Histone Retention Research Articles

17α-Ethynylestradiol alters testicular epigenetic profiles and histone-to-protamine exchange in mice

Spermatogenesis starts with the onset of puberty within the seminiferous epithelium of the testes. It is a complex process under intricate control of the endocrine system. Physiological regulations by steroid hormones in general and by estrogens in particular are due to their chemical nature prone to be disrupted by exogenous factors acting as endocrine disruptors (EDs). 17α-Ethynylestradiol (EE2) is an environmental pollutant with a confirmed ED activity and a well-known effect on spermatogenesis and chromatin remodeling in haploid germ cells. The aim of our study was to assess possible effects of two doses (2.5ng/ml; 2.5 μg/ml) of EE2 on both histone-to-protamine exchange and epigenetic profiles during spermatogenesis performing a multi/transgenerational study in mice. Our results demonstrated an impaired histone-to-protamine exchange with a significantly higher histone retention in sperm nuclei of exposed animals, when this process was accompanied by the changes of histone post-translational modifications (PTMs) abundancies with a prominent effect on H3K9Ac and partial changes in protamine 1 promoter methylation status. Furthermore, individual changes in molecular phenotypes were partially transmitted to subsequent generations, when no direct trans-generational effect was observed. Finally, the uncovered specific localization of the histone retention in sperm nuclei and their specific PTMs profile after EE2 exposure may indicate an estrogenic effect on sperm motility and early embryonic development via epigenetic mechanisms.

Constitutive Androstane Receptor Regulates Germ Cell Homeostasis, Sperm Quality, and Male Fertility via Akt-Foxo1 Pathway.

Male sexual function can be disrupted by exposure to exogenous compounds that cause testicular physiological alterations. The constitutive androstane receptor (Car) is a receptor for both endobiotics and xenobiotics involved in detoxification. However, its role in male fertility, particularly in regard to the reprotoxic effects of environmental pollutants, remains unclear. This study aims to investigate the role of the Car signaling pathway in male fertility. In vivo, in vitro, and pharmacological approaches are utilized in wild-type and Car-deficient mouse models. The results indicate that Car inhibition impaired male fertility due to altered sperm quality, specifically histone retention, which is correlated with an increased percentage of dying offspring in utero. The data highlighted interactions among Car, Akt, Foxo1, and histone acetylation. This study demonstrates that Car is crucial in germ cell homeostasis and male fertility. Further research on the Car signaling pathway is necessary to reveal unidentified causes of altered fertility and understand the harmful impact of environmental molecules on male fertility and offspring health.

P-066 A multi-bioassay approach to identify male gamete fertilization dysfunction

