Silencing transformer and transformer-2 in Zeugodacus cucurbitae causes defective sex determination with inviability of most pseudomales

transformer is a switch gene for sex determination in many insects, which cooperates with transformer-2 that is expressed in both sexes to regulate female differentiation, particularly in dipterans. Zeugodacus cucurbitae(Coquillett) is a very destructive pest worldwide, however, its sex determination pathway remains largely uncharacterized. Here, we show that the female sex ratio is sharply reduced with knockdown of either transformer or transformer-2 by RNA interference in early embryos of Z. cucurbitae. Most of the males grown from the embryos with transient transformer and transformer-2 suppression mated with wild-type females and produced mixed sex progeny, with one exception that produced only female progeny, and all of the few remaining males failed to mate with wild-type females and produced no progeny. The exceptional male and those males with mating failure were XX pseudomales as determined by the detection of Y chromosome-linked Maleness-on-the-Y, indicating that most XX pseudomales are not viable. The phenotypes of transformer and transformer-2 suggest that they play a key role in regulating sex determination and are required for female sexual development of Z. cucurbitae. Our results will be beneficial to the understanding of sex determination in Z. cucurbitae and can facilitate the development of genetic sexing strains for its biological control.

Sex Determination in Homo sapiens as a Multi-Step Process: Potential for Development of Variants and Sex Differences in Disease Risk

Reproduction via cis-binary mechanisms appears to have evolved fairly early in the evolution of complex organisms, and a system committed to prior to evolution of humans. While the evolution of a chromosomal-specific approach has been a successful strategy for survival of a large variety of species including humans, the fidelity of sex determination leading to 100% cis-binary outcomes is not achieved in many species, with evidence for homosexual or bisexual behaviour evident in more than 1500 species. Thus, such outcomes indicates that sex determination is a multi-step process and not a single event, and as such, could lead to the appearance of variants during the process which developed much earlier than humans. Variants could arise either due to intrinsic variation in the steps of determination, or also be influenced by environmental factors of a biological or psychological nature. In contrast to homosexual variants which do not require interventions such as hormone therapy or surgery, expression of gender dysphoria, is more based in psychology, but also has biological underpinnings and can be influenced by such hormonal interventions and surgery. While the numbers of those with gender dysphoria is small (~0.6% - 1.0% of population), the attention given to this issue raises the possibility of biological and psychological environmental factors impacting the emergence of some of those expressing gender dysphoria. Furthermore, transitioning from male-to-female or female-to-male can have consequences regarding disease risks latter in life, including the appearance of autoimmune diseases. This review will attempt to review some of the evidence regarding sex determination, discuss why the system has potentially not been improved upon during evolution, how a potential role for sex chromosome function on neurodevelopment may be central to variation in humans, and how commitment to the current strategy is likely integrated into other sex-related events such as puberty, pregnancy, and menopause to ensure species survival. It will also discuss how variants in sex determination could contribute to sex differences in disease risk and how epigenetic modifications could play a role in such risk. .

Maleness-on-the-Y(MoY) orthologue is a key regulator of male sex determination in Zeugodacus cucurbitae(Diptera:Tephritidae)

The initiation of sex differentiation in insects is regulated by primary sex determination signals.In the Medfly Ceratitis capitata and other tephritids,Maleness-on-the-Y(MoY) is the master gene for male sex determination.However,the primary signal in Zeugodacus cucurbitae(Coquillett),a very destructive tephritid pest across the world,remains ambiguous.In this study,we have isolated and characterized the Medfly MoY homolog in Z.cucurbitae,ZcMoY.ZcMOY protein shows high sequence conservation to its homologs in Bactrocera species.ZcMoY transcription begins and peaks at very early embryonic stages and then becomes undetectable except the testes and heads of day 1 male adults.Silencing ZcMoY in early embryos by RNAi causes abnormal external genitalia and interior reproductive organs,giving rise to intersexes and feminization of XY individuals.The expression pattern and knockdown phenotypes of ZcMoY indicate that ZcMoY plays a key role in regulating sex determination of Z.cucurbitae males.Our findings will help the understanding of sex determination in Z.cucurbitae and facilitate the development of genetic sexing strains in its biological control.

