Activation Of Mouse Oocytes Research Articles

In vitro Activation of Mouse Oocytes through Intracellular Ca2+ Regulation.

Background:Ca2+ signaling pathway is suggested to play an essential role in mediating oocyte maturation.Aims:The aim of this study was to evaluate intracellular Ca2+ of resistant immature oocytes that failed to resume meiosis following subsequent in vitro culture reach metaphase II after calcium ionophore A23187 activation.Settings and Design:This in vitro analytical experimental study was conducted at Animal Science Laboratory of Indonesian Medical Education and Research Institute (IMERI), Human Reproductive Infertility and Family Planning of IMERI, and Electrophysiology Imaging of Terpadu Laboratory, Faculty of Medicine, University of Indonesia.Methods:A total of 308 oocytes classed as resistant immature following in vitro culture were randomly allocated to control (n = 113) and treatment groups (n = 195). The oocyte activation group was exposed to A23187 solution for 15 min and then washed extensively. Maturation was evaluated by observing the first polar body extrusion 20‒24 h after A23187 exposure. Ca2+ imaging was conducted using a confocal laser scanning microscope to identify the dynamic of Ca2+ response.Statistical Analysis:SPSS 20, Chi-square, and Mann–Whitney U-test were used in this study.Results:Activation of resistant immature oocytes with A23187 significantly increased the number of oocyte maturation compared with the control group (P < 0.001). Furthermore, fluorescent intensity measurements exhibited a significant increase in the germinal vesicle stage when activated (P = 0.005), as well as the metaphase I stage, even though differences were not significant (P = 0.146).Conclusion:Artificial activation of resistant immature oocyte using chemical A23187/calcimycin was adequate to initiate meiosis progress.

Production of mouse offspring from inactivated spermatozoa using horse PLCζ mRNA

Improving artificial oocyte activation is essential for assisted reproduction or animal biotechnology that can obtain healthy offspring with a high success rate. Here, we examined whether intracytoplasmic injection of equine sperm-specific phospholipase C zeta (ePLCζ) mRNA, the PLCζ with the strongest oocyte activation potential in mammals, could improve the mouse oocyte activation rate and subsequent embryonic development using inactivated spermatozoa. mRNA of mouse PLCζ (mPLCζ) or ePLCζ were injected into mouse oocytes to determine the optimal mRNA concentration to maximize the oocyte activation rate and developmental rate of parthenogenetic embryos in vitro. Full-term development was examined using NaOH-treated inactive spermatozoa using the optimal activation method. We found that the most optimal ePLCζ mRNA concentration was 0.1 ng/µl for mouse oocyte activation, which was ten times stronger than mPLCζ mRNA. The concentration did not affect parthenogenetic embryo development in vitro. Relatively normal blastocysts were obtained with the same developmental rate (52–53% or 48–51%, respectively) when inactive spermatozoa were injected into activated oocytes using ePLCζ or mPLCζ mRNA injection. However, the birth rate after embryo transfer was slightly but significantly decreased in oocytes activated by ePLCζ mRNA (24%) compared to mPLCζ mRNA (37%) or strontium treatment (40%) activation. These results suggest that the higher activation rate does not always correlate the higher birth rate, and some mechanisms might exist in the oocyte activation process that could affect the later developmental stages like full-term development.

Synaptotagmin 1 regulates cortical granule exocytosis during mouse oocyte activation.

Synaptotagmin 1 (Syt1) is an abundant and important presynaptic vesicle protein that binds Ca2+ for the regulation of synaptic vesicle exocytosis. Our previous study reported its localization and function on spindle assembly in mouse oocyte meiotic maturation. The present study was designed to investigate the function of Syt1 during mouse oocyte activation and subsequent cortical granule exocytosis (CGE) using confocal microscopy, morpholinol-based knockdown and time-lapse live cell imaging. By employing live cell imaging, we first studied the dynamic process of CGE and calculated the time interval between [Ca2+]i rise and CGE after oocyte activation. We further showed that Syt1 was co-localized to cortical granules (CGs) at the oocyte cortex. After oocyte activation with SrCl2, the Syt1 distribution pattern was altered significantly, similar to the changes seen for the CGs. Knockdown of Syt1 inhibited [Ca2+]i oscillations, disrupted the F-actin distribution pattern and delayed the time of cortical reaction. In summary, as a synaptic vesicle protein and calcium sensor for exocytosis, Syt1 acts as an essential regulator in mouse oocyte activation events including the generation of Ca2+ signals and CGE.