Abstract Study question Can a multi-bioassay approach identify a specific male gamete dysfunction often overlooked by a standard semen analysis? Summary answer In cases of persistent fertilization failure, additional testing may assist in pinpointing underlying factors contributing to compromised sperm embryo developmental competence and tailor treatment. What is known already Conventional semen analysis, while informative, ineffectively captures the complete spectrum of male gamete dysfunction. Despite adequate sperm concentration and motility, some couples may still experience fertilization failure with Intracytoplasmic Sperm Injection (ICSI). This highlights the complexity of male infertility, that extends beyond standard parameters. In addressing sperm-related fertilization challenges, various assisted oocyte activation (AOA) methods have been proposed to trigger intracellular Ca2+ release, leading to calcium spikes and meiosis initiation. Study design, size, duration In the past 10 months, we identified couples (n = 50) with a history of poor ICSI fertilization, exhibiting suboptimal head morphology. Screening for acrosomal dysfunction involved assessing the presence of PLCxand related gene mutations. We measured the degree of chromatin compaction by histone-to-protamine ratio and evaluated sperm chromatin fragmentation (SCF). To correct for eventual acrosomal dysfunction, couples were counseled to undergo subsequent ICSI cycles with AOA. Clinical outcome was compared between historical and treatment cycles. Participants/materials, setting, methods Fifty men with a history of poor ICSI fertilization were included. A standard semen analysis was conducted according to WHO guidelines. The presence of PLCx was measured through immunofluorescent staining (normal threshold, ≥ 30%), corroborated by gene mutation detection though DNA-seq. Sperm histone-to-protamine ratio was evaluated by Aniline Blue (normal threshold, <15% histone retention). SCF was assessed utilizing the TUNEL assay (normal threshold, ≤15%). AOA was performed using ionomycin or recombinant PLCx protein. Main results and the role of chance Fifty men experienced a poor clinical outcome following ICSI with a fertilization of 7.4% (48/647), embryo implantation of 6.6% (1/15), clinical pregnancy of 6.6% (1/15), and pregnancy loss rate of 100% (1/1). Their semen analysis, interestingly, revealed the following semen parameters: 3.0±1 mL volume, 50.0±20 x106/mL concentration, 41.4±21% motility, and 1.9±1% normal morphology. Nonetheless, PLCx content was diminished, with values ranging from 2-27%. Genomic assessment confirmed mutations in PLCx, DPY19L2, SPATA16 and PICK1 genes, all implicated in sperm nuclear remodeling and acrosome formation. An elevated histone content at 29.7% was positively correlated with an increased SCF at 18.0% (R2=0.6, P<0.001). In subsequent cycles employing AOA, the normal fertilization increased to 42.3% (305/721), the embryo implantation reached 13.0% (9/69), the clinical pregnancy rose to 21.1% (8/38), and the delivery rate was 21.1% (8/38), without any pregnancy loss. (P<0.001). Limitations, reasons for caution While this study affirms the effectiveness of a multifaceted assessment in pinpointing specific sperm dysfunctions and customizing treatment, it is still a retrospective observation requiring independent validation in a larger population. Wider implications of the findings Semen analysis alone fails to address specific aspects of male gamete dysfunction. Incorporating additional bioassays to assess chromatin structure, integrity, and acrosomal function offers valuable insights for fertility management in tailoring personalized treatments. This comprehensive approach enhances the understanding of male reproductive health, maximizing the potential for successful clinical outcome. Trial registration number not applicable

FBXO24 modulates mRNA alternative splicing and MIWI degradation and is required for normal sperm formation and male fertility

Spermiogenesis is a critical, post-meiotic phase of male gametogenesis, in which the proper gene expression is essential for sperm maturation. However, the underFlying molecular mechanism that controls mRNA expression in the round spermatids remains elusive. Here, we identify that FBXO24, an orphan F-box protein, is highly expressed in the testis of humans and mice and interacts with the splicing factors (SRSF2, SRSF3, and SRSF9) to modulate the gene alternative splicing in the round spermatids. Genetic mutation of FBXO24 in mice causes many abnormal splicing events in round spermatids, thus affecting a large number of critical genes related to sperm formation that were dysregulated. Further molecular and phenotypical analyses revealed that FBXO24 deficiency results in aberrant histone retention, incomplete axonemes, oversized chromatoid body, and abnormal mitochondrial coiling along sperm flagella, ultimately leading to male sterility. In addition, we discovered that FBXO24 interacts with MIWI and SCF subunits and mediates the degradation of MIWI via K48-linked polyubiquitination. Furthermore, we show that FBXO24 depletion could lead to aberrant piRNA production in testes, which suggests FBXO24 is required for normal piRNA counts. Collectively, these data demonstrate that FBXO24 is essential for sperm formation by regulating mRNA alternative splicing and MIWI degradation during spermiogenesis.

FBXO24 modulates mRNA alternative splicing and MIWI degradation and is required for normal sperm formation and male fertility.

