Search Results (52)

High-throughput transcriptomic analyses have identified numerous candidate miRNA–mRNA and long non-coding RNA (lncRNA) regulatory networks in teleosts, but most remain without systematic functional validation or mechanistic definition. Here, by interrogating miRNA–lncRNA networks in rainbow trout (Oncorhynchus mykiss) gonads, we define their roles in meiotic progression and gonadal development. From preliminary screening, we identified lncRNA74687 as a central node and characterised its function. Subcellular localisation showed predominant nuclear enrichment of lncRNA74687 in gonadal cells. Dual-luciferase assays confirmed miR-15a-5p targeting of Cyclin E (CCNE1) and lncRNA74687. Functional studies showed that concurrent overexpression of lncRNA74687 and inhibition of miR-15a-5p synergistically increased the CCNE1 protein to maximal levels. 5-ethynyl-2′-deoxyuridine (EdU) assays showed that knockdown of lncRNA74687 and CCNE1 in rainbow trout gonadal (RTG-2) cells reduced proliferation by 36.4% and 41.2%, respectively (p < 0.05). Immunofluorescence indicated that lncRNA74687 increased Synaptonemal Complex Protein 1 (SYCP1) signalling 6.93-fold in gonadal cells via CCNE1. In vivo, lncRNA74687 knockdown increased miR-15a-5p expression 6.34-fold relative to the wild-type controls (p < 0.01). Transcriptomic profiling revealed broad downregulation of meiosis-related genes in lncRNA74687-deficient gonads, with the strongest reduction in mstrg1 expression, indicating a key role of lncRNA74687 in germ-cell meiotic progression. Together, these data show that lncRNA74687 enhances CCNE1 mRNA and the CCNE1 protein in rainbow trout by competitively binding miR-15a-5p. This lncRNA74687–miR-15a-5p–CCNE1 axis regulates gonadal cell proliferation and meiotic gene expression during gonadal development. Full article

Female gametogenesis is orchestrated by dynamic epigenetic modifications. In mammals, SETDB1, a histone H3K9 methyltransferase, is required for proper meiotic progression and early embryonic development. In Drosophila, the ortholog of SETDB1 plays a critical role in germ cell differentiation, transposon silencing, and the transcriptional repression of specific germline genes during oocyte fate determination. Moreover, Polycomb group (PcG) proteins in both mammals and Drosophila are essential for primary oocyte viability and meiosis, functioning through the silencing of early prophase I genes during later stages of prophase. While the repressive roles of epigenetic regulators in both Drosophila and mammalian oogenesis are well characterized, the functions of epigenetic activators remain less defined. Gene expression is controlled by the opposing activities of PcG and Trithorax group (TrxG) proteins, with the latter constituting a diverse family of chromatin remodelling factors that include H3K4 methyltransferases. In Drosophila, SET domain containing 1 (Set1)—the ortholog of mammalian SETD1A/B—acts as the primary regulator of global H3K4me2/3 levels. Set1 is critical for germline stem cell (GSC) self-renewal, functioning through both cell-autonomous and non-cell-autonomous mechanisms, with its depletion in the germline resulting in a progressive loss of GSC. More recently, Set1 has been implicated in germline cyst differentiation, although the mechanisms underlying this role remain poorly understood due to the complexity of the observed phenotypes. To investigate this, we analyzed ovaries from recently eclosed females in which Set1 and its highly conserved COMPASS partner, absent, small, or homeotic discs 2 (Ash2), were depleted—thus minimizing the confounding effects from GSC loss. We observed striking defects in both oocyte determination and Synaptonemal Complex (SC) integrity in one- to two-day-old females, within otherwise normal egg chambers. Interestingly, while defects in oocyte fate and oocyte–chromatin architecture were partially recovered in older egg chambers, SC integrity remained compromised. These findings suggest a critical window for SC assembly during germline cyst differentiation, after which this assembly cannot occur. Full article

Well-spread pachytene chromosomes are critical for studying the location of meiotic proteins along individual chromosomes. However, producing good spreads in species with large genomes is challenging due to the tangling of pachytene chromosomes. Existing protocols often fail to achieve proper separation of large chromosomes in spreads. Here, we describe in detail an improved protocol that ensures the effective separation of large pachytene chromosomes and demonstrates its suitability for protein immunodetection. To develop the protocol, pollen mother cells at the middle–late pachytene stage from Allium fistulosum, a species with a large genome and chromosomes, were used. The protocol involved three main steps: fixing anthers in Clark’s solution (ethanol–acetic acid, 3:1), digestion in an enzyme mixture, and gentle squashing in 45% acetic acid. A clear ZYP1 signal on all separated chromosomes was observed. The high quality of well-spread pachytene chromosomes obtained with the modified protocol allowed for the easy extraction of individual chromosomes for more precise detection and analysis of the proteins of interest. Full article

