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Reproductive Medicine and Biology Oct 2019Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this...
PURPOSE
Mouse in vitro spermatogenesis is possible with classical organ culture methods, by placing the testis tissue at the interphase between culture medium and air. In this condition, however, a tissue piece tends to round up to be compact, whose central region suffers from shortage of nutrients and oxygen. In this study, the authors improved the culture condition by spreading each tissue thin and flat, by which they were able to get better access to the oxygen and nutrients.
METHODS
Immature mouse testis tissues placed on agarose gel block were forced to spread flat by covering with a polydimethylsiloxane (PDMS) ceiling chip (PC chip). They were then cultured for weeks and evaluated by the transgene expression of , which reflects the progression of spermatogenesis.
RESULTS
Testis tissues covered with PC chip initiated and maintained spermatogenesis in its wider region than those without PC chip covering. Flow cytometric analysis demonstrated that the PC method yielded more numerous meiotic germ cells than those without PC. Immunohistochemical examination confirmed the authentic histological figure of spermatogenesis from spermatogonia up to round or elongating spermatids.
CONCLUSIONS
The PC chip method is simple and effective to improve the efficiency of in vitro spermatogenesis in the organ culture system.
PubMed: 31607796
DOI: 10.1002/rmb2.12291 -
Genetics and Molecular Research : GMR Mar 2015The current study investigated the relationship between the level of expression and tyrosine phosphorylation of the sperm protein 32 (sp32) and the activation of the...
The current study investigated the relationship between the level of expression and tyrosine phosphorylation of the sperm protein 32 (sp32) and the activation of the boar proacrosin/acrosin system. The acrosomal membrane proteins of boar sperm for use in different treatment experiments (i.e., fresh sperm, freezing and thawing, capacitation, and acrosome reaction) were separated, stained by Coomassie brilliant blue, and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot. The results showed that there were differences in the expression level of sp32 among capacitated, frozen-thawed, and post acrosomal reaction sperms. sp32 expression was higher and significantly higher in capacitated and post-acrosomal reaction sperms than in frozen-thawed sperms and fresh semen, respectively. The level of sp32 tyrosine phosphorylation was significantly different between the frozen-thawed sperms and sperms in the other experimental groups. However, bands with molecular masses of 38 to 170 ku in the fresh semen group were more noticeable, indicating that large acrosomal membrane proteins underwent modification and degradation during capacitation and the acrosomal reaction. As a proacrosin binding protein, sp32 shows upregulated expression and increase in tyrosine phosphorylation levels during the activation of the boar proacrosin/acrosin system.
Topics: Acrosin; Acrosome; Acrosome Reaction; Animals; Blotting, Western; Carrier Proteins; Cryopreservation; Enzyme Precursors; Male; Phosphorylation; Semen Preservation; Sperm Capacitation; Spermatozoa; Swine; Tyrosine
PubMed: 25867384
DOI: 10.4238/2015.March.27.23 -
Reproductive Biology and Endocrinology... Dec 2019Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and...
BACKGROUND
Capacitation involves physiological changes that spermatozoa must undergo in the female reproductive tract or in vitro to obtain the ability to bind, penetrate and fertilize the egg. Up to date, several methods have been developed to characterize this complex biological process. The goal of the presented study is to mutually compare several fluorescent techniques, check their ability to detect changes in molecular processes during the capacitation progress and determine their ability to predict the percentage of acrosome reacted (AR) sperm after the exposure to solubilized zona pellucida (ZP). The capacitation process was analyzed using four fluorescent techniques: 1. chlortetracycline (CTC) staining, 2. anti-acrosin antibody (ACR.2) assay, 3. anti-phosphotyrosine (pY) antibody assay, 4. fluorescein isothiocyanate-conjugated phalloidin (FITC-phall) assay. All these methods were tested using fluorescent microscopy and flow cytometry.
RESULTS
All selected methods are capable to detect the capacitation progress of boar sperm in vitro, but there are significant differences in their outcome when using fluorescent microscopy or flow cytometry experimental arrangements and subsequent statistical analysis (KW-ANOVA). Also, the ability to predict the absolute numbers of sperm which will undergo ZP-induced AR differ significantly (CTC and ACR.2 gave the best predictions).
