Developmental Ability Of Oocytes Research Articles

P-591 Follicular fluid-derived exosomes rejuvenate ovarian aging through miR-320a-3p-mediated FOXQ1 inhibition

Abstract Study question Dose the follicular fluid-derived exosomes rejuvenate ovarian aging? Summary answer Follicular fluid-derived exosomes efficiently rescued ovarian function in aged mice, which was achieved via transfer of miR-320-3p to granulosa cells and subsequent Foxq1 inhibition. What is known already The ovary enters the malfunctional aging phase earlier and faster than other organs and systems. Ovarian aging is mainly characterized by a progressive decline in oocyte quantity and quality, which ultimately leads to female infertility. Various therapies have been established to cope with age-related ovarian dysfunction, among which exosome-based therapy has been considered a promising strategy that can benefit ovarian functions via multiple pathways. Study design, size, duration Ovarian granulosa cells of young C57BL/6J mice (6-8 w) and old C57BL/6J mice (40 w) were cultured in vitro. The follicular fluid exosomes of young mice and the same amount of PBS were respectively injected into the ovarian sac of old mice. Participants/materials, setting, methods Cell proliferation and apoptosis were detected by CCK8, flow cytometry and Western Blot. Mitochondrial function was assessed by measuring mitochondrial membrane potential, ATP levels, mtDNA copy numbers. The number of ovarian follicles were observed by HE staining. Senescence and telomere related gene expression levels were detected by RT-qPCR. 2-cell rates and blastocyst formation rates were observed after in vitro fertilization. The model mice were directly cooped up to observe the pregnat and litter sizes. Main results and the role of chance The follicular fluid exosomes from young mice can promote the proliferation of ovarian granulosa cells from old mice and improve mitochondrial function. When the follicular fluid exosomes from young mice were injected into the ovarian sac of old mice, the number of primordial and growing follicles in the ovaries of old mice were increased and the developmental ability of oocytes was improved. Limitations, reasons for caution Findings in mouse. Wider implications of the findings This study suggests that young follicular fluid exosomes may be involved in ovarian senescence by transferring miR-320a-3p targeting FOXQ1 into ovarian granulosa cells of aged mice. Trial registration number no

78 Radio electric asymmetric conveyer treatment during prolonged cold storage of ovaries enhances developmental competence of oocytes in the domestic cat model

Hypothermic storage (4°C) of ovaries for long-distance transport holds great potential to expand access to fertility preservation in animals and humans (Duncan et al. 2016 Reproduction 152, 201-210). However, storage for prolonged periods (over 24h) leads to structural/functional changes in the ovarian tissue with a critical reduction of follicular viability and oocyte quality (Piras et al. 2018 Reprod. Biol. Endocrinol. 10, 16-76; Isachenko et al. 2009 Fertil. Steril. 91, 1556-1559). The radio electric asymmetric conveyer (REAC) is a novel technology platform for neuro and bio-modulation that optimize the ion fluxes and the mechanisms driving cellular asymmetry and polarization in biological structures (Maioli et al. 2016 Sci. Rep. 6, 28682). The REAC has already proved to be effective in promoting cell differentiation and reprogramming and to counteract the biological mechanisms linked to aging and the degenerative process (Berlinguer et al. 2017 Reprod. Biol. Endocrinol. 15, 11; Maioli et al. 2014 Age 36, 9-20). In the present study, the domestic cat was used as an experimental model to evaluate the effect of REAC treatment during ovary storage at 4°C for 48h on invitro developmental ability of oocytes retrieved from antral follicles. Ovaries harvested from healthy domestic queens during ovariectomy were randomly assigned to the REAC-treated (R: n=13) and untreated (C: n=13) groups. In detail, ovaries were maintained in 4mL of phosphate-buffered saline at 4°C for 48h. The REAC device was set at 2.4 GHz, and its conveyer electrodes were immersed into the phosphate-buffered saline. After 48h, ovaries were sliced to release cumulus-oocyte complexes, which were selected according to their morphological characteristics (Johnston et al. 1991 Biol. Reprod. 45, 898-906) for IVM (R: n=130; C: n=133). Matured oocytes were fertilised (IVF) with frozen-thawed epididymal spermatozoa and presumptive zygote were invitro cultured (IVC) for 7 days. On Day 2 and Day 7 of IVC, respectively, the number of embryos cleaved and developing to the blastocyst stage was determined. The IVM, IVF, and IVC were performed according to the procedure of Piras et al. (2018 Reprod. Biol. End. 16, 76). Data were analysed by chi-square test with STATA\IC 11.0. Maturation rate of oocytes did not differ between groups (R: n=59/130, 45.4%; C: n=66/133, 49.6%). Cleavage rate was higher (P<0.05) in the R group (n=34/59, 57.6%) compared with the C group (n=25/66, 37.9%). The percentages of blastocyst formation relative to the number of cleaved embryos (R: n=12/34, 35.3%; C: n=3/25 12.0%) and to the total number of MII oocytes (R: n=12/59, 20.4%; C: n=3/66, 4.5%) increased (P<0.05) after REAC treatment compared with the untreated counterpart. In conclusion, REAC treatment during cold storage of cat ovaries for 48h positively affected the quality of oocytes as assessed by invitro embryo production outcome. The REAC technology could provide a useful approach for the optimization of ovarian tissue transport conditions for fertility preservation especially for endangered species and patients with fertility-threatening conditions.

