Halloween genes AhCYP307A2 and AhCYP314A1 modulate last instar larva–pupa–adult transition, ovarian development and oogenesis in Agasicles hygrophila (Coleoptera:Chrysomelidae)

In insects,ecdysteroids are synthesized by genes of the Halloween family and play important roles in several key developmental events,including molting and metamorphosis.However,the roles of these genes in Agasicles hygrophila are still largely unknown.In this study,the expression patterns of the two Halloween genes AhCYP307A2 and AhCYP314A1 were determined by quantitative PCR(qPCR)at different developmental stages.Moreover,the functions of these two genes were explored using RNA interference(RNAi),and ovarian development was observed by dissecting the ovaries of A.hygrophila females.The qPCR results showed that AhCYP307A2 and AhCYP314A1 were highly expressed in last instar larvae and in adult females.In addition,AhCYP307A2 was also highly expressed in eggs and pupae but was markedly lower than in third-instar larvae and females.The RNAi results showed that the injection of dsAhCYP307A2 or dsAhCYP314A1 markedly inhibited their expression and the transcription levels of three related AhVgs.Knockdown of AhCYP307A2 or AhCYP314A1 significantly inhibited larval molting,impaired last instar larva–pupa–adult transition,delayed ovarian development,and stopped egg production(i.e.,no eggs were laid).These results indicate that AhCYP307A2 and AhCYP314A1 play important regulatory roles in last instar larva–pupa–adult transition and reproduction in A.hygrophila.

Ultrastructural study of oogenesis in Xiamen amphioxus

Using electron microscopic technique, ultrastructural characters of the oogonia and oocyte at the dif ferent phases of amphioxus are observed in detail in the present study.The squeezed out nucleolus, nucleolus-like bodies and yolk nucleus at the side of nucleus are the characters in the early egg cells of the first and secondary phase.The nucleus and cytoplasm of oocyte change obviously their morphology ad structure from large growth stage to mature stage (from Stage Ⅲ to Stage V). As a result, the dense distribution of nuclear pore and the exten sion and depression of nuclear envelope are observable.Mitochondria,Golgi complex,rough endoplasm reticular and annulate lamellae,and others in the cytoplasm join in the formation of yolk granular.Their morphology and number also change correspondingly with the development and maturation of the oocyte.These results will provide a whole base for the reproductive physiology and artificial propagation as well as the resource management of amphioxus.

Somatolactin Transcription during Oogenesis in Female Blue Gourami (<i>Trichogaster trichopterus</i>)

Somatolactin (SL), a specific pituitary hormone belonging to the prolactin (PRL) super family, is involved in background adaptation, osmoregulation, reproduction and fatty acid metabolism. The goal of this study was to examine the gene transcription of SL changes in the ovary of blue gourami females (Trichogaster trichopterus) during oogenesis by quantitative real-time polymerase chain reaction (Real-Time PCR). Somatolactin in the pituitary was higher in females at low vitellogenesis compared to females with oocytes in maturation, and the difference was significant (p < 0.05). No significant differences were found in mRNA levels between low and high vitellogenesis, and high vitellogenesis and maturation. The findings of this and previous studies demonstrate that SL, growth hormone (GH) and PRL are involved in oogenesis in blue gourami;however, considerably more studies are required in order to separate the functions of these hormones.

The Expression of vasa Gene during Zebrafish (Danio rerio) Oogenesis

vasa gene expression pattern during oogenesis of zebrafish was examined usingin situ hybridization and fluorescent quantitative RT-PCR. During zebrafish oogensis,vasa mRNA is expressed strongly and uniformly distributed in the cytoplasm in stage II oocytes, followed by a distribution among vacuome in stage III. Later in stage IV and V,vasa mRNA is enriched at the cortex and finally localized at the cortex. The fluorescent quantitative RT-PCR shows that the quantity ofvasa mRNA decreases from stage II to stage III, but remains relatively invariable from stage III to stage V. The observed differences invasa mRNA expression in the different stages of zebrafish oogenesis suggest thatvasa gene plays an important role during oogenesis. Key words vasa - zebrafish - fluorescent quantitative RT-PCR - oogenesis CLC number Q 952.6 Foundation item: Supported by the National Natural Science Foundation of China (30370744, 30150005)Biography: XIANG Fang (1979-), male, Master candidate, research direction: molecular development of animals.