The Effect of the Cyp19a1 Gene Methylation Modification on Temperature-dependent Sex Determination of Reeves' Turtle(Mauremys reevesii)

Temperature-dependent sex determination（TSD） is a type of environmental sex determination in which the sex of the embryos depends on the ambient temperature; however,the molecular mechanisms governing this process remain unknown.Aromatase,encoded by the cyp19a1 gene,which converts androgens into estrogens in animals,was considered to be the key gene for TSD.In this study,the 5＇-flanking region of the cyp19a1 gene in Reeves＇ turtle（Mauremys reevesii） was cloned,and the promoter region was identified using the luciferase reporter assay.Then the eggs of Reeves＇ turtle were incubated at different temperatures（26°C： male-biased temperature; 29°C： non-sex-biased temperature and 32°C： female-biased temperature）.During the thermosensitive period,the adrenal kidney gonad complexes（AKG） were sampled.DNA methylation analysis of the AKG samples showed that the promoter region of the cyp19a1 gene was significantly de-methylated in the female-biased temperature regime（P＆lt;0.01）.Quantitative analysis of the cyp19a1 gene and estrogen by q PCR and Elisa assay showed that the expression level of the cyp19a1 gene and estrogen content were both upregulated significantly at the female-biased temperature（P＆lt;0.01）.These results indicated that the de-methylation response of the cyp19a1 gene to incubation temperature,especially at the female-biased temperature,could lead to temperature-specific expression of aromatase and increased estrogen levels,which may further determine gonadal development in Reeves＇ turtle.These findings provide insights into the genetic mechanisms underlying TSD.

A Review of Sexual Determination and Differentiation in Crustacean

The prerequisite for monosex culture is to elucidate the molecular mechanism of sex determination and differentiation in crustaceans, as well as to explore the key genes that play a role in this process. Monosex culture technology based on crustacean economic species is of great importance in terms of genetic breeding and economic benefits of aquaculture. As a result, study into the mechanisms of sex determination and differentiation in crustaceans not only contributes to the current absence of basic theories of crustacean sexual mechanism, but also encourages technical innovation in aquaculture to increase overall economic efficiency. This study synthesizes and evaluates available research on sex determination and differentiation in crustaceans, and then provides recommendations for future research objectives and priorities in the field.

Sex Determination Mechanisms in Fish

In fish,sex determination(SD) system shows high variation. The SD mechanisms include environmental and genetic regulation. The research on SD system and related genes in intensively studied fish species was reviewed. Although some genes have been described as sex-related,only DMRT1bY can be considered as a master sex determination gene and none of them has been utilized in aquaculture. The variation of fish SD system,the importance of sex-related genes in evolution research and the relations between environmental factors and sex-related genes were also discussed. The fish sex determination mechanism remains largely unknown. Further research needs to be done considering the significance of fish SD studies in basic and applied aspects.

Sex determination and dif ferentiation in Aurelia sp.1: the absence of temperature dependence

Cnidarians, being regarded as ‘basal' metazoan animals, are considered to have relatively high plasticity in terms of sex reversal. In this study we used an experimental approach to demonstrate sexual diff erentiation and plasticity in benthic polyps and pelagic medusae of A urelia sp.1 maintained at dif ferent temperatures. Results indicated that in A urelia sp.1, sex diff erentiation has been determined at the polyp stage and that all medusae originating from a given polyp are, phenotypically, of the same sex. In addition, the sex of polyps budding from the same clone(either male or female) at dif ferent temperatures appears to be the same as that of the parent. The sex of medusae that had originated from a known-sex polyp was observed to remain the same as that of the parent, irrespective of differences in strobilation or rearing temperatures. These results indicate that the mechanism of sex determination of Aurelia sp.1. is not influenced by prevailing temperature regimes. A comparison of variability in terms of sexual plasticity of A urelia sp.1 with that of Hydrozoa and Anthozoa suggests that species characterized by a free-swimming medusa life stage have a high dispersal potential, which probably results in a lower rate of sex reversal.

