Computational analysis of bio-images by deep learning (DL) algorithms has made exceptional progress in recent years and has become much more accessible to non-specialists with the development of ready-to-use tools. The study of oogenesis mechanisms and female reproductive success in fish has also recently benefited from the development of efficient three-dimensional (3D) imaging protocols on entire ovaries. Such large datasets have a great potential for the generation of new quantitative data on oogenesis but are, however, complex to analyze due to imperfect fluorescent signals and the lack of efficient image analysis workflows. Here, we applied two open-source DL tools, Noise2Void and Cellpose, to analyze the oocyte content of medaka ovaries at larvae and adult stages. These tools were integrated into end-to-end analysis pipelines that include image pre-processing, cell segmentation, and image post-processing to filter and combine labels. Our pipelines thus provide effective solutions to accurately segment complex 3D images of entire ovaries with either irregular fluorescent staining or low autofluorescence signal. In the future, these pipelines will be applicable to extensive cellular phenotyping in fish for developmental or toxicology studies. Summary statement An accessible image analysis method for biologists, which includes easy-to-use deep learning algorithms, designed for accurate quantitative measurement of ovarian content from complex 3D fluorescent images.

An end-to-end pipeline based on open source deep learning tools for reliable analysis of complex 3D images of Medaka ovaries

MicroRNAs (miRNAs) can have tissue-specific expression and functions; they can originate from dedicated miRNA genes, from non-canonical miRNA genes, or from mirror-miRNA genes and can also experience post-transcriptional variations. It remains unclear, however, which mechanisms of miRNA production or modification are tissue-specific and the extent of their evolutionary conservation. To address these issues, we developed the software Prost! (PRocessing Of Short Transcripts), which, among other features, allows accurate quantification of mature miRNAs, takes into account post-transcriptional processing, such as nucleotide editing, and helps identify mirror-miRNAs. Here, we applied Prost! to annotate and analyze miRNAs in three-spined stickleback (Gasterosteus aculeatus), a model fish for evolutionary biology reported to have a miRNome larger than most teleost fish. Zebrafish (Danio rerio), a distantly related teleost with a well-known miRNome, served as comparator. Despite reports suggesting that stickleback had a large miRNome, results showed that stickleback has 277 evolutionary-conserved mir genes and 366 unique mature miRNAs (excluding mir430 gene replicates and the vaultRNA-derived mir733), similar to zebrafish. In addition, small RNA sequencing data from brain, heart, testis, and ovary in both stickleback and zebrafish identified suites of mature miRNAs that display organ-specific enrichment, which is, for many miRNAs, evolutionarily-conserved. These data also supported the hypothesis that evolutionarily-conserved, organ-specific mechanisms regulate miRNA post-transcriptional variations. In both stickleback and zebrafish, miR2188-5p was edited frequently with similar nucleotide editing patterns in the seed sequence in various tissues, and the editing rate was organ-specific with higher editing in the brain. In summary, Prost! is a critical new tool to identify and understand small RNAs and can help clarify a species miRNA biology, as shown here for an important fish model for the evolution of developmental mechanisms, and can provide insight into organ-specific expression and evolutionary-conserved miRNA post-transcriptional mechanisms.

https://www.biorxiv.org/content/biorxiv/early/2018/09/20/423533.full.pdf

miRNA analysis with Prost! reveals evolutionary conservation of organ-enriched expression and post-transcriptional modifications in three-spined stickleback and zebrafish

MicroRNAs play key roles during ovary development, with emerging evidence suggesting that miR-202-5p is specifically expressed in female animal gonads. Granulosa cells (GCs) are somatic cells that are closely related to the development of female gametes in mammalian ovaries. However, the biological roles of miR-202-5p in GCs remain unknown. Here, we show that miR-202-5p is specifically expressed in GCs and accumulates in extracellular vesicles (EVs) from large growth follicles in goat ovaries. In vitro assays showed that miR-202-5p induced apoptosis and suppressed the proliferation of goat GCs. We further revealed that miR-202-5p is a functional miRNA that targets the transforming growth factor-beta type II receptor (TGFβR2). MiR-202-5p attenuated TGF-β/SMAD signaling through the degradation of TGFβR2 at both the mRNA and protein level, decreasing p-SMAD3 levels in GCs. Moreover, we verified that steroidogenic factor 1 (SF1) is a transcriptional factor that binds to the promoters of miR-202 and cytochrome P450 family 19 subfamily A member 1 (CYP19A1) through luciferase reporter and chromatin immunoprecipitation (ChIP) assays. That contributed to positive correlation between miR-202-5p and CYP19A1 expression and estradiol (E2) release. Furthermore, SF1 repressed TGFβR2 and p-SMAD3 levels in GCs through the transactivation of miR-202-5p. Taken together, these results suggest a mechanism by which miR-202-5p regulates canonical TGF-β/SMAD signaling through targeting TGFβR2 in GCs. This provides insight into the transcriptional regulation of miR-202 and CYP19A1 during goat ovarian follicular development.

