Expression Of Protamine Research Articles

Sperm RNA quantity and PRM1, PRM2, and TH2B transcript levels reflect sperm characteristics and early embryonic development.

Spermatozoa have a highly complex RNA profile. Several of these transcripts are suggested as biomarkers for male infertility and contribute to early development. To analyze the differences between sperm RNA quantity and expression of protamine (PRM1 and PRM2) and testis-specific histone 2B (TH2B) genes, spermatozoa from 33 patients who enrolled in assisted reproduction treatment (ART) program were analyzed. Sperm RNA of teratozoospermic (T), oligoteratozoospermic (OT), and normozoospermic (N) samples was extracted, and the differences in transcript levels among the study groups were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The correlations of total RNA per spermatozoon and the expression of the transcripts were evaluated in relation to sperm characteristics and preimplantation embryo development. The mean (±standard deviation) RNA amount per spermatozoon was 28.48 (±23.03) femtogram in the overall group and was significantly higher in the OT group than that in N and T groups. Total sperm RNA and gene expression of PRM1 and PRM2 genes were related to preimplantation embryo development and developmental arrest. Specific sperm characteristics were correlated with the expressions of PRM1, PRM2, or TH2B genes. We conclude that the sperm RNA amount and composition are important factors and might influence early embryonic development and also differ in different cases of male infertility.

Somatic Cell Nuclear Transfer Using Freeze-Dried Protaminized Donor Nuclei.

Somatic cell nuclear transfer (SCNT) is the only nuclear reprogramming method that allows rewinding an adult nucleus into a totipotent state. As such, it offers excellent opportunities for the multiplication of elite genotypes or endangered animals, whose number have shrunk to below the threshold of safe existence. Disappointingly, SCNT efficiency is still low. Hence, it would be wise to store somatic cells from threatened animals in biobanks. We were the first to show that freeze-dried cells allow generating blastocysts upon SCNT. Only a few papers have been published on the topic since then, and viable offspring have not been produced. On the other hand, lyophilization of mammalian spermatozoa has made considerable progress, partially due to the physical stability that protamines provide to the genome. In our previous work, we have demonstrated that a somatic cell could be made more amenable to the oocyte reprogramming by the exogenous expression of human Protamine 1. Given that the protamine also provides natural protection against dehydration stress, we have combined the cell protaminization and lyophilization protocols. This chapter comprehensively describes the protocol for somatic cell protaminization, lyophilization, and its application in SCNT. We are confident that our protocol will be relevant for establishing somatic cells stocks amenable to reprogramming at low cost.

19 Exploring the expression of protamine 1 as a “xeroprotectant” in somatic cell nuclear transfer using sheep freeze-dried adult fibroblast

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Determining the expression levels of CSF-1 and OCT4, CREM-1, and protamine in testicular biopsies of adult Klinefelter patients: Their possible correlation with spermatogenesis.

Klinefelter syndrome (KS) is the most prevalent genetic disorder of infertile males. This study aimed to determine in Klinefelter patients (KS) the expression levels of spermatogenic markers and testicular growth factors that might predict spermatogenesis based on conventional testicular sperm extraction (TESE). The expression levels of the pre-meiotic (OCT4, CD9, GFR-α1, α-6-INTEGRIN, SALL4, C-KIT), meiotic (CREM-1), and post-meiotic (protamine) markers, as well as the colony stimulating factor-1 (CSF-1) were examined in testicular biopsies with and without mature sperm of KS and normal karyotype of azoospermic patients (AZO) with complete spermatogenesis. In the biopsies of AZO, the expression levels (fold of expression compared to the PPI of the same sample) of OCT4 were 9.68± 7.93, CREM 42.78± 28.22, CSF-1 3.07 ± 3.19, and protamine 78498.12 ± 73214.40. Biopsies from KS included 7 with sperm and 17 without sperm. Among the biopsies with sperm, the expression levels of OCT4 were 7.27± 9.29, CREM 3.13± 7.89, CSF-1 35.5 ± 48.01, and protamine 902.97 ± 2365.92. In 14 biopsies without sperm, we found low expression levels of OCT4, CREM and CSF-1, and no expression of protamine. However, in three of the biopsies without sperm that highly expressed OCT4 and CSF-1, the expression levels of CREM-1 and protamine were high. These results may be used for further consulting with patients considering repeating conventional TESE or micro TESE and cryopreservation for possible future in-vitro spermatogenesis.