Sex Determination in the Near Threatened Guadalupe Fur Seal: Molecular Markers and Their Potential Applications

The determination of sex by simple observation is often difficult in marine mammals, even in sexually dimorphic species. Moreover, there is often the practical necessity to determine sex in samples that have been collected without handling or observing the subjects. In these cases, a molecular assay for sex determination is required. We adapted an assay that targets the zinc-finger region and is based on a single primer pair, to the Guadalupe fur seal, a near threatened species that has a very limited breeding range. First, we validated the assay with a sample of pups in which we determined the sex by direct observation during handling;second, we compared it to a more recent assay, based on two different primer pairs and, finally, we evaluated the effect of DNA quantity on its reliability. The assay that we tested produced excellent results and was more reliable than the other one based on two primers. Reliable results were also obtained when only remarkably small quantities of DNA were amplified. These results show the potential use of this molecular assay in case of non-invasive sampling, an overly common situation when dealing with species of problematic conservation status.

Problems of Birds Sex Determination

Sex determination system in birds is characterized by a homo-(Neognatae) and heteromorphic (Paleognatae) sex chromosomes. Heterogametic sex is female (ZZ/ZW system). DMRT1 gene is a gene regarded as a main male sex determining factor in this group of animals. The question remains about the participation of other factors (HEMOGEN, AMH etc.) in appearance of testis, and the role of steroid hormones in formation of ovaries. Complete sex inversion is not typical for species with genotypic sex determination (GSD), although the effect of estrogen metabolites is noted for birds. For birds epigenetic mechanisms of regulation (methylation of DNA and non-coding RNA) have been described for sex controlling genes such as CYP19A1 and DMRT1.

Sturgeon (Acipenser gueldenstaedtii) Acclimatization to Optimal Growth Conditions—A Case Study of Adaptation, Nutrition, Reproduction, and Sex Determination

The development of new agricultural industries is an important challenge for a region’s economic establishment. The connection between a research department at MIGAL Institute and biotechnology in aquaculture is an example (case study) of the interrelationships that also exist in other departments. This article describes the research conducted by the institute supporting the development of the aquaculture industry in Israel through the introduction of a relatively new fish species—the Russian sturgeon (Acipenser gueldenstaedtii), with the relevant information to be used for acclimatization to aquaculture conditions in northern Israel. Many aspects related to bringing a new species to Israel and working with this new industry to study its adaptation to existing conditions in the north of the country influenced the economic success of this new type of precision agriculture. This article describes the research on various aspects affecting the successful acclimatization of Russian sturgeon in northern Israel: introduction, food development, reproduction, hormone systems that control reproduction and growth, gonadal development, and molecular markers for sex determination, differentiation, and economic assessment of caviar production.

Sex Chromatin in Peripheral Blood Neutrophils and Sex Determination

The objective of this paper is to review in what forms sex chromatin can appear in peripheral blood neutrophils and how sex determination can be done using sex chromatin appendages. Sex chromatin is an approximately 1 micron clump of chromatin seen usually at the periphery of female nuclei in certain tissues and called “Barr body” and as a drumstick in polymor phonuclear neutrophils nuclei in the blood smears. Sex chromatin is derived from one of the two X chromosomes in the female which replicates its deoxyribonucleic acid much later than the other and is thus positively heteropyknotic. In 1954, Davidson and Smith were the first to identify and report the presence of neutrophil drumsticks and nonspecific appendages and their differences in sexes. The inactive X chromosome in neutrophils appears in one of the five forms: drumsticks, racquet forms, sessile nodules, small clubs and minor lobes. Only drumstick appendage is sex-specific and considered for sex diagnosis. For sex determination, drumsticks are significantly higher in females than in males (p < 0.001).

