The renin angiotensin system (RAS) appears to influence male fertility at multiple levels. In this work, we analyzed the relationship between the RAS and DNA integrity. Fifty male volunteers were divided into two groups (25 each): control (DNA fragmentation ≤20%) and pathological (DNA fragmentation >20%) cases. Activities of five peptidases controlling RAS were measured fluorometrically: prolyl endopeptidase (which converts angiotensin [A] I and A II to A 1-7), neutral endopeptidase (NEP/CD10: A I to A 1-7), aminopeptidase N (APN/CD13: A III to A IV), aminopeptidase A (A II to A III) and aminopeptidase B (A III to A IV). Angiotensin-converting enzyme (A I to A II), APN/CD13 and NEP/CD10 were also assessed by semiquantitative cytometry and quantitative flow cytometry assays, as were the receptors of all RAS components: A II receptor type 1 (AT1R), A II receptor type 2 (AT2R), A IV receptor (AT4R or insulin-regulated aminopeptidase [IRAP]), (pro)renin receptor (PRR) and A 1-7 receptor or Mas receptor (MasR) None of the enzymes that regulate levels of RAS components, except for APN/CD13 (decrease in fragmented cells), showed significant differences between both groups. Micrographs of RAS receptors revealed no significant differences in immunolabeling patterns between normozoospermic and fragmented cells. Labeling of AT1R (94.3% normozoospermic vs 84.1% fragmented), AT4R (96.2% vs 95.3%) and MasR (97.4% vs 87.2%) was similar between the groups. AT2R (87.4% normozoospermic vs 63.1% fragmented) and PRR (96.4% vs 48.2%) were higher in non-fragmented spermatozoa. These findings suggest that fragmented DNA spermatozoa have a lower capacity to respond to bioactive RAS peptides.
Angiotensins ; DNA Fragmentation ; Humans ; Insulin ; Male ; Renin-Angiotensin System/physiology* ; Spermatozoa

The novel coronavirus disease (COVID-19) pandemic is emerging as a global health threat and shows a higher risk for men than women. Thus far, the studies on andrological consequences of COVID-19 are limited. To ascertain the consequences of COVID-19 on sperm parameters after recovery, we recruited 41 reproductive-aged male patients who had recovered from COVID-19, and analyzed their semen parameters and serum sex hormones at a median time of 56 days after hospital discharge. For longitudinal analysis, a second sampling was obtained from 22 of the 41 patients after a median time interval of 29 days from first sampling. Compared with controls who had not suffered from COVID-19, the total sperm count, sperm concentration, and percentages of motile and progressively motile spermatozoa in the patients were significantly lower at first sampling, while sperm vitality and morphology were not affected. The total sperm count, sperm concentration, and number of motile spermatozoa per ejaculate were significantly increased and the percentage of morphologically abnormal sperm was reduced at the second sampling compared with those at first in the 22 patients examined. Though there were higher prolactin and lower progesterone levels in patients at first sampling than those in controls, no significant alterations were detected for any sex hormones examined over time following COVID-19 recovery in the 22 patients. Although it should be interpreted carefully, these findings indicate an adverse but potentially reversible consequence of COVID-19 on sperm quality.
Adult ; Asthenozoospermia/virology* ; COVID-19/physiopathology* ; China ; Gonadal Steroid Hormones/blood* ; Humans ; Male ; Progesterone/blood* ; Prolactin/blood* ; SARS-CoV-2 ; Semen/physiology* ; Semen Analysis ; Sperm Count ; Sperm Motility ; Spermatozoa/physiology* ; Time Factors

Mammalian fertilization begins with the fusion of two specialized gametes, followed by major epigenetic remodeling leading to the formation of a totipotent embryo. During the development of the pre-implantation embryo, precise reprogramming progress is a prerequisite for avoiding developmental defects or embryonic lethality, but the underlying molecular mechanisms remain elusive. For the past few years, unprecedented breakthroughs have been made in mapping the regulatory network of dynamic epigenomes during mammalian early embryo development, taking advantage of multiple advances and innovations in low-input genome-wide chromatin analysis technologies. The aim of this review is to highlight the most recent progress in understanding the mechanisms of epigenetic remodeling during early embryogenesis in mammals, including DNA methylation, histone modifications, chromatin accessibility and 3D chromatin organization.
Animals ; Chromatin Assembly and Disassembly ; DNA Methylation ; DNA Transposable Elements ; Embryo, Mammalian ; Embryonic Development/genetics* ; Epigenesis, Genetic ; Epigenome ; Female ; Fertilization/physiology* ; Gene Expression Regulation, Developmental ; Histone Code ; Histones/metabolism* ; Male ; Mice ; Oocytes/metabolism* ; Spermatozoa/metabolism*

