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Komsky-Elbaz, Alisa, Dorit Kalo, and Zvi Roth. "Effect of aflatoxin B1 on bovine spermatozoa’s proteome and embryo’s transcriptome." Reproduction 160, no. 5 (November 2020): 709–23. http://dx.doi.org/10.1530/rep-20-0286.
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This study aims to evaluate the deleterious effect of the mycotoxin aflatoxin B1 (AFB1) on bull spermatozoa and the carryver effect on the developing embryo. Proteomic analysis of AFB1-treated spermatozoa revealed differential expression of proteins associated with biological processes and cellular pathways that involved in spermatozoon function, fertilization competence and embryonic development. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. To confirm this hypothesis, we have used the annexin V (AV) kit to separate the spermatozoa into apoptotic (AV+) and non-apoptotic (AV−) subpopulations which were found to correlate with high- and low DNA fragmentation, respectively. Fertilization with AV+ AFB1-treated spermatozoa, resulted in no blastocyst formation, whereas fertilization with AV− spermatozoa resulted in reduced cleavage rate and formation of genetically altered blastocysts (POU5F1 and SOX2). Microarray analysis of blastocysts derived from 10 µM AFB1-treated spermatozoa revealed differential expression of 345 genes that involved in cellular pathways such as embryo and placenta development, cell cycle, DNA repair and histone modification, and in signaling pathways, especially calcium signaling pathway. This is the first report on deleterious carrying over effects of AFB1 from the bovine spermatozoa to the formed embryo. Our findings suggest that aside from the damage caused by AFB1 to spermatozoa’s DNA integrity, additional damage mechanisms are involved.
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Komsky-Elbaz, A., D. Kalo, and Z. Roth. "Carryover effect of atrazine and its metabolite—from treated bovine spermatozoa to the embryo’s transcriptome†." Biology of Reproduction 104, no. 5 (February 24, 2021): 1162–80. http://dx.doi.org/10.1093/biolre/ioab027.
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Abstract Atrazine (ATZ) is an extensively used herbicide and ubiquitous environmental contaminant. ATZ and its metabolite, diaminochlorotriazine (DACT), cause several cellular and functional alterations in spermatozoa. We aimed to examine the effect of ATZ/DACT on spermatozoon DNA integrity, fertilization competence, embryonic development, and transcriptome profile of in vitro-produced embryos derived from fertilization with pre-exposed sperm. Bovine spermatozoa exposed to ATZ (0.1 or 1 μM) or DACT (1 or 10 μM) during in vitro capacitation were used for in vitro fertilization of untreated oocytes. Cleavage and blastocyst-formation rates were evaluated 42 h and 7 days postfertilization, respectively. The association between DNA fragmentation and apoptosis (annexin V kit) was determined. Fertilization competence of annexin-positive (AV+) and annexin-negative (AV−) spermatozoa was examined. Microarray analysis was performed for 7-day blastocysts. Intracytoplasmic sperm injection was performed with control (AV+, AV−) and DACT (AV+, AV−) spermatozoa. Cleavage rates did not differ between groups and blastocyst formation tended to be higher for AV− vs. AV+ in both control and DACT groups, suggesting that acrosome reaction, rather than DNA fragmentation, underlies the reduced cleavage. Transcriptomic analysis revealed 139 and 230 differentially expressed genes in blastocysts derived from ATZ- and DACT-exposed spermatozoa, respectively, relative to controls. Proteomic analysis shown differential expression of proteins in ATZ- or DACT-treated spermatozoa, in particular proteins related to cellular processes and biological pathways. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. Overall, the current study reveals a deleterious carryover effect of ATZ/DACT from the spermatozoa to the developing embryo.
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I’tishom, Reny, Doddy M. Soebadi, Aucky Hinting, Hamdani Lunardhi, and Rina Yudiwati. "IN VITRO FERTILITY TEST OF HUMAN SPERMATOZOA MEMBRANE PROTEIN FERTILIN BETA ANTIBODY IN MICE (Mus musculus Balb/c) AS IMMUNOCONTRACEPTIVE CANDIDATE." Folia Medica Indonesiana 52, no. 3 (August 14, 2017): 209. http://dx.doi.org/10.20473/fmi.v52i3.5453.
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One of the materials as potential candidates immunocontraception material is spermatozoa. Fertilin beta is spermatozoa membrane protein and is found only in mature spermatozoa and ejaculate, which serves as an adhesion molecule. Spermatozoa membrane protein that is used as an ingredient immunocontraception candidate, must have specific criteria that the specificity of spermatozoa, the role of antigen in the fertilization process, which includes the formation of immunogenicity sufficient antibody response has the potential to block fertilization. Antibodies against spermatozoa affect the stages before fertilization of the reproductive process and can hinder the development of the embryo after fertilization. Until now very little research data spermatozoa membrane protein as an ingredient immunocontraception are up to the test of experimental animals. The research objective is to prove the role of the resulting antibody induction of antibodies fertilin beta protein in the membrane of human spermatozoa induce agglutination and reduce motility thus reducing the number of in vitro fertilization. Research conducted at the IVF Laboratory, Department of Biology of Medicine, Faculty of Medicine, University of Airlangga. This research includes: Test the potential of antibody protein beta fertilin membrane of human spermatozoa and inhibit the role of antibodies in vitro fertilization in mice (Mus musculus Balb/c). In vitro studies have resulted in fertilization figure of 25% is smaller than the number that is equal to control fertilization of 58.7%, whereas previously the spermatozoa were incubated first with a beta membrane protein antibody fertilin human spermatozoa. While the percentage of inhibition of sperm to fertilize an oocyte by 33.75%. Potential imunokontraseptif considered effective if it decreased significantly (P <0.05) than the numbers fertilization in the treatment group compared with the control group. This shows fertilin beta membrane protein antibody has the ability to inhibit human spermatozoa to fertilize oocytes that reduce the number of fertilization.
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Peippo, J., M. Räty, K. Korhonen, M. Eronen, K. Kananen, T. Hurme, M. Halmekytö, and A. Mäki-Tanila. "Impact of in vitro fertilization of bovine oocytes with sex-sorted frozen–thawed spermatozoa on developmental kinetics, quality and sex ratio of developing embryos." Zygote 18, no. 3 (January 29, 2010): 185–94. http://dx.doi.org/10.1017/s0967199409990281.
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SummaryWe studied whether bovine embryos developing after in vitro fertilization (IVF) with sex-sorted spermatozoa differed in developmental kinetics, quality and sex ratio from embryos produced with unsorted spermatozoa. Abattoir-derived oocytes were fertilized with X-sorted, Y-sorted or unsorted spermatozoa from a single bull. To evaluate economical use of the sex-sorted spermatozoa, washed spermatozoa from a single straw (2 million spermatozoa) were used to fertilize each batch of collected oocytes without any further isolation steps. Concentration of the unsorted spermatozoa was adjusted accordingly. Fertilizations were assessed by staining sperm asters at 10 hpi and pronuclei at 20 hpi. Embryo development and morphological quality were monitored on days 2, 7, 8 and 9 of the development (IVF = day 0). All embryos were sexed using PCR. Following fertilization, penetration and subsequent cleavage rates were compromised in the X-sorted group compared with the Y-sorted and unsorted groups (penetration: 58.0% vs. 89.8% and 90.0%, cleavage: 65.3% vs. 81.5% and 75.0%). The use of the sex-sorted spermatozoa did not, however, reduce the proportion of transferable embryos (sex-sorted 29.6% vs. unsorted 27.7%) or their quality (quality 1: sex-sorted 36.0% vs. unsorted 19.9%). The Y-sorted spermatozoa produced more transferable embryos of better quality than the X-sorted spermatozoa (days 7–8: 31.9% vs. 26.4%, quality 1: 38.9% vs. 30.6%). On average, out of 10 transferable embryos, nine were of the predicted sex in the X- and Y-sorted spermatozoa groups. These results indicate that low numbers of X- and Y-sorted spermatozoa can be used successfully for female and male embryo production in vitro.
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Cannarella, Rossella, Rosita A. Condorelli, Aldo E. Calogero, and Sandro La Vignera. "Novel Insights on the Role of the Human Sperm Proteome." Protein & Peptide Letters 27, no. 12 (December 2, 2020): 1181–85. http://dx.doi.org/10.2174/0929866527666200505215921.
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: The spermatozoon has classically been seen only as a paternal DNA transporter into the oocyte, thus underestimating the entire contribution of the male gamete to the embryo development. The advancement of the research supports that not only the sperm genome, but the entire sperm transcriptome and proteome carry crucial information for fertilization and embryo development. : Altogether, 6871 proteins have been reported in spermatozoa so far. Their functional analysis has recently addressed to the sperm proteome a role in fertilization, preimplantation embryo development and paternal epigenetic inheritance. Targeted analysis of human spermatozoa is warranted to compile an evidence-based list of sperm-carried molecular targets in infertile patients.
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Limatola, Nunzia, Filip Vasilev, Jong Tai Chun, and Luigia Santella. "Sodium-mediated fast electrical depolarization does not prevent polyspermic fertilization in Paracentrotus lividus eggs." Zygote 27, no. 4 (August 2019): 241–49. http://dx.doi.org/10.1017/s0967199419000364.
