Sperm Regulation Research Articles

The regulation role of calcium channels in mammalian sperm function: a narrative review with a focus on humans and mice.

Mammalian sperm are characterized as specialized cells, as their transcriptional and translational processes are largely inactive. Emerging researches indicate that Ca2+ serves as a crucial second messenger in the modulation of various sperm physiological processes, such as capacitation, hyperactivation, and the acrosome reaction. Specifically, sperm-specific calcium channels, including CatSper, voltage-gated calcium channels (VGCCs), store-operated calcium channels (SOCCs), and cyclic nucleotide-gated (CNG) channels, are implicated in the regulation of calcium signaling in mammalian sperm. Calcium stores located in the sperm acrosomes, along with the IP3 receptors in the neck of the redundant nuclear envelope and the mitochondria in the tail, play significant roles in modulating intracellular Ca2+ levels in sperm. However, the functions and mechanisms of these calcium channels in modulating mammalian sperm physiological functions have not yet been well elucidated. Therefore, by focusing on humans and mice, this study aims to provide a comprehensive review of the current advancements in research regarding the roles of calcium signaling and associated calcium channels in regulating sperm function. This endeavor seeks to enhance the understanding of calcium signaling in sperm regulation and to facilitate the development of drugs for the treatment of infertility or as non-hormonal male contraceptives.

Ribonuclease Inhibitor 1 (RNH1) Regulates Sperm tsRNA Generation for Paternal Inheritance through Interacting with Angiogenin in the Caput Epididymis.

Environmental stressors can induce paternal epigenetic modifications that are a key determinant of the intergenerational inheritance of acquired phenotypes in mammals. Some of them can affect phenotypic expression through inducing changes in tRNA-derived small RNAs (tsRNAs), which modify paternal epigenetic regulation in sperm. However, it is unclear how these stressors can affect changes in the expression levels of tsRNAs and their related endonucleases in the male reproductive organs. We found that Ribonuclease inhibitor 1 (RNH1), an oxidation responder, interacts with ANG to regulate sperm tsRNA generation in the mouse caput epididymis. On the other hand, inflammation and oxidative stress induced by either lipopolysaccharide (LPS) or palmitate (PA) treatments weakened the RNH1-ANG interaction in the epididymal epithelial cells (EEC). Accordingly, ANG translocation increased from the nucleus to the cytoplasm, which led to ANG upregulation and increases in cytoplasmic tsRNA expression levels. In conclusion, as an antioxidant, RNH1 regulates tsRNA generation through targeting ANG in the mouse caput epididymis. Moreover, the tsRNA is an epigenetic factor in sperm that modulates paternal inheritance in offspring via the fertilization process.

Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

Detrimental effects of flame retardant, PBB153, exposure on sperm and future generations

In 1973, the Velsicol Chemical Company, which manufactured FireMaster, a brominated flame retardant, and NutriMaster, a nutritional supplement, mistakenly shipped hundreds of pounds of FireMaster to grain mills around Michigan where it was incorporated into animal feed and then into the food chain across the state. An estimated 6.5 million Michigan residents consumed polybrominated biphenyl (PBB)-laced animal products leading to one of the largest agricultural accidents in U.S. history. To date, there have been no studies investigating the effects of PBB on epigenetic regulation in sperm, which could explain some of the endocrine-related health effects observed among children of PBB-exposed parents. Fusing epidemiological approaches with a novel in vitro model of human spermatogenesis, we demonstrate that exposure to PBB153, the primary component of FireMaster, alters the epigenome in human spermatogenic cells. Using our novel stem cell-based spermatogenesis model, we show that PBB153 exposure decreases DNA methylation at regulatory elements controlling imprinted genes. Furthermore, PBB153 affects DNA methylation by reducing de novo DNA methyltransferase activity at increasing PBB153 concentrations as well as reducing maintenance DNA methyltransferase activity at the lowest tested PBB153 concentration. Additionally, PBB153 exposure alters the expression of genes critical to proper human development. Taken together, these results suggest that PBB153 exposure alters the epigenome by disrupting methyltransferase activity leading to defects in imprint establishment causing altered gene expression, which could contribute to health concerns in the children of men exposed to PBB153. While this chemical is toxic to those directly exposed, the results from this study indicate that the epigenetic repercussions may be detrimental to future generations. Above all, this model may be expanded to model a multitude of environmental exposures to elucidate the effect of various chemicals on germline epigenetics and how paternal exposure may impact the health of future generations.

