵母细胞比工蚁的小,两者的分化期卵母细胞体积之比约为1∶16。该结果表明工蚁向兵蚁转化过程中性腺进一步退化,兵蚁性腺极度退化使其丧失了成为补充繁殖蚁的可能性。该研究结果为兵蚁不能生殖提供组织学上的证据,同时又揭示了兵蚁和工蚁在潜在生殖能力差异方面的组织学基础。     

Bcl-2-like和Bax-like蛋白在白蚁生殖蚁和工蚁精子发生过程中的表达比较分析

为探讨凋亡调节因子Bcl-2和Bax蛋白对白蚁生殖蚁和工蚁性腺发育的影响, 揭示白蚁生殖品级与非生殖品级性腺发育的调节机理, 以尖唇散白蚁Reticulitermes aculabialis为研究对象, 运用免疫细胞化学定位方法对生殖蚁和工蚁精子发生过程中的Bcl-2和Bax蛋白表达进行了研究。结果显示： 生殖蚁和工蚁精子发生过程中从精原细胞至精子时期均有Bcl-2-like和Bax-like的阳性表达。生殖蚁的次级精母细胞、 精子细胞和精子中Bcl-2-like阳性表达率较高, 而在精原细胞和初级精母细胞中阳性率较低; 工蚁在次级精母细胞中最高, 在精原细胞和初级精母细胞中较低。除初级精母细胞期外, 工蚁生殖细胞其他发育阶段Bax-like阳性表达率均显著高于生殖蚁同一阶段生殖细胞。生殖蚁的生殖细胞在精原细胞、 初级精母细胞和次级精母细胞时期Bax-like阳性表达率较高, 发育至精子时期阳性率最低; 工蚁在次级精母细胞、 精子细胞和精子时期Bax-like表达率较高, 在初级精母细胞中表达率最低。在精子发生过程中, 生殖蚁生殖细胞Bax/Bcl表达量比值逐步下降; 而工蚁生殖细胞发育过程中Bax/Bcl表达量比值仅在次级精母细胞期下降, 其他发育时期均升高; 根据Bax/Bcl判断, 精原细胞和初级精母细胞是生殖蚁精子发生过程中主要的凋亡点, 而工蚁除了精原细胞和初级精母细胞外, 精子细胞和精子也是主要的凋亡目标。研究结果表明, 白蚁生殖细胞凋亡与其他动物一样受Bcl-2家族的调节, 在精子发生过程中Bcl-2-like和Bax-like表达具有动态变化规律, 正是这种变化调控生殖细胞在不同发育阶段的生或死; Bcl-2-like和Bax-like对生殖细胞凋亡调节不仅在精子发生中有非常重要的作用, 而且可能与工蚁品级的形成有关。     

尖唇散白蚁工蚁、兵蚁和繁殖蚁精子发生的比较研究

为探讨白蚁非生殖品级和生殖品级生殖细胞发育差异,采用组织学染色技术对尖唇散白蚁Reticulitermes aculabialis Tsaiet Hwang繁殖蚁、工蚁和兵蚁的精巢发育以及精子发生进行了显微观察和比较研究。结果发现3个品级间精巢发育的程度差异很大,三者精巢切面面积相对大小之比为:繁殖蚁∶工蚁=1.7∶1;繁殖蚁∶兵蚁=29.3∶1;工蚁∶兵蚁=17.1∶1。繁殖蚁和工蚁精巢管内有精子的形成,工蚁和繁殖蚁精子的发生都经历了精原细胞、初级精母细胞、次级精母细胞、精细胞和精子时期,但工蚁有大量次级精母细胞呈细胞凋亡状态。兵蚁生殖细胞发育仅有精原细胞、初级精母细胞、次级精母细胞,没有精细胞和精子产生。工蚁的生殖细胞显著小于同一时期繁殖蚁的生殖细胞,兵蚁的各时期生殖细胞均极显著小于繁殖蚁同一时期的生殖细胞。研究表明各品级之间生殖功能分化与生殖细胞发育有直接关系,工蚁有转化为补充繁殖蚁和兵蚁的能力;而兵蚁由于精巢极度退化不能产生精细胞和精子,因此是非生殖品级分化的终极形式,不具有转化成补充繁殖蚁或其它品级的能力。     

