Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome

Background Paternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit. Results Using proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation. Conclusions These data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Influence of the Season and Region Factor on Phosphoproteome of Stallion Epididymal Sperm

Epididymal maturation can be defined as a scope of changes occurring during epididymal transit that prepare spermatozoa to undergo capacitation. One of the most common post-translational modifications involved in the sperm maturation process and their ability to fertilise an oocyte is the phosphorylation of sperm proteins. The aim of this study was to compare tyrosine, serine, and threonine phosphorylation patterns of sperm proteins isolated from three subsequent segments of the stallion epididymis, during and out of the breeding season. Intensities of phosphorylation signals and phosphoproteins profiles varied in consecutive regions of the epididymis. However, significant differences in the phosphorylation status were demonstrated in case of endoplasmic reticulum chaperone BiP (75 and 32 kDa), protein disulfide-isomerase A3 (50 kDa), nesprin-1 (23 kDa), peroxiredoxin-5 (17 kDa), and protein bicaudal D homolog (15 kDa) for season x type of phosphorylated residues variables. Significant differences in the phosphorylation status were also demonstrated in case of endoplasmic reticulum chaperone BiP and albumin (61 kDa), protein disulfide-isomerase A3 (50 kDa), and protein bicaudal D homolog (15 kDa) for region x type of phosphorylated residues variables.

Caput Ligation Renders Immature Mouse Sperm Motile and Capable to Undergo cAMP-Dependent Phosphorylation

Mammalian sperm must undergo two post-testicular processes to become fertilization-competent: maturation in the male epididymis and capacitation in the female reproductive tract. While caput epididymal sperm are unable to move and have not yet acquired fertilization potential, sperm in the cauda epididymis have completed their maturation, can move actively, and have gained the ability to undergo capacitation in the female tract or in vitro. Due to the impossibility of mimicking sperm maturation in vitro, the molecular pathways underlying this process remain largely unknown. We aimed to investigate the use of caput epididymal ligation as a tool for the study of sperm maturation in mice. Our results indicate that after seven days of ligation, caput sperm gained motility and underwent molecular changes comparable with those observed for cauda mature sperm. Moreover, ligated caput sperm were able to activate pathways related to sperm capacitation. Despite these changes, ligated caput sperm were unable to fertilize in vitro. Our results suggest that transit through the epididymis is not required for the acquisition of motility and some capacitation-associated signaling but is essential for full epididymal maturation. Caput epididymal ligation is a useful tool for the study of the molecular pathways involved in the acquisition of sperm motility during maturation.

Kisspeptin Receptor on the Sperm Surface Reflects Epididymal Maturation in the Dog

Alongside the well-known central modulatory role, the Kisspeptin system, comprising Kiss1, its cleavage products (Kisspeptins), and Kisspeptin receptor (Kiss1R), was found to regulate gonadal functions in vertebrates; however, its functional role in the male gamete and its localization during maturation have been poorly understood. The present study analyzed Kisspeptin system in dog testis and spermatozoa recovered from different segments of the epididymis, with focus on Kiss1R on sperm surface alongside the maturation during epididymal transit, demonstrated by modification in sperm kinetic, morphology, and protamination. The proteins Kiss1 and Kiss1R were detected in dog testis. The receptor Kiss1R only was detected in total protein extracts from epididymis spermatozoa, whereas dot blot revealed Kiss1 immunoreactivity in the epidydimal fluid. An increase of the Kiss1R protein on sperm surface along the length of the epididymis, with spermatozoa in the tail showing plasma membrane integrity and Kiss1R protein (p < 0.05 vs. epididymis head and body) was observed by flow cytometry and further confirmed by epifluorescence microscopy and Western blot carried on sperm membrane preparations. In parallel, during the transit in the epididymis spermatozoa significantly modified their ability to move and the pattern of motility; a progressive increase in protaminization also occurred. In conclusion, Kisspeptin system was detected in dog testis and spermatozoa. Kiss1R trafficking toward plasma membrane along the length of the epididymis and Kiss1 in epididymal fluid suggested a new functional role of the Kisspeptin system in sperm maturation and storage.