An in silico analysis of human sperm genes associated with asthenozoospermia and its implication in male infertility.

Asthenozoospermia is the most common clinical symptom of male infertility. Molecular markers associated with asthenozoospermia spermatozoa are scarcely identified. The objective of this study was to screen the differentially expressed genes (DEGs) in asthenozoospermia spermatozoa and assess the underlying bioinformatics roles in regulation of sperm quality.Based on gene expression omnibus (GEO) database, the GSE22331, GSE1133, and GSE4193 expression profile data were downloaded. The DEGs of asthenozoospermia spermatozoa were identified. Germ cell specific genes in DEGs were further screened. Then, gene ontology (GO) and over-representation analysis of DEGs were performed, followed by protein-protein interaction (PPI) network analysis. Expressions of selected genes of TEX11, ADAMTS5, ASRGL1, GMCL1, PGK2, KLHL10 in normozoospermia and asthenozoospermia spermatozoa were identified using real time Reverse Transcription-Polymerase Chain Reaction (RT-PCR).A total of 1323 DEGs were identified, including 1140 down-regulated genes. Twenty one and 96 down-regulated genes were especially expressed in spermatogonia and round spermatids, suggesting their testicular origins and influences on sperm quality. Bioinformatics analysis showed enriched functions of ubiquitin-like protein transferase or protein binding activities in down-regulated genes. Expressions of selected genes were validated by RT-PCR, which was consistent with bioinformatical results.The present study provided a novel insight into the understanding of sperm quality, and a potential method and dataset for the diagnosis and assessment of sperm quality in the event of male infertility.

Sperm epigenome and its potential role in transgenerational inheritance

Epigenetics regulation is generally referred to DNA methylation, histone modification and non-coding RNA, which play important roles in chromatin management and gene expression. Recent studies have found that sperm genome is dynamically regulated by a variety of epigenetics mechanisms. More importantly, emerging evidences suggest that certain epigenetics information could be transmitted from sperm to offspring for limited generations. Here, we have overviewed the epigenetics mechanisms involved in spermatogenesis and after fertilization, and the potential underlining mechanisms of epigenetic inheritance. In the first part, we summarized the current understanding of the regulation of DNA methylation, histone modification and small non-coding RNA in spermatogenesis and early embryonic development. This part started from the establishment of sperm DNA methylome and the reprogramming of paternal DNA methylome after fertilization. We briefly described how DNA methylation is regulated by DNMT and TET proteins, and its role in silencing repetitive elements and imprinting genes. We also summarized the two rounds of reprogramming of DNA methylation after fertilization. During the first round of reprogramming, most DNA methylation signature from germ cells is erased, and somatic DNA methylome is then re-established. The second round occurring from primordial germ cells to germ cells, during which DNA methylation at imprinted genes is erased, and germ cell specific DNA methylome is then re-established. It is generally believed that the two rounds of reprogramming establish the proper DNA methylome for germ cell formation. We then reviewed the specific pattern and functional involvement of sperm histone modifications and histone variants. During sperm maturation, most histones undergo acetylation-mediated degradation and replaced by protamine, resulting in only ~10% genome with retained nucleosomes. The remaining nucleosomes contain canonic histones and various histone variants, including TH2A, TH2B, H3t, H3.3, etc. Importantly, sperm histones are also modified, such as H3K4me3, H3K27me3, H3K27ac and H4S1ph. Some modifications are deposited to developmental genes and imprinting loci, while others are thought to control sperm genome accessibility and compaction. Exemplified by transgenerational developmental defects caused by sperm H3K4me2 disruption, proper histone modification patterning in sperm is thought to be critical for early development. We also summarized the recent discoveries in the regulation of H3K4me3 and H3K27me3 from fertilized mouse oocytes to ICM, revealing a highly dynamic and regulated nature of histone modification during early development. The recent progresses of small non-coding RNA regulation in sperm are also included in this review. For instance, piRNAs are required for heterochromatin formation and gene silencing, especially retrotransposon silencing during spermatogenesis; certain miRNAs, like miR-290-miR295 and miRNA-34b-5p , are required for proper meiosis and cell cycle regulation during spermatogenesis; importantly, tRNA-derived small RNAs (tsRNAs) in sperm were found responsive to paternal diet and able to influence the offspring’s metabolism. In the second part, we focused on the environmental impacts on sperm epigenome and potential mechanisms of transgenerational inheritance. To date, several studies have implied that diet, pressure and chemical exposure can alter sperm epigenome, which could potentially be inherited through generations. For example, paternal diet is demonstrated to affect offspring’s metabolism. Paternal physiological stress, such as restraint stress or early trauma, could influence offspring’s metabolism or behavior through altered paternal sperm DNA methylation and miRNAs. Additionally, altered sperm DNA methylation has been observed in male rats suffered from utero undernourishment, which could contribute to the high risk of type II diabetes in the offspring. Taken together, we just started to understand the potential role of epigenetics involving in the transgenerational response to environmental changes. Future epigenomic investigations of sperm, as well as oocyte and zygote, should provide more mechanistic insights in how environment could influence the genome and how the effect might be inherited.