25th ANNIVERSARY OF CLONING BY SOMATIC-CELL NUCLEAR TRANSFER: Scientific and technological approaches to improve SCNT efficiency in farm animals and pets.

The birth of Dolly through somatic cell nuclear transfer (SCNT) was a major scientific breakthrough of the last century. Yet, while significant progress has been achieved across the technics required to reconstruct and in vitro culture nuclear transfer embryos, SCNT outcomes in terms of offspring production rates are still limited. Here, we provide a snapshot of the practical application of SCNT in farm animals and pets. Moreover, we suggest a path to improve SCNT through alternative strategies inspired by the physiological reprogramming in male and female gametes in preparation for the totipotency required after fertilization. Almost all papers on SCNT focused on nuclear reprogramming in the somatic cells after nuclear transfer. We believe that this is misleading, and even if it works sometimes, it does so in an uncontrolled way. Physiologically, the oocyte cytoplasm deploys nuclear reprogramming machinery specifically designed to address the male chromosome, the maternal alleles are prepared for totipotency earlier, during oocyte nuclear maturation. Significant advances have been made in remodeling somatic nuclei in vitro through the expression of protamines, thanks to a plethora of data available on spermatozoa epigenetic modifications. Missing are the data on large-scale nuclear reprogramming of the oocyte chromosomes. The main message our article conveys is that the next generation nuclear reprogramming strategies should be guided by insights from in-depth studies on epigenetic modifications in the gametes in preparation for fertilization.

ZFP628 Is a TAF4b-Interacting Transcription Factor Required for Mouse Spermiogenesis.

TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.

Protamine modification for fertility

Fertility During the final stage of spermatogenesis, small, positively charged proteins known as protamines tightly package DNA in the mature sperm. Itoh et al. found that mice deficient in the heat shock protein Hspa4l, which is implicated in spermatogenesis, produced sperm with malformed heads and were sterile. Hspa4l kept protamine 2 dephosphorylated at a specific serine residue. Expression of an unphosphorylatable protamine 2 mutant reversed the infertility of Hspa4l-deficient mice, suggesting a pathway that could be targeted for fertility or contraceptive purposes. Sci. Signal. 12 , eaao7232 (2019).

New monoclonal antibodies specific for mammalian protamines P1 and P2

ABSTRACTThe expression of protamines and the binding of these small arginine-rich proteins to DNA complete the process of spermatid chromatin reorganization and the global inactivation of the male’s haploid genome that occurs during the final stages of sperm development in mammals. While a number of anti-protamine antibodies have been created during the last 40 years, only a few have proven useful for detecting the presence of the protamines, determining the timing of their expression and deposition in chromatin, and investigating their structure and function in both maturing spermatids and sperm. The aim of this effort was to develop an additional set of monoclonal antibodies (MAbs) that not only recognize new P1 and P2 protamine epitopes but also work well as IHC reagents for detecting and identifying mammalian protamines in testicular tissue and ejaculated sperm. Using a combination of native and synthetic human protamines as antigens, 38 hybridoma clones recognizing human protamine P1 or P2 were generated. Antibodies produced by the 12 best clones were screened for selectivity by enzyme-linked immunosorbent assay, and two were found to recognize only human protamine P1 or P2, while a number of the others bound to both the human and mouse proteins. One MAb recognized every protamine tested. All the antibodies, including one recognizing stallion P1 and another recognizing stallion P2, bound to the native protamines in the chromatin of spermatids or sperm. While the majority labeled only elongating spermatids or sperm, several of the antibodies were found to also bind to the cytoplasm or nuclei of cells that lack protamine, which indicates these MAbs must recognize epitopes present in the protamines that are also found in other proteins. Thirteen overlapping human protamine P1 peptides were synthesized and subsequently used to identify the epitopes recognized by the six best antibodies.Abbreviations: BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; HCl: hydrochloric acid; IHC: immunohistochemistry; i.p: intraperitoneal; LIS: lithium diiodosalicylate; MAb: monoclonal antibody; PBS: phosphate buffered saline

