Pre-meiotic pairing of homologous chromosomes during Drosophila male meiosis

In the early stages of meiosis, maternal and paternal chromosomes pair with their homologous partner and recombine to ensure exchange of genetic information and proper segregation. These events can vary drastically between species and between males and females of the same species. In Drosophila, in contrast to females, males do not form synaptonemal complexes (SCs), do not recombine and have no crossing-over; yet, males are able to segregate their chromosomes properly. Here, we investigated the early steps of homologues pairing in Drosophila males. We found that homologues are not paired in germline stem cells (GSCs) and become paired in the mitotic region before meiotic entry, similarly to females. Surprisingly, male germline cells express SC proteins, which localize to centromeres and promote pairing. We further found that the SUN/KASH (LINC) complex and microtubules are required for homologues pairing as in females. Chromosome movements are however much slower than in females and we demonstrate that this slow dynamic is compensated in males by having longer cell cycles. In agreement, slowing down cell cycles was sufficient to rescue pairing-defective mutants in female meiosis. Our results demonstrate that although meiosis differs significantly between males and females, sex-specific cell cycle kinetics are integrated with similar molecular mechanisms to achieve proper homologues pairing.

Critical role of H3K27 methylation in cell fate determination of two cell lineages in male gametophyte

During angiosperm male gametogenesis, microspores divide to produce a vegetative cell (VC) and a male germline (MG), each with a distinct cell fate. How the MG cell/VC fate is determined remains largely unknown. Here, we report that H3K27me3 is essential for VC fate commitment and H3K27me3 erasure contributes to the MG cell fate initiation in male gametophyte of Arabidopsis. The VC-targeted H3K27me3 erasure disturbed the VC development and resulted in the VC fate shifting towards a gamete destination, which suggests that MG cells require H3K27me3 erasure for triggering the gamete cell fate. Multi-omics and cytologic analysis confirmed the occurrence of extensive cell identity transition due to H3K27me3 erasure. Therefore, we experimentally confirm that the MG cell/VC fate is epigenetically regulated. The H3K27 methylation plays a critical role in the guidance of MG cell/VC fate determination for pollen fertility in Arabidopsis. Our work also provides new evidences for two previous hypotheses that the germline cell fate is specified by the differential distribution of yet unknown determinant, and VC maintains the microspore's default program, namely the H3K27me3 setting, whereas MG requires reprogramming.

Comprehensive analysis of differences of N6-methyladenosine of lncRNAs between atrazine-induced and normal Xenopus laevis testis

Background Increasing evidence suggested N6-methyladenosine (m6A) modification is crucial for male germline development. However, m6A modification of lncRNAs gains a little attention in amphibians in recent years. Xenopus laevis (X. laevis) was chosen to be an ideal model organism for testing environmental endocrine disrupting chemicals (EDCs) exposure and resultant effects. Atrazine (AZ) as an endocrine disrupt can effect development of testis in amphibians. Our previous study revealed that m6A is a highly conserved modification across the species. Results The results of m6A sequences showed that m6A-methylated lncRNAs enriched in intergenic region in testes of X. laevis. We further examined the differential expression of lncRNAs m6A sites in testes of AZ-exposed and compared with that in animals from control group. The results indicated that up to 198 differentially methylated m6A sites were detected within 188 lncRNAs, in which 89 significantly up-methylated sites and 109 significantly down-methylated sites. Data from KEGG pathway analysis indicated that AZ-affected lncRNAs m6A sites were mainly involved in 10 pathways in which 3 mutual pathways were found in the result of differentially m6A-methylated mRNAs. Conclusions These findings suggested that differentially m6A-methylated lncRNAs and these 3 pathways may act on regulatory roles in abnormal testis development of AZ-exposed X. laevis. This study for the first time provides insights into the profile of lncRNAs m6A modifications in amphibian species.

Male-biased protein expression in primordial germ cells, identified through a comparative study of UAS vectors in Drosophila

In Drosophila, three types of UAS vectors (UASt, UASp, and UASz) are currently available for use with the Gal4-UAS system. They have been used successfully in somatic cells and germline cells from ovaries. However, it remains unclear whether they are functional in the germline cells of embryos, larvae, and adult testes. In this study, we found that all three types of UAS vectors were functional in the germline cells of embryos and larvae and that the UASt and UASz vectors were active in the germline of the distal tip region in adult testes. Moreover, we observed that protein expression from the UAS vectors was male-biased in germline cells of late embryos, whereas their respective mRNA expression levels were not. Furthermore, O-propargyl-puromycin (OPP) staining revealed that protein synthesis was male-biased in these germline cells. In addition, GO terms related to translation and ribosomal maturation were significantly enriched in the male germline. These observations show that translational activity is higher in male than in female germline cells. Therefore, we propose that male-biased protein synthesis may be responsible for the sex differences observed in the early germline.