Relationship between the number of cells surrounding oocytes and energy states of oocytes

Lipid content, ATP content, and histone acetylation are thought to reflect the energy state of cells. In addition, the energy state closely associates with growth and developmental ability of oocytes. Oocyte growth is accompanied by active proliferation of the surrounding granulosa cells (GCs), and GCs play a key role in the provision of energy substrates to the oocytes. In the present study, we first examined the relationship among the average number of GCs per follicle, the average number of cumulus cells (CCs) per oocyte, and the average lipid content in oocytes that developed in vivo within individual donor gilts. Second, we validated the relationship between the number of cells surrounding oocytes and the energy states of oocytes by using an IVC system of oocyte granulosa cell complexes (OGCs) derived from early antral follicles. We collected cumulus cells and oocyte complexes (COCs) from antral follicles (3–5 mm in diameter) and found that average lipid content in oocytes significantly correlated with the average number of both GCs/follicle and CCs/oocyte (P < 0.05). In the next series of experiments, we collected OGCs from early antral follicles (0.5–0.7 mm in diameter), and cultured them for 14 days, and then determined the cell number of OGCs, as well as the lipid content, ATP content, and acetylation level of H4K12 in oocytes grown in vitro. In addition, glucose consumption by OGCs was calculated from the sample media collected at Days 13 and 14. The lipid content of oocytes grown in vitro, significantly correlated with the number of cells surrounding the oocytes (P < 0.01) and with the level of glucose consumption by OGCs (P < 0.01). In addition, both ATP content and H4K12 acetylation levels of oocytes grown in vitro significantly correlated with the number of cells surrounding the oocytes (P < 0.05) and glucose consumption by OGCs (P < 0.05). In conclusion, the lipid content of oocytes depends on the number of cells surrounding the oocytes, and glucose uptake by OGCs is crucial for lipid content and ATP content, and H4K12 acetylation in oocytes.

Relationship between mitochondrial DNA copy number and SIRT1 expression in porcine oocytes.

