Male Gametogenesis Research Articles

In vitro reconstitution of the whole male germ-cell development from mouse pluripotent stem cells

<h2>Summary</h2> Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an <i>in vitro</i> reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes <i>in vivo</i> but also under an <i>in vitro</i> culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the <i>in vitro</i> male gametogenesis by PSCs.

Plasmodium falciparum Calcium-Dependent Protein Kinase 4 is Critical for Male Gametogenesis and Transmission to the Mosquito Vector.

ABSTRACTGametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium-independent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild-type and Plasmodium falciparum cdpk4− late gametocyte stages to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events, such as DNA replication, mRNA translation, and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation and thereby is critical for parasite transmission to the mosquito vector.

Sugar metabolism during pre- and post-fertilization events in plants under hightemperature stress.

High temperature challenges global crop production by limiting the growth and development of the reproductive structures and seed. It impairs the developmental stages of male and female gametogenesis, pollination, fertilization, endosperm formation and embryo development. Among these, the male reproductive processes are highly prone to abnormalities under high temperature at various stages of development. The disruption of source-sink balance is the main constraint for satisfactory growth of the reproductive structures which is disturbed at the level of sucrose import and utilization within the tissue. Seed development after fertilization is affected by modulation in the activity of enzymes involved in starch metabolism. In addition, the alteration in the seed-filling rate and its duration affects the seed weight and quality. The present review critically discusses the role of sugar metabolism in influencing the various stages of gamete and seed development under hightemperature stress. It also highlights the interaction of the sugars with hormones that mediate the transport of sugars to sink tissues. The role of transcription factors for the regulation of sugar availability under high temperature has also been discussed. Further, the omics-based systematic investigation has been suggested to understand the synergistic or antagonistic interactions between sugars, hormones and reactive oxygen species at various points of sucrose flow from source to sink under hightemperature stress.

Distinct roles of haspin in stem cell division and male gametogenesis

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

Sesame β-ketoacyl-acyl carrier protein synthase I regulates pollen development by interacting with an adenosine triphosphate-binding cassette transporter in transgenic Arabidopsis.

Male gametogenesis is an important biological process critical for seed formation and successful breeding. Understanding the molecular mechanisms of male fertility might facilitate hybrid breeding and increase crop yields. Sesame anther development is largely unknown. Here, a sesame β-ketoacyl-[acyl carrier protein] synthase I (SiKASI) was cloned and characterized as being involved in pollen and pollen wall development. Immunohistochemical analysis showed that the spatiotemporal expression of SiKASI protein was altered in sterile sesame anthers compared with fertile anthers. In addition, SiKASI overexpression in Arabidopsis caused male sterility. Cytological observations revealed defective microspore and pollen wall development in SiKASI-overexpressing plants. Aberrant lipid droplets were detected in the tapetal cells of SiKASI-overexpressing plants, and most of the microspores of transgenic plants contained few cytoplasmic inclusions, with irregular pollen wall components embedded on their surfaces. Moreover, the fatty acid metabolism and the expression of a sporopollenin biosynthesis-related gene set were altered in the anthers of SiKASI-overexpressing plants. Additionally, SiKASI interacted with an adenosine triphosphate (ATP)-binding cassette (ABC) transporter. Taken together, our findings suggested that SiKASI was crucial for fatty acid metabolism and might interact with ABCG18 for normal pollen fertility in Arabidopsis.

Photoperiod-independent testicular development in the model newt Pleurodeles waltl.

Urodele amphibian newts have unique biological properties in male gametogenesis, in addition to their extreme regenerative capacity. Male newts are able to regenerate new testes even after reaching sexual maturity and can possess multiple testes. Notably, these animals maintain primordial germ cell-like cells in a tissue adjacent to the testis. Spermatogenesis proceeds while synchronizing in a region-specific manner in the testis. However, the newt species that have been used most commonly require 2-3years to achieve sexual maturity, and spermatogenesis in these species shows seasonality. These traits have restricted the use of newts for studies on testicular development and spermatogenesis, and testis development in newts remains poorly characterized. Recently, the Iberian ribbed newt Pleurodeles waltl has been established as an emerging model organism. P.waltl reaches sexual maturity more quick after birth than do other newts and is capable of breeding year-round. Thus, P.waltl is expected to serve as an appealing experimental model for studying the mechanisms of male gametogenesis in the urodeles. In the present study, we use P.waltl to describe the entire developmental process of the newt testis from primordial gonad to maturity. Notably, the mature testes show synchronized progression of spermatogenesis along the anteroposterior axis. Additionally, we demonstrate that the process of spermatogenesis in P.waltl proceeds irrespective of day length. Our results show that P.waltl newts are a suitable model for investigating the process of testicular development. We also expect that these results will be useful for the maintenance of P.waltl bioresources.