Animals ; Antigens, Surface/genetics* ; Cell Communication/genetics* ; Copulation/physiology* ; Fallopian Tubes/metabolism* ; Female ; Fertilization/genetics* ; GPI-Linked Proteins/genetics* ; Gene Expression Regulation ; Genes, Reporter ; Green Fluorescent Proteins/metabolism* ; Litter Size ; Luminescent Proteins/metabolism* ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism* ; Reproduction/genetics* ; Signal Transduction ; Sperm Count ; Sperm Motility/genetics* ; Spermatozoa/metabolism* ; Uterus/metabolism*

Objective: This research was performed to evaluate the effect of tebuconazole (TBZ) on reproductive organs of male rats and to assess the protective role of combined essential trace elements in alleviating the detrimental effect of TBZ on male reproductive function. Methods: For this purpose, 48 rats were exposed to 100 mg/kg TBZ, TBZ supplemented with zinc (Zn), selenium (Se), copper (Cu), and iron (Fe), TBZ + (Se + Zn); TBZ + Cu; or TBZ + Fe. The experiment was conducted for 30 consecutive days. Results: TBZ caused a significant perturbation in mineral levels and reduction in reproductive organs weights, plasma testosterone level, and testicular antioxidant enzyme activities. The TBZ-treated group also showed a significant increase in sperm abnormalities (count, motility, and viability percent), plasma follicle-stimulating hormone and luteinizing hormone concentrations, lipid peroxidation, protein oxidation, and severe DNA degradation in comparison with the controls. Histopathologically, TBZ caused testis impairments. Conversely, treatment with trace elements, in combination or alone, improved the reproductive organ weights, sperm characteristics, TBZ-induced toxicity, and histopathological modifications in testis. Conclusion TBZ exerts significant harmful effects on male reproductive system. The concurrent administration of trace elements reduces testis dysfunction, fertility, and toxicity induced by TBZ.
Animal Feed/analysis* ; Animals ; Antioxidants/metabolism* ; Diet ; Dietary Supplements/analysis* ; Fungicides, Industrial/adverse effects* ; Male ; Minerals/metabolism* ; Mutagenicity Tests ; Rats ; Rats, Wistar ; Spermatozoa/physiology* ; Testis/physiology* ; Trace Elements/metabolism* ; Triazoles/adverse effects*

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER^® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER^® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER^® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.
Acrosome Reaction/physiology* ; Calcimycin/pharmacology* ; Calcium/pharmacology* ; Calcium Ionophores/pharmacology* ; Drug Delivery Systems ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/metabolism* ; Male ; Progesterone/pharmacology* ; Spermatozoa/metabolism* ; Zona Pellucida/metabolism*

Dysfunctional sperm maturation is the primary reason for the poor sperm motility and morphology in infertile men. Spermatozoa from infertile men were fractioned on three-layer density gradient (80%, 60%, and 40%). Fraction 1 (F1) refers to the least mature stage having the lowest density, whereas the fraction 4 (F4) includes the most dense and morphologically mature motile spermatozoa. Fraction 2 (F2) and fraction 3 (F3) represent the intermediate stages. Proteins were extracted and separated by 1-dimensional gel. Bands were digested with trypsin and analyzed on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. A total of 1585 proteins were detected in the four fractions of spermatozoa. A dysregulated protein turnover and protein folding may lead to accumulation of defective proteins or proteins that otherwise would have been eliminated during the process of maturation, resulting in the impairment of sperm function. Aberrant chaperone expression may be a major contributing factor to the defective sperm function. Androgen receptor was predicted as a transcription regulator in one of the networks and the affected pathways were chaperone-mediated stress response, proteosomal pathway, and sperm function. The downregulation of key pathways and proteins which compromises the fertilizing potential of spermatozoa may provide insight into the mechanisms that lead to male infertility.
Adult ; Cell Shape/physiology* ; Humans ; Infertility, Male/metabolism* ; Male ; Proteome/metabolism* ; Proteomics ; Signal Transduction/physiology* ; Sperm Motility/physiology* ; Spermatozoa/metabolism* ; Tandem Mass Spectrometry