Role of zinc trafficking in male fertility: from germ to sperm

What are the dynamics of zinc (Zn) trafficking in sperm, at the testicular, epididymal and ejaculate levels? Zn transporters are peculiarly expressed in the cells of the germ line and Zn uptake is maximal at the post-epididymal phase, where Zn is involved in the regulation of sperm functions. Zn is known to influence several phases of sperm life, from germ cell development to spermiation. Zn trafficking across the membrane is allowed by specific families of transporters known as the ZnTs, which are involved in effluent release, and the Zips, which mediate uptake. We enrolled 10 normozoospermic healthy participants in an infertility survey programme, as well as 5 patients affected by testicular germ cell cancer, and 18 patients presenting with obstructive azoospermia, without mutations of the CFTR gene, and undergoing assisted reproductive technologies. The research study was performed at our University Clinic. Semen samples, or biopsies or fine needle aspirates from the testis or epididymis, were obtained from each of the participants. Protein expression of main members of the ZnT and Zip families of Zn transporters was examined in human testis and epididymis samples by immunofluorescence. Quantification of sperm Zn content was performed by flow cytometry, atomic absorption spectrometry (AA) and autometallography. Intratubular cells of the germ line displayed a high redundancy of Zip family members involved in Zn uptake, while ZnT transporters were more represented in epididymis. Testicular and epididymal spermatozoa contained less Zn than ejaculated spermatozoa (2.56 ± 0.51 and 12.58 ± 3.16 versus 40.48 ± 12.71 ng Zn/10(6)cells, respectively). Gain of hypermotility and acrosomal reaction were significantly linked to the loss of Zn content in ejaculated spermatozoa. This was an ancillary study performed on a small cohort of normozoospermic subjects. Although these results clarify the Zn trafficking during different phases of sperm life, no conclusive information can be drawn about the fertilizing potential of sperm, and the overall pregnancy outcomes, after Zn supplementation. Our data disclose the dynamics of Zn trafficking during over the sperm lifespan. No external funding was sought or obtained for this study. No conflict of interest is declared.

Characterisation of Caenorhabditis elegans sperm transcriptome and proteome.