Redox regulation by TXNRD3 during epididymal maturation underlies capacitation-associated mitochondrial activation and sperm motility in mice

During epididymal transit, redox remodeling protects mammalian spermatozoa, preparing them for survival in the subsequent journey to fertilization. However, molecular mechanisms of the redox regulation in sperm development and maturation remain largely elusive. In this study, we report thioredoxin reductase 3 (TXNRD3) - a thioredoxin reductase family member particularly abundant in elongating spermatids at the site of mitochondrial sheath formation - contributes to regulate redox homeostasis in male reproduction. Using Txnrd3-/- mice, our biochemical, ultrastructural, and live cell imaging analyses revealed impairments in sperm morphology and motility in absence of TXNRD3. Absence of TXNRD3 alters redox status in both the head and tail during sperm maturation and capacitation, resulting in defective mitochondrial ultrastructure and activity under capacitating conditions. These findings provide insights into molecular mechanisms of redox homeostasis and bioenergetics during sperm maturation, capacitation, and fertilization.

Molecular perspective concerning fluoride and arsenic mediated disorders on epididymal maturation of spermatozoa: A concise review

Epididymis is a complex tubular structure of male reproductive system where spermatozoa undergo maturation and gain the fertilizing ability. Epididymal pseudostratified columnar epithelium with different cell types play imperative role by their secretory properties and enrich the luminal microenvironment necessary for achieving spermatozoal motility. During epididymal transit several secretory proteins like P26h, SPAG11, HSPD1 and many others are deposited on spermatozoal surface. At the same time spermatozoal proteins are also modified in this intraluminal milieu, which include cyritestin, fertilin, CE9 and others. Natural and anthropogenic activities disclose various environmental pollutants which affect different physiological systems of animals and human being. Likewise, reproductive system is also being affected. Fluoride causes structural alterations of caput and cauda segments of epididymis. Redox homeostasis and functional integrity are also altered due to diminished activities of SOD1, GR, Crisp2, Lrp2 and other important proteins. On the contrary arsenic affects mostly on cauda segment. Redox imbalance and functional amendment in epididymis have been observed with arsenic revelation as evidenced by altered genomic appearance of SOD, GST, catalase, Ddx3Y, VEGF and VEGFR2. This review is dealt with structure-function interplay in normal epididymal spermatozoal maturation along with subsequent complications developed under fluoride and arsenic toxicities.

Male obesity impacts DNA methylation reprogramming in sperm

Background Male obesity has profound effects on morbidity and mortality, but relatively little is known about the impact of obesity on gametes and the potential for adverse effects of male obesity to be passed to the next generation. DNA methylation contributes to gene regulation and is erased and re-established during gametogenesis. Throughout post-pubertal spermatogenesis, there are continual needs to both maintain established methylation and complete DNA methylation programming, even during epididymal maturation. This dynamic epigenetic landscape may confer increased vulnerability to environmental influences, including the obesogenic environment, that could disrupt reprogramming fidelity. Here we conducted an exploratory analysis that showed that overweight/obesity (n = 20) is associated with differences in mature spermatozoa DNA methylation profiles relative to controls with normal BMI (n = 47). Results We identified 3264 CpG sites in human sperm that are significantly associated with BMI (p < 0.05) using Infinium HumanMethylation450 BeadChips. These CpG sites were significantly overrepresented among genes involved in transcriptional regulation and misregulation in cancer, nervous system development, and stem cell pluripotency. Analysis of individual sperm using bisulfite sequencing of cloned alleles revealed that the methylation differences are present in a subset of sperm rather than being randomly distributed across all sperm. Conclusions Male obesity is associated with altered sperm DNA methylation profiles that appear to affect reprogramming fidelity in a subset of sperm, suggestive of an influence on the spermatogonia. Further work is required to determine the potential heritability of these DNA methylation alterations. If heritable, these changes have the potential to impede normal development.