红带锥蝽性别决定基因doublesex的克隆及表达分析

Doublesex是昆虫性别决定级联通路上的关键基因,其在控制昆虫性别分化中起着重要的作用。本研究旨在克隆、鉴定美洲锥虫病重要媒介红带锥蝽(Triatoma rubrofasciata)的性别决定基因doublesex(Trdsx),分析其不同发育时期的表达谱。基于红带锥蝽基因组数据库,比对Trdsx相关片段,分别以雌雄红带锥蝽cDNA为模板,采用PCR方法克隆Trdsx基因,利用生物信息软件对所得序列进行结构域预测、氨基酸序列比对和进化树分析。根据Trdsx特异性表达引物,利用real time RT-PCR方法研究其在红带锥蝽不同发育时期及雌雄个体的表达谱。从红带锥蝽雌、雄成虫中克隆获得一个雌、雄共有的Trdsx cDNA全长开放性读码框序列,命名为TrdsxB。TrdsxB长度为948 nt,编码315个氨基酸。结构域分析发现TrdsxB包含锌离子结合序列、具有保守的锌螯合残基C2H2C4的DM结构域和参与泛素介导的蛋白水解序列的UBA结构域。TrdsxB在卵中高表达,随后逐渐减少,在四期若虫降至最低,之后再次升高。TrdsxB雌、雄锥蝽中存在表达差异。本研究获得了红带锥蝽性别决定基因doublesex(TrdsxB)的全长开放性读码框序列,TrdsxB具有保守的结构域,其在雌、雄锥蝽体内存在表达差异。本研究结果将为红带锥蝽的性别调控机制和生物防制研究提供理论基础。

哺乳动物性别决定与性别控制研究进展

哺乳动物性别决定是一个由许多基因参与的级联调控过程。研究发现,参与哺乳动物性别决定的基因很多,比如Sry、Sox9、MIS、SF-1、Zfy等。通过研究哺乳动物的性别决定相关基因,结合现代分子生物学技术,如RNA干扰技术和基因工程技术等,有望从基因层面实现人为控制动物后代性别的目标。本文主要对哺乳动物性别决定相关基因和性别控制技术进行了综述,以期为畜牧生产中家畜的性别控制提供参考。

TLR7和TLR8在家畜性控技术中的研究进展

哺乳动物性别控制(sex control)技术在畜牧生产中有着重要的意义,在畜牧生产实践中,通过性别控制技术人类可以获得预想性别的优秀家畜后代,从而使家畜的泌乳性能、繁殖性能和生产性能发挥到最大。目前,传统的哺乳动物性别控制技术主要分为两类:受精前X/Y精子分离技术和受精后早期胚胎性别鉴定。这些方法都有各自的特点和局限性,如处理时间长、分离效率低、对精子活力和胚胎发育有损伤等,因此,这些技术有待进一步优化和完善。本文总结并分析了哺乳动物的传统性控技术,简述其各自特点及存在的问题。此外,利用激活Toll样受体7和Toll样受体8分离X/Y精子的新技术引起了广泛关注,与传统方法相比,该方法无需添加外源药物,具有高效、快速、无毒副作用等优点。尽管该技术已经成功应用于小鼠、羊、牛等动物的性别控制中,但在大规模应用上仍有一些问题需要进一步解决。本文总结了该技术的研究进展,分析了目前将该方法应用于实际生产中的可行性,并对该技术未来的发展趋势进行展望。