The present study assessed the effect of resveratrol on the expression of SIRT1 and mitochondrial quality and quantity in porcine oocytes. Supplementing the maturation medium with 20 µM resveratrol increased the expression of SIRT1, and enhanced mitochondrial functions, as observed from the increased ATP content and mitochondrial membrane potential. Addition of resveratrol also improved the ability of oocytes to develop into the blastocyst stage following activation. The effects of resveratrol on mitochondrial number were examined by comparing the mitochondrial DNA copy number (Mt number) between group of oocytes collected from the same donor gilt ovaries. Supplementing the maturation medium with only resveratrol did not affect the Mt number in the oocytes. However, supplementing the maturation medium with 10 µM MG132, a proteasome inhibitor, significantly increased the amount of ubiquitinated proteins and Mt number by 12 and 14%, respectively. In addition, when resveratrol was added to the medium containing MG132, the Mt number increased significantly by 39%, this effect was diminished by the addition of the SIRT1 inhibitor EX527. Furthermore, supplementing the medium with MG132 and EX527 did not affect Mt number. The mean SIRT1 expression in 20 oocytes was significantly and positively correlated with the Mt number in oocytes collected from the same donor. This study suggests that the expression of SIRT1 is associated with the Mt number in oocytes. In addition, activation of SIRT1 by resveratrol enhances the biosynthesis and degradation of mitochondria in oocytes, thereby replenishing and improving mitochondrial function and the developmental ability of oocytes.

Resveratrol Improves the Mitochondrial Function and Fertilization Outcome of Bovine Oocytes

The aim of the present study was to address the effect of resveratrol-mediated upregulation of sirtuin 1 (SIRT1) during oocyte maturation on mitochondrial function, the developmental ability of oocytes and on mechanisms responsible for blockage of polyspermic fertilization. Oocytes collected from slaughterhouse-derived ovaries were cultured in TCM-199 medium supplemented with 10% FCS and 0 or 20 µM resveratrol (Res). We examined the effect of Res on SIRT1 expression in in vitro-matured oocytes (Exp 1); fertilization and developmental ability (Exp 2); mitochondrial DNA copy number (Mt number), ATP content and mitochondrial membrane potential in matured oocytes (Exp 3); and the time required for proteinase to dissolve the zona pellucida following in vitro fertilization (as a marker of zona pellucida hardening), as well as on the distribution of cortical granules before and after fertilization (Exp 4). In Exp 1, the 20 µM Res treatment upregulated protein expression of SIRT1 in oocytes. In Exp 2, Res treatment improved the ratio of normal fertilization and the total cell number of blastocysts. In Exp 3, Res treatment significantly increased the ATP content in matured oocytes. Additionally, Res increased the overall Mt number and mitochondrial membrane potential, but the effect was donor-dependent. In Exp 4, Res-induced zona hardening improved the distribution and exocytosis of cortical granules after in vitro fertilization. In conclusion, Res improved the quality of oocytes by improving mitochondrial quantity and quality. In addition, Res added to the maturation medium enhanced SIRT1 protein expression in oocytes and improved fertilization via reinforcement of the mechanisms responsible for blockage of polyspermic fertilization.

