9-fold symmetry is not essential for centriole elongation and formation of new centriole-like structures.

As daughter centrioles assemble during G2, they recruit conserved Ana3/RTTN followed by its partner Rcd4/PPP1R35. Together, this contributes to the subsequent recruitment of Ana1/CEP295, required for the centriole's conversion to a centrosome. Here, we show that Rcd4/PPP1R35 is also required to maintain 9-fold centriole symmetry in the Drosophila male germline; its absence causes microtubule triplets to disperse into a reduced number of doublet or singlet microtubules. rcd4-null mutant spermatocytes display skinny centrioles that elongate normally and localize centriolar components correctly. Mutant spermatocytes also have centrioles of normal girth that splay at their proximal ends when induced to elongate by Ana1 overexpression. Skinny and splayed spermatid centrioles can still recruit a proximal centriole-like (PCL) structure marking a capability to initiate features of centriole duplication in developing sperm. Thus, stable 9-fold symmetry of microtubule triplets is not essential for centriole growth, correct longitudinal association of centriole components, and aspects of centriole duplication.

Genome-wide DNA methylation changes in human spermatogenesis.

Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.

Essential Role of COPII Proteins in Maintaining the Contractile Ring Anchoring to the Plasma Membrane during Cytokinesis in Drosophila Male Meiosis.

Coatomer Protein Complex-II (COPII) mediates anterograde vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Here, we report that the COPII coatomer complex is constructed dependent on a small GTPase, Sar1, in spermatocytes before and during Drosophila male meiosis. COPII-containing foci co-localized with transitional endoplasmic reticulum (tER)-Golgi units. They showed dynamic distribution along astral microtubules and accumulated around the spindle pole, but they were not localized on the cleavage furrow (CF) sites. The depletion of the four COPII coatomer subunits, Sec16, or Sar1 that regulate COPII assembly resulted in multinucleated cell production after meiosis, suggesting that cytokinesis failed in both or either of the meiotic divisions. Although contractile actomyosin and anilloseptin rings were formed once plasma membrane ingression was initiated, they were frequently removed from the plasma membrane during furrowing. We explored the factors conveyed toward the CF sites in the membrane via COPII-mediated vesicles. DE-cadherin-containing vesicles were formed depending on Sar1 and were accumulated in the cleavage sites. Furthermore, COPII depletion inhibited de novo plasma membrane insertion. These findings suggest that COPII vesicles supply the factors essential for the anchoring and/or constriction of the contractile rings at cleavage sites during male meiosis in Drosophila.

A Rad50-null mutation in mouse germ cells causes reduced DSB formation, abnormal DSB end resection and complete loss of germ cells.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

HDAC3 promotes Sertoli cell maturation and maintains the blood-testis barrier dynamics.

Germ cell development depends on the capacity of somatic Sertoli cells to undergo differentiation into a mature state and establish a germ cell-specific blood-testis barrier (BTB). The BTB structure confers an immunological barrier for meiotic and postmeiotic germ cells, and its dynamic permeability facilitates a transient movement of preleptotene spermatocytes through BTB to enter meiosis. However, the regulatory factors involved in Sertoli cell maturation and how BTB dynamics coordinate germ cell development remain unclear. Here, we found a histone deacetylase HDAC3 abundantly expresses in Sertoli cells and localizes in both cytoplasm and nucleus. Sertoli cell-specific Hdac3 knockout in mice causes infertility with compromised integrity of blood-testis barrier, leading to germ cells unable to traverse through BTB and an accumulation of preleptotene spermatocytes in juvenile testis. Mechanistically, nuclear HDAC3 regulates the expression program of Sertoli cell maturation genes, and cytoplasmic HDAC3 forms a complex with the gap junction protein Connexin 43 to modulate the BTB integrity and dynamics through regulating the distribution of tight junction proteins. Our findings identify HDAC3 as a critical regulator in promoting Sertoli cell maturation and maintaining the homeostasis of the blood-testis barrier.

Kinesin-7 CENP-E mediates chromosome alignment and spindle assembly checkpoint in meiosis I.