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SummaryDuring sea urchins fertilization, the activating spermatozoon triggers a series of physiological changes that transforms the quiescent egg into a dynamic zygote. It has been suggested that several of these egg activation events, e.g. sperm-induced plasma membrane depolarization and the Ca2+-linked cortical reaction, play additional roles to prevent the entry of supernumerary spermatozoa. In particular, the abrupt shift in egg membrane potential at fertilization, which is sustained by a Na+ influx, has been considered as a fast mechanism to block polyspermy. To test the relevance of the Na+-mediated fast electrical block to polyspermy, we fertilized sea urchin eggs in artificial seawater with a low concentration of Na+; nearly all the eggs were still monospermic, as judged by the number of Hoechst 33422-stained sperm. When fertilized in normal seawater, eggs that were pre-incubated in the low Na+ medium exhibited impaired elevation of the fertilization envelope. Nevertheless, these eggs manifested entry of a single spermatozoon, suggesting that the fertilization envelope was not the primary determinant of the block to polyspermy. Furthermore, we showed that the abnormal cleavage patterns displayed by eggs pre-incubated in low Na+, which were often considered a hallmark of polyspermy, were due to the alterations in the cortical actin filaments dynamics following fertilization, and not to the formation of multipolar spindles associated with supernumerary sperm centrosomes. Hence, our results suggested that Paracentrotus lividus eggs do not utilize Na+ to rapidly prevent additional spermatozoa from entering the egg, at variance with the hypothesis of an electrical fast block to polyspermy.
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FUKUMOTO, MAKOTO. "Fertilization in ascidians: apical processes and gamete fusion in Ciona intestinalis spermatozoa." Journal of Cell Science 89, no. 2 (February 1, 1988): 189–96. http://dx.doi.org/10.1242/jcs.89.2.189.
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This paper describes the fine structure of the spermatozoan apex and its morphological changes during the process of fertilization in Ciona intestinalis. At the apex of the head an acrosome is present in the form of a flattened vesicle containing moderately electron-dense material with an electron-dense plate in its centre. Contiguous to the acrosome, an apical substance is located in the anterior-most tip of the sperm head. The spermatozoa that bind to and penetrate the chorion of both normal and caffeine-treated eggs have an intact acrosome and do not show an observable alteration of the plasmalemma enclosing the apex of the head. On the other hand, spermatozoa in the perivitelline space of both normal and caffeine-treated eggs lack an acrosome. Instead of an acrosome, apical processes are observed at the apex of the spermatozoa in the perivitelline space of both normal and caffeine-treated eggs. Gamete fusion seems to occur between the egg membrane and some of these apical processes at the tip of the sperm head. The apical process reported here is thought to be analogous to the acrosomal process in other marine invertebrates. The apical processes that are induced in the perivitelline space have not been previously described in ascidians.
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Zuccotti, M., R. Yanagimachi, and H. Yanagimachi. "The ability of hamster oolemma to fuse with spermatozoa: its acquisition during oogenesis and loss after fertilization." Development 112, no. 1 (May 1, 1991): 143–52. http://dx.doi.org/10.1242/dev.112.1.143.
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To determine when the growing hamster oocyte gains the ability to fuse with the spermatozoon, oocytes at various stages of development were collected from ovaries, and zona-pellucida-free oocytes were inseminated in vitro with acrosome-reacted spermatozoa. Very small primary oocytes were unable to fuse with spermatozoa. Oocytes first became competent to fuse with spermatozoa when they had grown to about 20 microns in diameter. The acquisition of fusibility coincided with the first appearance of zona pellucida material and oolemma microvilli. The fusibility of the oolemma increased as the oocyte grew, reaching a maximum when the oocyte reached the metaphase of the second meiosis. The fusibility of the oolemma was reduced drastically after fertilization, and was lost completely by the 8-cell stage. The appearance and subsequent disappearance of a putative fusion-mediating molecule in the oolemma is proposed. Since this molecule is fairly resistant to proteinase digestion, at least in the hamster, it could be a cryptic protein or a glycolipid.
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O'Brien, Justine K., and Terri L. Roth. "Functional capacity and fertilizing longevity of frozen-thawed scimitar-horned oryx (Oryx dammah) spermatozoa in a heterologous in vitro fertilization system." Reproduction, Fertility and Development 12, no. 8 (2000): 413. http://dx.doi.org/10.1071/rd00105.
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This study was conducted to determine if cryopreservation and thawing reduces the quality of scimitar-horned oryx spermatozoa and thus might be responsible for sub-optimal artificial insemination (AI) efficiency. Functional capacity of frozen–thawed oryx spermatozoa was compared in a heterologous bovine in vitro fertilization (IVF) system after being prepared by four methods. Fertilizing longevity was also assessed after thawing and pre-incubating spermatozoa for 12 or 24 h before IVF. Sperm characteristics (viability, morphology, acrosomal and capacitation status) were superior for samples prepared by Percoll centrifugation and standard swim-up compared with microdrop swim-up and wash methods. Regardless of variation in sperm characteristics over time, fertilization success and embryo development were high and did not differ among treatments. Fertilization and cleavage success for spermatozoa pre-incubated for 12 h before IVF were comparable with that achieved with non-incubated spermatozoa. Even 24 h after thawing, spermatozoa were capable of fertilizing oocytes, but percentage fertilization and embryo cleavage were significantly lower than for spermatozoa pre-incubated for 12 h. Overall, functional capacity of oryx spermatozoa after thawing appears comparable with that of domestic bull spermatozoa. When used for AI, frozen—thawed oryx spermatozoa should be capable of fertilizing oocytes in females ovulating 12 or even 24 h after insemination, providing sperm transport mechanisms are adequate. The functional capacity and fertilizing longevity of oryx sperm after thawing is high, and therefore unlikely to be responsible for decreased AI efficiency in the scimitar-horned oryx.
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Adham, I. M., K. Nayernia, and W. Engel. "Spermatozoa lacking acrosin protein show delayed fertilization." Molecular Reproduction and Development 46, no. 3 (March 1997): 370–76. http://dx.doi.org/10.1002/(sici)1098-2795(199703)46:3<370::aid-mrd16>3.0.co;2-2.
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Sa, S. J., H. T. Cheong, B. K. Yang, and C. K. Park. "295 RELATIONSHIP BETWEEN PLASMINOGEN ACTIVATOR/PLASMIN SYSTEM AND IN VITRO FERTILIZATION ABILITY IN THE PIG." Reproduction, Fertility and Development 18, no. 2 (2006): 255. http://dx.doi.org/10.1071/rdv18n2ab295.
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Plasminogen activators (PAs) are serine proteases, known to be secreted by a large number of cell types, that convert plasminogen into plasmin. The present study was undertaken to identify PAs in porcine gametes and to investigate a possible role of plasminogen in fertilization in vitro in the pig. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG). To determine the changes of PA activities in porcine oocytes during maturation, the cumulus-oocyte complexes (COCs) were incubated in NCSU-33 in an atmosphere of 5% CO2 in air at 39�C for 0, 24, or 48 h. On the other hand, to investigate the release of PAs by boar spermatozoa, fresh spermatozoa were pre-incubated in fertilization medium (mTBM) for 0, 2, 4, or 6 h. After culture, 40 COCs, 40 cumulus-free oocytes, and 40 � 106 spermatozoa were separately put into microtubes containing 20 �L of sample buffer [5.0% (w:v) sodium dodecyl sulfate (SDS), 20% (v:v) glycerol, and 0.0025% (w:v) bromophenol blue in 0.125 M Tris-HCl buffer] and frozen at -80�C until used for analysis. PA activities in porcine oocytes and spermatozoa were quantified using SDS-PAGE, casein-agar zymography, and densitometry. Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). In the COCs cultured for 24-18 h, tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed. Also, PA activities increased as duration of culture increased. However, no uPA activity was detected in cumulus-free oocytes. In procine fresh spermatozoa, tPA, uPA, and tPA-PAI were observed. When spermatozoa were incubated for 2, 4, or 6 h in fertilization medium, the rate of acrosome reaction (AR) in spermatozoa increased as the duration of culture increased, but PA activities decreased gradually. However, PA activities in sperm-conditioned medium increased as duration of culture increased. On the other hand, to determine the effect of plasminogen on fertilization ability of porcine oocyte and spermatozoa, plasminogen (50 �g/mL) was added in fertilization medium. Addition of plasminogen to fertilization medium increased (P < 0.05) AR in spermatozoa and sperm binding to the zona pellucida (ZP), compared with control group. The ZP solubility (zona digestion time) was higher in medium with than that without plasminogen. When porcine oocytes and spermatozoa were co-incubated in fertilization medium with plasminogen, the polyspermic rate was lower in medium with than that without plasminogen. Also, plasminogen significantly (P < 0.05) increased formation rate of the male pronucleus in oocytes penetrated by spermatozoa. These results suggest that supplementing of plasminogen in fertilization medium may play a positive role in improving of fertilization ability in vitro in the pig.
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Vacquier, Victor D. "Activation of sea urchin spermatozoa during fertilization." Trends in Biochemical Sciences 11, no. 2 (February 1986): 77–81. http://dx.doi.org/10.1016/0968-0004(86)90270-7.
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Satake, N., A. K. Alhaider, W. V. Holt, and P. F. Watson. "312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION." Reproduction, Fertility and Development 19, no. 1 (2007): 272. http://dx.doi.org/10.1071/rdv19n1ab312.
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In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P < 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P < 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.
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O’Flaherty, Cristian. "Peroxiredoxin 6: The Protector of Male Fertility." Antioxidants 7, no. 12 (November 24, 2018): 173. http://dx.doi.org/10.3390/antiox7120173.
