Parthenogenetic Activation Of Oocytes Research Articles

Elevated chorionic gonadotropic hormone in transgenic mice induces parthenogenetic activation and ovarian teratomas

Both male and female reproductive functions are impacted by altered gonadotrophin secretion and action, which may also influence the development of endocrine tumours. To ascertain if chronic hypersecretion of human chorionic gonadotropin (hCG) contributes to the development of gonadal tumours, double transgenic (TG) mice that overexpress hCGα- and β-subunits were analysed. By the age of two months, ovarian tumours with characteristics of teratomas developed with 100% penetrance. Teratomas were also seen in wild-type ovaries orthotopically transplanted into TG mice, demonstrating an endocrine/paracrine mechanism for the hCG-induced ovarian tumorigenesis. Both in vitro and in vivo experiments showed oocyte parthenogenetic activation in TG females. In addition, ovaries showed reduced ovulatory gene expression, inhibited ERK1/2 phosphorylation, and impaired cumulus cell expansion. Hence, persistently high endocrine hCG activity causes parthenogenetic activation and development of ovarian teratomas, along with altered follicle development and impaired ERK1/2 signalling, offering a novel mechanism associated with the molecular pathogenesis of ovarian teratomas.

PLCζ can stably regulate Ca2+ fluctuations in early embryo

Phospholipase C zeta (PLCζ) is an important inducer of Ca2+ oscillations in mammalian sperm. To explore the influence of PLCζ on early embryonic Ca2+ fluctuations during sperm-egg binding, this study used PLCζ from sheep sperm to construct an early embryonic Ca2+ fluctuation model. First, sheep MII oocytes were cultivated and screened using microinjection technology. Then, a pEGFP-N1-PLCζ plasmid was constructed to activate oocytes in the test group. Ionomycin combined with 6-Dimethylaminopurine (6-DMAP) was used for the control group to explore the effects on early embryonic development and regulation of Ca2+ fluctuations during development. The results demonstrated that both the PLCζ and ionomycin combined with 6-DMAP activation methods induced sheep oocyte parthenogenetic activation and development in early embryos. In comparisons, the cleavage rate of ionomycin combined with 6-DMAP activation was significantly higher than that of PLCζ (60.9% ± 19.4% vs 76.1% ± 0.7%, respectively; p < 0.001), and the blastocyst rates were 16.2% ± 0.62% and 21.1% ± 0.92%, respectively (p < 0.05). Additionally, when comparing the distribution of Ca2+ in early embryos at different stages, Ca2+ in both treatment groups was mainly distributed in the cytoplasm, but the temporal pattern of Ca2+ fluctuations differed. PLCζ resulted in Ca2+ peaks that appeared at the cleavage and morula stages of early embryos, and Ca2+ returned to normal levels at the morula stage. However, the Ca2+ concentration after ionomycin combined with 6-DMAP activation was always much higher than that with PLCζ, and its single peak appeared later than in the PLCζ group. In summary, the PLCζ gene promoted stable regulatory effects on Ca2+ fluctuations at different stages during early embryonic development.

Effect of Ethanol on Parthenogenetic Activation and α-Tocopherol Supplementation during In Vitro Maturation on Developmental Competence of Summer-Collected Bovine Oocytes.

The use of α-tocopherol during in vitro maturation (IVM) is an alternative to minimize the adverse effects of heat stress on oocyte competence. However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 μM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 μM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 μM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.

The Cyclin B2/CDK1 Complex Conservatively Inhibits Separase Activity in Oocyte Meiosis II

Recently, we have reported that the cyclin B2/CDK1 complex regulates homologous chromosome segregation through inhibiting separase activity in oocyte meiosis I, which further elucidates the compensation of cyclin B2 on cyclin B1’s function in meiosis I. However, whether cyclin B2/CDK1 complex also negatively regulates separase activity during oocyte meiosis II remains unknown. In the present study, we investigated the function of cyclin B2 in meiosis II of oocyte. We found that stable cyclin B2 expression impeded segregation of sister chromatids after oocyte parthenogenetic activation. Consistently, stable cyclin B2 inhibited separase activation, while introduction of non-phosphorylatable separase mutant rescued chromatid separation in the stable cyclin B2-expressed oocytes. Therefore, the cyclin B2/CDK1 complex conservatively regulates separase activity via inhibitory phosphorylation of separase in both meiosis I and meiosis II of mouse oocyte.