MS1 is essential for male fertility by regulating the microsporocyte cell plate expansion in soybean.

Male sterility is an essential trait in hybrid seed production, especially for monoclinous and autogamous food crops. Soybean male-sterile ms1 mutant has been known for more than 50 years and could be instrumental in making hybrid seeds. However, the gene responsible for the male-sterile phenotype has remained unknown. Here, we report the map-based cloning and characterization of the MS1 gene in soybean. MS1 encodes a kinesin protein and localizes to the nucleus, where it is required for the male meiotic cytokinesis after telophase II. We further substantiated that MS1 colocalizes with microtubules and is essential for cell plate formation in soybean male gametogenesis through immunostaining. Both ms1 and CRISPR/Cas9 knockout mutants show complete male sterility but are otherwise phenotypically normal, making them perfect tools for producing hybrid seeds. The identification of MS1 has the practical potential for assembling the sterility system and speeding up hybrid soybean breeding.

Evidence of alterations in the learning and memory in offspring of stress-induced male rats.

There is extensive data pointing to offspring outcomes related to maternal life incidents, but there is less research concerning the association between paternal life events and progeny brain development and behaviour. As male gametogenesis is a continuous process, the incidences happening in life can modify the epigenetic regulation, altering the offspring's development and behaviour. The present study evaluates the effects of paternal stress during different life periods on their offspring's learning ability, memory, morphological and biochemical changes in the prefrontal cortex and hippocampus in the rat model. Four weeks' old male rats were subjected to five variable stressors at the rate of one per day. Stress received male rats were bred with naive female rats for 1 to 3 nights. The offspring's learning and memory were assessed by the Morris water maze test and automated Y maze. Following behavioural studies, offspring were euthanized to examine global DNA methylation, neurotransmitter levels, namely acetylcholine, glutamate in the hippocampus and frontal cortex. The offspring of stress-induced animals exhibited a delay in acquiring learning and defect in memory and altered global DNA methylation in the hippocampus (p=0.000124). There was significant reduction of acetylcholine and glutamate levels in hippocampus (p=0.000018, p=0.00001, respectively) and in prefrontal cortex (p=0.00001, p=0.00001, respectively). HPA axis of offspring was altered considerably (p=0.00001). The histomorphometry of the prefrontal cortex and different hippocampal regions revealed a statistically significant (p<0.05) reduction in neuronal numbers in the offspring of stressed animals compared to that of control. These impacts were markedly high in the offspring of fathers who received stress during both pubertal and adult periods. The findings of this study demonstrate that paternal stress can impact offspring learning and memory.

Proteomic Analysis of Generative and Vegetative Nuclei Reveals Molecular Characteristics of Pollen Cell Differentiation in Lily.

In plants, the cell fates of a vegetative cell (VC) and generative cell (GC) are determined after the asymmetric division of the haploid microspore. The VC exits the cell cycle and grows a pollen tube, while the GC undergoes further mitosis to produce two sperm cells for double fertilization. However, our understanding of the mechanisms underlying their fate differentiation remains limited. One major advantage of the nuclear proteome analysis is that it is the only method currently able to uncover the systemic differences between VC and GC due to GC being engulfed within the cytoplasm of VC, limiting the use of transcriptome. Here, we obtained pure preparations of the vegetative cell nuclei (VNs) and generative cell nuclei (GNs) from germinating lily pollens. Utilizing these high-purity VNs and GNs, we compared the differential nucleoproteins between them using state-of-the-art quantitative proteomic techniques. We identified 720 different amount proteins (DAPs) and grouped the results in 11 fate differentiation categories. Among them, we identified 29 transcription factors (TFs) and 10 cell fate determinants. Significant differences were found in the molecular activities of vegetative and reproductive nuclei. The TFs in VN mainly participate in pollen tube development. In comparison, the TFs in GN are mainly involved in cell differentiation and male gametogenesis. The identified novel TFs may play an important role in cell fate differentiation. Our data also indicate differences in nuclear pore complexes and epigenetic modifications: more nucleoporins synthesized in VN; more histone variants and chaperones; and structural maintenance of chromosome (SMC) proteins, chromatin remodelers, and DNA methylation-related proteins expressed in GN. The VC has active macromolecular metabolism and mRNA processing, while GC has active nucleic acid metabolism and translation. Moreover, the members of unfolded protein response (UPR) and programmed cell death accumulate in VN, and DNA damage repair is active in GN. Differences in the stress response of DAPs in VN vs. GN were also found. This study provides a further understanding of pollen cell differentiation mechanisms and also a sound basis for future studies of the molecular mechanisms behind cell fate differentiation.