Alternative splicing of ddx4 (DEAD-box helicase 4), a key germline marker gene, has been reported to generate sex-specific transcripts in zebrafish gonads. The biological functions and regulatory mechanisms of the ddx4 ovary-specific transcript (ddx4-L) during oogenesis remain unclear. In this study, we found that ddx4-L mutants, in which ddx4-L was specifically deleted, had enlarged ovaries but laid fewer eggs, along with having a lower fertilization rate compared to WT controls. RNA-seq analysis was performed to detect the changes in gene expression between WT and ddx4-L mutant ovaries. A total of 524 upregulated and 610 downregulated DEGs were identified. GO and GSEA enrichment analyses showed that genes involved in fertilization and reproduction biological processes were significantly downregulated. More specifically, we observed a remarkable reduction in Sycp1, a core component of synaptonemal complex, in ddx4-L mutant ovaries at both the mRNA and protein levels. In addition, the expressions of transposon elements, as well as the events of alternative splicing, alternative polyadenylation, and RNA editing, were analyzed based on the RNA-seq data. We found that the deletion of ddx4-L resulted in derepression of DNA transposons in zebrafish ovaries, possibly causing genome instability. In conclusion, our work demonstrates that the ovary-specific ddx4 transcript plays important roles in oocyte meiosis and DNA transposon repression, which extends our understanding of the biological functions and regulatory mechanisms of sex-specific alternative splicing in zebrafish oogenesis and reproduction. Full article

Among eukaryotes, there are many examples of partial genome elimination during ontogenesis. A striking example of this phenomenon is the loss of entire avian chromosomes during meiosis, called a germline-restricted chromosome (GRC). The GRC is absent in somatic tissues but present in germ cells. It has been established that a prophase I male GRC is usually represented by a univalent surrounded by heterochromatin. In the present study, an immunocytochemical analysis of zebra finch spermatocytes was performed to focus on some details of this chromosome’s organization. For the first time, it was shown that a prophase I GRC contains the HORMAD1 protein, which participates in the formation of a full axial element. This GRC axial element has signs of a delay of core protein loading, probably owing to peculiarities of meiotic silencing of chromatin. The presence of repressive marks (H3K9me3 and H3K27me3) and the lack of RNA polymerase II, typically associated with active transcription, indicate transcriptional inactivation in the GRC body, despite the known activity of some genes of the GRC. Nevertheless, RPA and RAD51 proteins were found at some GRC sites, indicating the formation and repair of double-strand breaks on this chromosome. Our results provide new insights into the meiotic behavior and structure of a GRC. Full article

Animals show diverse processes of gametogenesis in the evolutionary pathway. Here, we characterized the spermatogenic cells in the testis of the marine invertebrate Ciona robusta. Ciona sperm differentiate in a non-cystic type of testis, comprising many follicles with various sizes and stages of spermatogenic cells. In the space among follicles, we observed free cells that were recognized by antibody against Müllerian inhibiting substance, a marker for vertebrate Sertoli cells. We further categorized the spermatogenic cells into four round stages (RI to RIV) and three elongated stages (EI to EIII) by morphological and immunohistochemical criteria. An antibody against a vertebrate Vasa homolog recognized a few large spermatogonium-like cells (RI) near the basal wall of a follicle. Consistent with the period of meiosis, a synaptonemal complex protein SYCP3 was recognized from early spermatocytes (RII) to early spermatids (E1). Acetylated tubulins were detected in spermatids before flagellar elongation at the RIV stage and became distributed along the flagella. Electron microscopy showed that the free cells outside the testicular follicle possessed a characteristic of vertebrate Sertoli cells. These results would provide a basis for basic and comparative studies on the mechanism of spermatogenesis. Full article

The synaptonemal complex (SC) is a meiosis-specific multiprotein complex that forms between homologous chromosomes during prophase of meiosis I. Upon assembly, the SC mediates the synapses of the homologous chromosomes, leading to the formation of bivalents, and physically supports the formation of programmed double-strand breaks (DSBs) and their subsequent repair and maturation into crossovers (COs), which are essential for genome haploidization. Defects in the assembly of the SC or in the function of the associated meiotic recombination machinery can lead to meiotic arrest and human infertility. The majority of proteins and complexes involved in these processes are exclusively expressed during meiosis or harbor meiosis-specific subunits, although some have dual functions in somatic DNA repair and meiosis. Consistent with their functions, aberrant expression and malfunctioning of these genes have been associated with cancer development. In this review, we focus on the significance of the SC and their meiotic-associated proteins in human fertility, as well as how human genetic variants encoding for these proteins affect the meiotic process and contribute to infertility and cancer development. Full article