Alpha-lipoic acid improves the maturation and the developmental potential of goat oocytes in vitro.

Oxidative stress inevitably occurs during oocyte maturation in vitro. α-lipoic acid (α-LA) has a strong antioxidant capacity, but the effect of α-LA on parthenogenetic activation of oocytes was rarely reported. This study aims to investigate the effect of supplementing α-LA to in vitro maturation medium on the subsequent developmental ability of goat parthenogenetic embryos during oocytes maturation. In the study, the goat cumulus-oocyte complex was divided into the experimental (with 25μmol/L α-LA) and the control (without α-LA) groups. Oxidase expression was measured using RT-qPCR. After 18-22hr of maturation, the oocytes were then parthenogenetic activated. The total antioxidant capacity of embryos was measured after 0, 24, 48, 72 and 96hr of culture. Rates of oocyte maturation and the rates of development for parthenogenetic embryos in the α-LA group were significantly improved by 7.88% (p<.05) and 5.41% (p<.05) compared with those in the control group, respectively. After 24hr, the difference in total antioxidant capacity was extremely significant in both groups. An evident decrease in the control group and a minor decrease in the α-LA group were observed (p<.01). The ratio of inner cell mass cells to the total cell number of blastocysts in the α-LA group increased compared with that in the control group (p<.05) on day 8. α-LA significantly promoted the expression of SOD and GPX4 of parthenogenetic blastocysts and maturated oocytes. α-LA (25μmol/L) improved the maturation rate and the developmental competence of the parthenogenetic activation of oocytes, which might be mediated by maintaining the total antioxidant ability of oocytes during the culture period.

29 The effect of vitrification at the immature stage on DNA methylation in porcine oocytes and its relevance to subsequent embryo development

Oocyte vitrification is an important approach for invitro gene banking of female germplasm; however, in pigs, it hampers embryo development. In cattle, vitrification at the MII stage was reported to alter epigenetic status in oocytes and even in subsequently developing embryos (Chen et al. 2016 Theriogenology 86, 868-878). The present study investigated the effect of vitrification at the immature stage of porcine oocytes on DNA methylation status and its relevance to subsequent embryo development. Immature cumulus–oocyte complexes were vitrified in microdrops and warmed (vitrified group) or treated with cryoprotectant agents (17.5% ethylene glycol + 17.5% propylene glycol, CPA group) by our method (Appeltant et al. 2018 Cryobiology 85, 87-94). Then they were subjected to IVM, parthenogenetic activation (PA), and embryo culture. From each batch, a group of oocytes was processed without treatment (control group). Oocyte survival and polar body extrusion were recorded after IVM. Cleavage and blastocyst developmental rates were recorded on Day 2 and 6 of culture, respectively (Day 0=PA). In each replication, DNA methylation was assayed in representative oocytes at the MII stage after IVM and in embryos at the 2- to 4-cell stage on Day 2 by immunostaining with 5-methylcytosine (5mC). Relative fluorescent intensity of 5mC in the chromatin was compared among groups. The experiment was replicated 3 times. Data were analysed by ANOVA. After IVM, there was no significant difference among the control, CPA, and vitrified groups in terms of the percentage of live oocytes (99.3, 96.4, and 94.0%, respectively) or polar body extrusion (88.6, 86.9, and 79.6%, respectively). After PA of oocytes with a polar body, there was no difference between the control and CPA groups in the percentage of cleavage (84.1 and 80.7%, respectively) or blastocyst development of cleaved embryos (63.3 and 79.3%, respectively). However, in the vitrified group, cleavage and blastocyst development rates (46.6 and 33.5%, respectively) were reduced (P&amp;lt;0.05) compared with the other groups. The 5mC fluorescence in the DNA of oocytes at the MII stage in the CPA and vitrified groups were similar and significantly lower than that in the control group (0.88±0.02, 0.87±0.001, and 1.0±0.02, respectively) but higher than that in the negative control processed without primary antibody (0.33±0.02). In the embryos at the 2- to 4-cell stage, 5mC fluorescence was not significantly different among the control, CPA, and vitrified groups (1.0±0.1, 0.99±0.1, and 0.96±0.1, respectively) but was significantly higher than that of the negative control (0.36±0.04). In conclusion, CPA treatment reduced DNA methylation levels in oocytes. However, it was restored during early embryo development and did not affect blastocyst development. The results suggest that reduced DNA methylation in vitrified oocytes is caused by CPA but it may not be responsible for their reduced ability to develop to blastocysts.