圆唇散白蚁工蚁生殖可塑性相关的性腺发育和基因表达

【目的】为了探讨圆唇散白蚁Reticulitermes labralis雌性工蚁向补充生殖蚁转化过程中的卵巢发育特征以及卵母细胞从滞育到恢复生长发育的起点。【方法】观察圆唇散白蚁雌性工蚁从3龄-6龄-转化成补充生殖蚁发育过程中的卵巢和卵母细胞动态变化;在若蚁向原始生殖蚁和补充生殖蚁转化的转录组中筛选出与卵母细胞生长期相关的基因表达,利用qRT-PCR方法检测这些基因在工蚁向生殖蚁转化发育过程中的表达水平。【结果】圆唇散白蚁工蚁从低龄向老龄发育过程中卵巢逐渐增大;前补充生殖蚁卵巢长度和宽度分别是工蚁的约2和3倍,而前补充生殖蚁转化为补充生殖蚁之后,卵巢没有显著增大。工蚁转化为前补充生殖蚁之后,卵母细胞大小（长径）和滤泡细胞层厚度仍然没有显著改变;前补充生殖蚁转化成补充生殖蚁之后,卵母细胞大小和滤泡细胞层厚度显著增加。调控卵母细胞生长期发育的6个基因cyclin-dependent kinase 1, cell division cycle protein 20, G2/mitotic-specific cyclin-B3, G2/mitotic-specific cyclin-A, aurora kinase A和serine/threonine-protein kinasepolo在补充生殖蚁中的表达水平极显著增加,分别是前补充生殖蚁的约34, 62, 91, 36, 57和106倍。工蚁转化为前补充生殖蚁之后,仅G2/mitotic-specific cyclin-B3, G2/mitotic-specific cyclin-A和aurora kinase A表达水平显著增高,分别是工蚁的约3, 3和2倍。【结论】圆唇散白蚁工蚁卵巢发育的停滞发生在老龄期,卵母细胞发育的恢复和减数分裂启动从工蚁转化为补充生殖蚁之后开始。     

促性腺激素在白蚁脑中的免疫细胞化学定位

为研究昆虫卵泡刺激素 (FSH)和黄体生成素 (LH)的存在与分布 ,我们用免疫细胞化学染色技术对FSH和LH在尖唇散白蚁 (Reticulitermesaculabialis)脑中的分布进行了定位研究。结果显示 :FSH免疫阳性神经细胞及神经纤维分布在雌性繁殖蚁分飞期前脑的左叶、右叶及中部 ,雌性末龄若虫前脑的右叶 ,雄性繁殖蚁前脑的右叶 ;LH免疫阳性物质分布在雌、雄繁殖蚁前脑左叶 ;在工蚁和兵蚁中未见有FSH和LH免疫阳性物质存在。研究结果表明 :FSH和LH在白蚁脑中的存在具有发育阶段特异性 ,并且有可能像其在脊椎动物中那样参与调节性腺发育及精卵成熟 ;可能调节繁殖蚁、兵蚁、工蚁的产生     