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The spermatozoon is a terminal cell with the unique purpose of delivering the paternal genome to the oocyte during fertilization. Once spermatozoa enter into the female reproductive tract, they count on only the antioxidant protection that they received during spermatogenesis and epididymal maturation. Peroxiredoxins (PRDXs), particularly PRDX6, are important players in the antioxidant protection and regulation of reactive oxygen species (ROS) levels in spermatozoa. PRDX6, through its peroxidase and calcium-independent phospholipase A2 activities, plays a major role in the regulation of ROS to maintain viability and motility and allow the spermatozoon to achieve fertilizing ability during the complex process of capacitation. The absence of PRDX6 is sufficient to promote abnormal reproductive outcomes in mice that resemble what we observe in infertile men. Indeed, Prdx6−/− spermatozoa display low motility and severe DNA damage, which is translated into reduced ability to fertilize oocytes in vitro or produce a low number of pups compared to wild-type controls. This review focuses on the role of PRDX6 as the primary antioxidant enzyme that protects the spermatozoon from oxidative-stress-associated damages to protect the paternal genome and assure fertility.
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Zhang, Xue-Ying, Yi-Meng Xiong, Ya-Jing Tan, Li Wang, Rong Li, Yong Zhang, Xin-Mei Liu, et al. "Melatonin rescues impaired penetration ability of human spermatozoa induced by mitochondrial dysfunction." Reproduction 158, no. 5 (November 2019): 465–75. http://dx.doi.org/10.1530/rep-19-0231.
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Fertilization failure often occurs during in vitro fertilization (IVF) cycles despite apparently normal sperm and oocytes. Accumulating evidence suggests that mitochondria play crucial roles in the regulation of sperm function and male fertility. 3-Nitrophthalic acid (3-NPA) can induce oxidative stress in mitochondria, and melatonin, as an antioxidant, can improve mitochondrial function by reducing mitochondrial oxidative stress. The role of sperm mitochondrial dysfunction in fertilization failure during IVF is unclear. The present study revealed that spermatozoa with low, or poor, fertilization rates had swollen mitochondria, increased mitochondria-derived ROS, and attenuated mitochondrial respiratory capacity. 3-NPA treatment enhanced mitochondrial dysfunction in sperm. Spermatozoa with poor fertilization rates, and spermatozoa treated with 3-NPA, had reduced penetration ability. The concentration of melatonin was decreased in semen samples with low and poor fertilization rates. Melatonin, not only decreased excessive mitochondria-derived ROS, but also ‘rescued’ the reduced penetration capacity of spermatozoa treated with 3-NPA. Taken together, the study suggested that mitochondria-derived ROS and mitochondrial respiratory capacity are independent bio-markers for sperm dysfunction, and melatonin may be useful in improving sperm quality and overall male fertility.
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Shih, Ying-Fu, Shu-Ling Tzeng, Wen-Jung Chen, Chun-Chia Huang, Hsiu-Hui Chen, Tsung-Hsien Lee, and Maw-Sheng Lee. "Effects of Synthetic Serum Supplementation in Sperm Preparation Media on Sperm Capacitation and Function Test Results." Oxidative Medicine and Cellular Longevity 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/1027158.
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Albumin supplementation of culture media induces sperm capacitation in assisted reproduction technique cycles. Synthetic serum supplementation is clinically used to replace albumin for preventing transmission of infectious agents. However, the effects of synthetic serum supplementation on sperm capacitation have rarely been investigated. Spermatozoa from 30 men with normal basic semen analysis results were collected, divided into five aliquots, and cultured in capacitating conditions in four combinations of two synthetic serum supplements, serum substitute supplement (SSS) and serum protein substitute (SPS), and two fertilization media, Quinns Advantage™Fertilization (QF) and human tubular fluid (HTF) media. Reactive oxygen species (ROS) levels in spermatozoa were measured through chemiluminescence. Furthermore, acrosome reaction and western blotting for tyrosine phosphorylation were used to evaluate sperm capacitation. HTF+SSS had significantly higher ROS levels than QF+SPS did (11,725 ± 1,172 versus 6,278 ± 864 relative light units). In addition, the spermatozoa cultured in QF+SPS had lower motility, acrosome reaction rates, and tyrosine phosphorylation levels compared with those cultured in HTF+SSS. In conclusion, the effects of synthetic serum supplementation on sperm capacitation varied according to the combination of media. These differences may lead to variations in spermatozoon ROS levels, thus affecting sperm function test results.
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Mejía-Flores, Itzayana, Natalia Chiquete-Félix, Icela Palma-Lara, Salvador Uribe-Carvajal, and María de Lourdes Juárez-Mosqueda. "During capacitation in bull spermatozoa, actin and PLC-ζ undergo dynamic interactions." Zygote 25, no. 5 (September 20, 2017): 558–66. http://dx.doi.org/10.1017/s0967199417000260.
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SummaryThe migration pattern of sperm-specific phospholipase C-ζ (PLC-ζ) was followed and the role of this migration in actin cytoskeleton dynamics was determined. We investigated whether PLC-ζ exits sperm, opening the possibility that PLC-ζ is the ‘spermatozoidal activator factor’ (SOAF). As capacitation progresses, the highly dynamic actin cytoskeleton bound different proteins to regulate their location and activity. PLC-ζ participation at the start of fertilization was established. In non-capacitated spermatozoa, PLC-ζ is in the perinuclear theca (PT) and in the flagellum, therefore it was decided to determine whether bovine sperm actin interacts with PLC-ζ to direct its relocation as it progresses from non-capacitated (NC) to capacitated (C) and to acrosome-reacted (AR) spermatozoa. PLC-ζ interacted with actin in NC spermatozoa (100%), PLC-ζ levels decreased in C spermatozoa to 32% and in AR spermatozoa to 57% (P < 0.001). The level of actin/PLC-ζ interaction was twice as high in G-actin (P < 0.001) that reflected an increase in affinity. Upon reaching the AR spermatozoa, PLC-ζ was partially released from the cell. It was concluded that actin cytoskeleton dynamics control the migration of PLC-ζ during capacitation and leads to its partial release at AR spermatozoa. It is suggested that liberated PLC-ζ could reach the egg and favour fertilization.
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Bogliolo, L., P. Bonelli, L. Madau, M. T. Zedda, C. Santucciu, G. Leoni, S. Pau, and S. Ledda. "202FERTILIZING ABILITY OF EPIDIDYMAL CAT SPERMATOZOA AFTER CRYOPRESERVATION OR STORAGE AT 4°C." Reproduction, Fertility and Development 16, no. 2 (2004): 222. http://dx.doi.org/10.1071/rdv16n1ab202.
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The possibility of harvesting cat epididymal spermatozoa from excised testis represents a potentially important tool for preserving valuable genetic material from males that die unexpectedly. The purpose of this study was to evaluate plasma membrane integrity, acrosomal status and in vitro fertilizing capability of epididymal cat sperm after short- and long-term storage at 4°C and after cryopreservation. Spermatozoa were collected by flushing the cauda epididymis and the vasa deferentia removed from adult cats undergoing orchiectomy. Spermatozoa were split into three aliquots: A-fresh, B-frozen, and C-cooled. The A portion was immediately tested on the day of collection (control), the B portion was frozen in pellets in Test Yolk Buffer (TYB) with 8% glycerol, while the C portion was extended in TYB and stored at 4°C for 1 day, 2 days or 7 days. Cat oocytes recovered from minced ovaries were matured for 24h in TCM 199+1UI/mL hCG+0.5UI/mL FSH+0.3% BSA at 38.5°C in 5% CO2. In vitro fertilization (IVF) of in vitro-matured (IVM) oocytes was performed using spermatozoa from portions A, B or C in modified Tyrode’s solution supplemented with 0.6% BSA at 38.5°C in 5% in air. Before IVF, aliquots of sperm samples from the three portions were stained simultaneously with fluorescein isothiocyanate-labelled Pisum Sativum agglutinin (FITC-PSA) and propidium iodide to evaluate the percentage of plasma membrane-intact spermatozoa with acrosomes present. At least 200 spermatozoa were counted in duplicate for each sample. After 24h of incubation, to evaluate fertilization rate, the oocytes were stained with aceto-lacmoid. Oocytes with two pronuclei, presumably male and female, were classified as fertilized. The percentages of spermatozoa maintaining plasma membrane integrity and intact acrosomes after 1 day or 2 days of storage at 4°C were 75% and 65%, respectively, and were similar to that of fresh epididymal spermatozoa (78%). However, the percentages of spermatozoa with intact plasma membranes that had intact acrosomes after 7 days of storage at 4°C (52%) or after cryopreservation (48%) were significantly lower (P<0.01, ANOVA) than those of samples stored for 1 or 2 days at 4°C. As shown in the Table, fertilization rates of oocytes inseminated with fresh spermatozoa and spermatozoa stored for 1 or 2 days were similar. In contrast, the fertilization rates of oocytes inseminated with spermatozoa that had been cryopreserved or stored for 7 days were significantly lower than those obtained with spermatozoa used immediately after collection or storage for 1 day. From our results, we suggest that storage of epididymal spermatozoa at 4°C may be a potentially useful method for temporary storage of spermatozoa from endangered cats, and may allow more efficient use and long-distance transport of genetically important germplasm. This work was supported by MIUR (ex 40%). Table 1 In vitro fertilization of in vitro-matured cat oocytes with fresh, stored at 4°C and cryopreserved epididymal cat spermatozoa
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Malcolm, V., M. Marfil, M. Calvi, F. Rigali, M. Pugliese, J. Gutierrez, M. Panarace, and M. Medina. "365 COMPARISON OF IN VITRO FERTILIZING CAPACITY OF FROZEN - THAWED SEX-SORTED AND SEX-SORTED FROZEN - THAWED BULL SPERMATOZOA." Reproduction, Fertility and Development 19, no. 1 (2007): 298. http://dx.doi.org/10.1071/rdv19n1ab365.