Spermiogenesis is a critical, post-meiotic phase of male gametogenesis, in which the proper gene expression is essential for sperm maturation. However, the underFlying molecular mechanism that controls mRNA expression in the round spermatids remains elusive. Here, we identify that FBXO24, an orphan F-box protein, is highly expressed in the testis of humans and mice and interacts with the splicing factors (SRSF2, SRSF3, and SRSF9) to modulate the gene alternative splicing in the round spermatids. Genetic mutation of FBXO24 in mice causes many abnormal splicing events in round spermatids, thus affecting a large number of critical genes related to sperm formation that were dysregulated. Further molecular and phenotypical analyses revealed that FBXO24 deficiency results in aberrant histone retention, incomplete axonemes, oversized chromatoid body, and abnormal mitochondrial coiling along sperm flagella, ultimately leading to male sterility. In addition, we discovered that FBXO24 interacts with MIWI and SCF subunits and mediates the degradation of MIWI via K48-linked polyubiquitination. Furthermore, we show that FBXO24 depletion could lead to aberrant piRNA production in testes, which suggests FBXO24 is required for normal piRNA counts. Collectively, these data demonstrate that FBXO24 is essential for sperm formation by regulating mRNA alternative splicing and MIWI degradation during spermiogenesis.

Specialized replication of heterochromatin domains ensures self-templated chromatin assembly and epigenetic inheritance

Heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), spreads across large domains and can be epigenetically inherited in a self-propagating manner. Heterochromatin propagation depends upon a read-write mechanism, where the Clr4/Suv39h methyltransferase binds to preexisting trimethylated H3K9 (H3K9me3) and further deposits H3K9me. How the parental methylated histone template is preserved during DNA replication is not well understood. Here, we demonstrate using Schizosaccharomyces pombe that heterochromatic regions are specialized replication domains demarcated by their surrounding boundary elements. DNA replication throughout these domains is distinguished by an abundance of replisome components and is coordinated by Swi6/HP1. Although mutations in the replicative helicase subunit Mcm2 that affect histone binding impede the maintenance of a heterochromatin domain at an artificially targeted ectopic site, they have only a modest impact on heterochromatin propagation via the read-write mechanism at an endogenous site. Instead, our findings suggest a crucial role for the replication factor Mcl1 in retaining parental histones and promoting heterochromatin propagation via a mechanism involving the histone chaperone FACT. Engagement of FACT with heterochromatin requires boundary elements, which position the heterochromatic domain at the nuclear peripheral subdomain enriched for heterochromatin factors. Our findings highlight the importance of replisome components and boundary elements in creating a specialized environment for the retention of parental methylated histones, which facilitates epigenetic inheritance of heterochromatin.

Spermatozoa in mice lacking the nucleoporin NUP210L show defects in head shape and motility but not in nuclear compaction or histone replacement.

Biallelic loss-of-function mutation of NUP210L, encoding a testis-specific nucleoporin, has been reported in an infertile man whose spermatozoa show uncondensed heads and histone retention. Mice with a homozygous transgene intronic insertion in Nup210l were infertile but spermatozoa had condensed heads. Expression from this insertion allele is undefined, however, and residual NUP210L production could underlie the milder phenotype. To resolve this issue, we have created Nup210lem1Mjmm , a null allele of Nup210l, in the mouse. Nup210lem1Mjmm homozygotes show uniform mild anomalies of sperm head morphology and decreased motility, but nuclear compaction and histone removal appear unaffected. Thus, our mouse model does not support that NUP210L loss alone blocks spermatid nuclear compaction. Re-analyzing the patient's exome data, we identified a rare, potentially pathogenic, heterozygous variant in nucleoporin gene NUP153 (p.Pro485Leu), and showed that, in mouse and human, NUP210L and NUP153 colocalize at the caudal nuclear pole in elongating spermatids and spermatozoa. Unexpectedly, in round spermatids, NUP210L and NUP153 localisation differs between mouse (nucleoplasm) and human (nuclear periphery). Our data suggest two explanations for the increased phenotypic severity associated with NUP210L loss in human compared to mouse: a genetic variant in human NUP153 (p.Pro485Leu), and inter-species divergence in nuclear pore function in round spermatids.

Histone retention preserves epigenetic marks during heat stress-induced transcriptional memory in plants.

Plants often experience recurrent stressful events, for example, during heat waves. They can be primed by heat stress (HS) to improve the survival of more severe heat stress conditions. At certain genes, sustained expression is induced for several days beyond the initial heat stress. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), but it is unclear how this is maintained for extended periods. Here, we determined histone turnover by measuring the chromatin association of HS-induced histone H3.3. Genome-wide histone turnover was not homogenous; in particular, H3.3 was retained longer at heat stress memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, methylation loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation, but not vice versa. Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally mediated epigenetic modifications.