Nuclear quiescence and histone hyper-acetylation jointly improve protamine-mediated nuclear remodeling in sheep fibroblasts.

Recently we have demonstrated the possibility to replace histones with protamine, through the heterologous expression of human protamine 1 (hPrm1) gene in sheep fibroblasts. Here we have optimized protaminization of somatic nucleus by adjusting the best concentration and exposure time to trichostatin A (TSA) in serum-starved fibroblasts (nuclear quiescence), before expressing Prm1 gene. To stop cell proliferation, we starved cells in 0.5% FBS in MEM (“starved”—ST group), whereas in the Control group (CTR) the cells were cultured in 10% FBS in MEM. To find the most effective TSA concentration, we treated the cells with increasing concentrations of TSA in MEM + 10% FBS. Our results show that combination of cell culture conditions in 50 nM TSA, is more effective in terminating cell proliferation than ST and CTR groups (respectively 8%, 17.8% and 90.2% p<0.0001). Moreover, nuclear quiescence marker genes expression (Dicer1, Smarca 2, Ezh1 and Ddx39) confirmed that our culture conditions kept the cells in a nuclear quiescent state. Finally, ST and 50 nM TSA jointly increased the number of spermatid-like cell (39.4%) at higher rate compared to 25 nM TSA (20.4%, p<0.05) and 100 nM TSA (13.7%, p<0.05). To conclude, we have demonstrated that nuclear quiescence in ST cells and the open nuclear structure conferred by TSA resulted in an improved Prm1-mediated conversion of somatic nuclei into spermatid-like structures. This finding might improve nuclear reprogramming of somatic cells following nuclear transfer.

Effect of Heat Stress on Sperm DNA: Protamine Assessment in Ram Spermatozoa and Testicle.

Sperm DNA fragmentation is considered one of the main causes of male infertility. The most accepted causes of sperm DNA damage are deleterious actions of reactive oxygen species (ROS), defects in protamination, and apoptosis. Ram sperm are highly prone to those damages due to the high susceptibility to ROS and to oxidative stress caused by heat stress. We aimed to evaluate the effects of heat stress on the chromatin of ejaculated and epididymal sperm and the activation of apoptotic pathways in different cell types in ram testis. We observed higher percentages of ejaculated sperm with increased chromatin fragmentation in the heat stress group; a fact that was unexpectedly not observed in epididymal sperm. Heat stress group presented a higher percentage of spermatozoa with DNA fragmentation and increased number of mRNA copies of transitional protein 1. Epididymal sperm presented greater gene expression of protamine 1 on the 30th day of the spermatic cycle; however, no differences in protamine protein levels were observed in ejaculated sperm and testis. Localization of proapoptotic protein BAX or BCL2 in testis was not different. In conclusion, testicular heat stress increases ram sperm DNA fragmentation without changes in protamination and apoptotic patterns.

Perinatal exposure to 2,2',4'4' -Tetrabromodiphenyl ether induces testicular toxicity in adult rats.