CONCLUSIONS
Our study compared four largely used methods used to characterize capacitation process, highlighted their differences and showed that all are able to detect capacitation progress, CTC and ACR.2 are furthermore able to accurately predict the percentage of AR sperm after ZP-induced AR.
Topics: Acrosome Reaction; Animals; Calcium; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Fluorescent Dyes; Male; Microscopy, Fluorescence; Phalloidine; Sperm Capacitation; Spermatozoa; Sus scrofa; Zona Pellucida
PubMed: 31856844
DOI: 10.1186/s12958-019-0554-z -
Molecular Medicine Reports Nov 2016Male infertility is a complex, multifactorial and polygenic disease that contributes to ~50% cases of infertility. Previous studies have demonstrated that excess weight...
Male infertility is a complex, multifactorial and polygenic disease that contributes to ~50% cases of infertility. Previous studies have demonstrated that excess weight and obesity factors serve an important role in the development of male infertility. An increasing number of studies have reported that resveratrol may regulate the response of cells to specific stimuli that induce cell injury, as well as decrease germ cell apoptosis in mice or rats. In the present study, the semen quality and serum sex hormone levels were evaluated in 324 men, which included 73 underweight, 82 normal weight, 95 overweight and 74 obese men. All patients were referred to The Reproductive Medicine Center of Shanxi Women and Infants Hospital (Taiyuan, China) between January 2013 and January 2015. The aim of the present study was to investigate the effects of resveratrol treatment on the motility, plasma zinc concentration and acrosin activity of sperm from obese males. The sperm concentration, normal sperm morphology, semen volumes, DNA fragmentation rates and testosterone levels in men from the overweight and obese groups were markedly decreased when compared with men in the normal weight group. In addition, the progressive motility, seminal plasma zinc concentration and spermatozoa acrosin activity were notably decreased in the obese group compared with the normal weight group. However, estradiol levels were significantly increased in the overweight, obese and underweight groups compared with the normal weight group. Notably, semen samples from obese males with astenospermia treated with 0‑100 µmol/l resveratrol for 30 min demonstrated varying degrees of improvement in sperm motility. When these semen samples were treated with 30 µmol/l resveratrol, sperm motility improved when compared to other doses of resveratrol. Therefore, 30 µmol/l resveratrol was selected for further experiments. Upon treatment of semen samples with resveratrol (30 µmol/l) for 30 min, the seminal plasma zinc concentration and spermatozoa acrosin activity increased significantly in the experimental group compared with the control group. These data suggest that male obesity negatively impacts on male reproductive potential, not only through altering hormone levels, but also by directly altering sperm function. In addition, resveratrol may have a therapeutic and protective effect against obesity-induced abnormalities in semen.
Topics: Acrosin; Adult; Cell Survival; Cytoplasm; Gonadal Steroid Hormones; Humans; Infertility, Male; Male; Obesity; Overweight; Protective Agents; Resveratrol; Semen Analysis; Spermatozoa; Stilbenes; Zinc
PubMed: 27748829
DOI: 10.3892/mmr.2016.5840 -
International Journal of Peptide... 2021One of the most common gynecologic cancers is ovarian cancer and ranked third after the other two most common cancers: cervical and uterine. The highest mortality rate...