Differential sperm histone retention in normozoospermic ejaculates of infertile men negatively affects sperm functional competence and embryo quality.

The unique epigenetic architecture that sperm cells acquire during spermiogenesis by retaining <15% of either canonical or variant histone proteins in their genome is essential for normal embryogenesis. Whilst heterogeneous levels of retained histones are found in morphologically normal spermatozoa, their effect on reproductive outcomes is not fully understood. Processed spermatozoa (n=62) were tested for DNA integrity by sperm chromatin dispersion assay, and retained histones were extracted and subjected to dot-blot analysis. The impact of retained histone modifications in normozoospermic patients on sperm functional characteristics, embryo quality, metabolic signature in embryo spent culture medium and pregnancy outcome was studied. Dot-blot analysis showed heterogeneous levels of retained histones in the genome of normozoospermic ejaculates. Post-wash sperm yield was affected by an increase in H3K27Me3 and H4K20Me3 levels in the sperm chromatin (p<0.05). Also, spermatozoa with higher histone H3 retention had increased DNA damage (p<0.05). Spermatozoa from these cohorts, when injected into donor oocytes, correlated to a significant decrease in the fertilisation rate with an increase in sperm histone H3 (p<0.05) and H3K27Me3 (p<0.01). An increase in histone H3 negatively affected embryo quality (p<0.01) and clinical pregnancy outcome post-embryo transfer (p<0.05). On the other hand, spent culture medium metabolites assessed by high-resolution (800MHz) nuclear magnetic resonance showed an increased intensity of the amino acid methionine in the non-pregnant group than in the pregnant group (p<0.05) and a negative correlation with sperm histone H3 in the pregnant group (p<0.05). Histone retention in spermatozoa can be one of the factors behind the development of idiopathic male infertility. Such spermatozoa may influence embryonic behaviour and thereby affect the success rate of assisted reproductive technology procedures. These results, although descriptive in nature, warrant further research to address the underlying mechanisms behind these clinically important observations.

Sire modulates developmental kinetics and transcriptome of the bovine embryo.

The first week of gestation is a period of major pregnancy loss in cattle, this study reveals that the male plays a key role in regulating embryonic development during this time. The impact of sire on preimplantation embryonic development in cattle remains poorly understood. This study evaluated differences in embryos produced in vitro from sires with varying capacities to produce blastocysts. Sires classified as high (HP) and low performing (LP) based on their ability to produce embryos were used to better understand how sire regulates embryonic development. By monitoring development, it was determined that the most common arrest stage was the five- to six-cell stage. Embryos (four to six cells) from HP and LP sires were then analyzed for autophagic activity, where embryos for LP sires exhibited increased autophagy than HP-derived embryos. Transcriptome analysis of four-cell embryos found that embryos from LP sires might have issues in sperm mitochondrial clearance, histone retention, and DNA damage, while HP sires had increased expression of genes involved in transcription, chromosome segregation, and cell division. In conclusion, LP sires had an increased proportion of embryos arresting at the five- to six-cell stage, and these embryos had higher rates of cellular stress due to paternal contributions from the spermatozoon.

Micrococcal nuclease sequencing of porcine sperm suggests enriched co-location between retained histones and genomic regions related to semen quality and early embryo development.

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm's chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements.These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars.