The effect of autophagy after mouse oocyte activation test

The effect of autophagy after mouse oocyte activation test

Assisted oocyte activation significantly increases fertilization and pregnancy outcome in patients with low and total failed fertilization after intracytoplasmic sperm injection: a 17-year retrospective study

To investigate the extent to which assisted oocyte activation (AOA) improves clinical outcomes in patients diagnosed with oocyte activation deficiencies (OADs). Retrospective cohort study comparing AOA cycles and previous intracytoplasmic sperm injection (ICSI) cycles in couples experiencing low or total failed fertilization after ICSI. Importantly, the sperm-related oocyte-activating capacity was examined in all patients before AOA with the use of the mouse oocyte activation test (MOAT). Infertility center at a university hospital. A total of 122 couples with a history of low or total failed fertilization after ICSI. ICSI, MOAT, AOA, and embryo transfer. Fertilization, pregnancy, and live birth rates. MOAT revealed 19 patients with a sperm-related OAD (MOAT group 1), 56 patients with a diminished sperm-related oocyte-activating capacity (MOAT group 2), and 47 patients with a suspected oocyte-related OAD (MOAT group 3). AOA (191 cycles) significantly improved fertilization, pregnancy, and live birth rates in all MOAT groups compared with previous ICSI attempts (243 cycles). Fertilization rates after AOA were significantly different among MOAT groups 1 (70.1%), 2 (63.0%), and 3 (57.3%). Between MOAT group 1 and 3, significant differences in pregnancy (49.0% vs. 29.4%) and live birth (41.2% vs. 22.1%) rates were observed. In total, 225 embryo transfers resulted in 60 healthy live births following AOA. Patients undergoing diagnostic testing before AOA show a significant improvement in clinical outcomes compared with previous cycles. Our findings highlight that AOA should be reserved for patients with clear OADs.

Strontium fails to induce Ca2+ release and activation in human oocytes despite the presence of functional TRPV3 channels.