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Sperm sexing has become a world-wide technology, now available in many countries. The method has been incorporated into many reproductive technologies such as embryo production (Zhang et al. 2003 Theriogenology 60, 1657–1663), but sex-sorting is limited when bulls are located far from sorters or when only frozen semen is available. Previous studies on sexing frozen–thawed spermatozoa have been done in rams, which resulted in retention of the spermatozoan functional capacities (Hollinshead et al. 2004 Reproduction 127, 557–568). In vitro characteristics were assessed in bulls after sexing of thawed sperm (Hollinshead et al. 2004 Theriogenology 62, 958–968); however, the fertilizing capacity of frozen–thawed sex-sorted (FTSS) spermatozoa was not tested. The aim of the present study was to compare cleavage and embryo development rate among frozen–thawed (FT), sex-sorted frozen–thawed (SSFT), and FTSS bull spermatozoa. For FT, sperm were diluted to a final concentration of 60 × 106 sperm/mL, packaged in 0.5-mL straws, and frozen. In SSFT, spermatozoa were sex-sorted by flow cytometry following Schenk protocols (1999 Theriogenology 52, 1375–1391). Three × 106 spermatozoa were packaged into 0.25-mL straws and frozen. The final treatment (FTSS) consisted of thawing 6 to 10 frozen straws of 4 different bulls containing an average of 25 × 106 spermatozoa and centrifuging at 600g for 15 min at 21°C to extract cryodiluent. Spermatozoa were diluted and stained with Hoechst 33342 (stain concentration of 112.5 µM, the same used for SSFT treatment) following Schenk sexed-semen protocols (1999), sex-sorted by a flow cytometer, and collected in Tris-base extender containing 20% egg yolk. For each ejaculate frozen–thawed, SSFT and FTSS spermatozoa were prepared for oocyte in vitro fertilization. Also, semen from a bull routinely used as a control in the laboratory was added for a better comparison of results. Oocytes from a slaughterhouse were processed following standard in vitro fertilization procedures (Ferré 2002 Theriogenology 57, 664) 4 times for each bull, and comparison was made between treatments. Results were analyzed by ANOVA. No significant differences were observed among bulls (data not shown) (P > 0.05). Although embryo development rate was statistically different between sexed and non-sexed groups (P < 0.05), results showed that frozen–thawed bull spermatozoa can be sex-sorted and used for in vitro fertilization with comparable developmental rates comparable to those when frozen sexed semen is used (Table 1). This opens a new commercial window for cases where pre-selected sexed embryos from bulls that are not in AI centers are desired, also giving an opportunity for dead bulls. Nevertheless, since a large number of straws are necessary, further studies must be carried out to make this procedure more efficient and economically profitable. Table 1. Results of cleavage and embryo development rates between spermatozoa treatments
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Ganeco, Luciana Nakaghi, Irene Bastos Franceschini-Vicentini, and Laura Satiko Okada Nakaghi. "Structural analysis of fertilization in the fish Brycon orbignyanus." Zygote 17, no. 2 (May 2009): 93–99. http://dx.doi.org/10.1017/s0967199408005030.
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SummaryIn the present work, we analyzed the structure of oocytes and fertilized eggs of the piracanjuba fish (Brycon orbignyanus) under light and scanning electron microscopy. After inducing spawning, samples were collected at the moment of oocyte extrusion, when oocytes and semen were mixed (time 0), as well as at 10, 20 and 30 s after mixing, every minute up to 10 min, and then at 15 and 20 min. The oocytes are spherical, translucent and greenish with a mean diameter of 1.3 ± 0.11 mm. During the extrusion, cytoplasmic movement was observed in eggs towards the micropyle, characterizing the animal pole. At the moment of fertilization, the cortical cytoplasm showed a higher concentration of cortical alveoli at the animal pole than at the vegetal pole. The cortical alveoli breakdown promoted the elevation of the chorion with a consequent increase in egg diameter (1.95 ± 0.08 mm). The penetration of the spermatozoon promotes the formation of a fertilization cone of spherical external structure, which obstructs the opening of the micropyle. This structure acts as a main mechanism to avoid polyspermy, intercepting the access of supernumerary spermatozoa. Such studies about the reproductive biology of fish are important to species survival and conservation programmes.
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Payne, D. "Micro-assisted fertilization." Reproduction, Fertility and Development 7, no. 4 (1995): 831. http://dx.doi.org/10.1071/rd9950831.
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In couples who undergo routine in vitro fertilization (IVF), 17% experience significant problems with fertilization and many others are unable to have routine IVF because the quality of their semen is too poor. Often, the only options previously available to these couples were to use sperm donated by fertile men or to remain childless. Micromanipulative assisted fertilization techniques have improved the treatment of severe male factor infertility significantly and this paper provides a brief overview of the recent methodologies. Initially, techniques such as zona drilling and partial zona dissection, in which a hole or slit is placed in the zona pellucida, demonstrated that fertilization and pregnancies could be achieved with semen of very poor quality, but successes were sporadic. Later, subzonal injection of spermatozoa provided more consistent results with many units reporting pregnancies; however, relatively low rates of fertilization (14-34%) and high rates of polyspermy remained unresolved problems. The latest technique, the injection of a single spermatozoon into the oocyte cytoplasm, although technically difficult in animal models, proved to be highly successful in the human, restoring fertilization rates to those seen in routine IVF (65%) and producing good pregnancy rates from transferred embryos. Intracytoplasmic sperm injection has become the method of choice in the treatment of severe male factor infertility and preliminary data suggest that there is no increase in congenital abnormality among babies born after the transfer of injected oocytes.
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McCue, P., J. Kelly, S. Ashworth, D. Kleemann, and S. Walker. "249 EFFECT OF REFREEZING BULL SEMEN ON IVF SUCCESS RATE." Reproduction, Fertility and Development 17, no. 2 (2005): 275. http://dx.doi.org/10.1071/rdv17n2ab249.
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Cryopreserved semen from valuable sires may be available in limited quantities in some situations. A large percentage of the spermatozoa in a thawed straw is potentially wasted since a relatively small number of spermatozoa are required for most assisted reproduction techniques. The goal of this study was to determine the effect of dilution and refreezing of bull semen on fertilization and blastocyst development rates following in vitro fertilization. The hypothesis was that frozen bull semen that was thawed, diluted, and refrozen could be used successfully for IVF. Oocytes were harvested from cow ovaries collected from a local abattoir and matured in vitro for 24 hours. Ova were subsequently assigned to one of four in vitro fertilization treatment groups. Group 1 ova (n = 158) were fertilized with bull semen frozen at a concentration of 20 × 106 spermatozoa per 0.25 mL straw. Group 2 ova (n = 157) were fertilized with semen frozen at an initial concentration of 2 × 106 spermatozoa. Group 3 ova (n = 157) were fertilized with semen that had been thawed and refrozen at a concentration of 20 × 106 spermatozoa. Group 4 ova (n = 150) were fertilized with semen that had initially been frozen at a concentration of 20 × 106 spermatozoa and then thawed, diluted to a concentration of 2 × 106 spermatozoa, and refrozen. IVF was performed in a medium volume of 100 μL using 1 × 106 spermatozoa/mL. Cleavage and blastocyst rates were determined 2 days and 7 days, respectively after IVF. Cleavage rates following IVF was highest with semen frozen at 20 × 106 spermatozoa (89.9%), intermediate with semen frozen at 2 × 106 spermatozoa or refrozen at 20 × 106 spermatozoa (71.3% and 73.9%, respectively), and lowest with semen refrozen at 2 × 106 spermatozoa (38.7%) (P < 0.05). Blastocyst development rate was not significantly different (P > 0.05) among treatment groups. This study confirmed the hypothesis that refrozen bovine semen can be used successfully for in vitro fertilization. Although the overall IVF efficiency was lower using diluted refrozen semen, multiple IVF procedures could theoretically be performed over time from one initial straw. Consequently, if a limited amount of frozen semen is available, thawing of a single straw followed by dilution, re-allocation into multiple straws, and refreezing should be considered to facilitate the more efficient use of semen in future assisted reproduction endeavors. This study was supported by the PEG Program, Colorado State University.
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Li, Qiuyan, Jian Hou, Sheng Wang, Hong Guan, Nan Zhang, Yongfu Chen, and Xiaorong An. "Viable rabbits derived from oocytes by intracytoplasmic injection of spermatozoa from an infertile male." Zygote 17, no. 2 (May 2009): 157–62. http://dx.doi.org/10.1017/s0967199408005078.
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SummaryIntracytoplasmic sperm injection (ICSI) is an assisted fertilization technique and has been widely applied in human medicine to overcome some obstacles of infertility. However, this technology has not yet been used as a mainstream technique for animal production, including the rabbit, due to its limited success. The aim of this study was to improve ICSI techniques and establish an efficient ICSI method for rabbits. Spermatozoa used for ICSI were collected from mature New Zealand white male rabbits. They were washed two to three times with HEPES-buffered TCM199 containing 10% FBS and then mixed with 10% polyvinylpyrrolidone (PVP) prior to microinjection. Oocytes were harvested from superovulated donor rabbits after 14–15 h hCG treatment and were fertilized by microinjection of a single living spermatozoon into the ooplasm of each oocyte without additional activation treatment. A total of 317 injected oocytes resulted in the high survival rate of 86.1%. Among the surviving oocytes, 273 were placed into culture dishes for in vitro development. The fertilization, cleavage and blastocyst rates were 59.0%, 88.2% and 45.3% respectively. Furthermore, ICSI embryos were produced with spermatozoa from an infertile male rabbit, and 21 early-stage embryos (2-cell and 4-cell) were surgically transferred into the oviducts of two adult female rabbits. On day 31 after transfer, one out of the two recipients gave birth to two normal and healthy young rabbits. These results demonstrate that rabbit oocytes can be successfully activated and fertilized by the new ICSI protocol. Spermatozoa derived from infertile rabbits can successful fertilize oocytes and produce offspring by the simple ICSI technique.