Assessment of different stages of parthenogenetic embryos for production of embryonic stem cell like colonies

Parthenogenetic activation of oocytes has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. In this study, we described the production of goat parthenogenetic ES like cells from different stages of parthenogenetic embryos. We compared the source material on the pESCs outgrowth and culture. 8-16 cell stage embryos, morula, blastocysts and ICM (inner cell mass) from hatched blastocysts produced from chemical activation were cultured on goat fetal fibroblast monolayer in stem cell culture media. Colonies were passaged when signs of differentiation were visible. ESC like colonies were cultured on feeder cells in the presence of hLIF however, some ESC like colonies were also cultured in absence of hLIF for random differentiation. In our study, ESC like colony formation with ICM of hatched blastocyst was comparatively higher as compared to blastocysts while ESC like colony formation with blastocysts and ICM of hatched blastocyst was significantly higher as compared 8-16 cells and morula. It is observed that inner cell mass source is an important criterion for the ES like cells derivation in goats. Also, in comparison to the expanded blastocysts (80.10%), hatched blastocysts showed higher (86.06%) attachment rate and primary colony formation rate. ESC like colonies in absence of hLIF differentiated in vitro into epithelial like and neuronal like cells. Undifferentiated ESC like colonies stained positive for alkaline phosphatase, SSEA-3 and OCT-4.

A case of recurrent spontaneous parthenogenetic oocyte activation

Oocytes obtained during in vitro fertilization (IVF) cycles ideally are arrested in metaphase II (MII), which are characterized by the presence of a single polar body. They remain in arrest until after fertilization when time meiosis is resumed in a calcium-dependent manner. We present a case of a 23 year old gravida 0 with polycystic ovarian syndrome who presented for evaluation at our center after previously having failed two IVF cycles elsewhere. The patient subsequently underwent three controlled ovarian hyperstimulation cycles with either an antagonist or low dose luteal lupron down-regulation protocol. Vaginal oocyte retrieval (VOR) was performed 36 hours after administration of gonadotropin releasing hormone agonist (GnRHa) or human chorionic gonadotropin trigger. Cycle outcomes are demonstrated in Figure 1 . Oocytes were fertilized by intracytoplasmic sperm injection (ICSI) according to standard laboratory protocol. Embryos were cultured until day 7 of development. A substantial proportion of oocytes were noted to have one pronucleus (1PN) and two polar bodies (PB) at the time that the cumulus complex was denuded prior to ICSI ( Figure 2 ). The parthenogenetic oocytes were re-examined on the morning following VOR and were noted to have undergone cell division, with many that appeared similarly to cleavage stage embryos. In each cycle, there was complete embryo arrest prior to the blastocyst stage and no embryos were available for transfer. This case of recurrent parthenogenesis, accelerated cell division and complete developmental arrest highlights what potentially could be a rare genetic contribution to a cell-cycle regulatory deficiency. It has been suggested that defects in the c-mos/mitogen-activated protein kinase (MAPK) pathway in oocytes exhibit failure to arrest and premature activation ( 1 ). EMI2 is an additional regulatory factor that has been implicated in maintenance of arrest ( 2 ). It is unclear whether there is a failure of MII arrest due to functional deficiencies of MAPK and EMI2 or if there is an unidentified stimulus for escape which overrides these checkpoints. Attempts at fertilization of prematurely activated 1PNs have previously failed ( 3 ). Enhanced understanding of cell-cycle regulatory measures in oocytes and human preimplantation embryos may yield insight regarding diagnostic and therapeutic modalities for similar rare cases of spontaneous parthenogenesis.