STUDY QUESTIONAre the transient receptor potential cation channels vanilloid 3 (TRPV3) present and able to mediate strontium (Sr2+) induced artificial activation in human oocytes?SUMMARY ANSWERSr2+ did not induce Ca2+ rises or provoke activation in human oocytes, however, mRNA for the TRPV3 channel was present in metaphase II (MII) human oocytes after IVM and TRPV3 agonists induced Ca2+ rises and oocyte activation, demonstrating the channels were functional.WHAT IS KNOWN ALREADYSelective activation of TRPV3 by agonists induces Ca2+ entry and promotes mouse oocyte activation, and the absence of TRPV3 channels in mouse oocytes prevents Sr2+ mediated artificial activation. Sr2+ is sometimes used to overcome fertilization failure after ICSI in the clinic, but its efficiency is still controversial and the mechanism(s) of how it mediates the Ca2+ flux has not been studied yet in human.STUDY DESIGN, SIZE, DURATIONThe protein distribution (n = 10) and mRNA expression level (n = 19) of the TRPV3 channels was investigated in human MII oocytes after IVM. The Sr2+ (10 mM) and TRPV3 agonists (200 μM 2-aminoethoxydiphenyl borate [2-APB] and 200 μM carvacrol)-induced Ca2+ response was analyzed in human (n = 15, n = 16 and n = 16, respectively) and mouse oocytes (n = 15, n = 19 and n = 26, respectively). The subsequent embryonic developmental potential following the parthenogenetic activation using these three agents was recorded in human (n = 10, n = 9 and n = 9, respectively) and mouse (n = 20 per agent) oocytes, by determining pronucleus, or 2-cell and blastocyst formation rates.PARTICIPANTS/MATERIALS, SETTING, METHODSMII oocytes from B6D2F1 mice (6–10 weeks old) as well as human IVM oocytes and IVO oocytes (from patients aged 25–38 years old) with aggregates of smooth endoplasmic reticulum clusters were used. The expression of TRPV3 channels was determined by immunofluorescence staining with confocal microscopy and RT-PCR, and the temporal evolution of intracellular Ca2+ concentration was measured by time-lapse imaging after exposure to Sr2+ and TRPV3 agonists (2-APB and carvacrol). Artificial activation efficiency was assessed using these agents.MAIN RESULTS AND THE ROLE OF CHANCESr2+ did not promote Ca2+ oscillations or provoke activation in human oocytes. Transcripts of TRPV3 channels were present in IVM MII human oocytes. TRPV3 protein was expressed and distributed throughout the ooplasm of human oocytes, rather than particularly concentrated in plasma membrane as observed in mouse MII oocytes. Both agonists of TRPV3 (2-APB and carvacrol), promoted a single Ca2+ transient and activated a comparable percentage of more than half of the exposed human oocytes (P > 0.05). The agonist 2-APB was also efficient in activating mouse oocytes, however, significantly fewer mouse oocytes responded to carvacrol than 2-APB in both the Ca2+ analysis and activation test (P < 0.001).LIMITATIONS REASONS FOR CAUTIONThe availability of fresh IVO matured oocytes in human was limited. Data from TRPV3 knockout model are not included.WIDER IMPLICATIONS OF THE FINDINGSThe benefit of clinical application using Sr2+ to overcome fertilization failure after ICSI requires further validation.STUDY FUNDING/COMPETING INTEREST(S)This study was supported by FWO-Vlaanderen, China Scholarship Council and Special Research Fund from Ghent University (Bijzonder Onderzoeksfonds, BOF). No competing interests are declared.

Human sperm handling in intracytoplasmic sperm injection processes: In vitro studies on mouse oocyte activation, embryo development competence and sperm oxidation-reduction potential.

Polyvinylpyrrolidone (PVP) and hyaluronic acid (HA) are routinely used in handling spermatozoa for intracytoplasmic sperm injection (ICSI). As there are still concerns about possible adverse effects on the embryo, this study investigated sperm handling in a mouse ICSI model to (i) evaluate oocyte activation after injection of spermatozoa selected for rotational or linear motion in PVP; (ii) assess the effect of sperm selection in PVP, HA and medium on oocyte activation; (iii) examine the effects of PVP and HA on parthenogenetic oocyte activation and embryo development; and (iv) assess the oxidation-reduction potential (ORP) of spermatozoa exposed to PVP, HA or medium. Oocyte activation was higher when spermatozoa exhibited rotational motion rather than linear motion (79% vs. 52%; p=.05). There was no difference in oocyte activation and embryo development after parthenogenetic oocyte activation after sperm injection using PVP, HA or medium-incubated spermatozoa. PVP-selected spermatozoa exhibited lower (p<.0001) ORP levels than using HA. Thus, results indicate that the sperm handling method and the type of medium used impact ICSI outcomes. Overall, sperm incubation in PVP, HA and medium yields similar outcomes with regard to oocyte activation and embryo development. However, PVP provides more antioxidative protection than HA and should therefore be preferred for sperm manipulation.

Detection of Separase Activity Using a Cleavage Sensor in Live Mouse Oocytes.