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Sutovsky, Peter. "Sperm proteasome and fertilization." REPRODUCTION 142, no. 1 (July 2011): 1–14. http://dx.doi.org/10.1530/rep-11-0041.
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The omnipresent ubiquitin–proteasome system (UPS) is an ATP-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. The current knowledge of UPS involvement in the process of sperm penetration through vitelline coat (VC) during human and animal fertilization is reviewed in this study, with attention also being given to sperm capacitation and acrosome reaction/exocytosis. In ascidians, spermatozoa release ubiquitin-activating and conjugating enzymes, proteasomes, and unconjugated ubiquitin to first ubiquitinate and then degrade the sperm receptor on the VC; in echinoderms and mammals, the VC (zona pellucida/ZP in mammals) is ubiquitinated during oogenesis and the sperm receptor degraded during fertilization. Various proteasomal subunits and associated enzymes have been detected in spermatozoa and localized to sperm acrosome and other sperm structures. By using specific fluorometric substrates, proteasome-specific proteolytic and deubiquitinating activities can be measured in live, intact spermatozoa and in sperm protein extracts. The requirement of proteasomal proteolysis during fertilization has been documented by the application of various proteasome-specific inhibitors and antibodies. A similar effect was achieved by depletion of sperm-surface ATP. Degradation of VC/ZP-associated sperm receptor proteins by sperm-borne proteasomes has been demonstrated in ascidians and sea urchins. On the applied side, polyspermy has been ameliorated by modulating sperm-associated deubiquitinating enzymes. Diagnostic and therapeutic applications could emerge in human reproductive medicine. Altogether, the studies on sperm proteasome indicate that animal fertilization is controlled in part by a unique, gamete associated, extracellular UPS.
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Hsu, Meng-Chieh, Jyun-Yuan Wang, Yue-Jia Lee, De-Shien Jong, Kuan-Hao Tsui, and Chih-Hsien Chiu. "Kisspeptin modulates fertilization capacity of mouse spermatozoa." REPRODUCTION 147, no. 6 (June 2014): 835–45. http://dx.doi.org/10.1530/rep-13-0368.
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Kisspeptin acts as an upstream regulator of the hypothalamus–pituitary–gonad axis, which is one of the main regulatory systems for mammalian reproduction.Kiss1and its receptorKiss1r(also known as G protein-coupled receptor 54 (Gpr54)) are expressed in various organs, but their functions are not well understood. The purpose of this study was to investigate the expression profiles and functions of kisspeptin and KISS1R in the reproductive tissues of imprinting control region mice. To identify the expression pattern and location of kisspeptin and KISS1R in gonads, testes and ovarian tissues were examined by immunohistochemical or immunofluorescent staining. Kisspeptin and KISS1R were expressed primarily in Leydig cells and seminiferous tubules respectively. KISS1R was specifically localized in the acrosomal region of spermatids and mature spermatozoa. Kisspeptin, but not KISS1R, was expressed in the cumulus–oocyte complex and oviductal epithelium of ovarian and oviductal tissues. The sperm intracellular calcium concentrations significantly increased in response to treatment with kisspeptin 10 in Fluo-4-loaded sperm. The IVF rates decreased after treatment of sperm with the kisspeptin antagonist peptide 234. These results suggest that kisspeptin and KISS1R might be involved in the fertilization process in the female reproductive tract. In summary, this study indicates that kisspeptin and KISS1R are expressed in female and male gametes, respectively, and in mouse reproductive tissues. These data strongly suggest that the kisspeptin system could regulate mammalian fertilization and reproduction.
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Anzalone, D., M. Czernik, L. Palazzese, Y. Ressaissi, P. Scapolo, and P. Loi. "119 Short spermatozoa-oocyte co-incubation improves outcomes of IVF in sheep." Reproduction, Fertility and Development 32, no. 2 (2020): 186. http://dx.doi.org/10.1071/rdv32n2ab119.
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The assisted reproductive technique IVF is routinely applied in humans and large animals, both to boost reproductive performance and also for basic research. Despite its value, IVF has seen very little progress in the last two decades and relies on established paradigms, such as overnight sperm-egg co-incubation. However, the long exposure of oocytes to spermatozoa in a dish increases the risk of polyspermy and could be detrimental for early stages of embryonic development. We identified a time window within which fertilization occurs, in order to reduce the length of sperm-egg co-incubation and optimize the procedure, comparing polyspermy rate and embryo development after short (shIVF) and overnight (o/nIVF) spermatozoa-oocyte co-incubation. A total of 666 invitro-matured sheep oocytes were co-incubated with spermatozoa in IVF medium (synthetic oviductal fluid (SOF) with 20% oestrus sheep serum and 16 µM isoproterenol). First, small batches of oocytes were collected every 30min to check for the presence of a fertilizing spermatozoon. To assess this, cumulus cells were removed and presumptive fertilized oocytes were fixed and stained with propidium iodide for nuclei and Pisum sativum agglutinin for zona pellucida (ZP) detection, respectively. Then, pronuclear formation (PN) and embryo development were evaluated after 16h (PN), 24h (2 cells), and 7 days of culture (blastocyst). The oocytes that were not cleaved at 24h were stained for DNA content with Hoechst 33342. Furthermore, we evaluated embryo quality by counting cells of 8-day blastocysts after differential staining of inner cell mass (ICM) and trophectoderm (TE). We found that spermatozoa reach the ZP no earlier than 90min from the beginning of co-incubation and achieve fertilization within 4h. Polyspermic fertilization (>2PN) was lower in shIVF (6.5%) than in o/nIVF (17.8%; P=0.006). This proportion of polyspermy was maintained between groups in noncleaved oocytes at 24h from fertilization. Likewise, cleavage and blastocyst rate were higher in shIVF compared with the o/n-IVF group (2-cells: 48.3% vs. 31.6%, P=0.001; blastocyst: 29.4% vs. 20.5%, P=0.046, respectively). Differential staining of blastocysts revealed no significant difference in cell number between the blastocysts of the two groups. This work demonstrates that 4h of sperm-egg interaction are sufficient to achieve fertilization, reduce polyspermy, and improve the rate of embryos reaching blastocyst stage without compromising embryo quality.
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Kashiwazaki, N., Y. Seita, M. Shino, S. Hisamatsu, and T. Inomata. "279 LIVE RAT OFFSPRING FROM CRYOPRESERVED EJACULATED SPERMATOZOA THROUGH IN VITRO FERTILIZATION." Reproduction, Fertility and Development 18, no. 2 (2006): 247. http://dx.doi.org/10.1071/rdv18n2ab279.
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We have previously reported successful cryopreservation of epididymal rat spermatozoa (Nakatsukasa et al. 2001 Reproduction 122, 463). However, the procedure for cryopreservation of rat spermatozoa has a disadvantage; a male has to be euthanized for collection of spermatozoa from its epididymides. Obtaining ejaculated spermatozoa repeatedly from the same male could be useful for cryopreservation of invaluable spermatozoa which carry mutations including transgenes. The objective of the present study was to develop a reliable system for cryopreservation of ejaculated rat spermatozoa and efficient production of offspring from the cryopreserved spermatozoa. Matured Wistar females were mated with three males of the same strain, and killed by cervical dislocation after formation of vaginal plugs. The uteri of mated females were excised and flushed with freezing medium containing 23.0% egg yolk, 8.0% lactose, and 0.7% Equex STM to recover ejaculated spermatozoa. The semen samples were loaded into 0.25-mL straws. The straws were cooled to 5.0�C at 0.5�C/min in a programmable freezer and then exposed to liquid nitrogen (LN) vapor at 4 cm (-150�C) above the LN level for 15 min. The straws were then plunged into LN and stored for at least a week. The straws were thawed in a 37.0�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL into a 200-�L droplet of R1ECM and then pre-incubated for 5 h. Ovulated oocytes collected from superovulated females were introduced into the droplet and co-cultured for 10 h for in vitro fertilization (IVF). The oocytes were denuded and examined for the presence of two pronuclei (2PN) microscopically. The denuded oocytes with 2PN were transferred into the oviducts of pseudo-pregnant females. The rates of sperm motility at recovery, post-thaw, and the initiation of IVF (after pre-incubation) were 57 � 6%, 24 � 5%, and 18 � 3%, respectively. After co-culture, 46 (14%) of the total 329 co-cultured oocytes were confirmed to contain 2PN. A total of the 44 putative zygotes were transferred to five recipients, and a total of 21 live young (48%) were born from all of the transferred recipients. We were able to produce zygotes and offspring derived from cryopreserved ejaculated spermatozoa of all three males used in the present study. In conclusion, the cryopreservation system for ejaculated rat spermatozoa used in the present study is a workable protocol for banking of valuable genetic resources of laboratory rats. Further studies on the IVF procedure with cryopreserved ejaculated spermatozoa in the rat are needed to improve the fertilization rate.
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Mei, Si, Panyu Chen, Cheuk-Lun Lee, Weie Zhao, Ying Wang, Kevin K. W. Lam, Pak-Chung Ho, William S. B. Yeung, Cong Fang, and Philip C. N. Chiu. "The role of galectin-3 in spermatozoa-zona pellucida binding and its association with fertilization in vitro." Molecular Human Reproduction 25, no. 8 (June 13, 2019): 458–70. http://dx.doi.org/10.1093/molehr/gaz030.