In eukaryotes, meiosis is the genetic basis for sexual reproduction, which is important for chromosome stability and species evolution. The defects in meiosis usually lead to chromosome aneuploidy, reduced gamete number, and genetic diseases, but the pathogenic mechanisms are not well clarified. Kinesin-7 CENP-E is a key regulator in chromosome alignment and spindle assembly checkpoint in cell division. However, the functions and mechanisms of CENP-E in male meiosis remain largely unknown. In this study, we have revealed that the CENP-E gene was highly expressed in the rat testis. CENP-E inhibition influences chromosome alignment and spindle organization in metaphase I spermatocytes. We have found that a portion of misaligned homologous chromosomes is located at the spindle poles after CENP-E inhibition, which further activates the spindle assembly checkpoint during the metaphase-to-anaphase transition in rat spermatocytes. Furthermore, CENP-E depletion leads to abnormal spermatogenesis, reduced sperm count, and abnormal sperm head structure. Our findings have elucidated that CENP-E is essential for homologous chromosome alignment and spindle assembly checkpoint in spermatocytes, which further contribute to chromosome stability and sperm cell quality during spermatogenesis.

Lycopene inhibits apoptosis of mouse spermatocytes in varicocele via miR-23a/b-induced downregulation of PROK2.

Context The varicocele is the leading cause of male infertility and can impair sperm quality and testicular function through various mechanisms. In our previous study, we found that lycopene could attenuate hypoxia-induced testicular injury. Aims To illustrate the detailed mechanism of lycopene on spermatocytes. Methods The effect of lycopene on GC-2 cells under hypoxia were detected by flow cytometry and western blot assay. miR-seq was used to determine miRNA expression in varicocele rat model testes. The function of miR-23a/b were determined by flow cytometry and western blot assay. Key results We demonstrate that lycopene could alleviate hypoxia-induced GC-2 cell apoptosis and could elevate miR-23a/b expression of the hypoxia model in vivo and in vitro . The miR-23a and -23b mimics could reduce the hypoxia-induced GC-2 cell apoptosis. Both miR-23a and -23b could directly bind with prokineticin 2 (PROK2) mRNA and downregulate its expression. Conclusions Lycopene could attenuate hypoxia-induced spermatocyte injury through the miR-23a/b-PROK2 pathway. Implications Lycopene may be an effective treatment for varicocele to improve testicular impairment.

C. elegans spermatocyte divisions show a weak spindle checkpoint response.

Male meiotic division exhibits two consecutive chromosome separation events without apparent pausing. Several studies have shown that spermatocyte divisions are not stringently regulated as in mitotic cells. In this study, we investigated the role of the canonical spindle assembly (SAC) pathway in Caenorhabditis elegans spermatogenesis. We found the intensity of chromosome-associated outer kinetochore protein BUB-1 and SAC effector MDF-1 oscillates between the two divisions. However, the SAC target securin is degraded during the first division and remains undetectable for the second division. Inhibition of proteasome-dependent protein degradation did not affect the progression of the second division but stopped the first division at metaphase. Perturbation of spindle integrity did not affect the duration of meiosis II, and only slightly lengthened meiosis I. Our results demonstrate that male meiosis II is independent of SAC regulation, and male meiosis I exhibits only weak checkpoint response.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies.

OBJECTIVE: To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). DESIGN: Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. SETTING: A laboratory study. PATIENTS: Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). INTERVENTION: None. MAIN OUTCOME MEASURES: Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. RESULTS: Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. CONCLUSION: This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and in vitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

Cell-type specific and differential expression of LINC-RSAS long noncoding RNA declines in the testes during ageing of the rat.