Since 1965, polybrominated diphenyl ethers (PBDEs) have been used internationally as flame-retardant additives. PBDEs were recently withdrawn from commerce in North America and Europe due to their environmental persistence, bioaccumulative properties and endocrine-disrupting effects. Generations exposed perinatally to the highest environmental doses of PBDE account for one-fifth of the total United States population. While, toxicity of PBDE for the male reproductive system has been demonstrated in several human and animal studies, the long-lasting effects of perinatal exposures on male reproduction are still poorly understood. In this study, pregnant Wistar rats were exposed to 0.2mg/kg 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) from gestation day 8 until postnatal day 21. Male reproductive outcomes were analyzed on postnatal day 120 in offspring. Exposed animals had significantly smaller testes, displayed decreased sperm production per testis weight, had significantly increased percentage of morphologically abnormal spermatozoa, and showed an increase in spermatozoa head size. Perinatal BDE-47 exposure led to significant changes in testes transcriptome, including suppression of genes essential for spermatogenesis and activation of immune response genes. In particular, we observed a 4-fold average decrease in expression of protamine and transition protein genes in testes, suggesting that histone-protamine exchange may be dysregulated during spermatogenesis, resulting in an aberrant sperm epigenome. The possibility of long-lasting effects of developmental PBDE exposures calls for additional studies to build a foundation for the development of preventive and protective interventions against the environmentally-induced decline in fertility.

Novel insights into the role of cell-free seminal mRNAs on semen quality and cryotolerance of spermatozoa in bulls (Bos taurus).

The aim of the present study was to ascertain the effectiveness of seminal plasma mRNAs as markers to assess the reproductive performance of bulls. Semen samples (33 ejaculates) from 11 bulls were evaluated for sperm kinematic and functional parameters. Total RNA was isolated from cell-free seminal (cfs) using TRIzol LS reagent and the concentration of cfs-RNA was 24.4±2.3µgmL-1 seminal plasma. The cfs-RNA was fragmented to a size of 25-500bp. Of the cfs-mRNAs screened using real time PCR, expression of protamine 1 (PRM1) was positively (P<0.05) associated with the mitochondrial membrane potential of raw semen, whereas expression of Fas Ligand (FASLG) was negatively (P<0.05) associated with sperm velocity, membrane integrity and chromatin distribution in post-thaw semen samples. The percentage of Type A spermatozoa (amplitude of lateral movement of head >2.5μm and straightness >85%) in raw semen was positively (P<0.05) associated with bone morphogenetic protein 2 (BMP2), ubiquitin conjugating enzyme E2D3 (UBE2D3), tumour-associated necrotic factor-associated death domain (TRADD) and caspase-3 (CASP3) expression. Nerve growth factor (NGF) expression was positively (P<0.05) associated with the maintenance of post-thaw functional membrane integrity in spermatozoa and could be used to assess the cryotolerance of bull semen. In conclusion, the expression of cfs mRNAs can be used to assess the reproductive performance of males and to predict the sensitivity of spermatozoa to cryoinjury.

Remodeling somatic nuclei via exogenous expression of protamine 1 to create spermatid-like structures for somatic nuclear transfer.

This protocol describes how to convert the chromatin structure of sheep and mouse somatic cells into spermatid-like nuclei through the heterologous expression of the protamine 1 gene (Prm1). Furthermore, we also provide step-by-step instructions for somatic cell nuclear transfer (SCNT) of Prm1-remodeled somatic nuclei in sheep oocytes. There is evidence that changing the organization of a somatic cell nucleus with that which mirrors the spermatozoon nucleus leads to better nuclear reprogramming. The protocol may have further potential application in determining the protamine and histone footprints of the whole genome; obtaining 'gametes' from somatic cells; and furthering understanding of the molecular mechanisms regulating the maintenance of DNA methylation in imprinted control regions during male gametogenesis. The protocol is straightforward, and it requires 4 weeks from the establishment of the cell lines to their transfection and the production of cloned blastocysts. It is necessary for researchers to have experience in cell biology and embryology, with basic skills in molecular biology, to carry out the protocol.