IncR26319/miR-2834/EndophilinA axis regulates oogenesis of the silkworm,Bombyx mori

Oocyte maturation is critical for insect reproduction.Vitellogenesis,the timely production and uptake of vitellogenin(Vg),is crucial for female fecundity.Vg is synthesized in fat body and absorbed by the oocytes through endocytosis during insect oogenesis.In the silkworm,Bombyx mori,we discovered that a nucleus-enriched long-noncoding RNA(lncRNA)lncR26319 regulates Endophilin A(EndoA)-a member of the endophilin family of endocytic proteins-through competitive binding to miR-2834.The lncR26319-miR-2834-EndoA axis was required for Vg endocytosis in the silkworm;loss of EndoA or overexpression ofmiR-2834significantlyreduced eggnumbers invirginmoths.Inaddition,accumulation of miR-2834 resulted in pupal and adult deformation and reduced fecundity in females.The expression of Vg,30-kDa(30K)protein,and egg-specific protein(Esp)decreased after knockdown of EndoA or overexpression of miR-2834,while knockdown of miR-2834 had an opposite effect on the expression of Vg,30K protein gene,and Esp.These results suggest that the lncR26319-miR-2834-EndoA axis contributes to the endocytic activity in theVguptake and leads to the normal progression of oogenesis in the silkworm.Thus,miR-2834 and EndoA are crucial for female reproduction and could be potential targets for new pest management strategies in lepidopterans.

Ultrastructure of Oogenesis in Dryopteris crassirhizoma Nakai

Abstract: The ultrastructure of oogenesis in Dryopteris crassirhizoma Nakai has been investigated using transmission electron microscopy. The nucleus in the young egg is rounded with an uneven outline. As it develops, it becomes amoeboid and extends nuclear protrusions that are not only sac-like nuclear evaginations like those often seen in the oogenesis of other ferns, but also mushroom-like and finger-like, with an opening at their end allowing the nucleolus material to flow out from the openings. This has not been observed previously. The nuclear protrusions differ from Dryopteris filix-mas (L.) Schott. in the absence of sheets of nuclear membrane in the form of a closed ring. As the egg matures, the nucleus transforms into a tuber-like structure with a smooth surface, lying transversely in the egg cell. In the immature egg, vesicles almost encircle the nucleus twice and are most remarkable. In the maturing egg, the vesicles are distributed at the periphery, except for at the top of the egg, and affect the formation of the separation cavity and extra egg membrane. Simultaneously, vesicles from the venter canal cell move to the egg and take part in the formation of separation cavity and extra egg membrane. In the mature egg, a large number of small vesicles containing fragments of lamellae or osmiophilic material emerge from the cytoplasm. The origin of these vesicles is obscure. Irregular plastids containing a cylindrical starch grain dedifferentiated progressively. Mitochondria seem to have been undeveloped during the process, but return to normal at later stages of oogenesis. There is a high frequency of ribosomes in the mature egg. Microtubules, rarely seen in the eggs of D. filix-mas (L.) Schott. and Pteridium aquilinum (L.) Kuhn, have been observed inside the plasmalemma of the maturing egg in D. crassirhizoma.

Differential expression and characterization analysis of a new gene with WD domains in fish oogenesis

A new gene with WD domains is cloned and characterized according to its differential transcription and expression between previtellogenic oocytes (phase I oocytes) and fully-grown oocytes (phase V oocytes) from natural gynogenetic silver crucian carp (Carassius auratus gibelio)by using the combinative methods of suppressive subtraction hybridization, SMART cDNA synthelowed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of Antranslated region and an AATAAA polyadenylation signal. Because it has 92% homology to STRAP (serine-threonine kinase receptor-associated protein), a recently reported gene, we named it FSTRAP (fish STRAP). Virtual Northern blotting indicated that the FSTRAP was transcribed in fully-grown oocytes (phase V oocytes), but not in previtellogenic oocytes (phase I oocytes).RT-PCR analysis showed that FSTRAP was transcribed in brain, heart, kidney, muscle, ovary,spleen and testis, but not in liver. And its mRNA could be detected in the oocytes from phase II to phase V. Western blotting also showed that FSTRAP protein could be detected in brain, heart,kidney, muscle, ovary, spleen and testis except liver. Results of Western blotting on various oocytes were also similar to the RT-PCR data. FSTRAP protein was not expressed in the previtelIogenic oocytes. Its expression initiated from phase II oocytes after vitellogenesis, and was consistent with the mRNA transcription.