Long noncoding RNAs (lncRNAs) have emerged as major regulators of gene expression, chromatin structure, epigenetic changes, post-transcriptional processing of RNAs, translation of mRNAs into proteins as well as contributing to the process of ageing. Ageing is a universal, slow, progressive change in almost all physiological processes of organisms after attaining reproductive maturity and often associated with age-related diseases. Mammalian testes contain various cell-types, vast reservoir of transcriptome complexity, produce haploid male gametes for reproduction and testosterone for development and maintenance of male sexual characters as well as contribute genetic variation to the species. We report age-related decline in expression and cellular localization of Long intergenic noncoding repeat-rich sense-antisense (LINC-RSAS) RNA in the testes and its major cell-types such as primary spermatocytes, Leydig cells and Sertoli cells during ageing of the rat. LINC-RSAS expression in testes increased from immature (4-weeks) to adult (16- and 44-weeks) and declined from adult (44-weeks) to nearly-old (70-weeks) rats. Genomic DNA methylation in the testes showed a similar pattern. Cell-type specific higher expression of LINC-RSAS was observed in primary spermatocytes (pachytene cells), Leydig cells and Sertoli cells of testes of adult rats. Over-expression of LINC-RSAS in cultured human cell lines revealed its possible role in cell-cycle control and apoptosis. We propose that LINC-RSAS expression is involved in molecular physiology of primary spermatocytes, Leydig cells and Sertoli cells of adult testes and its decline is associated with diminishing function of testes during ageing of the rat.

The male pachynema-specific protein MAPS drives phase separation in vitro and regulates sex body formation and chromatin behaviors in vivo.

Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps-/- pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. MapsΔ2-9/Δ2-9 male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps-/- mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.

Balanced spatiotemporal arrangements of histone H3 and H4 posttranslational modifications are necessary for meiotic prophase I chromosome organization.

Dynamic nuclear architecture and chromatin organizations are the key features of the mid-prophase I in mammalian meiosis. The chromatin undergoes major changes, including meiosis-specific spatiotemporal arrangements and remodeling, the establishment of chromatin loop-axis structure, pairing, and crossing over between homologous chromosomes, any deficiencies in these events may induce genome instability, subsequently leading to failure to produce gametes and infertility. Despite the significance of chromatin structure, little is known about the location of chromatin marks and the necessity of their balance during meiosis prophase I. Here, we show a thorough cytological study of the surface-spread meiotic chromosomes of mouse spermatocytes for H3K9,14,18,23,27,36, H4K12,16 acetylation, and H3K4,9,27,36 methylation. Active acetylation and methylation marks on H3 and H4, such as H3K9ac, H3K14ac, H3K18ac, H3K36ac, H3K56ac, H4K12ac, H4K16ac, and H3K36me3 exhibited pan-nuclear localization away from heterochromatin. In comparison, repressive marks like H3K9me3 and H3K27me3 are localized to heterochromatin. Further, taking advantage of the delivery of small-molecule chemical inhibitors methotrexate (heterochromatin enhancer), heterochromatin inhibitor, anacardic acid (histone acetyltransferase inhibitor), trichostatin A (histone deacetylase inhibitor), IOX1 (JmjC demethylases inhibitor), and AZ505 (methyltransferase inhibitor) in seminiferous tubules through the rete testis route, revealed that alteration in histone modifications enhanced the centromere mislocalization, chromosome breakage, altered meiotic recombination and reduced sperm count. Specifically, IOX1 and AZ505 treatment shows severe meiotic phenotypes, including altering chromosome axis length and chromatin loop size via transcriptional regulation of meiosis-specific genes. Our findings highlight the importance of balanced chromatin modifications in meiotic prophase I chromosome organization and instability.

PTBP2 binds to a testis-specific long noncoding RNA, Tesra, and activates transcription of the Prss42/Tessp-2 gene.

Long noncoding RNAs (lncRNAs) have recently been proved to be functional in the testis. Tesra, a testis-specific lncRNA, was suggested to activate the transcription of Prss42/Tessp-2, a gene that is involved in meiotic progression, in mouse spermatocytes. To reveal the molecular mechanism underlying the activation, we searched for Tesra-binding proteins by a Ribotrap assay followed by LC-MS/MS analysis and identified polypyrimidine tract binding protein 2 (PTBP2) as a candidate. Analysis of public RNA-seq data and our qRT-PCR results indicated that Ptbp2 mRNA showed an expression pattern similar to the expression patterns of Tesra and Prss42/Tessp-2 during testis development. Moreover, PTBP2 was found to be associated with Tesra in testicular germ cells by RNA immunoprecipitation. To evaluate the effect of PTBP2 on the Prss42/Tessp-2 promoter, we established an in vitro reporter gene assay system in which Tesra expression could be induced by the Tet-on system and thereby Prss42/Tessp-2 promoter activity could be increased. In this system, the Prss42/Tessp-2 promoter activity was significantly decreased by the knockdown of PTBP2. These results suggest that PTBP2 contributes to Prss42/Tessp-2 transcriptional activation by Tesra in spermatocytes. The finding provides a precious example of a molecular mechanism of testis lncRNA functioning in spermatogenesis.