Closely related mammalian species often have differences in chromosome number and morphology, but there is still a debate about how these differences relate to reproductive isolation. To study the role of chromosome rearrangements in speciation, we used the gray voles in the Alexandromys genus as a model. These voles have a high level of chromosome polymorphism and substantial karyotypic divergence. We investigated testis histology and meiotic chromosome behavior in the captive-bred colonies of Alexandromys maximowiczii, Alexandromys mujanensis, two chromosome races of Alexandromys evoronensis, and their interracial and interspecies hybrids, to explore the relationship between karyotypic differences and male hybrid sterility. We found that the seminiferous tubules of the males of the parental species and the interracial hybrids, which were simple heterozygotes for one or more chromosome rearrangements, contained germ cells at all stages of spermatogenesis, indicating their potential fertility. Their meiotic cells displayed orderly chromosome synapsis and recombination. In contrast, all interspecies male hybrids, which were complex heterozygotes for a series of chromosome rearrangements, showed signs of complete sterility. Their spermatogenesis was mainly arrested at the zygotene- or pachytene-like stages due to the formation of complex multivalent chains, which caused extended chromosome asynapsis. The asynapsis led to the silencing of unsynapsed chromatin. We suggest that chromosome asynapsis is the main cause of meiotic arrest and male sterility in the interspecies hybrids of East Asian voles. Full article

Homologous recombination (HR) is essential for meiosis in most sexually reproducing organisms, where it is induced upon entry into meiotic prophase. Meiotic HR is conducted by the collaborative effort of proteins responsible for DNA double-strand break repair and those produced specifically during meiosis. The Hop2-Mnd1 complex was originally identified as a meiosis-specific factor that is indispensable for successful meiosis in budding yeast. Later, it was found that Hop2-Mnd1 is conserved from yeasts to humans, playing essential roles in meiosis. Accumulating evidence suggests that Hop2-Mnd1 promotes RecA-like recombinases towards homology search/strand exchange. This review summarizes studies on the mechanism of the Hop2-Mnd1 complex in promoting HR and beyond. Full article

The basic causes of postzygotic isolation can be elucidated if gametogenesis is studied, which is a drastically different process in males and females. As a step toward clarifying this problem, we obtained an experimental inbred lineage of the eastern mole vole Ellobius tancrei, whose founder animals were animals with identical diploid numbers 2n = 50 but with different Robertsonian translocations (Rb), namely 2Rb4.12 and 2Rb9.13 in the female and 2Rb.2.18 and 2Rb5.9 in the male. Here, we analyzed strictly inbred hybrids (F1, fertile and F10, sterile) using immunocytochemical methods in order to study spermatocytes during the meiotic prophase I. Previously, the presence of trivalents was assumed to have no significant effect on spermatogenesis and fertility in hybrids, but we demonstrated that spermatogenesis might be disturbed due to the cumulative effects of the retarded synapses of Rb bivalents as well as trivalents and their associations with XX sex bivalents. Alterations in the number of gametes due to the described processes led to a decrease in reproductive capacity up to sterility and can be examined as a mechanism for reproductive isolation, thus starting speciation. Full article

Transcription is known to be substage-specific in meiotic prophase I. If transcription is reactivated in the mid pachytene stage in mammals when synapsis is completed, then this process is observed in the zygotene stage in insects. The process of transcriptional reactivation has been studied in a small number of different taxa of invertebrates and vertebrates. Here, for the first time, we investigate synapsis and transcription in prophase I in the European river lamprey Lampetra fluviatilis (Petromyzontiformes, Cyclostomata), which is representative of jawless vertebrates that diverged from the main branch of vertebrates between 535 and 462 million years ago. We found that not all chromosomes complete synapsis in telomeric regions. Rounded structures were detected in chromatin and in some synaptonemal complexes, but their nature could not be determined conclusively. An analysis of RNA polymerase II distribution led to the conclusion that transcriptional reactivation in lamprey prophase I is not associated with the completion of chromosome synapsis. Monomethylated histone H3K4 is localized in meiotic chromatin throughout prophase I, and this pattern has not been previously detected in animals. Thus, the findings made it possible to identify synaptic and epigenetic patterns specific to this group and to expand knowledge about chromatin epigenetics in prophase I. Full article