276 ACTIVATION OF ROUND SPERMATID-INJECTED OOCYTES USING PHOSPHOLIPASE C ZETA IN PIGS

In several mammalian species, the generation of offspring by round spermatid injection has been reported. However, in domestic species, including pigs, no one has reported success to date. One of the reasons is that round spermatid-injected oocytes require artificial stimuli for oocyte activation, but the developmental ability of the oocytes is low in pigs, suggesting that a more optimal activation protocol is needed. During fertilization, a sperm-derived factor induces repetitive increases in intracellular calcium, known as calcium oscillations. It is now acknowledged that phospholipase C zeta (PLCζ) has an essential role in inducing calcium oscillations, not only in mammals, but also in several other vertebrates. Therefore, if PLCζ is used as a stimulus for oocyte activation, the efficiency of oocyte activation can be improved. Recently, we found that equine PLCζ (ePLCζ) has higher activity than those of other mammalian species to be studied. In the present study, we examined whether injection of ePLCζ complementary RNA (cRNA) improves the activation of round spermatid-injected oocytes in pigs. First, we examined whether ePLCζ is expressed in round spermatids. Porcine round spermatids were isolated from adult testes, and immunostaining using anti-PLCζ antibody was carried out. The PLCζ was localised at the head and tail in mature sperm, and a part of the round spermatid was also stained. Next, we evaluated the developmental ability of round spermatid-injected oocytes activated by different protocols (electrical pulses v. injection of ePLCζ cRNA). The cytoplasts were then injected with round spermatids. One hour later, the oocytes were divided into two groups. In group 1, the oocytes were activated by a direct current pulse (150 V mm–1 and 60 µs). In group 2, the oocytes were injected with ePLCζ cRNA as follows: the reagent (0.1 µg µL–1) was diluted in injection buffer [100 mM KCl and 10 mM HEPES (pH = 7.0)], loaded into glass micropipettes by aspiration, and delivered to the ooplasm by pneumatic pressure (Narishige, Tokyo, Japan). Each oocyte received 3 to 10 pL (1 to 3% of the total volume of the oocyte). After the stimulations, oocytes were cultured in PZM-5 under 38.5°C in a humidified incubator (95% air, 5% CO2). In the ePLCζ-injected group, rates of pronuclear formation (n = 22/32, 68.8%) and blastocysts (n = 2/43, 4.7%) were higher than those in the electrical pulse-treated group (n = 9/41, 22%; and n = 0/51, 0%, respectively; P &lt; 0.05). In conclusion, our data suggest that injection of PLCζ is effective for activation of round spermatid-injected oocytes in pigs.

Exposure of in vitro-matured porcine oocytes to SYBR-14 and fluorescence impairs their developmental capacity

Staining with Hoechst 33342 followed by ultraviolet irradiation is frequently used to aid or confirm the enucleation of recipient oocytes in porcine somatic cell nuclear transfer programs. However, the procedure has a clearly deleterious effect on the developmental ability of oocytes. This study evaluated the effectiveness of a longer-wavelength fluorochrome (SYBR-14) for visualizing maternal chromosomes in in vitro-matured porcine oocytes and the effects of this dye in combination with fluorescence excitation on the subsequent in vitro fertilization and embryo development of the oocytes. In the first experiment, the oocytes were exposed to different concentrations (1, 3, 5 and 7μg/mL) of SYBR-14 at different incubation times (5, 10 and 30min) in a 4×3 factorial design. The optimal condition for proper metaphase-II plate and first polar body visualization was a 10-min incubation with 5μg/mL of SYBR-14. In the second experiment, the degeneration rate of the oocytes 18h after exposure to SYBR-14 (5μg/mL for 10min) and fluorescence excitation for 5 or 30s was significantly higher (p<0.002) than that obtained for non-exposed oocytes. The fertilization parameters were not influenced by the treatments. The cleavage and blastocyst rates during culture were lower (p<0.001) for the oocytes exposed to SYBR-14 and fluorescence than for those in the non-exposed group. These results indicate that the exposure of mature oocytes to SYBR-14 and fluorescence for periods as short as 5s increased the rate of oocyte degeneration and limited their subsequent developmental competence.

Effects of Hoechst 33342 staining and ultraviolet irradiation on the developmental competence of in vitro-matured porcine oocytes