TAF7L regulates early stages of male germ cell development in the rat.

Male germ cell development is dependent on the orchestrated regulation of gene networks. TATA-box binding protein associated factors (TAFs) facilitate interactions of TATA-binding protein with the TATA element, which is known to coordinate gene transcription during organogenesis. TAF7 like (Taf7l) is situated on the X chromosome and has been implicated in testis development. We examined the biology of TAF7L in testis development using the rat. Taf7l was prominently expressed in preleptotene to leptotene spermatocytes. To study the impact of TAF7L on the testis we generated a global loss-of-function rat model using CRISPR/Cas9 genome editing. Exon 3 of the Taf7l gene was targeted. A founder was generated possessing a 110 bp deletion within the Taf7l locus, which resulted in a frameshift and the premature appearance of a stop codon. The mutation was effectively transmitted through the germline. Deficits in TAF7L did not adversely affect pregnancy or postnatal survival. However, the Taf7l disruption resulted in male infertility due to compromised testis development and failed sperm production. Mutant germ cells suffer meiotic arrest at late zygotene/early pachynema stages, with defects in sex body formation. This testis phenotype was more pronounced than previously described for the subfertile Taf7l null mouse. We conclude that TAF7L is essential for male germ cell development in the rat.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice.

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

A postmeiotically bifurcated roadmap of honeybee spermatogenesis marked by phylogenetically restricted genes.

Haploid males of hymenopteran species produce gametes through an abortive meiosis I followed by meiosis II that can either be symmetric or asymmetric in different species. Thus, one spermatocyte could give rise to two spermatids with either equal or unequal amounts of cytoplasm. It is currently unknown what molecular features accompany these postmeiotic sperm cells especially in species with asymmetric meiosis II such as bees. Here we present testis single-cell RNA sequencing datasets from the honeybee (Apis mellifera) drones of 3 and 14 days after emergence (3d and 14d). We show that, while 3d testes exhibit active, ongoing spermatogenesis, 14d testes only have late-stage spermatids. We identify a postmeiotic bifurcation in the transcriptional roadmap during spermatogenesis, with cells progressing toward the annotated spermatids (SPT) and small spermatids (sSPT), respectively. Despite an overall similarity in their transcriptomic profiles, sSPTs express the fewest genes and the least RNA content among all the sperm cell types. Intriguingly, sSPTs exhibit a relatively high expression level for Hymenoptera-restricted genes and a high mutation load, suggesting that the special meiosis II during spermatogenesis in the honeybee is accompanied by phylogenetically young gene activities.

Gss deficiency causes age-related fertility impairment via ROS-triggered ferroptosis in the testes of mice.

Glutathione synthetase (GSS) catalyzes the final step in the synthesis of glutathione (GSH), a well-established antioxidant. Research on the specific roles of the Gss gene during spermatogenesis remains limited due to the intricate structure of testis. In this study, we identified pachytene spermatocytes as the primary site of GSS expression and generated a mouse model with postnatal deletion of Gss using Stra8-Cre (S8) to investigate the role of GSS in germ cells. The impact of Gss knockout on reducing male fertility is age-dependent and caused by ferroptosis in the testis. The 2-month-old S8/Gss-/- male mice exhibited normal fertility, due to a compensatory increase in GPX4, which prevented the accumulation of ROS. With aging, there was a decline in GPX4 and an increase in ALOX15 levels observed in 8-month-old S8/Gss-/- mice, resulting in the accumulation of ROS, lipid peroxidation, and ultimately testicular ferroptosis. We found that testicular ferroptosis did not affect spermatogonia, but caused meiosis disruption and acrosome heterotopia. Then the resulting aberrant sperm showed lower concentration and abnormal morphology, leading to reduced fertility. Furthermore, these injuries could be functionally rescued by inhibiting ferroptosis through intraperitoneal injection of GSH or Fer-1. In summary, Gss in germ cells play a crucial role in the resistance to oxidative stress injury in aged mice. Our findings deepen the understanding of ferroptosis during spermatogenesis and suggest that inhibiting ferroptosis may be a potential strategy for the treatment of male infertility.