Proteomic and genetic dissection of testis-specific histone 2B in infertile men reveals its contribution to meiosis and sperm motility

To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men. Case-control study. Basic science laboratory. Fertile and infertile men. Not applicable. The histone and protamine status of sperm was studied by aniline blue and chromomycin A3 staining, respectively. Testis-specific histone 2B, total H2B, and phosphorylated TSH2B (pTSH2B) were estimated by Western blot analysis. The frequency of genetic polymorphisms and rare variants in H2BC1 was studied by Sanger sequencing. Phosphosites on TSH2B in sperm were identified by reverse-phase high-performance liquid chromatography purification of TSH2B followed by mass spectrometric analysis. Aniline blue and chromomycin A3 staining revealed significantly higher histone retention and low protamine in sperm of infertile men. Sperm TSH2B and total H2B levels were significantly lower in oligozoospermic and oligoasthenozoospermic men (in both groups). The TSH2B levels were comparable in asthenozoospermic men; however, the pTSH2B level was significantly low. The H2BC1 gene sequencing identified 6 variants, of which 2 are rare variants (rs368672899 and rs544942090) and 4 (rs4711096, rs4712959, rs4712960 and rs4712961) are single nucleotide polymorphisms. Minor allele frequency of 5'-untranslated region variant rs4711096 was significantly lower in infertile men (OR = 0.65). The rare nonsynonymous variant, rs368672899, p.Ser5Pro was seen in 1 oligoasthenoteratozoospermic individual. Interestingly, mass spectrometric analysis identified a site on TSH2B to bear a phosphate group in the sperm of fertile men. Our study reveals a defect in the replacement of somatic histones with testis-specific variants in infertile men. Chromatin compaction positively correlates with sperm motility, which is suggestive of its utility in diagnostic semen analysis of infertile individuals. Our observations with TSH2B and its cognate gene in sperm of infertile men indicate an essential role for TSH2B in meiosis and its phosphorylation in sperm motility, respectively.

P-028 Microfluidics as a methodology for sperm selection. A prospective functional study

Abstract Study question Is microfluidics an optimal technique to improve the sperm selection process in comparison with standard techniques like Density Gradient Centrifugation or Swim-up? Summary answer A significant increase in sperm quality was obtained when microfluidics was compared to density gradient centrifugation but improvement evidence regarding swim-up is not yet demonstrated. What is known already Assisted reproduction clinics for in vitro fertilization treatments have developed several techniques to perform sperm selection, being density gradient centrifugation (DGC) and swim-up the most widely used. However, sperm selection is a procedure that requires bulky and expensive equipment, long waiting times and gamete manipulation, which results in cellular stress. The SwimCount Harvester is a microfluidic-based device capable of performing sperm selection and overcoming the problems of other sorting systems. In our study, we analyzed several sperm quality parameters between these three sperm selection techniques. Study design, size, duration This was a prospective, cohort and observational study including 111 semen samples from patients and donors (mean age 33,7±9,3 years) between February 2021 and January 2022. The semen sample from each patient or donor was divided into two volumes, one part, the sperm selection was performed using the SwimCount Harvester and the other part using DGC or Swim-up. These sperm selection techniques were used to isolate sperm based on fluid dynamics and cell motility. Participants/materials, setting, methods Fresh ejaculate and sperm selected samples from each patient were analyzed according to the 2010 WHO-criteria to assess concentration, motility, morphology and vitality, using automatic image analysis. The excessive histone retention indicating defective chromatin compaction was assessed using aniline blue staining. Sperm chromatin fragmentation (SCF) was assessed by TUNEL on at least 20.000 sperm using flow cytometry. Kruskal-Wallis test was performed in order to assess statistical differences of the variables between the sperm selection methods. Main results and the role of chance The SwimCount Harvester was compared to DGC (n = 95). Ejaculated sperm yielded an average concentration of 53,2±34,2x106/mL. After DGC and SwimCount Harvester, the sperm concentration was 11,1±8,8 and 12,5±11,2x106/mL, respectively. The motility of fresh sperm sample improved from 41,9±10,4% to 71,6±10,6% after DGC and 76,8±10,0% after SwimCount Harvester (P &amp;lt; 0,05). The percentage of normal sperm increased from 2,1±1,2%, for the fresh samples, to 3,5±1,4% and 4,2±1,6% for the samples processed by DGC and microfluidics, respectively (P &amp;lt; 0,05). The percentage of live sperm increased from 74,0±8,1% and 77,5±8,7% in fresh sperm and after DGC, respectively to 85,9±9,0% after using microfluidics (P &amp;lt; 0,05). In the same way, the normal sperm chromatine structure percentage increased from 67,4±7,5% to 75,4±7,9% for the sperm samples selected by DGC and 77,7±8,9% when the SwimCount Harvester was used (P &amp;lt; 0,05). A decrease in SCF was observed from 12,9±8,4% in samples selected by DGC to 10,4±5,1% in raw samples (P &amp;gt; 0,05). However, after sperm selection using SwimCount Harvester, SCF fell to 4,6±4,1%, showing significant differences between both sperm selection methods (P &amp;lt; 0,05). Similar results were obtained for oligozoospermic samples (n = 6). When the SwimCount Harvester was compared with the Swim-up (n = 10), non-significant improvements were observed for all the parameters studied due to the reduced sample size. Limitations, reasons for caution The database of samples processed using swim-up and oligozoospermic samples is too small to draw reliable conclusions. Although significantly better results are obtained in sperm samples selected by the SwimCount Harvester with respect to DGC, a clinical study using the microfluidic device in assisted reproduction cycles has to be performed. Wider implications of the findings The SwimCount Harvester, in addition to significantly improving sperm selection and quality, is a reliable alternative to integrate numerous laboratory steps into a single automated procedure, reducing workload, the amount of culture media and equipment used, gamete handling and the stress that produces. Moreover, microfluidics may eliminate inter-laboratory variability. Trial registration number This project has received funding from the Eurostars-2 joint program with co-funding from CDTI and the European Union's Horizon 2020 Research and Innovation Framework Program E! 113740/ CIIP-20201009.