Separase proteolytically removes cohesin complexes from sister chromatid arms in meiosis I, which is essential for chromosome segregation. Regulation of separase activity is essential for proper cell cycle progression and correct chromosome segregation. Onset of endogenous separase activity has not yet been observed in live oocytes.We describe here a method for detecting separase activity in mouse oocytes in vivo. This method utilizes a previously described cleavage sensor made up of H2B-mCherry fused with Scc1(107-268aa)-YFP. The cleavage sensor is loaded on the chromosomes through its H2B-tag, and the signal from both mCherry and YFP is visible. Upon separase activation the Scc1 fragment is cleaved and YFP dissociates from the chromosomes. The change in the ratio between mCherry and YFP fluorescence intensity is a readout of separase activity.

Application of auxin-inducible degron technology to mouse oocyte activation with PLCζ.

In mammals, spermatozoa activate oocytes by triggering a series of intracellular Ca2+ oscillations with phospholipase C zeta (PLCζ), a sperm-borne oocyte-activating factor. Because the introduction of PLCζ alone can induce oocyte activation, it might be a promising reagent for assisted reproductive technologies. To test this possibility, we injected human PLCζ (hPLCζ) mRNA into mouse oocytes at different concentrations. We observed the oocyte activation and subsequent embryonic development. Efficient oocyte activation and embryonic development to the blastocyst stage was achieved only with a limited range of mRNA concentrations (0.1 ng/μl). Higher concentrations of mRNA caused developmental arrest of most embryos, suggesting that excessive PLCζ protein might be harmful at this stage. In a second series of experiments, we aimed to regulate the PLCζ protein concentration in oocytes by applying auxin-inducible degron (AID) technology that allows rapid degradation of the target protein tagged with AID induced by auxin. Injection of the hPLCζ protein tagged with AID and enhanced green fluorescent protein (hPLCζ-AID-EGFP) demonstrated that high EGFP expression levels at the late 1-cell stage were efficiently reduced by auxin treatment, suggesting efficient hPLCζ degradation by this system. Furthermore, the defective development observed with higher concentrations of hPLCζ-AID-EGFP mRNA was rescued following auxin treatment. Full-term offspring were obtained by round spermatid injection with optimized hPLCζ-AID activation. Our results indicate that this AID technology can be applied to regulate the protein levels in mouse oocytes and that our optimized PLCζ system could be used for assisted fertilization in mammals.

The role of L-type calcium channels in mouse oocyte maturation, activation and early embryonic development

Calcium ion fluctuation is closely related to the transformation of cell cycle. However, little is known about the function of L-type calcium channel in mammalian oocyte and embryo development. We thus studied the roles of L-type calcium channel in mouse oocyte meiotic maturation, parthenogenetic activation and early embryonic development. We used the antagonist Amlodipine to block L-type calcium channel. Oocytes or zygotes were cultured to different time points with 0 μM, 10 μM, 30 μM and 50 μM Amlodipine. Then we checked the rate of first polar body extrusion, spindle formation, asymmetric division parthenogenetic activation and early embryo cleavage. The results showed that Amlodipine treatment did not affect germinal vesicle breakdown, but caused disruption of cytoskeleton organization, symmetric division, formation of mature oocytes with a large polar body, or reduced the first polar body extrusion, depending on its concentrations. Amlodipine treatment also resulted in decreased parthenogenetic activation and arrested early embryonic development. Overall, these data suggest that proper function of L-type calcium channel is critical for oocyte maturation, activation, and early embryonic development.

Activation of mouse oocytes after intracytoplasmic injection of human sperm with linear and rotational motion

During the journey through the female reproductive tract, sperm exhibit a variety of motility modes and swimming patterns including linear and rotational motion. Currently, only those sperm that show rotational motion are chosen for intracytoplasmic sperm injection (ICSI). Whether this leads to better oocyte activation and embryo development competence after ICSI, however, is unknown. The objective of this study was to compare the activation of mouse oocytes after ICSI of human sperm with linear and rotational motion.