FLA14 is required for pollen development and preventing premature pollen germination under high humidity in Arabidopsis

BackgroundAs an important subfamily of arabinogalactan proteins (AGPs), fasciclin-like AGPs (FLAs) contribute to various aspects of growth, development and adaptation, yet their function remains largely elusive. Despite the diversity of FLAs, only two members, Arabidopsis FLA3 and rice MTR1, are reported to be involved in sexual reproduction. In this study, another Arabidopsis FLA-encoding gene, FLA14, was identified, and its role was investigated.ResultsArabidopsis FLA14 was found to be a pollen grain-specific gene. Expression results from fusion with green fluorescent protein showed that FLA14 was localized along the cell membrane and in Hechtian strands. A loss-of-function mutant of FLA14 showed no discernible defects during male gametogenesis, but precocious pollen germination occurred inside the mature anthers under high moisture conditions. Overexpression of FLA14 caused 39.2% abnormal pollen grains with a shrunken and withered appearance, leading to largely reduced fertility with short mature siliques and lower seed set. Cytological and ultramicroscopic observation showed that ectopic expression of FLA14 caused disruption at the uninucleate stage, resulting in either collapsed pollen with absent intine or pollen of normal appearance but with a thickened intine.ConclusionsTaken together, our data suggest a role for FLA14 in pollen development and preventing premature pollen germination inside the anthers under high relative humidity in Arabidopsis.

The PRAME family of cancer testis antigens is essential for germline development and gametogenesis†.

Preferentially expressed antigen in melanoma (PRAME) belongs to a group of cancer/testis antigens that are predominately expressed in the testis and a variety of tumors, and are involved in immunity and reproduction. Much of the attention on PRAME has centered on cancer biology as PRAME is a prognostic biomarker for a wide range of cancers and a potential immunotherapeutic target. Less information is available about the PRAME family's function (s) during gametogenesis and in the overall reproduction process. Here, we review the current knowledge of the PRAME gene family and its function in germline development and gametogenesis. Members of the PRAME family are leucine rich repeat proteins, localized in nucleus and cytoplasm, with multifaceted roles in germ cells. As transcriptional regulators, the PRAME family proteins are involved in germline development, particularly in the maintenance of embryonic stem cell pluripotency, development of primordial germ cells, and differentiation/proliferation of spermatogenic and oogenic cells. The PRAME family proteins are also enriched in cytoplasmic organelles, such as rough endoplasmic reticulum, Golgi vesicle, germinal granules, centrioles, and play a role in the formation of the acrosome and sperm tail during spermiogenesis. The PRAME gene family remains transcriptionally active in the germline throughout the entire life cycle and is essential for gametogenesis, with some members specific to either male or female germ cells, while others are involved in both male and female gametogenesis. A potential molecular mechanism that underlies the function of PRAME, and is shared by gametogenesis and oncogenesis is also discussed.

Engineered sex ratio distortion by X-shredding in the global agricultural pest Ceratitis capitata

BackgroundGenetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits.ResultsHere, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%).ConclusionWe provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.

The essential role of Dnmt1 in gametogenesis in the large milkweed bug Oncopeltus fasciatus.

Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise thequestion as to how a gene that is so critical to fitness across multiple insect species is able to diverge widely across the insect tree of life.

Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA.

piRNAs are small non-coding RNAs required to maintain genome integrity and preserve RNA homeostasis during male gametogenesis. In murine adult testes, the highest levels of piRNAs are present in the pachytene stage of meiosis, but their mode of action and function remain incompletely understood. We previously reported that BTBD18 binds to 50 pachytene piRNA-producing loci. Here we show that spermatozoa in gene-edited mice lacking a BTBD18 targeted pachytene piRNA cluster on Chr18 have severe sperm head dysmorphology, poor motility, impaired acrosome exocytosis, zona pellucida penetration and are sterile. The mutant phenotype arises from aberrant formation of proacrosomal vesicles, distortion of the trans-Golgi network, and up-regulation of GOLGA2 transcripts and protein associated with acrosome dysgenesis. Collectively, our findings reveal central role of pachytene piRNAs in controlling spermiogenesis and male fertility.