Loss of Prm1 leads to defective chromatin protamination, impaired PRM2 processing, reduced sperm motility and subfertility in male mice.

ABSTRACTOne of the key events during spermiogenesis is the hypercondensation of chromatin by substitution of the majority of histones by protamines. In humans and mice, protamine 1 (PRM1/Prm1) and protamine 2 (PRM2/Prm2) are expressed in a species-specific ratio. Using CRISPR-Cas9-mediated gene editing, we generated Prm1-deficient mice and demonstrated that Prm1+/− mice were subfertile, whereas Prm1−/− mice were infertile. Prm1−/− and Prm2−/− sperm showed high levels of reactive oxygen species-mediated DNA damage and increased histone retention. In contrast, Prm1+/− sperm displayed only moderate DNA damage. The majority of Prm1+/− sperm were CMA3 positive, indicating protamine-deficient chromatin, although this was not the result of increased histone retention in Prm1+/− sperm. However, sperm from Prm1+/− and Prm1−/− mice contained high levels of incompletely processed PRM2. Furthermore, the PRM1:PRM2 ratio was skewed from 1:2 in wild type to 1:5 in Prm1+/− animals. Our results reveal that PRM1 is required for proper PRM2 processing to produce mature PRM2, which, together with PRM1, is able to hypercondense DNA. Thus, the species-specific PRM1:PRM2 ratio has to be precisely controlled in order to retain full fertility.

The Cif proteins from Wolbachia prophage WO modify sperm genome integrity to establish cytoplasmic incompatibility.

Inherited microorganisms can selfishly manipulate host reproduction to drive through populations. In Drosophila melanogaster, germline expression of the native Wolbachia prophage WO proteins CifA and CifB cause cytoplasmic incompatibility (CI) in which embryos from infected males and uninfected females suffer catastrophic mitotic defects and lethality; however, in infected females, CifA expression rescues the embryonic lethality and thus imparts a fitness advantage to the maternally transmitted Wolbachia. Despite widespread relevance to sex determination, evolution, and vector control, the mechanisms underlying when and how CI impairs male reproduction remain unknown and a topic of debate. Here, we use cytochemical, microscopic, and transgenic assays in D. melanogaster to demonstrate that CifA and CifB proteins of wMel localize to nuclear DNA throughout the process of spermatogenesis. Cif proteins cause abnormal histone retention in elongating spermatids and protamine deficiency in mature sperms that travel to the female reproductive tract with Cif proteins. Notably, protamine gene knockouts enhance wild-type CI. In ovaries, CifA localizes to germ cell nuclei and cytoplasm of early-stage egg chambers; however, Cifs are absent in late-stage oocytes and subsequently in fertilized embryos. Finally, CI and rescue are contingent upon a newly annotated CifA bipartite nuclear localization sequence. Together, our results strongly support the Host modification model of CI in which Cifs initially modify the paternal and maternal gametes to bestow CI-defining embryonic lethality and rescue.