先天性巨结肠患儿术后肠道组织中CAD、SOX2的表达水平及其临床意义

目的探讨先天性巨结肠(HD)患儿术后肠道组织蛋白酶D(CAD)、性别决定区Y框蛋白2(SOX2)的表达水平及其临床意义。方法选取2015年5月至2019年3月郑州大学第一附属医院收治的56例HD患儿结肠组织(HD组)和23例因肠道梗阻或穿孔实施造瘘手术获取的结肠组织标本(对照组)。采用免疫组化、Western-blot技术检测结肠组织中CAD、SOX2的表达水平,并采用Pearson线性相关分析法分析CAD、SOX2表达与病变肠段肌间神经丛直径、神经节细胞数的关系。结果HD患儿的狭窄段组织中CAD蛋白、SOX2蛋白阳性表达率分别为7.14%、12.50%,移行段肠组织中CAD蛋白、SOX2蛋白阳性表达率分别为32.14%、35.71%,明显低于对照组的78.26%、82.61%,而狭窄段组织中CAD蛋白、SOX2蛋白阳性表达率明显低于移行段,差异均有统计学意义(P<0.05);HD患儿的狭窄段肠组织中肌间神经丛直径、神经节细胞数目分别为(30.66±5.14)μm、(0.83±0.24)个/视野,移行段肠组织中肌间神经丛直径、神经节细胞数目分别为(31.20±5.83)μm、(1.94±0.56)个/视野,明显短(少)于对照组的(54.29±8.50)μm、(5.40±1.84)个/视野,狭窄段肠组织中肌间神经丛直径、神经节细胞数目明显短(少)于移行段,差异均有统计学意义(P<0.05);Pearson相关分析结果显示,HD患儿狭窄段、移行段肠组织中肌间神经丛直径、神经节细胞数目与CAD蛋白、SOX2蛋白表达强度均呈显著正相关(P<0.05)。结论HD患儿病变结肠组织中CAD蛋白、SOX2蛋白的表达强度下调,可能与肌间神经丛直径、神经节细胞数目的减少有关。

电动汽车充/换电装置的选址定容改建优化策略

针对电动汽车在快速发展中面临的问题,在改建现有设施的思路下,以现有的加油站和停车场作为候选地点,基于M/M/a排队系统,通过构建充/换电装置选址定容模型,将需求分配和充电站定容融入遗传算法进行仿真求解。优化的目标是最小化换电站建设总成本、充电站建设总成本、未覆盖需求的总惩罚成本以及用户排队时间总成本之和。在算例中对单位惩罚成本、充电桩功率、覆盖范围、单位换电站建设成本进行敏感性分析。结果表明:在公共充电站内安装的充电桩并不是功率越大越好,应同时兼顾经济性和适用性。同时,政府可以采用补贴等形式鼓励企业建设换电站。

SOX7靶向ERK1/2/PD-L1通路抑制结直肠癌血管生成

目的探讨性别决定区Y框蛋白7(SOX7)对结直肠癌血管生成的影响及潜在作用机制。方法应用免疫荧光检测结直肠癌患者组织样本中SOX7表达水平,之后通过裸鼠、转染SOX7 mimic的人结直肠癌细胞系SW480细胞和人脐静脉内皮细胞(HUVEC)共培养进一步研究。用Western-blot验证SOX7与ERK1/2/PD-L1对结直肠癌细胞的相关蛋白表达的影响。用CCK8检测SOX7与ERK1/2/PD-L1对HUVEC增殖的影响。通过体外内皮细胞成管实验测定SOX7与ERK1/2/PD-L1对肿瘤血管生成的影响。结果SOX7在人结直肠癌组织中表达被抑制(P<0.01),同时SOX7的过表达抑制了小鼠体内肿瘤生长(P<0.01)。SW480细胞中SOX7的过表达抑制了ERK1/2、c-Jun的表达,并在ERK1/2的激动剂Senkyunolide I的作用下上调了SW480细胞的ERK1/2、c-Jun蛋白表达(P<0.01),逆转了SOX7对SW480细胞中ERK1/2、c-Jun蛋白表达的影响(P<0.01)。HUVEC中SOX7抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,Senkyunolide I上调了HUVEC的PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,并逆转了SOX7对HUVEC中上述相关蛋白表达的影响(P<0.01)。PD-1/PD-L1 Inhibitor 3抑制了PD-L1、V-EGFR2、p-PI3K、HIF-1α的蛋白表达,SOX7过表达在PD-1/PD-L1 Inhibitor 3的影响下并没有表现出抑制作用。CCK8实验结果显示SOX7过表达显著抑制了HUVEC的增殖能力,Senkyunolide I作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显上升,PD-1/PD-L1 Inhibitor 3作用下的两组HUVEC增殖能力较SOX7 NC组与SOX7 mimic组明显下降,以上均有明显统计学差异(P<0.01)。成管实验结果显示SOX7过表达抑制了HUVEC的血管生成,Senkyunolide I强烈加速了血管生成,而PD-1/PD-L1 Inhibitor 3血管生成则被显著抑制,以上均有明显统计学差异(P<0.01)。结论SOX7通过ERK1/2/PD-L1通路抑制结直肠肿瘤的增殖和血管生成,SOX7可能是晚期CRC患者临床治疗中潜在的抗血管生成靶点。