Effect of Endoplasmic Reticulum (ER) Stress Inhibitor Treatment during Parthenogenetic Activation on the Apoptosis and In Vitro Development of Parthenogenetic Porcine Embryos.

We investigate the effect of endoplasmic reticulum (ER) stress inhibitor treatment during parthenogenetic activation of oocytes on the ER stress generation, apoptosis, and in vitro development of parthenogenetic porcine embryos. Porcine in vitro matured oocytes were activated by 1) electric stimulus (E) or 2) E+10 μM Ca-ionophore (A23187) treatment (EC). Oocytes were then treated by ER stress inhibitors such as salubrinal (200 nM) and tauroursodeoxychloic acid (TUDCA, 100 μM) for 3 h prior to in vitro culture. Parthenogenetic embryos were sampled to analyze ER stress and apoptosis at the 1-cell and blastocyst stages. The x-box binding protein 1 (Xbp1) mRNA and ER stress-associated genes were analyzed by RT-PCR or RT-qPCR. Apoptotic gene expression was analyzed by RT-PCR. At the 1-cell stage, although no difference was observed in Xbp1 splicing among treatments, BiP transcription level in the E group was significantly reduced by salubrinal treatment, and GRP94 and ATF4 transcription levels in EC group were significantly reduced by all treatments (p<0.05) compared to control. In the EC group, both apoptotic genes were reduced by ER stress inhibitor treatments compared to control (p<0.05) except Caspase-3 gene by TUDCA treatment. These results suggest that the treatment of ER stress inhibitor during parthenogenetic activation can reduce ER stress, and thereby reduce apoptosis and promote in vitro development of porcine parthenogenetic embryos.

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.

Parthenogenetic Activation of Oocytes.

Numerous studies have been initiated to investigate the influence of maternal and paternal genomes on early mammalian development. For this type of study, parthenogenetic embryos provide a unique source of preimplantation and early postimplantation embryos that (by definition) develop in the absence of any contribution from a male gamete. Parthenogenetic activation is used for biochemical and morphological studies of oocytes during fertilization and early development and is a critical component of the cloning procedure. This protocol describes the activation of oocytes using ethanol. Parthenogenesis can also be induced by exposure of unfertilized oocytes to strontium-containing medium.

Enhanced in vitro maturation of canine oocytes by oviduct epithelial cell co-culture