ABAP1 Plays a Role in the Differentiation of Male and Female Gametes in Arabidopsis thaliana.

The correct development of a diploid sporophyte body and a haploid gametophyte relies on a strict coordination between cell divisions in space and time. During plant reproduction, these divisions have to be temporally and spatially coordinated with cell differentiation processes, to ensure a successful fertilization. Armadillo BTB Arabidopsis protein 1 (ABAP1) is a plant exclusive protein that has been previously reported to control proliferative cell divisions during leaf growth in Arabidopsis. Here, we show that ABAP1 binds to different transcription factors that regulate male and female gametophyte differentiation, repressing their target genes expression. During male gametogenesis, the ABAP1-TCP16 complex represses CDT1b transcription, and consequently regulates microspore first asymmetric mitosis. In the female gametogenesis, the ABAP1-ADAP complex represses EDA24-like transcription, regulating polar nuclei fusion to form the central cell. Therefore, besides its function during vegetative development, this work shows that ABAP1 is also involved in differentiation processes during plant reproduction, by having a dual role in regulating both the first asymmetric cell division of male gametophyte and the cell differentiation (or cell fusion) of female gametophyte.

SAP130 and CSN1 interact and regulate male gametogenesis in Arabidopsis thaliana.

COP9 signalosome (CSN) is a nuclear complex composed of eight distinct subunits that governs vast developmental processes in Arabidopsis thaliana (L.) Heynh. The null alleles of csn mutants display pleiotropic phenotypes that result in seedling lethality. To date, several partially complemented transgenic plants, expressing the particular CSN subunit in its corresponding null mutant allele, were utilized to bypass seedling lethality and investigate CSN regulation at later stages of development. One such transgenic plant corresponding to CSN1 subunit, fus6/CSN1-3-4, accumulates wild-type level of CSN1 and displays normal plant architecture at vegetative stage. Here we show through histological analyses that fus6/CSN1-3-4 plants display impairment of pollen development at the bicellular stage. This defect is identical to that observed in RNAi plants of SAP130, encoding a subunit of the multiprotein splicing factor SF3b. We further dissected the previously reported interaction between CSN1 and SAP130, to reveal that approximately 100 amino-acid residues located at the N-terminal end of CSN1 (CSN1NN) were essential for this interaction. In silico structure modeling demonstrated that CSN1NN could swing out towards SAP130 to dock onto its Helical Insertion protruding from the structure. These results support our model that CSN1 embeds itself within CSN protein complex through its C-terminal half and reaches out to targets through its N-terminal portion of the protein. Taken together, this is the first report to document the identical loss-of-function phenotypes of CSN1 and SAP130 during male gametogenesis. Thus, we propose that SAP130 and CSN1 coordinately regulate development of male reproductive organs.

Plant Epigenetics: Propelling DNA Methylation Variation across the Cell Cycle

DNA methylation is reconfigured during male reproduction in plants, but little is known regarding the mechanisms controlling these epigenetic dynamics. New research highlights how the cell cycle can influence DNA methylation dynamics observed during male gametogenesis and may induce epigenetic variation in clonally propagated plants.

COPII genes SEC31A/B are essential for gametogenesis and interchangeable in pollen development in Arabidopsis

SummaryIn eukaryotes, coat protein complex II (COPII) vesicles mediate anterograde traffic from the endoplasmic reticulum to the Golgi apparatus. Compared to yeasts, plants have multiple COPII coat proteins; however, the functional diversity among them is less well understood. SEC31A and SEC31B are outer coat proteins found in COPII vesicles in Arabidopsis. In this study, we explored the function of SEC31A and compared it with that of SEC31B from various perspectives. SEC31A was widely expressed, but at a significantly lower level than SEC31B. SEC31A‐mCherry and SEC31B‐GFP exhibited a high co‐localization rate in pollen, but a lower rate in growing pollen tubes. The sec31a single mutant exhibited normal growth. SEC31A expression driven by the SEC31B promoter rescued the pollen abortion and infertility observed in sec31b. A sec31asec31b double mutant was unavailable due to lethality of the sec31asec31b gametophyte. Transmission electron microscopy revealed that one quarter of male gametogenesis was arrested at the uninuclear microspore stage, while confocal laser scanning microscopy showed that 1/4 female gametophyte development was suspended at the functional megaspore stage in sec31a‐1/+sec31b‐3/+ plants. Our study highlights the essential role of SEC31A/B in gametogenesis and their interchangeable functions in pollen development.