哺乳动物初级性别决定机制研究进展

哺乳动物的性别决定是发生在胚胎发育早期的一个重要事件,是动物繁殖潜力形成的关键环节。近年来,随着高通量测序技术的发展,单细胞水平转录组、蛋白组和空间转录组等检测方法快速更新迭代,细胞谱系追踪和基因精准操控等技术取得突破,对哺乳动物性别决定和性腺发育的机制研究取得了显著进展。伴随畜禽养殖对特定性别动物需求的持续增加,基于性别决定机制开发新型性别控制技术的研究也取得了一系列的突破。随着动物福利要求升高,出生后的性别控制技术应用受限增加,性别决定阶段进行性别控制的技术在畜禽生产领域展现了巨大的应用潜力。作者通过分析性别决定相关的经典实验,结合遗传缺陷病例,总结了哺乳动物性别决定的调控网络,并通过对性别决定相关基因表达操纵试验中出现的性别逆转现象和性别特异性分化过程的研究,串联起整个哺乳动物初级性别决定的调控网络;追踪了基于性别决定机制、利用基因编辑技术而开发的性别控制技术的进展和发展趋势,力求通过总结近年来对哺乳动物性别决定机制的研究进展,为创新家畜性别控制技术提供理论参考。

硬骨鱼类性别调控对大刺鳅性逆转的启示

鱼类的性别控制受遗传性别决定和环境性别决定两种机制的影响,对水产养殖具有重要意义。与其他脊椎动物相比,鱼类在性别分化和性腺发育方面可塑性强。硬骨鱼类雌雄个体的大小和生长速度存在显著的两性差异,培育和养殖体型大、生长速度快的单性群体有利于提高经济效益。大刺鳅是一种具有高营养价值和经济价值的淡水养殖鱼类,雄性个体比雌性生长速度更快、体型更大,因此培育全雄大刺鳅的经济效益更高。野生大刺鳅雌雄比例约为1∶1,但在人工养殖条件下易出现高雌性率的现象。目前尚不清楚何种因素引起大刺鳅的雌性化,也未见成功诱导大刺鳅生理雌鱼发生性逆转的报道,这极大制约了大刺鳅养殖产业的发展和经济效益的提高。目前关于大刺鳅的研究主要集中在形态学、生理学、繁殖生物学和遗传多样性等方面,关于大刺鳅性别决定与性别分化的研究较为薄弱,需从其他硬骨鱼类的性别决定机制、性别调控方式和性别控制育种技术中发掘思路。基于硬骨鱼类的性别决定机制,综述了硬骨鱼类性别控制的几种方式,主要包括遗传因素调控(如性染色体、常染色体上的性别决定基因)和环境因素调控(如温度、外源激素、饵料等)。旨在为解析人工养殖大刺鳅高雌化的原因及潜在作用机制提供思路,同时为大刺鳅单性种群的繁殖与保持、优良品种的选育和种质资源保护提供重要的理论依据,以期更好地了解大刺鳅性腺发育和生殖机制,解决现阶段养殖大刺鳅雄性率低的瓶颈,为大刺鳅养殖业可持续发展提供保障。