Vitamin D deficiency is a common health issue around the world. We therefore evaluated the associations of semen quality with both serum and seminal plasma vitamin D levels and studied the mechanisms underlying these by incubating spermatozoa with 1,25(OH)₂D In vitro. Two hundred and twenty-two men were included in our study. Vitamin D was detected using an electrochemiluminescence method. Spermatozoa used for In vitro experiments were isolated by density gradient centrifugation. Positive relationships of serum 25(OH)D with semen volume and seminal plasma fructose were identified. Seminal plasma 25(OH)D level showed no relationship with serum 25(OH)D level, while it was inversely associated with sperm concentration and positively correlated with semen volume and sperm kinetic values. In vitro, sperm kinetic parameters increased after incubation with 1,25(OH)₂D, especially upon incubation for 30 min with it at a concentration of 0.1 nmol l^-1. Under these incubation conditions, the upward migration of spermatozoa increased remarkably with increasing adenosine triphosphate (ATP) concentration. The concentration of cyclic adenosine monophosphate (cAMP) and the activity of protein kinase A (PKA) were both elevated, and the PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89) reversed the increase of ATP production. The concentrations of cytoplasmic calcium ions and nicotinamide adenine dinucleotide (NADH) were both enhanced, while mitochondrial calcium uniporter (MCU) inhibitor, Ruthenium 360 (Ru360) did not reverse the increase of ATP production. Therefore, seminal plasma vitamin D may be involved in regulating sperm motility, and 1,25(OH)₂D may enhance sperm motility by promoting the synthesis of ATP both through the cAMP/PKA pathway and the increase in intracellular calcium ions.
Adenosine Triphosphate/metabolism* ; Adult ; Calcium/metabolism* ; Cyclic AMP/metabolism* ; Cyclic AMP-Dependent Protein Kinases/metabolism* ; Humans ; Male ; Semen/metabolism* ; Semen Analysis ; Signal Transduction/physiology* ; Sperm Motility/physiology* ; Spermatozoa/metabolism* ; Vitamin D/pharmacology* ; Vitamin D Deficiency/blood* ; Wit and Humor as Topic ; Young Adult

Objective: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.
Acrosome ; enzymology ; Animals ; Blotting, Western ; Cricetinae ; Enzyme-Linked Immunosorbent Assay ; Epididymis ; Female ; Fertilization ; physiology ; Free Radical Scavengers ; metabolism ; Humans ; Hyaluronic Acid ; metabolism ; Male ; Muramidase ; analysis ; physiology ; Pichia ; Plasmids ; metabolism ; Recombinant Proteins ; analysis ; metabolism ; Sperm-Ovum Interactions ; physiology ; Spermatozoa ; enzymology ; Testis

Nanos2, a member of the Nanos2 gene family, is a specific gene in male germ cells and encodes an evolutionarily conserved RNA binding protein expressed in male primordial germ cells (PGCs) during the embryonic period as well as in the spermatogonial stem cells (SSCs) of the testis. In the embryonic period, Nanos2 promotes the development of male PGCs and inhibits them from meiosis. In the process of spermatogenesis, Nanos2 suppresses the differentiation of SSCs in the testis and maintains the stability of the SSC pool. The knockout of Nanos2 may cause the disappearance of germ cells and sterility in male mice while its overexpression in the testis may lead to accumulation of SSCs in seminiferous tubules. Besides, Nanos2 is involved in the degradation of specific RNAs and possibly associated with some diseases of the male reproductive system. This review focuses on the recent progress in the studies of Nanos2 in the male reproductive system.
Animals ; Cell Differentiation ; Gene Knockout Techniques ; Male ; Meiosis ; Mice ; RNA ; metabolism ; RNA-Binding Proteins ; genetics ; metabolism ; Spermatogenesis ; physiology ; Spermatogonia ; Spermatozoa ; Testis ; cytology