黑胸散白蚁工蚁与工蚁型补充生殖蚁体内抗氧化酶和解毒酶的活性及基因表达变化

【目的】社会性白蚁的工蚁可以转化成补充生殖蚁参与生殖,提高自身适合度。本研究旨在探究工蚁转变成补充生殖蚁后,与氧化应激抗衰老相关的抗氧化酶和解毒酶活性的变化,为揭示生殖蚁的繁殖和抗衰老机制提供参考。【方法】利用生化方法分别测定黑胸散白蚁Reticulitermes chinensis工蚁、工蚁型补充生殖蚁和原始生殖蚁体内的2种抗氧化酶过氧化氢酶(catalase, CAT)和超氧化物歧化酶(superoxide dismutase, SOD)与4种解毒酶酸性磷酸酶(acid phosphatase, ACP)、碱性磷酸酶(alkaline phosphatase, AKP)、羧酸酯酶(carboxylesterase, CarE)和细胞色素P450(cytochrome P450, CYP450)的酶活性;同时利用qRT-PCR检测这些酶对应的基因RsCAT, RsSOD, RsACP, RsCYP450和RsCarE的表达量。【结果】与雌性工蚁相比,黑胸散白蚁工蚁型补充生殖蚁体内的CAT, SOD, ACP, AKP和CarE活性显著上升,分别达到雌性工蚁的5.82, 1.41, 1.39, 2.27和2.70倍,CYP450活性在两品级间没有显著差异;RsCAT, RsSOD, RsACP, RsCarE和RsCYP450的表达量也显著增加。补充生殖蚁体内CAT和ACP活性显著高于原始生殖蚁的,而SOD和AKP活性在两品级间没有显著差异;雌雄补充生殖蚁RsCAT的相对表达量分别是原始生殖蚁的5.68和3.60倍,RsACP的相对表达量分别是原始生殖蚁的81.12和46.72倍。【结论】黑胸散白蚁工蚁由非生殖品级转化为生殖品级以后,体内抗氧化酶和解毒酶的酶活力和基因表达水平显著升高,从一定程度上揭示了生殖品级的抗衰老机制。     

雌激素受体在白蚁卵发生过程中的免疫细胞化学定位（简报）

雌激素受体（Estrogen receptor,ER）是一种与性腺发育和精、卵成熟有直接重要关系的性激素受体,不仅在哺乳动物中已经证实与卵子的发生有关系,近年来还发现雌激素受体也存在于雌性文昌鱼早期卵原细胞、卵黄生成期卵母细胞和成熟期卵母细胞中：Osada等在扇贝的卵巢中发现有ER免疫阳性物质存在于卵黄形成期的卵母细胞中,认为ER是基本的类固醇激素受体,有着古老的进化历史。有关ER在昆虫性腺发育成熟中的作用,迄今在国内外都未见报道。白蚁的卵子发育是否也像脊椎动物那样受ER的调节,这个问题尚不清楚。     

DDX4 (VASA) is conserved in germ cell development in marsupials and monotremes

DDX4 (VASA) is an RNA helicase expressed in the germ cells of all animals. To gain greater insight into the role of this gene in mammalian germ cell development, we characterized DDX4 in both a marsupial (the tammar wallaby) and a monotreme (the platypus). DDX4 is highly conserved between eutherian, marsupial, and monotreme mammals. DDX4 protein is absent from tammar fetal germ cells but is present from Day 1 postpartum in both sexes. The distribution of DDX4 protein during oogenesis and spermatogenesis in the tammar is similar to eutherians. Female tammar germ cells contain DDX4 protein throughout all stages of postnatal oogenesis. In males, DDX4 is in gonocytes, and during spermatogenesis it is present in spermatocytes and round spermatids. A similar distribution of DDX4 occurs in the platypus during spermatogenesis. There are several DDX4 isoforms in the tammar, resulting from both pre- and posttranslational modifications. DDX4 in marsupials and monotremes has multiple splice variants and polyadenylation motifs. Using in silico analyses of genomic databases, we found that these previously unreported splice variants also occur in eutherians. In addition, several elements implicated in the control of Ddx4 expression in the mouse, including RGG (arginine-glycine-glycine) and dimethylation of arginine motifs and CpG islands within the Ddx4 promoter, are also highly conserved. Collectively these data suggest that DDX4 is essential for the regulation of germ cell proliferation and differentiation across all three extant mammalian groups-eutherians, marsupials, and monotremes.     