Hoechst 33342 (H342) in combination with ultraviolet (UV) irradiation is frequently used to assist the enucleation of porcine oocytes in somatic cell nuclear transfer programs. This work evaluated the effects of H342 (5 μg/mL for 12 min) staining and/or exposure to UV irradiation on fertilisability and developmental capacity of porcine oocytes matured in vitro. In Experiment 1, a total of 1388 mature oocytes were distributed in the following groups: Group 1: oocytes without treatment (Control), Group 2: oocytes stained with H342, Group 3: oocytes stained with H342 and UV irradiated for 30 sec, and Group 4: oocytes UV irradiated for 30 sec. Oocytes from each group were exposed to thawed spermatozoa and cultured for 18 h to assess fertilization parameters or for 7 d to evaluate embryo development. Sperm penetration (P < 0.001) and monospermy (P < 0.04) were lower in oocytes exposed to H342/UV (80.7 ± 4.5% and 30.7 ± 5.4%, respectively) than in oocytes from the control group (94.9 ± 4.3 and 50.0 ± 4.9, respectively). The oocytes exposed to H342/UV showed lower (P < 0.001) cleavage (49.8 ± 2.9%) and blastocyst (7.7 ± 2.9%) rates than oocytes from the other groups (range: 73.8 ± 2.9% to 77.7 ± 2.9% and 22.3 ± 2.9% to 30.9 ± 3.0%, respectively). Experiment 2 was designed to evaluate the effect of shorter UV irradiation (5 sec). A total of 1835 mature oocytes were separated into the same groups as those of Experiment 1. The fertilization parameters and the cleavage rates were not influenced by the different treatments. However, the oocytes exposed to H342 and UV irradiation for 5 sec showed a lower (P < 0.02) rate of blastocyst formation (15.2 ± 4.5%) than the oocytes from other groups (range: 26.1 ± 4.5% to 30.7 ± 4.5%). In conclusion, our results demonstrate that the combination of H342 staining with UV irradiation has a clear deleterious effect on the developmental ability of oocytes, with the effects being more intense with increased exposure to UV irradiation.

Comparison of Two Different Schemes of Once-weekly Ovum Pick Up in Dairy Heifers

To compare two different schemes, continuous scheme (CS) and discontinuous scheme (DCS), of once-weekly ovum pick up (OPU) with ultrasound-guided follicular puncture technique, Holstein heifers were randomly divided into two groups of five. After characterization of their two normal estrous cycles, the heifers were subjected to consecutive 20 weeks of once-weekly OPU under two schemes: the CS (one week interval between continuous OPU, total 100 OPU sessions performed) and the DCS (OPU fixed to the day 3 and day 10 of each estrus). Then, the status of ovaries and artificial insemination results were observed. On oocyte yield, the total number of punctured follicles using DCS was lower than that using CS, but the mean numbers of punctured follicles and recovered oocytes per session were higher in DCS than CS group. So the total number of recovered oocytes was similar in both groups. There were also no differences in the quality of recovered oocytes, nor in the developmental ability of oocytes fertilized in vitro between groups. The heifers in the DCS group showed regular estrous cycles with stable estrous signs through the periods of before, during, and after OPU, while those in CS group showed longer estrous cycles and less estrous signs during and/or after OPU compared with before period. Furthermore, the mean number of inseminations required for obtaining pregnancy after completion of the experiments was lower in DCS than CS group. The research demonstrates that similar quantity and quality oocytes can be achieved, and the side effects on donors are lower in DCS that needs fewer OPUs than CS group, and DCS is superior to CS.

In vitro Fertilization and Development of Pig Oocytes Inseminated with Boar Sperm by Different Sperm Washing Media after Thawing of the Frozen Straws

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing 500 µl mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to 24°C over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were resuspended in a lactose-egg yolk and N-acetyl-D- glucosamine (LEN) diluent to contain 1×10 9 sperm/ml and cooled to 5°C over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with 2×10 7 sperm concentration. Oocytes were coincubated for 6 h in 500 µl mTBM fertilization. At 6 h after IVF, oocytes were transferred into 500 µl NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 2 : 164-167)

Cytoplasmic changes in relation to nuclear maturation and early embryo developmental potential of porcine oocytes: effects of gonadotropins, cumulus cells, follicular size, and protein synthesis inhibition.