Comparative proteomic analysis of kinesin-8B deficient Plasmodium berghei during gametogenesis

Plasmodium blood stages, responsible for human to vector transmission, termed gametocytes, are the precursor cells that develop into gametes in the mosquito. Male gametogenesis works as a bottleneck for the parasite life cycle, where, during a peculiar and rapid exflagellation, a male gametocyte produces 8 intracellular axonemes that generate by budding 8 motile gametes. Understanding the molecular mechanisms of gametogenesis is key to design strategies for controlling malaria transmission. In the rodent P. berghei, the microtubule-based motor kinesin-8B (PbKIN8B) is essential for flagellum assembly during male gametogenesis and its gene disruption impacts on completion of the parasitic life cycle. In efforts to improve our knowledge about male gametogenesis, we performed an iTRAQ-based quantitative proteomic comparison of P. berghei mutants with disrupted kinesin-8B gene (ΔPbkin8B) and wild type parasites. During the 15 min of gametogenesis, ΔPbkin8B parasites exhibited important motor protein dysregulation that suggests an essential role of PbKIN8B for the correct interaction or integration of axonemal proteins within the growing axoneme. The energy metabolism of ΔPbkin8B mutants was further affected, as well as the response to stress proteins, protein synthesis, as well as chromatin organisation and DNA processes, although endomitoses seemed to occur. SignificanceMalaria continues to be a global scourge, mainly in subtropical and tropical areas. The disease is caused by parasites from the Plasmodium genus. Plasmodium life cycle alternates between female Anopheles mosquitoes and vertebrate hosts through bites. Gametocytes are the parasite blood forms responsible for transmission from vertebrates to vectors. Inside the mosquito midgut, after stimulation, male and female gametocytes transform into gametes resulting in fertilization. During male gametogenesis, one gametocyte generates eight intracytoplasmic axonemes that generate, by budding, flagellated motile gametes involving a process termed exflagellation. Sexual development has a central role in ensuring malaria transmission. However, molecular data on male gametogenesis and particularly on intracytoplasmic axoneme assembly are still lacking. Since rodent malaria parasites permit the combination of in vivo and in vitro experiments and reverse genetic studies, our group investigated the molecular events in rodent P. berghei gametogenesis. The P. berghei motor ATPase kinesin-8B is proposed as an important component for male gametogenesis. We generated Pbkin8B gene-disrupted gametocytes (ΔPbkin8B) that were morphologically similar to the wild- type (WT) parasites. However, in mutants, male gametogenesis is impaired, male gametocytes are disabled in their ability to assemble axonemes and to exflagellate to release gametes, reducing fertilization drastically. Using a comparative quantitative proteomic analysis, we associated the nonfunctional axoneme of the mutants with the abnormal differential expression of proteins essential to axoneme organisation and stability. We also observed a differential dysregulation of proteins involved in protein biosynthesis and degradation, chromatin organisation and DNA processes in ΔPbkin8B parasites, although DNA condensation, mitotic spindle formation and endomitoses seem to occur. This is the first functional proteomic study of a kinesin gene-disrupted Plasmodium parasite providing new insights into Plasmodium male gametogenesis.

Resetting FLOWERING LOCUS C Expression After Vernalization Is Just Activation in the Early Embryo by a Different Name.

The reproductive success of many plants depends on their capacity to respond appropriately to their environment. One environmental cue that triggers flowering is the extended cold of winter, which promotes the transition from vegetative to reproductive growth in a response known as vernalization. In annual plants of the Brassicaceae, the floral repressor, FLOWERING LOCUS C (FLC), is downregulated by exposure to low temperatures. Repression is initiated during winter cold and then maintained as the temperature rises, allowing plants to complete their life cycle during spring and summer. The two stages of FLC repression, initiation and maintenance, are distinguished by different chromatin states at the FLC locus. Initiation involves the removal of active chromatin marks and the deposition of the repressive mark H3K27me3 over a few nucleosomes in the initiation zone, also known as the nucleation region. H3K27me3 then spreads to cover the entire locus, in a replication dependent manner, to maintain FLC repression. FLC is released from repression in the next generation, allowing progeny of a vernalized plant to respond to winter. Activation of FLC in this generation has been termed resetting to denote the restoration of the pre-vernalized state in the progeny of a vernalized plant. It has been assumed that resetting must differ from the activation of FLC expression in progeny of plants that have not experienced winter cold. Considering that there is now strong evidence indicating that chromatin undergoes major modifications during both male and female gametogenesis, it is time to challenge this assumption.