Dual roles of juvenile hormone signaling during early oogenesis in Drosophila

Juvenile hormone(JH)signaling plays crucial roles in insect metamorphosis and reproduction.Function of JH signaling in germline stem cells(GSCs)remains largely unknown.Here,we found that the number of GSCs significantly declined in the ovaries of Met,Gee and JHAMT mutants.Then we inhibited JH signaling in selected cell types of ovaries by expressing Met and Gee or Kr-hl double-stranded RNAs(dsRNAs)using different Gal4 drivers.Blocking of JH signaling in muscle cells has no effect on GSC numbers.Blocking of JH signaling in cap cells reduced GSCs cells.Inductive expression of Met and Gee dsRNA but not Kr-hl by Nos-Gal4 increased GSC cells.These results indicate that JH signaling plays an important role in GSC maintenance.

Molecular and functional characterization of the American cockroach, Periplaneta americana, Rab5： the first exopterygotan low molecular weight ovarian GTPase during oogenesis

The small Rab GTPases are key regulators of membrane vesicle trafficking. Ovaries of Periplaneta americana （Linnaeus） （Blattodea： Blattidae） have small molecular weight GTP/ATP-binding proteins during early and late vitellogenic periods of oogenesis. However, the identification and characterization of the detected proteins have not been yet reported. Herein, we cloned a cDNA encoding Rab5 from the American cockroach, P. americana, ovaries （PamRab5）. It comprises 796 bp, encoding a protein of 213 amino acid residues with a predicted molecular weight of 23.5 kDa. PamRab5 exists as a single-copy gene in the P. americana genome, as revealed by Southern blot analysis. An approximate 2.6 kb ovarian mRNA was transcribed especially at high levels in the previtellogenic ovaries, detected by Northern blot analysis. The muscle and head tissues also showed high levels of PamRab5 transcript. PamRab5 protein was localized, via immunofluorescence labeling, to germline-derived cells of the oocytes, very early during oocyte differentiation. Immunoblotting detected a ~25 kDa signal as a membrane-associated form revealed after application of detergent in the extraction buffer, and 23 kDa as a cytosolic form consistent with the predicted molecular weight from amino acid sequence in different tissues including ovary, muscles and head. The PamRab5 during late vitellogenic periods is required to regulate the endocytotic machinery during oogenesis in this cockroach. This is the first report on Rab5 from a hemimetabolan, and presents an inaugural step in probing the molecular premises of insect oocyte endocytotic trafficking important for oogenesis and embryonic development.

Specific Expression of ZP3 During Mouse Oogenesis by in situ Hybridization

The zona pellucida of murine ovum is composed of three sulfatel glycoproteins designated as ZP1, ZP2 and ZP3.During oogenesis these three glycoproteins are coordinately synthesized and secreted to form zona pellucida.Among them ZP3 has been operated as a sperm receptor and acrosomal reaction inducer.

Caenorhabditis elegans homologue of Fam210 is required for oogenesis and reproduction

Mitochondria are the central hub for many metabolic processes,including the citric acid cycle,oxidative phosphorylation,and fatty acid oxidation.Recent studies have identified a new mitochondrial protein family,Fam210,that regulates bone metabolism and red cell development in vertebrates.The model organism Caenorhabditis elegans has a Fam210 gene,y56a3a.22,but it lacks both bones and red blood cells.In this study,we report that Y56A3A.22 plays a crucial role in regulating mitochondrial protein homeostasis and reproduction.The nematode y56a3a.22 is expressed in various tissues,including the intestine,muscle,hypodermis,and germline,and its encoded protein is predominantly localized in mitochondria.y56a3a.22 deletion mutants are sterile owing to impaired oogenesis.Loss of Y56A3A.22 induced mitochondrial unfolded protein response(UPRmt),which is mediated through the ATFS-1-dependent pathway,in tissues such as the intestine,germline,hypodermis,and vulval muscle.We further show that infertility and UPRmt induces by Y56A3A.22 deficiency are not attributed to systemic iron deficiency.Together,our study reveals an important role of Y56A3A.22 in regulating mitochondrial protein homeostasis and oogenesis and provides a new genetic tool for exploring the mechanisms regulating mitochondrial metabolism and reproduction as well as the fundamental role of the Fam210 family.