https://www.mdpi.com/2073-4409/9/2/445/pdf

Transactivation of miR-202-5p by Steroidogenic Factor 1 (SF1) Induces Apoptosis in Goat Granulosa Cells by Targeting TGFβR2.

Trehalose biosynthesis enzyme homologues in plants contain two families, trehalose-6-phosphate synthases (TPSs) and trehalose-6-phosphate phosphatases (TPPs). Both families participate in trehalose synthesis and a variety of stress-resistance processes. Here, nine BdTPS and ten BdTPP genes were identified based on the Brachypodium distachyon genome, and all genes were classified into three classes. The Class I and Class II members differed substantially in gene structures, conserved motifs, and protein sequence identities, implying varied gene functions. Gene duplication analysis showed that one BdTPS gene pair and four BdTPP gene pairs are formed by duplication events. The value of Ka/Ks (non-synonymous/synonymous) was less than 1, suggesting purifying selection in these gene families. The cis-elements and gene interaction network prediction showed that many family members may be involved in stress responses. The quantitative real-time reverse transcription (qRT-PCR) results further supported that most BdTPSs responded to at least one stress or abscisic acid (ABA) treatment, whereas over half of BdTPPs were downregulated after stress treatment, implying that BdTPSs play a more important role in stress responses than BdTPPs. This work provides a foundation for the genome-wide identification of the B. distachyon TPS–TPP gene families and a frame for further studies of these gene families in abiotic stress responses.

https://www.mdpi.com/2223-7747/8/10/362/pdf

Genome-Wide Identification, Evolution, and Expression Analysis of TPS and TPP Gene Families in Brachypodium distachyon

Female gamete production relies on coordinated molecular and cellular processes that occur in the ovary throughout oogenesis. In fish, as in other vertebrates, these processes have been extensively studied both in terms of endocrine/paracrine regulation and protein expression and activity. The role of small non-coding RNAs in the regulation of animal reproduction remains however largely unknown and poorly investigated, despite a growing interest for the importance of miRNAs in a wide variety of biological processes. Here, we analyzed the role of miR-202, a miRNA predominantly expressed in male and female gonads in several vertebrate species. We studied its expression in the medaka ovary and generated a mutant line (using CRISPR/Cas9 genome engineering) to determine its importance for reproductive success with special interest for egg production. Our results show that miR-202-5p is the biologically active form of the miRNA and that it is expressed in granulosa cells and in the unfertilized egg. The knock out (KO) of miR-202 resulted in a strong phenotype both in terms of number and quality of eggs produced. Mutant females exhibited either no egg production or produced a drastically reduced number of eggs that could not be fertilized, ultimately leading to no reproductive success. We quantified the size distribution of the oocytes in the ovary of KO females and performed a genome-wide transcriptomic analysis approach to identified dysregulated molecular pathways. Together, cellular and molecular analyses indicate that lack of miR-202 impairs the early steps of oogenesis/folliculogenesis and decreases the number of large (i.e. vitellogenic) follicles, ultimately leading to dramatically reduced female fecundity. This study sheds new light on the regulatory mechanisms that control the early steps of follicular development and provides the first in vivo functional evidence that an ovarian-predominant microRNA may have a major role in female reproduction.

https://www.biorxiv.org/content/biorxiv/early/2018/03/22/287359.full.pdf

MicroRNA-202 (miR-202) controls female fecundity by regulating medaka oogenesis

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MicroRNA-202 (miR-202) controls female fecundity by regulating medaka oogenesis