PD36-05 LOSS-OF-FUNCTION IN PHF7 CAUSE MALE INFERTILITY BY IMPAIRING HISTONE-TO-PROTAMINE EXCHANGE DURING SPERMIOGENESIS

You have accessJournal of UrologyCME1 May 2022PD36-05 LOSS-OF-FUNCTION IN PHF7 CAUSE MALE INFERTILITY BY IMPAIRING HISTONE-TO-PROTAMINE EXCHANGE DURING SPERMIOGENESIS Jianxing Cheng, Mengyang Cao, Zhongjie Zheng, and Haocheng Lin Jianxing ChengJianxing Cheng More articles by this author , Mengyang CaoMengyang Cao More articles by this author , Zhongjie ZhengZhongjie Zheng More articles by this author , and Haocheng LinHaocheng Lin More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002594.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Introduction: Recently, studies shows that PHF7 is specifically expressed in elongating spermatid during spermiogenesis, however whether it results infertility in human are still unclear as well as the mechanism. Objective: To investigate the role of PHF7 on male infertility and explore its mechanism. METHODS: The blood and testicular samples from azoospermia patients were collected and used for genome sequencing and immunohistology. The PHF7 knockout mice model were established and used for animal study. RT-PCR, Immunohistology, GST-pulldown and western blotting were used for mechanism exploring. RESULTS: Our results revealed that expression level of PHF7 in patients with male infertility was significant less than control. Two heterozygous mutations were also identified in azoospemia patients. PHF7 knockout mice show defective spermatogenesis at a later spermiogenic stage with abnormal sperm morphology with histone retention. CONCLUSIONS: Our study identified PHF7 mutations as etiological factors in male infertility and reveal its mechanism for regulating the histone-to-protamine exchange during spermiogenesis. Source of Funding: Peking University Clinical Medicine +X Youth Special Program (PKU2018LCXQ019) © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e634 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jianxing Cheng More articles by this author Mengyang Cao More articles by this author Zhongjie Zheng More articles by this author Haocheng Lin More articles by this author Expand All Advertisement PDF DownloadLoading ...

Role of epigenetic transgenerational inheritance in generational toxicology.

Many environmental toxicants have been shown to be associated with the transgenerational inheritance of increased disease susceptibility. This review describes the generational toxicity of some of these chemicals and their role in the induction of epigenetic transgenerational inheritance of disease. Epigenetic factors include DNA methylation, histone modifications, retention of histones in sperm, changes to chromatin structure, and expression of non-coding RNAs. For toxicant-induced epigenetic transgenerational inheritance to occur, exposure to a toxicant must result in epigenetic changes to germ cells (sperm or eggs) since it is the germ cells that carry molecular information to subsequent generations. In addition, the epigenetic changes induced in transgenerational generation animals must cause alterations in gene expression in these animals’ somatic cells. In some cases of generational toxicology, negligible changes are seen in the directly exposed generations, but increased disease rates are seen in transgenerational descendants. Governmental policies regulating toxicant exposure should take generational effects into account. A new approach that takes into consideration generational toxicity will be needed to protect our future populations.

Epigenetic Transgenerational Inheritance of the Effects of Obesogen Exposure.