New Understandings on Folliculogenesis/Oogenesis Regulation in Mouse as Revealed by Conditional Knockout

In comparison to conventional knockout technology and in vitro research methods, conditional gene knockout has remarkable advantages. In the past decade, especially during the past five years, conditional knockout approaches have been used to study the regulation of folliculogenesis, follicle growth, oocyte maturation and other major reproductive events. In this review, we summarize the recent findings about folliculogenesis/oogenesis regulation, including the functions of four signaling cascades or glycoprotein domains that have been extensively studied by conditional gene deletion. Several other still fragmented areas of related work are introduced which are awaiting clarification. We have also discussed the future potential of this technology in clarifying gene functions in reproductive biology.

Cortisol safeguards oogenesis by promoting follicular cell survival

The role of glucocorticoids in oogenesis remains to be elucidated. cyp11c1 encodes the key enzyme involved in the synthesis of cortisol, the major glucocorticoid in teleosts. In our previous study, we mutated cyp11c1 in tilapia and analyzed its role in spermatogenesis. In this study, we analyzed its role in oogenesis. cyp11c1^(+/-)XX tilapia showed normal ovarian morphology but poor egg quality, as indicated by the mortality of embryos before 3 d post fertilization, which could be partially rescued by the supplement of exogenous cortisol to the mother fish. Transcriptome analyses revealed reduced expression of maternal genes in the eggs of the cyp11c1^(+/-)XX fish. The cyp11c1^(-/-)females showed impaired vitellogenesis and arrested oogenesis due to significantly decreased serum cortisol. Further analyses revealed decreased serum E2 level and expression of amh, an important regulator of follicular cell development, and increased follicular cell apoptosis in the ovaries of cyp11c1^(-/-)XX fish, which could be rescued by supplement of either exogenous cortisol or E2. Luciferase assays revealed a direct regulation of cortisol and E2 on amh transcription via GRs or ESRs. Taken together, our results demonstrate that cortisol safeguards oogenesis by promoting follicular cell survival probably via Amh signaling.

C30F12.4 influences oogenesis, fat metabolism, and lifespan in C. elegans

Reproduction, fat metabolism, and longevity are inter- twined regulatory axes; recent studies in C. elegans have provided evidence that these processes are directly coupled. However, the mechanisms by which they are coupled and the reproductive signals modu- lating fat metabolism and lifaspan are poorly under- stood. Here, we find that an oogenesis-enriched gene, c30PI2.4, is specifically expressed and located in germ cells and early embryos; when the gene is knocked out, oogenesis is disrupted and brood size is decreased. In addition to the reproductive phenotype, we find that the loss of c30f12.4 alters fat metabolism, resulting in decreased fat storage and smaller lipid droplets. Mean- while, c30f12.4 mutant worms display a shortened lifaspan. Our results highlight an important role for c30f12.4 in regulating reproduction, fat homeostasis, and aging in C. elegans, which helps us to better understand the relationship between these processes.

Mitotic chromosome mis-segregation mediated by Separase phosphosite mutation leads to aneuploidy and impaired oogenesis

Aneuploidy is the leading genetic cause of human fertility failure. It is considered results from errors in meiotic and post-zygotic mitotic chromosome segregation. Using a

Review of the impact of heat stress on reproductive performance of sheep

Heat stress significantly impairs reproduction of sheep,and under current climatic conditions is a significant risk to the efficiency of the meat and wool production,with the impact increasing as global temperatures rise.Evidence from field studies and studies conducted using environmental chambers demonstrate the effects of hot temperatures(≥32℃)on components of ewe fertility(oestrus,fertilisation,embryo survival and lambing)are most destructive when experienced from 5 d before until 5 d after oestrus.Temperature controlled studies also demonstrate that ram fertility,as measured by rates of fertilisation and embryo survival,is reduced when mating occurs during the period 14 to 50 d post-heating.However,the contribution of the ram to heat induced reductions in flock fertility is difficult to determine accurately.Based primarily on temperature controlled studies,it is clear that sustained exposure to high temperatures(≥32℃)during pregnancy reduces lamb birthweight and will,therefore,decrease lamb survival under field conditions.It is concluded that both ewe and ram reproduction is affected by relatively modest levels of heat stress(≥32℃)and this is a concern given that a significant proportion of the global sheep population experiences heat stress of this magnitude around mating and during pregnancy.Despite this,strategies to limit the impacts of the climate on the homeothermy,behaviour,resource use and reproduction of extensively grazed sheep are limited,and there is an urgency to improve knowledge and to develop husbandry practices to limit these impacts.