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AbstractHuman spermatozoa can fertilize an oocyte only after post-testicular maturation and capacitation. These processes involve dynamic modification and reorganization of the sperm plasma membrane, which allow them to bind to the zona pellucida (ZP) of the oocyte. Defective sperm-ZP binding is one of the major causes of male subfertility. Galectin-3 is a secretory lectin in human seminal plasma well known for its action on cell adhesion. The aim of this study was to determine the role of galectin-3 in spermatozoa-ZP interaction and its association with fertilization rate in clinical assisted reproduction. Our studies revealed that the acrosomal region of ejaculated and capacitated spermatozoa possess strong galectin-3 immunoreactivity, which is much stronger than that of epididymal spermatozoa. Expression of galectin-3 can also be detected on seminal plasma-derived extracellular vesicles (EVs) and can be transferred to the sperm surface. Blocking of sperm surface galectin-3 function by antibody or carbohydrate substrate reduced the ZP-binding capacity of spermatozoa. Purified galectin-3 is capable of binding to ZP, indicating that galectin-3 may serve as a cross-linking bridge between ZP glycans and sperm surface glycoproteins. Galectin-3 levels in seminal plasma-derived EVs were positively associated with fertilization rates. These results suggest that galectin-3 in EVs is transferred to the sperm surface during post-testicular maturation and plays a crucial role in spermatozoa-ZP binding after capacitation. Reduced galectin-3 expression in seminal plasma-derived EVs may be a cause behind a low fertilization rate. Further studies with more clinical samples are required to confirm the relationship between galectin-3 levels and IVF outcomes.
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Aoki, Yusuke, Akira Tsujimura, Kazuhiro Kaseda, Masaru Okabe, Keizo Tokuhiro, Tomoe Ohta, Moira K. O'Bryan, et al. "Haprin ‐deficient spermatozoa are incapable of in vitro fertilization." Molecular Reproduction and Development 87, no. 5 (April 20, 2020): 534–41. http://dx.doi.org/10.1002/mrd.23344.
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Islam, M. Sadiqul, O. Kawase, S. Hase, M. Hoshi, and M. Matsumoto. "PKA activation in concert with ARIS and asterosap induces the acrosome reaction in starfish." Zygote 14, no. 4 (November 2006): 329–40. http://dx.doi.org/10.1017/s0967199406003881.
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SummaryThe acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.
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Herrero, MB, JM Viggiano, Martinez S. Perez, and Gimeno MF de. "Effects of nitric oxide synthase inhibitors on the outcome of in vitro fertilization in the mouse." Reproduction, Fertility and Development 8, no. 2 (1996): 301. http://dx.doi.org/10.1071/rd9960301.
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The effect of three nitric oxide (NO) synthase inhibitors, L-NG-nitro-arginine (NO2Arg), NG-Nitro-L-arginine methyl ester (L-NAME) and aminoguanidine, on in vitro fertilization in the mouse was examined. Mouse epididymal spermatozoa were capacitated in a medium with or without NO synthase inhibitors. Oocytes were inseminated and the percentage of oocytes with two pronuclei was scored after an 8-h incubation. NO2Arg and L-NAME, but not aminoguanidine, inhibited fertilization. L-NAME inhibited fertilization in a dose-dependent manner, and its effects were stereospecific. The inhibitory effect was neutralized by L-arginine but not by D-arginine. Moreover, D-NAME did not inhibit fertilization. The results suggest that NO synthase activity (presumably of the constitutive type is necessary for spermatozoa to display their full fertilizing ability.
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Sano, H., K. Matsuura, K. Naruse, and H. Funahashi. "215 APPLICATION OFA MICROFLUIDIC SPERM SORTER TO THE IN VITRO FERTILIZATION OF PORCINE OOCYTES." Reproduction, Fertility and Development 20, no. 1 (2008): 187. http://dx.doi.org/10.1071/rdv20n1ab215.
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In porcine IVF, polyspermy is a persistent obstacle to the efficient production of normal embryos in vitro. A microfluidic sperm sorter (MFSS; Strex Inc., Osaka, Japan) has been developed to isolate motile human spermatozoa (from diluted semen by 2 laminar flows in the microchannel). The motile spermatozoa can gradually accumulate in a chamber of the MFSS. In addition, we have succeeded in reducing the incidence of polyspermic penetration by a transient coincubation of oocytes with spermatozoa in the presence of caffeine, followed by culture of the oocytes in the absence of caffeine (2004 Reproduction 128, 789–800). If porcine oocytes could be cocultured with motile spermatozoa in the chamber of the MFSS, the gradual accumulation of motile spermatozoa might further improve the efficiency of production of monospermic fertilized porcine embryos. The current study was undertaken to apply the MFSS technology to porcine IVF. The sperm-rich fraction from 3 Berkshire boars was diluted at 1 � 108 cells mL–1 with modified Modena containing 20% seminal fluid, cooled to 15�C for 4 h, and kept at the same temperature until use within 2 days. Stored, diluted semen with greater than 80% viability was flowed with Tyrode lactate-HEPES-polyvinyl alcohol medium for 5 or 10 min at room temperature. The concentration and viability of spermatozoa mixed in the flowed medium were determined. In the next experiment, a diluted semen sample was flowed with modified TCM-199 containing 5 mm caffeine for 5 min at room temperature. Before flowing, porcine IVM oocytes were put into the chamber of the MFSS, where motile spermatozoa will accumulate with modified TCM-199 containing 5 mm caffeine. After flowing for 5 min, those oocytes were transferred into a 500-µL droplet of caffeine-free modified TCM-199, and culture was continued for 8 h. Statistical analyses were carried out by ANOVA and Fisher's PLSD post hoc test. After flowing for 5 min, the concentration of spermatozoa recollected from the chamber was 5.7 � 105 cells mL–1. The viability of recollected spermatozoa was significantly higher than the original flowed semen. However, when spermatozoa were flowed for 10 min, the viability of recollected spermatozoa was not different from the original flowed semen, whereas the concentration of recollected spermatozoa was 9.7 � 105 cells mL–1. When IVM oocytes were cocultured with spermatozoa gradually accumulated in the chamber of MFSS for 5 min, 35% (22 to 48%) of oocytes were penetrated, and 95% (90 to 100%) of the penetrated oocytes were monospermic. These observations demonstrate that the MFSS can separate penetrable boar spermatozoa with a high viability and sufficient concentration for IVF. Furthermore, the current data suggest the possibility of improving the efficiency of monospermic fertilization of porcine IVM oocytes by a transient coculture with MFSS.
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Marques, V. Alonso, L. R. Goulart, and A. E. D. Feliciano Silva. "Variations of protein profiles and calcium and phospholipase A2 concentrations in thawed bovine semen and their relation to acrosome reaction." Genetics and Molecular Biology 23, no. 4 (December 2000): 825–29. http://dx.doi.org/10.1590/s1415-47572000000400020.
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Just as calcium plays an integral role in acrosome capacitation and reaction, several spermatozoon proteins have been reported as binding to the ovum at fertilization. We examined the relationship between thawed bovine semen protein profiles, seminal plasma calcium ion concentration, spermatozoon phospholipase A2 (PLA2) activity and acrosome reaction. Electrophoretic profile analysis of spermatozoa and bovine seminal plasma proteins (total and membrane) revealed qualitative and quantitative differences among bulls. Variations in PLA2 and seminal plasma calcium concentration indicated genetic diversity among individuals. A 15.7-kDa membrane protein was significantly correlated (r = 0.71) with acrosome reaction, which in turn has been associated with in vivo fertility.
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Morozumi, Kazuto, Hiroyuki Tateno, Kaoru Yanagida, Haruo Katayose, Yujiroh Kamiguchi, and Akira Sato. "Chromosomal analysis of mouse spermatozoa following physical and chemical treatments that are effective in inactivating HIV." Zygote 12, no. 4 (November 2004): 339–44. http://dx.doi.org/10.1017/s0967199404002989.
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Human immunodeficiency virus (HIV) can be inactivated by heating at 56 °C for 30 min, treating with 50% ethanol at room temperature for 10 min, or treating with 2% sodium hypochlorite solution (NaClO) at room temperature for 60 min. Using a mouse model, we evaluated the risk of generating chromosome damage in spermatozoa following these treatments. The spermatozoa were all dead after the treatments. Although 41.3% of oocytes injected with ethanol-treated spermatozoa successfully activated, none of the oocytes injected with heated or NaClO-treated spermatozoa activated. When artificial stimulation with strontium was used, the fertilization of oocytes with heated or ethanol-treated spermatozoa was completely rescued. Sperm nuclei treated with NaClO neither decondensed nor developed to a male pronucleus. The incidences of structural chromosome aberrations in 1-cell zygotes derived from the heated spermatozoa (45.6%) and ethanol-treated spermatozoa (91.2%) were significantly higher than those in the matched controls (5.5% and 10.5%, respectively). Further study is needed to develop a methodology for the protection of spermatozoa against chromosome damage or the separation of damaged spermatozoa before intracytoplasmic sperm injection.
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Osaki, Shigeru, Kenji Matsumura, Ken Yamamoto, Takashi Miyano, Masashi Miyake, and Seishiro Kato. "Fertilization of bovine oocytes grown in vitro." Reproduction, Fertility and Development 9, no. 8 (1997): 781. http://dx.doi.org/10.1071/r97044.