UNLABELLED
One of the most common gynecologic cancers is ovarian cancer and ranked third after the other two most common cancers: cervical and uterine. The highest mortality rate has been observed in the case of ovarian cancer. To treat ovarian cancer, an immune-informatics approach was used to design a multi-epitope vaccine (MEV) structure. Epitopes prediction of the cancer testis antigens (NY-ESO-1), A-Kinase anchor protein (AKAP4), Acrosin binding protein (ACRBP), Piwi-like protein (PIWIL3), and cancer testis antigen 2 (LAGE-1) was done. Non-toxic, highly antigenic, non-allergenic, and overlapping epitopes were shortlisted for vaccine construction. Chosen T-cell epitopes displayed a robust binding attraction with their corresponding Human Leukocyte Antigen (HLA) alleles demonstrated 97.59% of population coverage. The vaccine peptide was established by uniting three key constituents, comprising the 14 epitopes of CD8 + cytotoxic T lymphocytes (CTLs), 5 helper epitopes, and the adjuvant. For the generation of the effective response of CD4 + cells towards the T-helper cells, granulocyte-macrophage-colony-stimulating factor (GM-CSF) was applied. With the addition of adjuvants and linkers, the construct size was 547 amino acids. The developed MEV structure was predicted to be antigenic, non-toxic, non-allergenic, and firm in nature. I-tasser anticipated the 3D construction of MEV. Moreover, disulfide engineering further enhanced the stability of the final vaccine protein. In-silico cloning and vaccine codon optimization were done to analyze the up-regulation of its expression. The outcomes established the vaccine's immunogenicity and safety profile, besides its aptitude to encourage both humoral and cellular immune responses. The offered vaccine, grounded on our in-silico investigation, may be considered for ovarian cancer immunotherapy.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s10989-021-10294-w.
PubMed: 34690620
DOI: 10.1007/s10989-021-10294-w -
Biomedical Reports May 2022The aim of the present study was to investigate the influence of millimeter-wave electromagnetic (MW) irradiation on normal and pathological human sperm , and to...
The aim of the present study was to investigate the influence of millimeter-wave electromagnetic (MW) irradiation on normal and pathological human sperm , and to evaluate a possible role of polyamines (PA) in this process. The stability of sperm membranes, the number of apoptotic gametes, and the content of seminal plasma PA in the ejaculates of fertile and subfertile men were compared before and after short-term MW electromagnetic exposure . The ejaculate samples were collected from healthy donors [n=25, age 22-38 years old (y.o.), average age 30.6±1.1 y.o. (mean ± SEM)] and from subfertile men (n=78, age 25-48 y.o., average age 34.1±0.8 y.o.) and exposed to MW radiation. The electromagnetic field had a wavelength of 7.1 mm, a frequency of 42.194 GHz and an exposure time of 20 min. The fragility of sperm membranes was evaluated by their resistance to sodium chloride solution (Milovanov test) and to acetic acid (Joel test). Acrosin activity was assayed spectrophotometrically. Apoptosis was determined by the externalization of phosphatidylserine on the outer side of the sperm membrane and propidium iodide staining. The PA levels were determined by agar gel electrophoretic fractionation. An increase in the resistance of sperm membranes, a decrease in acrosin activity, a decrease in the number of apoptotic gametes and a decrease in the seminal plasma PA concentrations were found after exposure of the native human sperm to low-intensity MW irradiation. Two types of reactions were revealed for the subfertile samples. The results revealed positive bio-effects of specific microwaves on the human semen and the participation of PA in the realization of these effects.
PubMed: 35386108
DOI: 10.3892/br.2022.1521 -
Reproductive Medicine and Biology 2022ML-2-3 is a novel tetracyclic iridoid derived from Bentham leaves. This compound has anti-trypanosomal and anti-leishmanial effects. In this study, the authors...
PURPOSE
ML-2-3 is a novel tetracyclic iridoid derived from Bentham leaves. This compound has anti-trypanosomal and anti-leishmanial effects. In this study, the authors investigated effects of ML-2-3 on in vitro fertilization (IVF) rates, motility, and acrosome reaction of the mouse sperm.
METHODS
IVF was performed using sperm from BALB/cByJJcl mice treated with ML-2-3. Computer-assisted sperm analysis (CASA) was performed on the sperm of C57BL/6J mice to investigate sperm motility. The effect of ML-2-3 on the acrosome reaction was examined by observing the fluorescence of sperm labeled with the acrosin-EGFP transgene.
RESULTS
ML-2-3 improved IVF in BALB/cByJJcl mice with low fertilization rates. The optimum concentration of ML-2-3 in sperm pre-culture medium was 20 M, and no significant toxicity of ML-2-3 was observed in developing embryos at this concentration. ML-2-3 affected sperm motility but not the acrosome reaction. ML-2-3 increased the IVF rate of mouse sperm that had been refrigerated for 3 days.