Mitochondrial Functionality in Male Fertility: From Spermatogenesis to Fertilization

Mitochondria are structurally and functionally distinct organelles that produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS), to provide energy to spermatozoa. They can also produce reactive oxidation species (ROS). While a moderate concentration of ROS is critical for tyrosine phosphorylation in cholesterol efflux, sperm–egg interaction, and fertilization, excessive ROS generation is associated with male infertility. Moreover, mitochondria participate in diverse processes ranging from spermatogenesis to fertilization to regulate male fertility. This review aimed to summarize the roles of mitochondria in male fertility depending on the sperm developmental stage (from male reproductive tract to female reproductive tract). Moreover, mitochondria are also involved in testosterone production, regulation of proton secretion into the lumen to maintain an acidic condition in the epididymis, and sperm DNA condensation during epididymal maturation. We also established the new signaling pathway using previous proteomic data associated with male fertility, to understand the overall role of mitochondria in male fertility. The pathway revealed that male infertility is associated with a loss of mitochondrial proteins in spermatozoa, which induces low sperm motility, reduces OXPHOS activity, and results in male infertility.

The calcium-sensing receptor is essential for calcium and bicarbonate sensitivity in human spermatozoa

Context The calcium-sensing receptor (CaSR) is essential to maintain a stable calcium concentration in serum. Spermatozoa are exposed to immense changes in concentrations of CaSR ligands such as calcium, magnesium, and spermine during epididymal maturation, in the ejaculate, and in the female reproductive environment. However, the role of CaSR in human spermatozoa is unknown. Objective and design We identified CaSR in human spermatozoa and characterized the response to CaSR agonists on intracellular calcium, acrosome reaction, and cAMP in spermatozoa from men with either loss-of-function or gain-of-function mutations in CASR and healthy donors. Results CaSR is expressed in human spermatozoa and is essential for sensing extracellular Ca 2+ and Mg 2+. Activators of CaSR augmented the effect of sperm activating signals such as the response to HCO3- and the acrosome reaction, while spermatozoa from men with a loss-of-function mutation in CASR had a diminished response to HCO3-, lower progesterone-mediated calcium influx, and were less likely to undergo the acrosome reaction in response to progesterone or Ca 2+. CaSR activation increased cAMP through soluble adenylyl cyclase (sAC) activity and increased calcium influx through CatSper. Moreover, external Ca 2+ or Mg 2+ was indispensable for HCO3- activation of sAC. Two male patients with CASR loss-of-function mutation in exon 3 present with normal sperm counts and motility, while a patient with a loss-of-function mutation in exon 7 had low sperm count, motility, and morphology. Conclusion CaSR is important for the sensing of Ca 2+, Mg 2+, and HCO3 - in spermatozoa, and loss-of-function may impair male sperm function.

The Cytoplasmic Droplets of Rat Epididymal Spermatozoa Show Diminution of Glycosyl Transferases During Spermatozoa Epididymal Maturation

The cytoplasmic droplet of the epididymal spermatozoon is a small localized outpouching of cytoplasm of the tail and is of unknown significance. Electron microscopy revealed flattened saccular elements of almost exclusive membranous components. In this study, a simple, reproducible method for the isolation of saccular elements from the caput and the cauda of rat epididymal spermatozoa is presented. Using exogenous glycosylation assays and immunocytochemistry for light and electron microscopy, we confirm the presence of three Golgi markers in the saccular elements of rat cytoplasmic droplets, namely, a-2,6-sialyltransferase, b- 1,4-galactosyltransferase and TGN-38 (trans-Golgi network protein). The variation in galactosyltransferase activity of isolated saccular elements in response to in vitro trypsinization was similar to that observed in the rat liver Golgi apparatus. Electrophoresis and immunoblotting analyses revealed polypeptide modifications in the saccular elements during spermatozoa epididymal transit. In addition, a significant decline in the content of galactosyltransferase and sialyltransferase occurred in the saccular elements during spermatozoa transit from the caput to the cauda epididymides. The content of TGN-38, however, did not show any significant change, suggesting that the modification of the content of the saccular elements is selective. Considering the glycosylating ability of the saccular elements and that the diminution of glycosyltransferase activities in cytoplasmic droplets coincides with the maturation of spermatozoa during epididymal transit, we suggest a potential role for the saccular elements of cytoplasmic droplets in the maturation of spermatozoa during their transit through the epididymis.