Exploring the reproductive ecology of the tropical semifossorial snake Ninia atrata

Based on histological analyses and field studies,this research describes the reproductive ecology of a population of Ninia atrata snakes inhabiting an oil palm plantation.Furthermore,through a multivariate approach,we explored the main drivers of reproductive output in N.atrata.Results showed that prey abundance and food intake were crucial variables contributing to reproductive output.Multiple linear regression models showed that neonates had high sensitivity(R2=55.29%)to extreme changes in climate,which was strongly related to slug and snail abundance variability and microhabitat quality.Reproductive cycles were markedly different between the sexes,being continuous in males and cyclical in females.Despite this variation,reproductive cycles at the population level were seasonal semi-synchronous.Constant recruitment of neonates all year,multiple clutches,high mating frequency,and continuous sperm production characterized the reproductive phenology of N.atrata.In addition,a significant number of previtellogenic females presented oviductal sperm as well as uterine scars,suggesting a high precocity in the species.The main drivers of reproductive output also differed between the sexes.In females,clutch size and secondary follicle variability were highly related to stomach bolus volume,fat body area,and body mass.In males,height of piles of palm leaves and body mass,rather than intrinsic reproductive traits,were the main drivers of sperm production.Nevertheless,in both cases,the relationship between body mass,prey abundance,and food intake suggests that N.atrata follows the income breeding strategy to compensate for reproductive costs and to maximize fitness.

Identification of chorion genes and RNA interference-mediated functional characterization of chorion-1 in Plutella xylostella

Choriogenesis is the last step of insect oogenesis,a process by which the chorion polypeptides are produced by the follicular cells and deposited on the surface of oocytes in order to provide a highly specialized protective barrier to the embryo.The essential features of chorion genes have yet to be clearly understood in the diamondback moth,Plutella xylostella,a worldwide Lepidoptera pest attacking cruciferous crops and wild plants.In this study,complete sequences for 15 putative chorion genes were identified,and grouped into A and B classes.Phylogenetic analysis revealed that both classes were highly conserved and within each,branches are also species-specific.Chorion genes from each class were located in pairs on scaffolds of the P.xylostella genome,some of which shared the common promoter regulatory region.All chorion genes were highly specifically expressed in the P.xylostella adult females,mostly in the ovary with full yolk,which is a crucial period to build the shells of the eggs.RNAi-based knockdown of chorion-1,which is located on the Px_scaffold 6 alone,although had no effect on yolk deposition,resulted in smaller eggs and sharply reduced hatchability.Additionally,inhibition of PxCho-1 expression caused a less dense arrangement of the columnar layers,reduced exochorion roughness and shorter microvilli.Our study provides the foundation for exploring molecular mechanisms of female reproduction in P.xylostella,and for making use of chorion genes as the potential genetic-based molecular target to better control this economically important pest.

Intracellular compartmentation of CTP synthase in Drosophila

Compartmentation is essential for the localization of biological processes within a eukaryotic cell. ATP synthase localizes to organelles such as mitochondria and chloroplasts. By contrast, little is known about the subcellular distribution of CTP synthase, the critical enzyme in the production of CTP, a high-energy molecule similar to ATP. Here I describe the identification of a novel intracellular structure con- taining CTP synthase, termed the cytoophidium, in Drosophila cells. I find that cytoophidia are present in all major cell types in the ovary and exist in a wide range of tissues such as brain, gut, trachea, testis, accessory gland, salivary gland and lymph gland. In addition, I find CTP synthase-containing cytoophidia in other fruit fly species. The observation of compartmentation of CTP synthase now permits a broad range of questions to be addressed concerning not only the structure and function of cytoophidia but also the organization and regulation of CTP synthesis.

Recent advances in mammalian reproductive biology

Reproductive biology is a uniquely important topic since it is about germ cells, which are central for transmitting genetic information from generation to generation. In this review, we discuss recent advances in mammalian germ cell development,including preimplantation development, fetal germ cell development and postnatal development of oocytes and sperm. We also discuss the etiologies of female and male infertility and describe the emerging technologies for studying reproductive biology such as gene editing and single-cell technologies.