Parthenogenetic activation and developmental potential of mouse oocytes after intracytoplasmic injection (ICSI) of PVP (polyvinylpyrrolidone) and HA (hyaluronic acid)

Parthenogenetic activation and developmental potential of mouse oocytes after intracytoplasmic injection (ICSI) of PVP (polyvinylpyrrolidone) and HA (hyaluronic acid)

Oocyte activation and latent HIV-1 reactivation: AMPK as a common mechanism of action linking the beginnings of life and the potential eradication of HIV-1

In all mammalian species studied to date, the initiation of oocyte activation is orchestrated through alterations in intracellular calcium (Ca2+) signaling. Upon sperm binding to the oocyte plasma membrane, a sperm-associated phospholipase C (PLC) isoform, PLC zeta (PLCζ), is released into the oocyte cytoplasm. PLCζ hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG), which activates protein kinase C (PKC), and inositol 1,4,5-trisphosphate (IP3), which induces the release of Ca2+ from endoplasmic reticulum (ER) Ca2+ stores. Subsequent Ca2+ oscillations are generated that drive oocyte activation to completion. Ca2+ ionophores such as ionomycin have been successfully used to induce artificial human oocyte activation, facilitating fertilization during intra-cytoplasmic sperm injection (ICSI) procedures. Early studies have also demonstrated that the PKC activator phorbol 12-myristate 13-acetate (PMA) acts synergistically with Ca2+ ionophores to induce parthenogenetic activation of mouse oocytes. Interestingly, the Ca2+-induced signaling cascade characterizing sperm or chemically-induced oocyte activation, i.e. the “shock and live” approach, bears a striking resemblance to the reactivation of latently infected HIV-1 viral reservoirs via the so called “shock and kill” approach, a method currently being pursued to eradicate HIV-1 from infected individuals. PMA and ionomycin combined, used as positive controls in HIV-1 latency reversal studies, have been shown to be extremely efficient in reactivating latent HIV-1 in CD4+ memory T cells by inducing T cell activation. Similar to oocyte activation, T cell activation by PMA and ionomycin induces an increase in intracellular Ca2+ concentrations and activation of DAG, PKC, and downstream Ca2+-dependent signaling pathways necessary for proviral transcription. Interestingly, AMPK, a master regulator of cell metabolism that is activated thorough the induction of cellular stress (e.g. increase in Ca2+ concentration, reactive oxygen species generation, increase in AMP/ATP ratio) is essential for oocyte maturation, T cell activation, and mitochondrial function. In addition to the AMPK kinase LKB1, CaMKK2, a Ca2+/calmodulin-dependent kinase that also activates AMPK, is present in and activated on T cell activation and is also present in mouse oocytes and persists until the zygote and two-cell stages. It is our hypothesis that AMPK activation represents a central node linking T cell activation-induced latent HIV-1 reactivation and both physiological and artificial oocyte activation. We further propose the novel observation that various compounds that have been shown to reactivate latent HIV-1 (e.g. PMA, ionomycin, metformin, bryostatin, resveratrol, etc.) or activate oocytes (PMA, ionomycin, ethanol, puromycin, etc.) either alone or in combination likely do so via stress-induced activation of AMPK.

Rab3A, Rab27A, and Rab35 regulate different events during mouse oocyte meiotic maturation and activation.

Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions.

Effect of two assisted oocyte activation protocols used to overcome fertilization failure on the activation potential and calcium releasing pattern

To assess the effect of two assisted oocyte activation (AOA) protocols with the use of two calcium (Ca(2+)) ionophores, ionomycin and A23187 (calcimycin), on the intracellular Ca(2+) level in mouse and human oocytes and the fertilization rates. Comparison of two Ca(2+) ionophores, ionomycin and A23187, regarding their capacity to increase the intracellular Ca(2+) level and to support subsequent oocyte activation and development. University hospital research laboratory. Patients undergoing intracytoplasmic sperm injection (ICSI) treatment and B6D2F1 mice. Assisted oocyte activation and microinjection of mouse and human oocytes with sperm. Measurement of the fertilizing and Ca(2+)-releasing ability of human sperm. Ionomycin was more potent than A23187 in provoking Ca(2+) increases in both mouse and human oocytes with significantly higher amplitude and area under the receiver operating characteristic curve. The oocyte activation rate was significantly higher when mouse oocytes were activated with the use of the ionomycin- rather than the A23187-based AOA protocol. Furthermore, oocyte activation rate was higher when human in vitro matured oocytes were activated with the ionomycin-based AOA protocol, but the difference did not reach statistical significance. In both mouse and human oocytes, the AOA protocol that used ionomycin was more efficient than the one that used A23187. Bearing in mind that mammalian fertilization is successful when the total dose of Ca(2+) released reaches a minimal threshold, the use of ionomycin for human AOA might be justified instead of the use of A23187.

