foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes.The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system,where most males initially develop juvenile ovaries before committing to male fate.In species with chromosomal sex determination,gonadal somatic cells are recognized as key determinants of germ cell fate.Notably,the loss of germ cells in zebrafish leads to masculinization,implying that germ cells harbor an intrinsic feminization signal.However,the specific signal triggering oogenesis in zebrafish remains unclear.In the present study,we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells.Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males.Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis,leading to premature spermatogenesis during early gonadal development.Furthermore,while mutation of the pro-male gene dmrt1 led to fertile female differentiation,simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis,with a large proportion of germ cells arrested as germline stem cells,highlighting the crucial role of foxl2l in oogenesis.Overall,this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

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.

Ultrastructure of Oogenesis in Osmunda cinnamomea var. asiatica

The ultrastructure of oogenesis in the fern Osmunda cinnamomea L. var. asiatica Fernald has been studied by electron microscopy. During oogenesis, numerous vesicles not only moved towards the periphery, but also were arranged in line along the inside of plasmalemma, and in addition aggregated outside the plasmalemma by exocytosis. They released or excreted osmiophilic material. It was observed that a few vesicles containing lamellar osmiophilic material situated closely along the plasmalemma, seemed to break open. Simultaneously, a separation cavity between egg cell and archegonium wall formed. Its width was broader than the other advanced ferns reported previously, and an extra egg membrane occurred outside around the plasmalemma of the egg, its thickness being greater than in Pteridium and Dryopteris. Amyloplasts around the nucleus were filled with large triangular semicircular or subelliptical starch grains, but as the egg matured they progressively decreased. Nucleus was large and flattened, and paired membrane in two to three couples laid within the nucleus, close and parallel to the nuclear membrane. No nuclear evagination was observed. Mitochondria seemed to have been undeveloped, but finally recovered normally.

Dynamic Distribution of Glycoconjugates During Oogenesis of Atractomorpha sinensis

The dynamic distribution of three different glycoconjugates in oocytes and follicle cells during the oogenesis of Atractomorpha sinensis were detected using biotin-labeled Peanut Agglutinin （PNA）, Soy Bean Agglutinin （SBA） and Ulex Europaeus Agglutinin I （UEA-I） lectins. The results showed that during oogenesis there was no distribution of the UEA-I receptor. The receptors of PNA and SBA were found to be dependent on developmental stage and present different distribution patterns accordingly. The binding sites of the two lectins indicated the presence of different sugars （PNA for Galβ1,3GalNAc and SBA for GalNAc） and showed considerable variation during oogenesis. PNA and SBA receptors first appeared at the oocyte growth phase, the PNA receptors then disappeared gradually and the SBA receptors exhibited the greatest expression. At the early phase of yolk formation, PNA and SBA receptors were located just at the brim of ooplasm, which was the region of vitellin formation. However at the later phase of yolk formation, neither of the two receptors was detected. In the mature egg, PNA and SBA receptors were distributed again on the vitellin membrane and the eggshell. The two receptors were also widely distributed in the follicular cells, showing similar distribution variation to the oocytes. The results indicate that the change and modification of the two receptors may be greatly related to the growth of oocytes, the preparation for yolk formation, the differentiation of follicular cells and the maturation of oocytes. The glycoconjugates on the vitellin membrane probably play important roles in sperm and egg recognition. The two lectins bound moderately or strongly to the eggshell, which indicates that the eggshell of A. sinensis contains the GalNAc and GalβGalNAc glycoproteins.

Study on the Ultrastructural of Oogenesis of Bullacta exarata

The ultrastructure of oogenesis of Bullacta exarata was studied with transmission electron microscope.The result showed that the oogenesis of Bullacta exarata could be divided into four stages,for example,oogonium,early vitellogenic oocyte,mid vitellogenic oocyte and end vitellogenic oocyte.Besides a large and round nucleus,oogonium mainly contains mitochondria,sacs of golgi and microvillie appears in this stage.At the early stage of vitellogenesis,the organelles are developed,and a great deal of nuages like materials appear in oocyte cytoplasm.At the mid stage of vitellogenesis,the nucleus forms pseudopodia liked extension.The organelles including mitochondria,golgi complex and endoplasmic reticulum are well developed,and lots of yolk granule and lipid droplet are formed in the cytoplasm.The exchange of material between oocytes and follicle cells is intense in this stage.At the end stage of vitellogenesis,the oocytes contain fewer numbers of the organelles.The changes of structure and significance in oocytes,and the exchange of material between oocytes and follicle cells are also discussed.