Canine-assisted reproductive techniques have been successful for several years; however, the lack of an oocyte in vitro maturation system has limited their application. The aim of this study was to evaluate the effect of canine oviduct epithelial cells (cOECs) on canine oocyte maturation in vitro. Specifically, the method used for isolation of cOECs did not affect the expression of epithelial markers, E-cadherin and cytokeratin, on fresh, cultured and cryopreserved cells. Moreover, BrdU analysis showed that cOECs cultured in Medium 171 supplemented with mammary epithelial growth supplement were more proliferative than counterparts in advanced Dulbecco's modified Eagle medium or Medium 199. Maturation rate of canine oocytes collected from bitches at diestrus was significantly increased when oocytes were co-cultured with either fresh, cultured or frozen/thawed cOECs (13.23 ± 1.15%, 10.38 ± 4.89%, or 10.54 ± 2.96%, respectively) than that of control oocytes cultured without cOECs (2.48 ± 2.16%, p < 0.05). Additionally, the number of oocytes collected from bitches at estrus the reached metaphase II was increased ∼4 fold in co-culture with fresh, cultured, or frozen/thawed cOECs (47.2 ± 3.82%, 45.4 ± 7.34%, and 46.9 ± 1.51%, respectively) as compared with oocytes cultured without cOECs (11.9 ± 3.18%, p < 0.05). Nuclear maturation was further confirmed by assessing the formation of normal metaphase-II spindles, whereas cytoplasmic maturation was confirmed by inducing parthenogenetic oocyte activation. Embryonic development to the 8-cell stage was similar between in vivo and in vitro matured oocytes. These results suggested that co-culturing immature canine oocytes with cOECs facilitated canine oocyte maturation and early stages of embryonic development.

NLRP2 controls age-associated maternal fertility.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are well-known for their key roles in the immune system. Ectopically expressed NLRP2 in immortalized cell lines assembles an inflammasome and inhibits activation of the proinflammatory transcription factor NF-κB, but the physiological roles of NLRP2 are unknown. Here, we show that Nlrp2-deficient mice were born with expected Mendelian ratios and that Nlrp2 was dispensable for innate and adaptive immunity. The observation that Nlrp2 was exclusively expressed in oocytes led us to explore the role of Nlrp2 in parthenogenetic activation of oocytes. Remarkably, unlike oocytes of young adult Nlrp2-deficient mice, activated oocytes of mature adult mice developed slower and largely failed to reach the blastocyst stage. In agreement, we noted strikingly declining reproductive rates in vivo with progressing age of female Nlrp2-deficient mice. This work identifies Nlrp2 as a critical regulator of oocyte quality and suggests that NLRP2 variants with reduced activity may contribute to maternal age-associated fertility loss in humans.

FSH stimulation of anestrous cats improves oocyte quality and development of parthenogenetic embryos

In the domestic cat, the efficiency of in vitro embryo production systems is negatively affected during the nonbreeding season. The objective of this research was to evaluate the effect of FSH stimulation in anestrous cats, on quality of cumulus-oocyte complexes (COCs) and in vitro developmental competence after parthenogenetic activation. To accomplish this purpose, anestrous cats were grouped into: (1) FSH treated (serial doses of 5 mg of porcine FSH each, every 24 hours, for 4 days) and (2) untreated control. The COCs were classified morphologically and a proportion of grade I and II COCs was used for expression analysis of FSHR, LHCGR, EGFR, PTGS2, EGR1, GDF9, and GATM by RT-qPCR. In addition, another proportion of grade I and II COCs was matured in vitro and used for parthenogenetic activation. After 8 days in culture, blastocyst and hatching blastocyst rates were assessed, and the expression of OCT4, SOX2, NANOG, CDX2, and GATA6 was evaluated. The COCs in the FSH group had an enhanced quality, a higher expression of LHCGR and a lower expression of GATM than did COCs from the control group (P < 0.05). Furthermore, embryos in the FSH group had increased blastocyst and hatching blastocyst rates, and those embryos had a higher expression of OCT4 and GATA than their counterparts from the control group (P < 0.05). In conclusion, ovarian stimulation of anestrous cats with FSH improved quality and increased the expression of LHCGR in COCs. The enhanced in vitro developmental competence, after parthenogenetic activation of oocytes from FSH-treated cats, coincided with an increased expression of OCT4 and GATA6 in blastocysts and hatching blastocysts.

Effect of Follicle Size on In Vitro Maturation of Pre-Pubertal Porcine Cumulus Oocyte Complexes.