Morphological and biochemical changes indicative of cytoplasmic maturation in relation to nuclear maturation progression and early embryo developmental potential was studied. Fluorescently labeled microfilaments and cortical granules were visualized by using laser scanning confocal microscopy. The mitogen-activated protein (MAP) kinase phosphorylation and cyclin B1 levels were revealed by Western blot. With the maturation of oocytes, cortical granules and microfilaments were localized at the cell cortex. A cortical granule-free domain (CGFD) and an actin-thickening area were observed over both the MII spindle of a mature oocyte and chromosomes of a nocodazole-treated oocyte, suggesting that chromosomes, but not the spindle, determined the localization of CGFD and actin-thickening area. In oocytes that are incompetent to resume meiosis, as indicated by the failure of germinal vesicle breakdown (GVBD), peripheral localization of cortical granules and microfilaments, phosphorylation of MAP kinase and synthesis of cyclin B1 did not occur after 44 hr in vitro. These cytoplasmic changes were also blocked when GVBD of meiotically competent oocytes was inhibited by cycloheximide. Culture of oocytes in a chemically defined medium showed that biological factors such as gonadotropins, cumulus cells and follicle size affected both nuclear and cytoplasmic maturation as well as embryo developmental potential. Absence of gonadotropins or removal of cumulus cells alone did not significantly influence GVBD or cyclin B1 levels, but decreased the final maturation and developmental ability of oocytes. A combination of gonadotropin absence and cumulus removal decreased GVBD, MAP kinase phosphorylation and embryo development. A high proportion of oocytes derived from small follicles were able to resume meiosis, synthesize cyclin B(1), phosphorylate MAP kinase and translocate CGs, but their maturation and embryo developmental ability were limited. Removal of cumulus cells from small follicle-derived oocytes severely affected their ability to undergo cytoplasmic and nuclear maturation.

Development of Automated Nuclear Transplantation System.

In nuclear transplantation, especially in enucleation, rotating the oocyte to a certain position has been an important work. Therefore, we developed an oocyte rotation system by using electrostatic force, which is known to be excellent in handling minute objects. This system has become the basic technology for the automated nuclear transplantation system. Mouse oocyles were rotated at a speed of about 60 deg/s by electrostatic phenomena (i.e. electrorotation and dielectrophoresis), and the direction of rotation could be reversed. In order to confirm the electrostatic effect on the fertilization and developmental ability of oocytes, we performed IVF and nuclear transplantation with rotated oocytes. The result was that the fertilization and developmental ability of the rotated oocytes was the same as that of the control, proving that rotation by electrostatic force does not influence the fertilization and development ability of oocytes.

Developmental Ability and Normality of Mouse Oocytes Reconstructed by Pronuclear Transfer

Objectives: It is suggested that there is a possibility to improve the quality and the developmental ability of oocytes from old patients by microinjection of intact ooplasm of young donors (Mol Hum Reprod, 1998;4:269–280). Whole exchange of cytoplasm between patient and donor oocytes can be completed by using the pronuclear (PN) transfer technology, and this may maximize the improving of oocytes quality. This study was conducted to know the safety of the PN transfer technology using mice oocytes as an animal model.

The developmental ability of human oocytes penetrated at the germinal vesicle stage after insemination in vitro

This study demonstrates that sperm penetration into the ooplasm occurs at high frequency in germinal vesicle (GV) stage human oocytes which failed to resume meiosis after ovulation induction in cycles of ovarian hyperstimulation for in-vitro fertilization. The capacity of the immature human oocyte to prevent polyspermic penetration at the cell surface level was suggested by the finding that despite the presence of numerous spermatozoa within the zona pellucida and on the oocyte surface within 3 h after insemination, all normal-appearing GV stage oocytes examined in this study were penetrated by a single spermatozoon. This notion was also supported by scanning confocal microscopic analysis of oocytes double-stained for DNA and cortical granules which showed highly localized regions of cortical granule-free cytoplasm in proximity to the penetrated spermatozoon. The developmental ability of these oocytes was assessed by culture in vitro. The results show that oocytes penetrated by a spermatozoon at the GV stage resume meiosis, develop the capacity to decondense sperm DNA, abstrict both first and second polar bodies, and form a male pronucleus from the spermatozoon which enters the oocyte prior to the resumption of meiotic maturation. After penetration, sperm nuclei rapidly migrate to the centre of the oocyte and become juxtaposed with the germinal vesicle, suggesting the presence of a cellular mechanism which permits directed movement within the cytoplasm. The developmental ability of these oocytes and the normality of the resulting embryos are discussed.