BackgroundAlthough sperm is transcriptionally and translationally quiescent, complex populations of RNAs, including mRNAs and non-coding RNAs, exist in sperm. Previous microarray analysis of germ cell mutants identified hundreds of sperm genes in Caenorhabditis elegans. To take a more comprehensive view on C. elegans sperm genes, here, we isolate highly pure sperm cells and employ high-throughput technologies to obtain sperm transcriptome and proteome.ResultsFirst, sperm transcriptome consists of considerable amounts of non-coding RNAs, many of which have not been annotated and may play functional roles during spermatogenesis. Second, apart from kinases/phosphatases as previously reported, ion binding proteins are also enriched in sperm, underlying the crucial roles of intracellular ions in post-translational regulation in sperm. Third, while the majority of sperm genes/proteins have low abundance, a small number of sperm genes/proteins are hugely enriched in sperm, implying that sperm only rely on a small set of proteins for post-translational regulation. Lastly, by extensive RNAi screening of sperm enriched genes, we identified a few genes that control fertility. Our further analysis reveals a tight correlation between sperm transcriptome and sperm small RNAome, suggesting that the endogenous siRNAs strongly repress sperm genes. This leads to an idea that the inefficient RNAi screening of sperm genes, a phenomenon currently with unknown causes, might result from the competition between the endogenous RNAi pathway and the exogenous RNAi pathway.ConclusionsTogether, the obtained sperm transcriptome and proteome serve as valuable resources to systematically study spermatogenesis in C. elegans.

Lactoferrin expression and secretion in the stallion epididymis

Lactoferrin is one of the most abundant proteins secreted by the stallion epididymis, but its cellular localization and regulation remain unknown. This study was designed to address the following objectives: (1) identify the epididymal cell types producing lactoferrin in pre-pubertal, peri-pubertal and post-pubertal animals; (2) demonstrate that lactoferrin binds to stallion sperm; and (3) determine if testosterone and estradiol regulate lactoferrin secretion in vitro. Using an immunohistochemical method, lactoferrin was localized in the cytoplasm of principal cells in the corpus and cauda of peri- and post-pubertal animals. The epididymis of pre-pubertal animals did not express lactoferrin. Immunolabeling of lactoferrin was also observed on the mid-piece and tail of the sperm. The role of estradiol and testosterone in regulating secretion of lactoferrin in the post-pubertal epididymis was investigated using tissue culture methods. Lactoferrin concentration in the culture media was determined by validated enzyme-linked immunosorbent assays (ELISA). Testosterone did not increase the concentration of lactoferrin in the media in any epididymal region. In contrast, estradiol-17β significantly increased the concentration of lactoferrin in the media containing tissue from the cauda. In conclusion, the expression of lactoferrin was found in the cytoplasm of principal cells in the corpus and cauda of the epididymis in peri- and post-pubertal stallions but not pre-pubertal stallions. Furthermore, lactoferrin binds to sperm, suggesting a biological role for protection or regulation of sperm in the corpus and cauda. In addition, estrogen appears to regulate lactoferrin secretion in the cauda of the epididymis in post-pubertal stallions.

Effect of Tumor Necrosis Factor‐α on the Intracellular Ca2+ Homeostasis in Human Sperm

Inflammation and genital infections promote the increase in leukocytes, pro-inflammatory cytokines, and oxygen reactive species, impairing sperm functions such as motility, capacitation, and acrosome reaction. All these functions are primarily regulated by cytoplasmic concentration of Ca(2+) ([Ca(2+) ]cyto ). This study evaluated the effect of tumor necrosis factor (TNF)-α on the [Ca(2+) ]cyto and its regulation in human sperm. Sperm loaded with fura-2 were incubated with or without TNF-α (0-500 pg/mL) from 0 to 120 min. After incubation, the basal [Ca(2+) ]cyto and membrane permeability to Ca(2+) were evaluated by spectrofluorometry, before and after Ca(2+) addition to the extracellular medium. Without TNF-α, the addition of Ca(2+) promotes an transitory increase in [Ca(2+) ]cyto in the spermatozoa, that returns in a few minutes to a basal level, indicating calcium regulation activation. TNF-α decreases the Ca(2+) permeation and increases the basal level of [Ca(2+) ]cyto after a Ca(2+) pulse (P < 0.04); affecting calcium regulation in a way that is time and concentration dependent. TNF-α effect was partially prevented by the addition of an antioxidant (butylated hydroxytoluene) (P < 0.03). Tumor necrosis factor-α decreases membrane permeability to Ca(2+) and affects Ca(2+) regulation in sperm cells in vitro, probably via lipid peroxidation, which may explain the decrease in sperm fertilizing capacity during inflammatory and infectious processes.