Very small follicles (<3.0mm diameter) are over-represented on the surface of ovaries of non-cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence invitro. This study examined the effect of follicle size on the nuclear maturation (n=608), the potential of parthenogenetic activation (n=243) and the cyclic AMP (cAMP) content of pre-pubertal porcine oocytes (n=480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5-4.0mm) or large (4.5-6.0mm) ovarian follicles were cultured for 0, 28 and 46h. After 46h of IVM, a greater proportion of oocytes from 4.5- to 6.0-mm follicles reach metaphase II (MII) compared with those from follicles with 2.5-4.0mm of diameter (96.1 vs 77.0%, respectively; p<0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p<0.05). At 28h, the IVM medium with oocytes from large follicles contained significantly more 17ß-oestradiol (E2 ) than the medium with oocytes from small follicles (5.55 vs 3.45ng/ml, respectively; p<0.05) and at 46h, the medium with oocytes from small follicles contained significantly more progesterone (P4 ) than the medium with oocytes from large follicles (276.7 vs 108.2ng/ml, respectively, p<0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP-mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0mm are more suitable for invitro studies.

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro.

Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.

284 INTRACELLULAR PATHWAYS MEDIATING DIRECT EFFECTS OF PROLACTIN AND GROWTH HORMONE ON BOVINE METAPHASE-II OOCYTES AGING IN VITRO

Maturation of mammalian oocytes coincides with various aging processes, which negatively affect the oocyte quality. Cellular and molecular changes in matured oocytes aging in vivo and in vitro are very similar, suggesting similarities in the underlying mechanisms. The goal of the present research was to study direct effects of prolactin (PRL) and growth hormone (GH) on bovine metaphase-II (M-II) oocytes aging in vitro and intracellular pathways mediating these effects. Cumulus-enclosed oocytes were matured for 20 h in TCM 199 containing 10% fetal calf serum (FCS), 10 µg mL–1 of porcine FSH, and 10 µg mL–1 of ovine LH. After IVM, oocytes were set free from cumulus and denuded oocytes (DO) were cultured for 44 h in the aging medium consisting of TCM 199 supplemented with 10% FCS in the absence of PRL and GH (Control), then in the presence of either 50 ng mL–1 of bovine PRL or 10 ng mL–1 of bovine GH or protein kinase inhibitors. The following inhibitors were added: (1) PP2 (an inhibitor of Src-family tyrosine kinases, 20 µM), (2) U0126 (a MEK inhibitor, 20 µM), and (3) triciribine (an inhibitor of Akt kinase, 50 µM). At the end of culture, the state of the nuclear material of oocytes and embryos was evaluated by the method of Tarkowski. The number of oocytes undergone spontaneous parthenogenetic activation was determined by summarising the numbers of embryos cleaved and oocytes reached anaphase-II to pronucleus stages. The data from 4 replicates were analysed by ANOVA. During aging in the control medium, the rate of M-II oocytes with destructive changes of chromosomes (decondensation, clumping, fragmentation) increased from 20.3 ± 1.7 to 65.1 ± 3.5% (P &lt; 0.001) and was unaffected by PRL and GH. At the same time, the frequency of oocyte parthenogenetic activation (18.7 ± 4.9%) was reduced (P &lt; 0.001) by both PRL and GH (up to 6.0 ± 2.9 and 5.9 ± 2.4%, respectively). The inhibitor of Src-family tyrosine kinases PP2 eliminated (at least P &lt; 0.05) the supporting effects of PRL and GH on the meiotic arrest at M-II. The MEK inhibitor U0126 also abolished the effect of PRL (but not that of GH), increasing the frequency of the oocyte activation from 5.9 ± 2.3 to 21.6 ± 1.9% (P &lt; 0.01). Concurrently, both inhibitors did not affect the meiotic arrest in the control group. The inhibitor of Akt kinase triciribine did not influence the frequency of the oocyte activation in the PRL- and GH-treated groups. Meanwhile, triciribine decreased the rate of M-II oocytes with chromosome abnormalities in the control medium (from 71.2 ± 1.7 to 47.0 ± 2.7%; P &lt; 0.001). When added to the aging medium, PRL enhanced the triciribine effect, while GH suppressed it (P &lt; 0.01). Thus, PRL and GH can directly support the meiotic arrest at M-II in aging oocytes by activating MAP kinases or Src-family tyrosine kinases. Besides, GH may enhance the chromosome destruction in DOs through activation of Akt kinase.The research was supported by RFBR (No. 13-04-01888).