Obesity and metabolic disorders have become a worldwide pandemic affecting millions of people. Although obesity is a multifaceted disease, there is growing evidence supporting the obesogen hypothesis, which proposes that exposure to a subset of endocrine disrupting chemicals (EDCs), known as obesogens, promotes obesity. While these effects can be observed in vitro using cell models, in vivo and human epidemiological studies have strengthened this hypothesis. Evidence from animal models showed that the effects of obesogen exposure can be inherited transgenerationally through at least the F4 generation. Transgenerational effects of EDC exposure predispose future generations to undesirable phenotypic traits and diseases, including obesity and related metabolic disorders. The exact mechanisms through which phenotypic traits are passed from an exposed organism to their offspring, without altering the primary DNA sequence, remain largely unknown. Recent research has provided strong evidence suggesting that a variety of epigenetic mechanisms may underlie transgenerational inheritance. These include differential DNA methylation, histone methylation, histone retention, the expression and/or deposition of non-coding RNAs and large-scale alterations in chromatin structure and organization. This review highlights the most recent advances in the field of epigenetics with respect to the transgenerational effects of environmental obesogens. We highlight throughout the paper the strengths and weaknesses of the evidence for proposed mechanisms underlying transgenerational inheritance and why none of these is sufficient to fully explain the phenomenon. We propose that changes in higher order chromatin organization and structure may be a plausible explanation for how some disease predispositions are heritable through multiple generations, including those that were not exposed. A solid understanding of these possible mechanisms is essential to fully understanding how environmental exposures can lead to inherited susceptibility to diseases such as obesity.

Label-Free Evaluation of Chromatin Condensation in Human Normal Morphology Sperm Using Raman Spectroscopy

Chromatin condensation is one of the main factors essential for sperm function. Evaluation of chromatin condensation by current methods render the assessed sperm unsuitable for assisted reproduction. We examined the Raman spectra of normal morphology sperm to determine whether a non-invasive confocal Raman spectroscopy can detect spectral differences between groups having different levels of chromatin condensation. Semen samples from 85 donors who underwent ICSI were obtained. Chromomycin A3, aniline blue and acridine orange staining were performed to evaluate the protamine deficiency, histone retention and DNA fragmentation respectively. Raman spectra were obtained from 50 normal morphology sperm for each donor. Spectral analysis was performed using home written programs in LabVIEW software and samples were grouped based on chromomycin A3 staining. Raman peaks intensities at 670 cm-1, 731 cm-1, 785 cm-1, 858 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 were significantly correlated with at least one of the sperm staining methods. The median intensity of the Raman peaks at 670 cm-1, 731 cm-1, 785 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 show a significant difference between the CMA3≤41 and CMA3>41groups. The Raman spectroscopic measurements represent a promising diagnostic tool that has the ability to label-free detect sperm with chromatin abnormalities, such as improper chromatin condensation and DNA fragmentation to a certain degree similar to that of the existing staining techniques at the individual cell level.

Integration of sperm ncRNA-directed DNA methylation and DNA methylation-directed histone retention in epigenetic transgenerational inheritance

BackgroundEnvironmentally induced epigenetic transgenerational inheritance of pathology and phenotypic variation has been demonstrated in all organisms investigated from plants to humans. This non-genetic form of inheritance is mediated through epigenetic alterations in the sperm and/or egg to subsequent generations. Although the combined regulation of differential DNA methylated regions (DMR), non-coding RNA (ncRNA), and differential histone retention (DHR) have been shown to occur, the integration of these different epigenetic processes remains to be elucidated. The current study was designed to examine the integration of the different epigenetic processes.ResultsA rat model of transiently exposed F0 generation gestating females to the agricultural fungicide vinclozolin or pesticide DDT (dichloro-diphenyl-trichloroethane) was used to acquire the sperm from adult males in the subsequent F1 generation offspring, F2 generation grand offspring, and F3 generation great-grand offspring. The F1 generation sperm ncRNA had substantial overlap with the F1, F2 and F3 generation DMRs, suggesting a potential role for RNA-directed DNA methylation. The DMRs also had significant overlap with the DHRs, suggesting potential DNA methylation-directed histone retention. In addition, a high percentage of DMRs induced in the F1 generation sperm were maintained in subsequent generations.ConclusionsMany of the DMRs, ncRNA, and DHRs were colocalized to the same chromosomal location regions. Observations suggest an integration of DMRs, ncRNA, and DHRs in part involve RNA-directed DNA methylation and DNA methylation-directed histone retention in epigenetic transgenerational inheritance.