Germ cell survival in C. elegans and C. remanei is affected when the dead box rna helicases Vbh‐1 or Cre‐VBH‐1 are silenced

The Vasa family of proteins comprises several conserved DEAD box RNA helicases important for mRNA regulation whose exact function in the germline is still unknown. In Caenorhabditis elegans, there are six known members of the Vasa family, and all of them are associated with P granules. One of these proteins, VBH‐1, is important for oogenesis, spermatogenesis, embryo development, and the oocyte/sperm switch in this nematode. We decided to extend our previous work in C. elegans to sibling species Caenorhabditis remanei to understand what is the function of the VBH‐1 homolog in this gonochoristic species. We found that Cre‐VBH‐1 is present in the cytoplasm of germ cells and it remains associated with P granules throughout the life cycle of C. remanei. Several aspects between VBH‐1 and Cre‐VBH‐1 function are conserved like their role during oogenesis, spermatogenesis, and embryonic development. However, Cre‐vbh‐1 silencing in C. remanei had a stronger effect on spermatogenesis and spermatid activation than in C. elegans. An unexpected finding was that silencing of vbh‐1 in the C. elegans caused a decrease in germ cell apoptosis in the hermaphrodite gonad, while silencing of Cre‐vbh‐1 in C. remanei elicited germ cell apoptosis in the male gonad. These data suggest that VBH‐1 might play a role in germ cell survival in both species albeit it appears to have an opposite role in each one. genesis 1–18 2012. © 2012 Wiley Periodicals, Inc.     

Gonadal development and sex determination in mouse

The development of primordial germ cells and gonads are determinants of reproductive health and fertility. Although the gonadal development process is similar for both genders, the gender-determining process and the mechanism of development of female and male gonads have different molecular mechanisms. Spermatogenesis and oogenesis are also included in this process for a healthy gonadal development. Many specific molecular signaling pathways play role in oogenesis and spermatogenesis and it is important to know at which stage these factors are effective, to understand the mechanism of a healthy gonadal development. With this review, we defined the importance of stage specific genes expressing during the events such as oogenesis and spermatogenesis with the prenatal and postnatal gonadal development. It will be important to know about the cellular signals involved in the control of the gonadal development.     

The Fog-3 Gene and Regulation of Cell Fate in the Germ Line of Caenorhabditis Elegans

In the nematode Caenorhabditis elegans, germ cells normally adopt one of three fates: mitosis, spermatogenesis or oogenesis. We have identified and characterized the gene fog-3, which is required for germ cells to differentiate as sperm rather than as oocytes. Analysis of double mutants suggests that fog-3 is absolutely required for spermatogenesis and acts at the end of the regulatory hierarchy controlling sex determination for the germ line. By contrast, mutations in fog-3 do not alter the sexual identity of other tissues. We also have characterized the null phenotype of fog-1, another gene required for spermatogenesis; we demonstrate that it too controls the sexual identity of germ cells but not of other tissues. Finally, we have studied the interaction of these two fog genes with gld-1, a gene required for germ cells to undergo oogenesis rather than mitosis. On the basis of these results, we propose that germ-cell fate might be controlled by a set of inhibitory interactions among genes that specify one of three fates: mitosis, spermatogenesis or oogenesis. Such a regulatory network would link the adoption of one germ-cell fate to the suppression of the other two.     

Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

Essential functions of microRNAs in animal reproductive organs

The development of the reproductive organs and the gametogenesis in animals are complex and multistage processes that require the precise and efficient regulation. In this review, we summarized the recent findings about the essential functions of microRNAs (miRNAs) in the regulation of gene expression during the differentiation of germ cells in spermatogenesis and oogenesis. Most likely, main and common functions of the conserved and highly expressed miRNAs in the male and female reproductive organs of mammals are the control of differentiation and proliferation of cells. Also we discussed a possible involvement of germline-expressed miRNAs in the formation of reproductive barriers and speciation.     