Murine and Human Spermatids Are Characterized by Numerous, Newly Synthesized and Differentially Expressed Transcription Factors and Bromodomain-Containing Proteins.

Much of spermatid differentiation takes place in the absence of active transcription, but in the early phase, large amounts of mRNA are synthesized, translationally repressed, and stored. Most nucleosomal histones are then degraded, and chromatin is repackaged by protamines. For both transcription and the histone-to-protamine transition in differentiating spermatids, chromatin must be opened. This raises the question of whether two different processes exist. It is conceivable that for initiation of the histone-to-protamine transition, the already accessible, actively transcribed chromatin regions are utilized or vice versa. We analyzed the enrichment of different canonical TATA-box-binding, protein-associated factors and their variants in murine spermatids, diverse bromodomain-containing proteins, and components of the Polycomb repressive complexes PRC1 and PRC2 using quantitative PCR. We compared the enrichment of corresponding proteins in human and murine spermatids and analyzed the time frame of postmeiotic transcription and expression of histones, transition proteins, and protamines in human and murine spermatids using immunohistology. We correlated the expression of different transcription factors and bromodomain-containing proteins and the pattern of acetylated histones to active transcription and to the histone-to-protamine transition in both human and murine spermatids. Our findings suggest that differentiating spermatids use both common and specific features to open chromatin first for transcription and subsequently for histone-to-protamine transition.

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures.

SummaryProtamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer.

Expression of sperm-specific protamines impairs bacterial and eukaryotic cell proliferation.

Protamines are the predominant nuclear proteins in testicular spermatids and ejaculated spermatozoa. During spermiogenesis, protamine-DNA interaction induces a higher-order chromatin packaging which finally results in a complete transcriptional stop in elongating spermatids. Although numerous studies investigated the role of protamines in male fertility, to date, no study is available that investigates protamine function, particularly transcriptional silencing, in non-germ cells. Transcriptional stop due to the high binding affinity of arginine-rich protamines to the negatively charged DNA backbone, however, may be induced in somatic cells and may result in suppressing cell division in tumor cells. In the present study, we therefore analyzed whether a protamine-mediated chromatin condensation in somatic cancer cell lines can stop gene expression and arrest cancer cell proliferation. In contrast to terminally differentiated sperm, cancer cells represent immortalized cells that have modulated natural mechanisms for the regulation of apoptosis and cell proliferation. We expressed human protamines in two fast-growing cell systems, E. coli and HeLa cells. In both cases, protamine expression significantly attenuated cell proliferation when compared with control cells. To our knowledge, this is the first study that demonstrates a stop of cell proliferation in both E. coli and HeLa cells by protamine expression. Follow-up studies on the molecular effect of protamines on proliferative cells may, in the future, open new avenues to investigate effective and specific treatments of cancer cells.