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Early antral follicles 0·5–0·7 mm in diameter were dissected from bovine ovaries, and oocyte–cumulus complexes with pieces of parietal granulosa (OCCGs) were then collected from the follicles. The OCCGs containing oocytes of 90–99 m diameter (94·7 ± 2·8 µm, n = 196) were selected and embedded in collagen gels and cultured for 14 days in TCM199 containing 10% fetal calf serum and 4 mM hypoxanthine. From cultured OCCGs, 144 surviving oocytes were recovered, of which 53 were granulosa cell-enclosed oocytes and 91 were denuded oocytes. The mean diameter of the surviving oocytes was 114·2 ± 8·4 µm, significantly larger than that measured before culture (P < 0·05). The granulosa cell-enclosed oocytes and denuded oocytes were further cultured for maturation for 24 h. After culture, 72% (38/53) of the granulosa cell-enclosed oocytes and 59% (54/91) of the denuded oocytes showed normal morphology. These oocytes were then inseminated with bovine spermatozoa. After 29 h of insemination, all of the denuded oocytes had degenerated, while 32% (12/38) of the granulosa cell-enclosed oocytes showed normal morphology. Of the 12 oocytes, 5 were penetrated by spermatozoa, and 2 formed both male and female pronuclei. These results demonstrate for the first time that bovine oocytes grown in vitro acquire meiotic competence and can be penetrated by spermatozoa.
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Glogowski, J., I. Babiak, K. Goryczko, and S. Dobosz. "Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm." Reproduction, Fertility and Development 8, no. 8 (1996): 1179. http://dx.doi.org/10.1071/rd9961179.
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Milt of brown, rainbow and brook trout was cryopreserved. Activity of aspartate aminotransferase (AspAT) and acid phosphatase was assayed both in supernatants and in spermatozoa obtained from thawed sperm samples; additionally, post-thaw motility was evaluated. Enzyme activities differed according to fish species and were strongly affected by the type of cryoprotectant used. The activity in supernatants was usually higher than that in spermatozoa because of protein leakage from injured cells. AspAT activity in cryopreserved spermatozoa correlated positively with fertilization success in all three species. There was a negative correlation between activity of extracellular (supernatant) AspAT and fertilization rates in variants with dimethyl sulfoxide and dimethylacetamide-based extenders. The motility of thawed sperm, determined microscopically, provided some information on the cryopreservation efficiency of trout milt.
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Kuroda, K., M. Fukushima, M. Miyake, and H. Harayama. "303 FERTILIZATION-RELATED PARAMETERS OF FROZEN - THAWED SPERMATOZOA FROM SUBFERTILE JAPANESE BLACK CATTLE." Reproduction, Fertility and Development 19, no. 1 (2007): 266. http://dx.doi.org/10.1071/rdv19n1ab303.
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The subfertility derived from male factors is a problem of concern in domestic animals, because it could cause a disintegration of the breeding system and large economic losses, particularly when the subfertility affects genetically superior male animals. Therefore, it is urgent that causal factors of male subfertility be determined. Recently, an increasing number of subfertile bulls have been found among Japanese Black cattle, which is a representative breed of Japanese beef cattle. The purpose of the present study was to elucidate causal factors of male subfertility in Japanese Black cattle. Frozen–thawed spermatozoa from 8 subfertile (S1-S8) and 7 fertile (F1–F7, control) bulls were used for the assessment of fertilization-related parameters. The data obtained from each subfertile bull in the following experiments were individually compared with the mean values of the fertile bull group. In Experiment 1, sperm motility was observed in samples that were frozen-thawed and subsequently washed in PBS. Many spermatozoa (higher than 65%) exhibited flagellar movement in all samples from fertile and subfertile bulls. However, the percentages of progressively motile spermatozoa from 2 subfertile bulls were significantly lower (S2: 6%; S7: 7%; P < 0.05, ANOVA and Tukey's multiple range tests) than those from fertile bulls (average: 37%). Moreover, rapidly progressive movement was not observed in the spermatozoa from 4 subfertile bulls (S1, S2, S6, and S7). These data suggest abnormality in the motility system of sperm flagella in these 4 subfertile bulls. In Experiment 2, the capacitation–acrosome reaction state of frozen–thawed spermatozoa was examined by the CTC-staining assay. More than 50% of the frozen–thawed spermatozoa from 4 subfertile bulls (S5–S8) were prematurely progressing in the capacitation state immediately after washing and resuspension in the medium lacking CaCl2. Moreover, the addition of CaCl2 to the medium induced acrosomal loss in these sperm samples (percentages of spermatozoa without the acrosome: 36–49%). These findings indicate the occurrence of premature capacitation and a spontaneous acrosome reaction in spermatozoa from these 4 subfertile bulls. In Experiment 3, the in vitro fertilizing ability of frozen–thawed spermatozoa was evaluated by the IVF test. The percentages of fertilized eggs with both male and female pronuclei or developmental rates of fertilized eggs to the 2-cell or 4-cell stage were significantly lower in the spermatozoa from S6 to S8 bulls than in those from fertile bulls (P < 0.05, chi-squared tests). This may suggest that spermatozoa from these 3 subfertile bulls hardly accomplish the normal fertilization process. In summary, low progressive motility and low in vitro fertilizing ability because of premature capacitation were found in the spermatozoa from subfertile bulls. It is therefore possible that these are causal factors for the subfertility of male Japanese Black cattle.
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Ahuja, K. K., and D. J. Gilburt. "Involvement of sperm sulphatases in early sperm-zona interactions in the hamster." Journal of Cell Science 78, no. 1 (October 1, 1985): 247–61. http://dx.doi.org/10.1242/jcs.78.1.247.
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Sulphatase preparations from Abalone entrails, the limpet Patella vulgata and ox liver, as well as artificial substrates for these enzymes, were used in the hamster in vitro fertilization system to study the possible roles of sperm sulphatases in sperm-zona pellucida interactions. p-nitrophenyl sulphate, p-nitrocatechol sulphate, ascorbate 2-sulphate, as well as D-glucopyranose and D-galactopyranose, both sulphated at the 3, 4 or 6 position but not the 2 position, inhibited fertilization in a dose-dependent manner. Sperm-egg fusion was not inhibited by the substrates used and eggs pre-treated with sulphates could readily be fertilized. Sperm motility and therefore viability was unaffected by inhibitory concentrations of substrates as determined by rhodamine 123 labelling of motile spermatozoa. Acrosomal integrity of rhodamine-labelled spermatozoa was assessed and found to be unaffected by inhibitory levels of substrates. Fertilization was inhibited by high concentrations of the two molluscan sulphatases but not by purified ox liver sulphatase. Pre-treatment of eggs with these enzymes did not prevent fertilization. Long-term exposure of hamster oocytes to N-acetylhexosaminidase or limpet sulphatase caused thinning and distension of the zona pellucida but these changes were not observed with the ox liver sulphatase. The results suggest that a glycosulphatase is probably released from hamster spermatozoa during sperm-egg adhesion and, or, penetration. If sperm-egg adhesion molecules are sulphated, the commercially available sulphatases would be unsuitable for their characterization.
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Bernecic, Naomi C., Simon P. de Graaf, Tamara Leahy, and Bart M. Gadella. "HDL mediates reverse cholesterol transport from ram spermatozoa and induces hyperactivated motility." Biology of Reproduction 104, no. 6 (March 1, 2021): 1271–81. http://dx.doi.org/10.1093/biolre/ioab035.
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Abstract Reverse cholesterol transport or cholesterol efflux is part of an extensive plasma membrane remodeling process in spermatozoa that is imperative for fertilization. For ram spermatozoa, sheep serum is well known to support in vitro fertilization (IVF), but knowledge of its explicit role is limited. Though, it is postulated to elicit cholesterol efflux owing to the presence of high-density lipoproteins (HDLs) that interact with transmembrane cholesterol transporters, such as adenosinetriphosphate (ATP)-binding cassette transporter A1 (ABCA1) and scavenger receptor class B, type I (SR-BI). In this study, we report that both sheep serum and HDLs were able to elicit cholesterol efflux alone by up to 20–40% (as measured by the boron dipyrromethene (BODIPY)-cholesterol assay). Furthermore, when the antagonists glibenclamide and valspodar were used to inhibit the function of ABCA1 and SR-BI or ABCA1 alone, respectively, cholesterol efflux was only marginally reduced (8–15%). Nevertheless, it is likely that in ram spermatozoa, a specific facilitated pathway of cholesterol efflux is involved in the interaction between cholesterol acceptors and transporters. Interestingly, exposure to HDLs also induced hyperactivated motility, another critical event required for successful fertilization. Taken together, this study details the first report of the dual action of HDLs on ram spermatozoa, providing both an insight into the intricacy of events leading up to fertilization in vivo as well as demonstrating the possible application of HDL supplementation in media for IVF.
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Giordano, Roberto, Anna Rosa Magnano, Germana Zaccagnini, Carmine Pittoggi, Nicola Moscufo, Rodolfo Lorenzini, and Corrado Spadafora. "Reverse Transcriptase Activity in Mature Spermatozoa of Mouse." Journal of Cell Biology 148, no. 6 (March 20, 2000): 1107–14. http://dx.doi.org/10.1083/jcb.148.6.1107.
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We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.
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Vázquez, J. M., E. Martínez, J. Roca, P. Coy, and L. M. Pastor. "Acrosome reaction of boar spermatozoa in homologous in vitro fertilization." Molecular Reproduction and Development 36, no. 1 (September 1993): 84–88. http://dx.doi.org/10.1002/mrd.1080360112.
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Shalgi, R., A. Matityahu, S. J. Gaunt, and R. Jones. "Antigens on rat spermatozoa with a potential role in fertilization." Molecular Reproduction and Development 25, no. 3 (March 1990): 286–96. http://dx.doi.org/10.1002/mrd.1080250311.