Maturation-associated Dbf4 expression is essential for mouse zygotic DNA replication.

Cdc7 is an S-phase-promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one-cell embryos at zygotic S-phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one-cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi-mediated knockdown of maternal Dbf4 expression prevents the maturation-associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one-cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3' untranslated region (3'UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.

Cdh1- and Cdc20-independent APC activity in mouse oocytes

Cdh1- and Cdc20-independent APC activity in mouse oocytes

MP68-11 SUCCESS RATES OF NATURAL CONCEPTION AND INTRA-UTERINE INSEMINATION IN MEN WITH SEVERELY ABNORMAL STRICT MORPHOLOGY (

INTRODUCTION AND OBJECTIVES: It has been established that increasing maternal age leads to a higher risk of infertility and miscarriage. On the other hand, the recent rise in birth rates to older fathers is of concern because of evidence that advanced paternal age might be associated with an increased risk of pregnancy and various disorders of the newborn. However there is no clearly accepted consensus on the implication of advancing male age. To examine the question of whether advanced paternal age impact fertility, we focused on oocyte activating ability of the spermatozoa and investigated age related change of this ability using mouse oocyte activation test (MOAT). METHODS: MOAT, which was first proposed in 1995 by Rybouchkin et al., was used in the present study. After obtaining institutional review board approval, the infertile sperms were obtained from male infertile patients between October 2012 and September 2013. As the control, fertile sperms were obtained from donors with proven fertility. Human sperms were injected into mouse oocytes by a piezo-driven unit and these oocytes were incubated at 37 C under 5% CO2 in air. Approximately 10 hours after injection, the oocytes were observed under an microscope and were rotated to determine whether or not a second polar body was present. An oocyte with the second polar body was recorded 0activated0. We evaluated the activated rates of each generation of male infertile patients and the controls. RESULTS: Semen samples from 77 male infertile patients and 15 control people were available for analysis. In the control group, the activated rates of the oocytes were 82.0% at <30 years, 80.0% at 30-34 years, 77.0% at 35-39 years, 70.0% at 40-44 years and 70.0% at 45-50 years, whereas in male infertile group 69.0% at <30 years, 68.8% at 30-34 years, 62.2% at 35-39 years, 54.8% at 40-44 years and 47.3% at 45-50 years. In both group, there was an age-associated decrease in the rate of activated oocytes. The decrease was more remarkable in male infertile group compared to the control group. CONCLUSIONS: Oocyte activation ability of spermatozoa appears to decrease with aging, in particular, among subfertile patients. This suggests that we need immediate assessment and appropriate intervention when handling male infertile patients.