42 CONVERSION OF THE CHROMATIN OF SOMATIC CELLS INTO SPERMATID-LIKE STRUCTURES

The post-meiotic phase of spermatogenesis is characterised by a radical reorganization of the chromatin, leading to its nucleosomal to toroid transition. The replacement of histones with protamine is a gradual process regulated to the hierarchical translation of repressed mRNAs leading to the following events: incorporation of testis-specific histone variants and general histone hyperacetylation, bromodomain proteins, transition proteins, concluded by protamine incorporation on DNA. In this work, we tested whether the induced expression of human protamine 1 (PR1) in sheep somatic cells could induce a protamine/toroid conformation of interphase nuclei. Sheep adult fibroblasts (SAF) were cultured in DMEM with 10% fetal bovine serum from second to eighth passage. Then, SAF at 80% confluence were transfected with 3 μg of pPR1-red fluorescent protein (RFP) and pRFP (CTR) with lipofectamine. At 4 h post-transfection, cells were treated with 5 nM trichostatin A (TSA; histone deacethylase inhibitor) for an additional 16 h. Transfected cells (visualised through the RFP tag) were analysed for nuclear morphology (transmission electron microscopy), PR1-RFP expression (confocal microscope, RT-PCR, Western blot), cytofluorimeter, DNA damage (comet assay, pH2A.X immune-detection), and chromatin immune-precipitation assays (ChIP). Moreover, to visualise the histone/protamine exchange, we transfected with PR1 mouse GFP-H2B fibroblasts. Protaminized cells were used as donors for nuclear transfer (NT) and TH2B (testis/oocyte-specific histone normally present in male pronucleus, a marker of reprogramming) was detected in pronuclear stage of NT zygotes. The χ2 test was used for statistical analyses. We demonstrated that PR1 translocates into the nuclei and gradually compacts them into elongating spermatid-like structures in 48 h; TSA treatment facilitates the process [TSA 83.3% (50/60); without TSA 55.2%; 58/105; P &lt; 0.0003]. A complete histone-to-protamine exchange was also visualised in GFP-H2B nuclei (mouse fibroblasts) 40h after PR1 transfection. Cytofluorimetric analysis demonstrated that protamine incorporation occurs in any cell cycle stage and without DNA breaks. Next, protamine-protamine binding was excluded by ChIP analysis, which confirmed protamine-DNA binding. Finally, protaminized nuclei transplanted into enucleated oocytes incorporated maternal histone TH2B (3/8), whereas no signal was detected in control cells (0/9), suggesting that protaminized nuclei are better remodelled. We conclude that the induced expression of PR1 forces the somatic chromatin to acquire a structure overlapping elongated spermatid/spermatozoa, a conformation that perfectly matches the nuclear reprogramming machinery of the oocyte. Further work will determine whether protaminized cells are better reprogrammed upon nuclear transfer.

A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells.

To culture the in vitro mouse embryonic stem cells (mESCs) and to direct their differentiation to germ-line cells; in present study we used a vector backbone containing the fusion construct Stra8-EGFP to select differentiated ES cells that entered meiosis. Retinoic acid was used to differentiate embryonic stem cells to germ cells. A fragment of Stra8 gene promoter (-1400 to +7) was inserted in ScaI/HindIII multiple cloning site of pEGFP-1 vector. The electroporation was done on embryonic stem cells and positive colonies were selected as puromycin-resistant after three weeks of treatment with puromycin. All-trans retinoic acid (RA) was used for differentiation of mESCs at final concentration of 10(-)5M. The expression of protamine 1 (Prm1) gene was checked as post meiotic marker in differentiated mESCs after 5, 10, 15, 21 and 30 days after RA induction. The PCR amplification by specific primers for Stra8-EGFP fusion gene was detected in DNA sample from mESCs after electroporation and puromycin treatment. GFP-positive mESC colonies were observed after 72 hr RA induction. The protamine 1 gene was expressed after 21 days of RA induction. In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

An acute exposure to glyphosate-based herbicide alters aromatase levels in testis and sperm nuclear quality

Roundup is the major pesticide used in agriculture worldwide; it is a glyphosate-based herbicide. Its molecular effects are studied following an acute exposure (0.5%) of fifteen 60-day-old male rats during an 8-day period. Endocrine (aromatase, estrogen and androgen receptors, Gper1 in testicular and sperm mRNAs) and testicular functions (organ weights, sperm parameters and expression of the blood–testis barrier markers) were monitored at days 68, 87, and 122 after treatment, spermiogenesis and spermatogenesis. The major disruption is an increase of aromatase mRNA levels at least by 50% in treated rats at all times, as well as the aromatase protein. We have also shown a similar increase of Gper1 expression at day 122 and a light modification of BTB markers. A rise of abnormal sperm morphology and a decrease of the expression of protamine 1 and histone 1 testicular in epididymal sperm are observed despite a normal sperm concentration and motility.

Chd5 orchestrates chromatin remodelling during sperm development.

One of the most remarkable chromatin remodelling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodelling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying this process of extensive chromatin remodelling.