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Mate, KE, and JC Rodger. "Capacitation and the acrosome reaction in marsupial spermatozoa." Reproduction, Fertility and Development 8, no. 4 (1996): 595. http://dx.doi.org/10.1071/rd9960595.
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Although yet to be established definitively, it appears that marsupial spermatozoa require a process of capacitation and that the mechanisms involved may be quite different between the Australian and American species. For Australian species, failure to induce this functional event in culture has meant that in vitro fertilization (IVF) is yet to be achieved. However, in the American species with paired spermatozoa, IVF and subsequent embryo development have been obtained under quite simple culture conditions. Our understanding of the interactions of marsupial spermatozoa with the female tract, and in particular the oviduct, the most likely site of capacitation, is discussed. Although the acrosome reaction (AR) is an equally critical event in marsupial fertilization it appears to be regulated quite differently. The uniquely stable character of the marsupial acrosome is examined as well as our current understanding of the regulation of the marsupial sperm AR in vivo and in vitro.
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Qu, Fei, Xiaoqian Ying, Wei Guo, Qiangsu Guo, Guowu Chen, Yue Liu, and Zhide Ding. "The role of Zn-α2 glycoprotein in sperm motility is mediated by changes in cyclic AMP." Reproduction 134, no. 4 (October 2007): 569–76. http://dx.doi.org/10.1530/rep-07-0145.
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Sperm motility is essential for male reproduction or natural fertilization. The cyclic AMP (cAMP)/cAMP-dependent protein kinase A (PKA) signaling pathway is generally recognized as one of the significant signaling pathways in the regulation of mammalian spermatozoan motility. Since Zn-α2-glycoprotein (ZAG) activity in mammalian adipose tissue is mediated via the β3-adrenoreceptor, with upregulation of the cAMP pathway, we hypothesize that ZAG may play the same role in sperm motility regulation, a new factor of regulation of sperm motility. Therefore, the gene encoding human ZAG was cloned and polyclonal antibodies were generated, and then laser scanning confocal microscopy and flow cytometry were employed to identify this protein in human spermatozoa. The results showed that ZAG protein was mostly localized on the pre-equatorial region covering the acrosome, neck, and middle piece of the flagellum of spermatozoa. Furthermore, using computer-assisted sperm analysis, we found that anti-human ZAG antibodies could significantly reduce the motility of human swim-up spermatozoa after 90- or 120-min incubation (P<0.05 andP<0.01 respectively), together with the decreasing of intracellular cAMP and PKA levels. In conclusion, these data suggest that ZAG is present in human spermatozoa and may be involved in the regulation of sperm motility via the cAMP/PKA signaling pathway.
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Kato, H., T. Koda, M. Kishimoto, T. Mitani, K. Matsumoto, K. Saeki, Y. Hosoi, and A. Iritani. "324 EFFECT OF AGING ON AMOUNTS OF DNA METHYLTRANSFERASE mRNA IN MOUSE SPERMATOZOA." Reproduction, Fertility and Development 19, no. 1 (2007): 277. http://dx.doi.org/10.1071/rdv19n1ab324.
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The spermatozoon is a specially differentiated cell designed to carry a haploid male genome into an oocyte at fertilization. It recently was reported that a matured spermatozoon contains several kinds of mRNAs and these are delivered into the oocyte at fertilization (Ostermeier et al. 2004 Nature 429, 154). The physiological role of paternally derived mRNAs is not clear; however, there is a report that the DNA methyltransferase (Dnmt) mRNA level in spermatozoa from male rats exposed to ethanol was significantly reduced (Bielawski et al. 2002 Alcohl. Clin. Res. 26, 347–351). The reduction of mRNA levels of Dnmt in spermatozoa would lead to altered epigenetic modification of the genome. Because factors such as age may affect spermatozoa mRNA levels, this study evaluated the effect of individual aging on the expression levels of Dnmts during spermatogenesis. This was accomplished by determining expression levels of Dnmts in the whole testis and in spermatozoa from young and aged mice by quantitative reverse-transcription-PCR. Seven- (young) and 68- (aged) week-old C57BL/6N male mice (n = 3/group) were sacrificed by cervical dislocation and whole testes and matured spermatozoa were collected. Total RNA was extracted and purified from each sample. In this study, 5 Dnmts (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a reference gene, were examined for expression levels in whole testis and spermatozoa using SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Shiga, Japan) and the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Real-Time PCR runs for each Dnmt and GAPDH were repeated 3 times using different RNA batches from different individuals. The GAPDH expression level was used to normalize the expression levels of each Dnmt. Data were analyzed by Student's t-test. Relative expression levels of each Dnmt in testis from aged males compared to that of young males were 0.94, 1.15, 0.91, 1.15, and 1.14 (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l, respectively). There was no difference in the expression levels of the 5 Dnmts examined between testes from aged and young males. On the other hand, the relative amounts of each Dnmt mRNA in spermatozoa from aged males compared to that of young males were 0.87, 0.01, 0.54, 1.07, and 1.75 (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l, respectively). There was a significant reduction (P < 0.05) in the amount of Dnmt1p mRNA. The reason why the amount of Dnmt1p mRNA in spermatozoa from aged male mice showed such reduction is not clear. There was no difference in the relative expression levels of Dnmt1p in testis irrespective of male age. Dnmt1p is only translated in the spermatocyte during the pachytene stage in meiosis and its physiological role is not clear. To elucidate this male, age-related reduction of the amount of Dnmt1p mRNA in spermatozoa would clarify part of physiological role of Dnmt1p. This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan.
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Heuwieser, W., X. Yang, S. Jiang, and R. H. Foote. "A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects." Molecular Reproduction and Development 33, no. 4 (December 1992): 489–91. http://dx.doi.org/10.1002/mrd.1080330416.
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Rasweiler, John J., Nilima K. Badwaik, and Kiranmayi V. Mechineni. "Selectivity in the transport of spermatozoa to oviductal reservoirs in the menstruating fruit bat, Carollia perspicillata." REPRODUCTION 140, no. 5 (November 2010): 743–57. http://dx.doi.org/10.1530/rep-10-0130.
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To better document the timing of ovulation and fertilization, female reproductive tracts were collected every 12 h from captive-bred fruit bats (Carollia perspicillata) on days 1–3 postcoitum and examined histologically. This also permitted observations on sperm transport, storage, and disposition. As the animals had previously been sexually segregated, most had been cycling and possessed menstrual uteri at the time of collection. Menstruation is periovulatory in this species. A widespread, headfirst orientation of spermatozoa to the uterine mucosa was observed in specimens apparently collected soon after insemination. Thereafter, however, this relationship was limited in most cases to the area around the entrance of each uterotubal junction (UTJ). A small number of spermatozoa also colonized the UTJs, which functioned as temporary sperm reservoirs on days 1–2. AlthoughC. perspicillatais monovular, no consistent differences were observed between the two oviducts in the pattern of sperm storage and release. Very few sperm were ever observed in the isthmus or ampulla (the site of fertilization). Menstrual debris (including fine particulate matter) and leukocytes present in the uterine cavity in most tracts did not gain access to the UTJ with the spermatozoa. Smooth muscle and abundant elastic fibers in the wall of the intramural UTJ, as well as receptors on its luminal epithelial cells, may play roles in the selective transport of spermatozoa to the fertilization site. While some spermatozoa are phagocytosed in the uterine lumen or by epithelial cells in the UTJ, the fate of most is probably expulsion into the vagina.
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Dunbar, BS, S. Avery, V. Lee, S. Prasad, D. Schwahn, E. Schwoebel, S. Skinner, and B. Wilkins. "The mammalian zona pellucida: its biochemistry, immunochemistry, molecular biology, and developmental expression." Reproduction, Fertility and Development 6, no. 3 (1994): 331. http://dx.doi.org/10.1071/rd9940331.
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Many studies of the molecular and biochemical aspects of mammalian fertilization have focused on the interaction of the spermatozoa with the zona pellucida (ZP). The zona pellucida, a unique extracellular matrix surrounding the mammalian oocyte, is formed during ovarian follicular development. Following ovulation of the mature ovum, the spermatozoa must bind to and penetrate this matrix before the fertilization process is completed and the male and female genetic information combine. Although numerous models for this interaction have been proposed, the complete process has yet to be elucidated. The precise mechanisms by which these interactions occur also vary markedly among different mammalian species, making it more difficult to establish a unified model. To a great extent, the study of the molecules involved in these interactions have been limited because small numbers of female gametes are available for these studies. The recent development of techniques to isolate large numbers of zonae pellucidae as well as advances in immunological and molecular biology techniques have permitted the detailed characterization of ZP proteins. Although there is a paucity of information on the post-translational modification and extracellular processing of these molecules which result in matrix formation, a number of properties have been elucidated allowing better correlation between the structure and function of different ZP proteins among species. This review reflects these studies in relation to protein nomenclature and the molecular complexity of ZP antigens.
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Park, Yoo-Jin, and Myung-Geol Pang. "Mitochondrial Functionality in Male Fertility: From Spermatogenesis to Fertilization." Antioxidants 10, no. 1 (January 12, 2021): 98. http://dx.doi.org/10.3390/antiox10010098.
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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.
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Gómez, M. C., J. W. Catt, L. Gillan, G. Evans, and W. M. C. Maxwell. "Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection." Reproduction, Fertility and Development 9, no. 7 (1997): 665. http://dx.doi.org/10.1071/r96122.
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This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitrofertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30°C or 39°C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30°C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.