Two distinct mechanisms lead to either oocyte or spermatocyte decrease in C. elegans after whole developmental exposure to γ-rays.

Wildlife is subject to various sources of pollution, including ionizing radiation. Adverse effects can impact the survival, growth, or reproduction of organisms, later affecting population dynamics. In invertebrates, reproduction, which directly impacts population dynamics, has been found to be the most radiosensitive endpoint. Understanding the underlying molecular pathways inducing this reproduction decrease can help to comprehend species-specific differences in radiosensitivity. From our previous studies, we found that decrease in reproduction is life stage dependent in the roundworm Caenorhabditis elegans, possibly resulting from an accumulation of damages during germ cell development and gamete differentiation. To go further, we used the same experimental design to assess more precisely the molecular determinants of reproductive toxicity, primarily decreases in gamete number. As before, worms were chronically exposed to 50 mGy·h-1 external gamma ionizing radiation throughout different developmental periods (namely embryogenesis, gametogenesis, and full development). To enable cross species extrapolation, conserved molecular pathways across invertebrates and vertebrates were analysed: apoptosis and MAP kinase Ras/ERK (MPK-1), both involved in reproduction and stress responses. Our results showed that these pathways are life-stage dependent, resulting from an accumulation of damages upon chronic exposure to IR throughout the life development. The Ras/ERK pathway was activated in our conditions in the pachytene region of the gonad where it regulates cell fate including apoptosis, but not in the ovulation zone, where it controls oocyte maturation and ovulation. Additionally, assessment of germ cell proliferation via Ras/ERK pathway showed no effect. Finally, a functional analysis of apoptosis revealed that while the decrease of the ovulation rate is caused by DNA-damaged induced apoptosis, this process does not occur in spermatocytes. Thus, sperm decrease seems to be mediated via another mechanism, probably a decrease in germ cell proliferation speed that needs further investigation to better characterize sex-specific responses to IR exposure. These results are of main importance to describe radio-induced reprotoxic effects and contribute as weight of evidence for the AOP #396 "Deposition of ionizing energy leads to population decline via impaired meiosis".

Chromosome-specific maturation of the epigenome in the Drosophila male germline.

Spermatogenesis in the Drosophila male germline proceeds through a unique transcriptional program controlled both by germline-specific transcription factors and by testis-specific versions of core transcriptional machinery. This program includes the activation of genes on the heterochromatic Y chromosome, and reduced transcription from the X chromosome, but how expression from these sex chromosomes is regulated has not been defined. To resolve this, we profiled active chromatin features in the testes from wildtype and meiotic arrest mutants and integrate this with single-cell gene expression data from the Fly Cell Atlas. These data assign the timing of promoter activation for genes with germline-enriched expression throughout spermatogenesis, and general alterations of promoter regulation in germline cells. By profiling both active RNA polymerase II and histone modifications in isolated spermatocytes, we detail widespread patterns associated with regulation of the sex chromosomes. Our results demonstrate that the X chromosome is not enriched for silencing histone modifications, implying that sex chromosome inactivation does not occur in the Drosophila male germline. Instead, a lack of dosage compensation in spermatocytes accounts for the reduced expression from this chromosome. Finally, profiling uncovers dramatic ubiquitinylation of histone H2A and lysine-16 acetylation of histone H4 across the Y chromosome in spermatocytes that may contribute to the activation of this heterochromatic chromosome.

Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase.

During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.