MP68-10 OOCYTE ACTIVATING ABILITY OF THE SPERMATOZOA APPEARS TO DECREASE WITH AGING AMONG SUBFERTILE PATIENTS

You have accessJournal of UrologyInfertility: Evaluation1 Apr 2014MP68-10 OOCYTE ACTIVATING ABILITY OF THE SPERMATOZOA APPEARS TO DECREASE WITH AGING AMONG SUBFERTILE PATIENTS Takeshi Shin, Mai Fukushima, Toshiyuki Iwahata, Keisuke Suzuki, Yuko Sadaoka, Shigeyuki Ota, Masashi Iijima, Ryo Sato, Kojiro Nishio, Makoto Kawaguchi, Yoshitomo Kobori, Yoshio Ashizawa, Hiroshi Yagi, Shigehiro Soh, Gaku Arai, and Hiroshi Okada Takeshi ShinTakeshi Shin More articles by this author , Mai FukushimaMai Fukushima More articles by this author , Toshiyuki IwahataToshiyuki Iwahata More articles by this author , Keisuke SuzukiKeisuke Suzuki More articles by this author , Yuko SadaokaYuko Sadaoka More articles by this author , Shigeyuki OtaShigeyuki Ota More articles by this author , Masashi IijimaMasashi Iijima More articles by this author , Ryo SatoRyo Sato More articles by this author , Kojiro NishioKojiro Nishio More articles by this author , Makoto KawaguchiMakoto Kawaguchi More articles by this author , Yoshitomo KoboriYoshitomo Kobori More articles by this author , Yoshio AshizawaYoshio Ashizawa More articles by this author , Hiroshi YagiHiroshi Yagi More articles by this author , Shigehiro SohShigehiro Soh More articles by this author , Gaku AraiGaku Arai More articles by this author , and Hiroshi OkadaHiroshi Okada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.2189AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES It has been established that increasing maternal age leads to a higher risk of infertility and miscarriage. On the other hand, the recent rise in birth rates to older fathers is of concern because of evidence that advanced paternal age might be associated with an increased risk of pregnancy and various disorders of the newborn. However there is no clearly accepted consensus on the implication of advancing male age. To examine the question of whether advanced paternal age impact fertility, we focused on oocyte activating ability of the spermatozoa and investigated age related change of this ability using mouse oocyte activation test (MOAT). METHODS MOAT, which was first proposed in 1995 by Rybouchkin et al., was used in the present study. After obtaining institutional review board approval, the infertile sperms were obtained from male infertile patients between October 2012 and September 2013. As the control, fertile sperms were obtained from donors with proven fertility. Human sperms were injected into mouse oocytes by a piezo-driven unit and these oocytes were incubated at 37°C under 5% CO2 in air. Approximately 10 hours after injection, the oocytes were observed under an microscope and were rotated to determine whether or not a second polar body was present. An oocyte with the second polar body was recorded ′activated′. We evaluated the activated rates of each generation of male infertile patients and the controls. RESULTS Semen samples from 77 male infertile patients and 15 control people were available for analysis. In the control group, the activated rates of the oocytes were 82.0% at <30 years, 80.0% at 30-34 years, 77.0% at 35-39 years, 70.0% at 40-44 years and 70.0% at 45-50 years, whereas in male infertile group 69.0% at <30 years, 68.8% at 30-34 years, 62.2% at 35-39 years, 54.8% at 40-44 years and 47.3% at 45-50 years. In both group, there was an age-associated decrease in the rate of activated oocytes. The decrease was more remarkable in male infertile group compared to the control group. CONCLUSIONS Oocyte activation ability of spermatozoa appears to decrease with aging, in particular, among subfertile patients. This suggests that we need immediate assessment and appropriate intervention when handling male infertile patients. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e802 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Takeshi Shin More articles by this author Mai Fukushima More articles by this author Toshiyuki Iwahata More articles by this author Keisuke Suzuki More articles by this author Yuko Sadaoka More articles by this author Shigeyuki Ota More articles by this author Masashi Iijima More articles by this author Ryo Sato More articles by this author Kojiro Nishio More articles by this author Makoto Kawaguchi More articles by this author Yoshitomo Kobori More articles by this author Yoshio Ashizawa More articles by this author Hiroshi Yagi More articles by this author Shigehiro Soh More articles by this author Gaku Arai More articles by this author Hiroshi Okada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Analysis of calcium oscillations pattern triggered by human sperm showing failed or low fertilization

Failed fertilization after ICSI has been mainly attributed to an oocyte activation deficiency. In such cases, we can evaluate the activation capacity by injecting human sperm into mouse oocytes (mouse oocyte activation test, MOAT). We determined the calcium oscillations pattern after injection of these sperm cells into both mouse and donated human oocytes to elucidate the cause of failed fertilization.