Aquaporins in sperm osmoadaptation: an emerging role for volume regulation

Upon ejaculation, mammalian sperm experience a natural osmotic decrease during male to female reproductive tract transition. This hypo-osmotic exposure not only activates sperm motility, but also poses potential harm to sperm structure and function by inducing unwanted cell swelling. In this physiological context, regulatory volume decrease (RVD) is the major mechanism that protects cells from detrimental swelling, and is essential to sperm survival and normal function. Aquaporins are selective water channels that enable rapid water transport across cell membranes. Aquaporins have been implicated in sperm osmoregulation. Recent discoveries show that Aquaporin-3 (AQP3), a water channel protein, is localized in sperm tail membranes and that AQP3 mutant sperm show defects in volume regulation and excessive cell swelling upon physiological hypotonic stress in the female reproductive tract, thereby highlighting the importance of AQP3 in the postcopulatory sperm RVD process. In this paper, we discuss current knowledge, remaining questions and hypotheses about the function and mechanismic basis of aquaporins for volume regulation in sperm and other cell types.

Shedding Light on Sperm pHertility

The acquisition of fertilization capacity by sperm is regulated by intracellular pH (pH(i)), but the transport pathways that regulate pH(i) are not well understood. Lishko et al. (2010) now report that Hv1, the voltage-sensitive proton channel, is present in human sperm and is an important regulator of the functional maturation of sperm.

2P-151 精子鞭毛波形調節におけるNCSファミリータンパク質カラクシンの機能(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

2P-151 精子鞭毛波形調節におけるNCSファミリータンパク質カラクシンの機能(細胞生物的課題(接着,運動,骨格,伝達,膜),第47回日本生物物理学会年会)

Subtractive analysis of S. franciscanus and S. purpuratus ovary mRNA: what kinds of genes determine species-specificity?

Our goal is to identify and understand points of regulation in sperm–egg recognition as well as in steps of early development. These processes are species-specific and are the key to understanding speciation. In both vertebrates and invertebrates, the interaction of the sperm and egg displays a wide range of species-specificity. The questions we would like to answer are: What kinds of molecules determine the specificity and control the fertilisation process? Are early steps in development regulated in a species-specific manner?As an approach to identifying genes that determine species-specificity, in two different species, S. purpuratus (S. p.) and S. franciscanus (S. f.), we used a new subtractive hybridisation method known as RDA (representational difference analysis) (Lisitsyn et al., 1993; Hubank et al., 1994). Several species-specific clones were isolated from S. f. ovary mRNA by this method using mRNA from another species but the same genus of sea urchin, S. p. Four different clones were obtained and the species-specificity of the sequence was confirmed by hybridisation. One of them has four tandem EGF repeats and is homologous to the S. p. EGF-II gene (Yang et al., 1989) and A. crassispina EGIP (exogastrula inducing protein) gene (Ishihara et al., 1982). The first three EGF repeats (EGF 1–3) have 60% similarity among these species, but the fourth EGF domain (EGF 4) is highly divergent. The EGF-II protein is believed to be involved in the signalling events of early development, because purified EGF causes exogastrulation when it is added to the seawater prior to gastrulation (Ishihara et al., 1982). Recombinant EGF 3 from S. f. induces exogastrulation in both S. f. and S. p.