Genetic control of sex determination in the germ line of Caenorhabditis elegans

The nematode Caenorhabditis elegans normally exists as one of two sexes: self-fertilizing hermaphrodite or male. Development as hermaphrodite or male requires the differentiation of each tissue in a sex-specific way. In this review, I discuss the genetic control of sex determination in a single tissue of C. elegans: the germ line. Sex determination in the germ line depends on the action of two types of genes:--those that act globally in all tissues to direct male or female development and those that act only in the germ line to specify either spermatogenesis or oogenesis. First, I consider a tissue-specific sex-determining gene, fog-1, which promotes spermatogenesis in the germ line. Second, I consider the regulation of the hermaphrodite pattern of germ-line gametogenesis where first sperm and then oocytes are produced.     

Apoptose et spermatogenèse

Onset of spermatogenesis is associated with a wave of apoptosis, which limits its efficacy during the first cycles in most mammals. After the first cycles, the actual efficacy of spermatogenesis always remains below the theoretical yield. Among the germinal cells, spermatogonia are the main targets of physiological apoptosis. This physiological apoptosis partly depends on the relationships between germ cells and Sertoli cells. The impact of the Sertoli cell/germ cell number ratio on the efficacy of spermatogenesis is well accepted, the concept of density-dependent regulation in the seminiferous tubule was proposed in the early eighties. Since the steps of spermatogenesis require a continuous progression of the cell cycle rather than an arrest, germ cells might therefore be more sensitive to apoptosis. This may also lead to severe disturbances between proliferation and cell death. The first experiments designed to elucidate the mechanisms of germ cell apoptosis were based on hormonal deprivation or cryptorchidism. However, the link between hormonal or cellular action and cell survival remained to be established. Analysis of signal transduction pathways involved in germ cell apoptosis and their regulation were the next steps. The involvement of bcl-2 family genes has been confirmed, although the expression of some of its members remains more controversial. Data derived from overexpression of some genes (Bcl-2, Bcl-xl) or resulting from gene inactivation (Bax) at the testicular level have highlighted the role of these genes in the control of germ cell apoptosis and have also provided some evidence for the strict requirement for density-dependent regulation of spermatogenesis. More recently, variations in the pattern of expression of these genes or proteins helped to explain some of the discrepancies in the literature. The place of the Fas/Fas ligand system during the first cycle of spermatogenesis remains a matter of debate, with controversies concerning the precise site of expression of this oncogene and its receptor. Conversely, its role in the testis after chemotoxic or radiotoxic treatments is well established. However, the normal fertility of animals with a spontaneous inactivation of Fas or Fas L genes does not support a physiological role of these factors during spermatogenesis. While factors involved in TNF/TNF R1 (Tumor Necrosis Factor) are under study, some data have been reported concerning the role of TRAIL (TNFalpha Related Apoptosis Inducing Ligand) and its active or decoy receptors in the testis. Among the oncogenes which may modulate the apoptotic process, Kit/Stem Cell Factor is particularly interesting, as Kit is expressed in some germ cells and Leydig cells, whereas SCF is expressed by Sertoli cells. Its impact during gonadal development and in the survival and proliferation of differentiated spermatogonia has been clearly established. Using a transgenic mice model, in which the Kit gene was inactivated by the insertion of a nls-lacZ sequence in its first exon, we showed that one single copy of the gene was unable to sustain physiological spermatogenesis and fertility in male mice. Our results also suggest that the Kit gene might be expressed at different steps of spermatogenesis, with different signal transduction pathways and biological actions. Finally, analysis of the signal transduction pathways involved in testicular apoptosis and their mechanisms of control is one of the key steps to a better understanding of both impairment of spermatogenesis and the pathogenesis of certain germ cell tumours.