Identification of Z-OTU protein during zebrafish oogenesis and early embryogenesis

Zebrafish（Danio rerio） Z-OTU,containing OTU and TUDOR domains,was predicted to be a member of OTU-related protease,a family of the deubiquitylating enzymes（DUBs）.A previous report from our laboratory clearly describes the expression patterns of z-otu mRNA.Here,we characterized the Z-OTU protein during zebrafish oogenesis and early embryogenesis.After prokaryotic expression,the recombinant protein of the OTU domain and GST was purified and injected into rabbits to obtain the polyclonal antibody-anti-Z-OTU,which was used for immunohistochemistry in zebrafish ovaries and embryos.Interestingly,obvious differences existed between the expression patterns of z-otu mRNA and its protein during oogenesis and early embryogenesis.In stage I oocytes,z-otu mRNA was detected in cytoplasm while its protein existed in the germinal vesicle.In addition,its protein was distributed during entire oogenesis,while mRNA was not detected in oocytes at stage IV or mature oocytes.The z-otu mRNA disappeared after midblastula transition（MBT） and its protein gradually decreased after this stage.We inferred that Z-OTU protein,like other OTU-related protease with DUB activity,was required for germinal vesicle breakdown of oocytes during meiosis,germinal vesicle migration,and embryo cleavage maintenance.

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.

Oogenesis in summer females of the rice water weevil,Lissorhoptrus oryzophilus Kuschel (Coleoptera: Curculionidae),in southern Zhejiang,China

The rice water weevil, Lissorhoptrus oryzophilus Kuschel, has two generations in southern Zhejiang, China. To determine oogenesis in first-generation females （summer females） and its relations to temperature, females were collected from a rice field in early and mid-July and reared on young rice plants at 28, 31 and 34 ℃ in the laboratory. Percentage of females having oocytes, number of oocytes of different stages （stage-Ⅰ, from early previtellogenesis to middle vitellogenesis; stage-Ⅱ, late vitellogenesis; and matute-oocyte stage）, and length of ovarioles were determined every 10 d of feeding. At each temperature, oogenesis took place in over 40% of females after 20-40 d of feeding, but only 0.0-3.3 stage-Ⅰ, 0.0-0.8 stage-Ⅱ and 0.0-1.1 mature oocytes were observed at each observation date. Temperature had significant effect on number of stage-Ⅰ oocytes but not on number of stage-Ⅱ and mature oocytes in early July females; temperature had no significant effect on number of oocytes of either stage in mid-July females. Conclusively, in southern Zhejiang, summer L. oryzophilus females have great potential to become reproductive on rice, but their oogenesis activity is very low, with the overall procedures little affected by temperature.

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.

Lethal（2）giant larvae is required in the follicle cells for formation of the initial AP asymmetry and the oocyte polarity during Drosophila oogenesis

The intricately regulated differentiation of the somatic follicle cell lineages into distinct subpopulations with specific functions plays an essential role in Drosophila egg development. At early oogenesis, induction of the stalk cells generates the first anteroposterior （AP） asymmetry in the egg chamber by inducing the posterior localization of the oocyte. Later, the properly specified posterior follicle cells signal to polarize the oocyte along the AP and dorsoventral （DV） axes at mid-oogenesis. Here, we show that lethal（2）giant larvae （lgl）, a Drosophila tumor suppressor gene, is required in the follicle cells for the differentiation of both stalk cells and posterior follicle cells. Loss-of-function mutations in lgl cause oocyte mispositioning in the younger one of the fused chambers, due to lack of the stalk. Removal oflgl function from the posterior follicle cells using the FLP/FRT system results in loss of the oocyte polarity that is elicited by the failure of those posterior cells to differentiate normally. Thus, we provide the first demonstration that lgl is implicated in the formation of the initial AP asymmetry and the patterning of the AP and DV axes in the oocyte by acting in the specification of a subset of somatic follicle cells.

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 synthesis and RACE-PCR. The full-length cDNA is 1870 bp. Its 5' untranslated region is 210 bp, followed by an open reading frame of 990 bp, which has the typical vertebrate initiator codon of AN-NATG. The open reading frame encodes a protein with 329 amino acids. It has 670 bp of 3' untranslated 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 previtellogenic 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.

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.

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.