This brief review is focused on the viviparous lizard Zootoca vivipara (Lichtenstein, 1823), of the family Lacertidae, which possesses female heterogamety and multiple sex chromosomes (male 2n = 36, Z₁Z₁Z₂Z₂/Z₁Z₂W, female 2n = 35, with variable W sex chromosome). Multiple sex chromosomes and their changes may influence meiosis and the female meiotic drive, and they may play a role in reproductive isolation. In two cryptic taxa of Z. vivipara with different W sex chromosomes, meiosis during early spermatogenesis and oogenesis proceeds normally, without any disturbances, with the formation of haploid spermatocytes, and in female meiosis with the formation of synaptonemal complexes (SCs) and the lampbrush chromosomes. In females, the SC number was constantly equal to 19 (according to the SC length, 16 SC autosomal bivalents plus three presumed SC sex chromosome elements). No variability in the chromosomes at the early stages of meiotic prophase I, and no significant disturbances in the chromosome segregation at the anaphase–telophase I stage, have been discovered, and haploid oocytes (n = 17) at the metaphase II stage have been revealed. There should be a factor/factors that maintain the multiple sex chromosomes, their equal transmission, and the course of meiosis in these cryptic forms of Z. vivipara. Full article

Vipera berus is the species with the largest range of snakes on Earth and one of the largest among reptiles in general. It is also the only snake species found in the Arctic Circle. Vipera berus is the most involved species of the genus Vipera in the process of interspecific hybridization in nature. The taxonomy of the genus Vipera is based on molecular markers and morphology and requires clarification using SC-karyotyping. This work is a detailed comparative study of the somatic and meiotic karyotypes of V. berus, with special attention to DNA and protein markers associated with synaptonemal complexes. The karyotype of V. berus is a remarkable example of a bimodal karyotype containing both 16 large macrochromosomes and 20 microchromosomes. We traced the stages of the asynchronous assembly of both types of bivalents. The number of crossing-over sites per pachytene nucleus, the localization of the nucleolar organizer, and the unique heterochromatin block on the autosomal bivalent 6—an important marker—were determined. Our results show that the average number of crossing-over sites per pachytene nucleus is 49.5, and the number of MLH1 sites per bivalent 1 reached 11, which is comparable to several species of agamas. Full article

Nonhomologous chromosome interactions take place in both somatic and meiotic cells. Prior to this study, we had discovered special contacts through the SYCP3 (synaptonemal complex protein 3) filament between the short arms of nonhomologous acrocentrics at the pachytene stage in the Alay mole vole, and these contacts demonstrate several patterns from proximity to the complete fusion stage. Here, we investigated the nonhomologous chromosome contacts in meiotic prophase I. It turned out that such contacts do not introduce changes into the classic distribution of DNA double-strand breaks. It is noteworthy that not all meiotic contacts were localized in the H3k9me3-positive heterochromatic environment. Both in the mid zygotene and in the early–mid diplotene, three types of contacts (proximity, touching, and anchoring/tethering) were observed, whereas fusion seems to be characteristic only for pachytene. The number of contacts in the mid pachytene is significantly higher than that in the zygotene, and the distance between centromeres in nonhomologous contacts is also the smallest in mid pachytene for all types of contacts. Thus, this work provides a new insight into the behavior of meiotic contacts during prophase I and points to avenues of further research. Full article

Understanding meiotic crossover (CO) variation in crops like bread wheat (Triticum aestivum L.) is necessary as COs are essential to create new, original and powerful combinations of genes for traits of agronomical interest. We cytogenetically characterized a set of wheat aneuploid lines missing part or all of chromosome 3B to identify the most influential regions for chiasma formation located on this chromosome. We showed that deletion of the short arm did not change the total number of chiasmata genome-wide, whereas this latter was reduced by ~35% while deleting the long arm. Contrary to what was hypothesized in a previous study, deletion of the long arm does not disturb the initiation of the synaptonemal complex (SC) in early meiotic stages. However, progression of the SC is abnormal, and we never observed its completion when the long arm is deleted. By studying six different deletion lines (missing different parts of the long arm), we revealed that at least two genes located in both the proximal (C-3BL2-0.22) and distal (3BL7-0.63-1.00) deletion bins are involved in the control of chiasmata, each deletion reducing the number of chiasmata by ~15%. We combined sequence analyses of deletion bins with RNA-Seq data derived from meiotic tissues and identified a set of genes for which at least the homoeologous copy on chromosome 3B is expressed and which are involved in DNA processing. Among these genes, eight (CAP-E1/E2, DUO1, MLH1, MPK4, MUS81, RTEL1, SYN4, ZIP4) are known to be involved in the recombination pathway. Full article