CONCLUSIONS
ML-2-3 can improve the outcome of IVF and motility without inducing the acrosome reaction in mice. These effects of ML-2-3 on sperm behaviors are different from those of the similar drugs.
PubMed: 35414765
DOI: 10.1002/rmb2.12455 -
Andrology Mar 2019Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease...
BACKGROUND AND OBJECTIVES
Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins.
MATERIALS AND METHODS
The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, AEBSF. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native PAGE, co-immunoprecipitation, and mass spectrometry, with interrogation of protein-protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin-containing complex in sperm function.
RESULTS
Testisin surface expression increased significantly in capacitated spermatozoa (p < 0.001), increased further following acrosome reaction (p < 0.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes; co-immunoprecipitation revealed interactions of testisin with a multitude of zona pellucida-binding proteins, including ZPBP, ZAN, acrosin, several heat-shock proteins, and components of the TCP1 complex.
CONCLUSION
Testisin appears to form part of the zona pellucida-binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.
Topics: Acrosome Reaction; Animals; Female; Horses; Male; Protein Binding; Serine Endopeptidases; Sperm Capacitation; Spermatozoa; Zona Pellucida
PubMed: 30549223
DOI: 10.1111/andr.12569 -
Molecular and Cellular Biochemistry Dec 2015Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events....
Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads comprised 54, 50, 45, and 38-19 kDa polypeptides. The objective of this study is to identify and characterize the 45 kDa (OMC45) polypeptide, to define its role in binding acrosomal hydrolases, and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI-TOF-TOF yielded eight peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X-100-permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti-OMC45 and anti-TEKT3. The OMC45 polypeptide was solubilized by radio-immunoprecipitation assay buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti-OMC45 immunoprecipitation pellet. An identical blot stained with anti-TEKT3 exhibited the presence of TEKT3 polypeptide in the anti-OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide and that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N-linked and O-linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N-acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid membrane complex.
Topics: Acrosin; Acrosome; Acrosome Reaction; Animals; Cattle; Databases, Protein; Glycoproteins; Glycosylation; Male; Membrane Proteins; Molecular Weight; Protein Binding; Protein Processing, Post-Translational; Proteomics; Solubility; Sperm Capacitation; alpha-N-Acetylgalactosaminidase
PubMed: 26268136
DOI: 10.1007/s11010-015-2534-8 -
International Journal of Molecular... Dec 2019This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during...
This study sought to identify and localize SLO1 channels in boar spermatozoa by immunoblotting and immunofluorescence, and to determine their physiological role during in vitro sperm capacitation. Sperm samples from 14 boars were incubated in a capacitation medium for 300 min in the presence of paxilline (PAX), a specific SLO1-channel blocker, added either at 0 min or after 240 min of incubation. Negative controls were incubated in capacitation medium, and positive controls in capacitation medium plus tetraethyl ammonium (TEA), a general K-channel blocker, also added at 0 min or after 240 min of incubation. In all samples, acrosome exocytosis was triggered with progesterone after 240 min of incubation. Sperm motility and kinematics, integrity of plasma and acrosome membranes, membrane lipid disorder, intracellular calcium levels and acrosin activity were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. In boar spermatozoa, SLO1 channels were found to have 80 kDa and be localized in the anterior postacrosomal region and the mid and principal piece of the tail; their specific blockage through PAX resulted in altered calcium levels and acrosome exocytosis. As expected, TEA blocker impaired in vitro sperm capacitation, by altering sperm motility and kinematics and calcium levels. In conclusion, SLO1 channels are crucial for the acrosome exocytosis induced by progesterone in in vitro capacitated boar spermatozoa.
Topics: Acrosome; Acrosome Reaction; Animals; Biomechanical Phenomena; Calcium; Exocytosis; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Male; Membrane Lipids; Potassium Channels; Progesterone; Sperm Capacitation; Sperm Motility; Spermatozoa; Sus scrofa; Swine
PubMed: 31847486
DOI: 10.3390/ijms20246330