Characterization of germline development and identification of genes associated with germline specification in pineapple

Understanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species.

Centromere function in asymmetric cell division in Drosophila female and male germline stem cells

The centromere is the constricted chromosomal region required for the correct separation of the genetic material at cell division. The kinetochore protein complex assembles at the centromere and captures microtubules emanating from the centrosome to orchestrate chromosome segregation in mitosis and meiosis. Asymmetric cell division (ACD) is a special type of mitosis that generates two daughter cells with different fates. Epigenetic mechanisms operating at the centromere have been proposed to contribute to ACD. Recent studies have shown that an asymmetric distribution of CENP-A—the centromere-specific histone H3 variant—between sister chromatids can bias chromosome segregation in ACD. In stem cells, this leads to non-random sister chromatid segregation, which can affect cell fate. These findings support the ‘silent sister' hypothesis, according to which the mechanisms of ACD are epigenetically regulated through centromeres. Here, we review the recent data implicating centromeres in ACDs and cell fate in Drosophila melanogaster female and male germline stem cells.

Spd-2 Gene Duplication Suggests Cell-Type Specific Assembly Mechanisms of Pericentriolar Material

Centrosomes are multi-protein complexes that function as the major microtubule organizing center (MTOC) for the cell. While centrosomes play tissue-specific MTOC functions, little is known about how particular centrosome proteins are regulated across cell types to achieve these different functions. To investigate this cell type-specific diversity, we searched for gene duplications of centrosome genes in the Drosophila lineage with the aim of identifying centrosome gene duplications where each copy evolved for specialized functions. Through in depth functional analysis of a Spd-2 gene duplication in the Willistoni group, we discovered differences in the regulation of PCM in somatic and male germline cells. The parental gene, Spd-2A, is expressed in somatic cells, where it can function to organize pericentriolar material (PCM) and the mitotic spindle in larval brain neuroblasts. Spd-2A is absent during male meiosis, and even when ectopically expressed in spermatocytes it fails to rescue PCM and spindle organization. In contrast, the new gene duplicate, Spd-2B, is expressed specifically in spermatocytes. During male meiosis, Spd-2B localizes to centrosomes, organizes PCM and spindles, and is sufficient for proper male fertility. Experiments using chimeric transgenes reveal that differences in the C-terminal tails of Spd-2A and Spd-2B are responsible for these functional changes. Thus, Spd-2A and Spd-2B have evolved complementary functions by specializing for distinct subsets of cells. Together, our results demonstrate that somatic cells and male germline cells have fundamentally different requirements for PCM, suggesting that PCM proteins such as Spd-2 is differentially regulated across cell types to satisfy distinct requirements.

dRTEL1 is essential for the maintenance of Drosophila male germline stem cells

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, we identify dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1, as a novel regulator of male germline stem cells (GSCs). Our genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, our findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1.

Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1).

During the development of the male germline, spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing and regenerating mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs. However, an enrichment of Lhx1 expression in spermatogonia-like cells generated in vitro has not been reported so far. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including a timed combination of the SIRT1 inhibitor Ex-527, the DNA methyltransferase inhibitor RG-108 and the electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of the PGC to gonocyte differentiation process. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3β and MEK (termed 2i) with leukemia inhibitory factor (LIF) (termed 2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes. This was achieved by the removal of 2iL and a specific schedule of 2 partial medium replacements per day with alternating 8-hour and 16-hour intervals over a period of 32 days. Combination of this new cell culture protocol with the previously reported chemical intervention in ES cells changed the population-averaged expression of spermatogonia- and gonocyte-specific genes during the differentiation process and increased the population-averaged gene expression of Lhx1 in the resulting CSMs compared to CSMs without chemical intervention. Furthermore, we detected single CSMs with a strong nuclear LHX1/5 protein signal only in the chemical intervention group. Our results provide the first experimental evidence for the generation of CSMs with an enrichment of Lhx1 expression in vitro. We propose that further investigation of the CSMs generated with this in vitro system may provide new insights into male germline and stem cell development.