290 3-DIMENSIONAL VISUALIZATION OF BOVINE OOCYTE FERTILIZATION BY CONFOCAL LASER SCANNING MICROSCOPY

Cattle can serve as a model organism to resolve central questions in mammalian reproduction that cannot be clarified in the mouse model due to notable species-specific peculiarities of early mouse embryogenesis. As part of a project on structural, molecular, and functional deficiencies of bovine oocytes, we started to systematically investigate fertilization and the onset of embryo development in vitro by 3-dimensional multicolor fluorescence microscopy. We are using 3D visualisation as key approach to clarify the multiple parallel and sequential processes and events of fertilization as well as to identify and classify errors and failures. Moreover, we aim to gain insights into the mechanisms of aberrations by linking processes at the cellular and the molecular level. We studied class I and II oocytes collected from slaughterhouse ovaries and matured for 23 h in vitro. Oocytes were fixed at different times from 4 to 12 h postinsemination with formaldehyde in a microtubule-stabilising buffer containing taxol in such a way that the 3-dimensional cell architecture was maintained, and were stained for DNA, microtubules, and f-actin microfilaments. In addition, serine 10-phosphorylated histone H3 was used as a marker for chromosome condensation and the spindle midbody. For 3-dimensional imaging of the oocytes in toto, confocal serial sections were captured at 1-µm intervals using a 40× objective (NA = 1.3). For imaging details, we used a high spatial sampling density (pixel size: 50 × 50 nm, z-step size: 200 nm) close to the Nyquist criterion and image restoration by maximum likelihood estimation (MLE) deconvolution. A series of more than 500 three-dimensional snapshots of fertilized oocytes at different points in time gives a first detailed view on the spatial and temporal course of the sperm entry, the formation of the paternal pronucleus and the sperm aster, completion of oocyte meiosis and the formation of the maternal pronucleus, as well as dynamic changes of the cytoskeleton. Moreover, we can document a spectrum of abnormalities including spontaneous parthenogenetic oocyte activation, polyspermy, and aberrations of meiosis I and II. The latter include irregular spindle formation and chromosome segregation, the occurrence of chromatin bridges and abnormal spindle positioning and rotation (e.g. leading to nonextrusion of a first or a second polar body or the extrusion of two second polar bodies). Our microscopic investigation in the bovine system contributes to unraveling the origins of irregular cleavage, aneuploidy, and mosaicism in mammals. Three-dimensional high-speed microscopy of oocytes and zygotes in affordable timeframes could be of great value in improving the differential diagnosis of oocyte and sperm dysfunction, as well as in identifying and dissecting problems, limitations, and potential risks of reproductive technologies (ART).This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).

Changes of spontaneous parthenogenetic activation and development potential of golden hamster oocytes during the aging process

The golden hamster is an excellent animal experimental model for oocyte research. The hamster oocytes are very useful in clinical examination of human spermatozoan activity. Non-fertile oocytes can lead to time-dependent processes of aging, which will affect the results of human spermatozoa examination. As a consequence there is a need to investigate the aging and anti-aging processes of golden hamster oocytes. In order to study the aging processes and parthenogenetic activation of golden hamster oocytes, in vivo oocytes, oocytes cultured with or without cumulus cells, and oocytes treated with Trichostatin A (TSA) or caffeine were collected and investigated. We found that: (1) spontaneous parthenogenetic activation, developmental potential (cleavage rate), and zona pellucida (ZP) hardening undergo age-dependent changes in in vivo, in vitro, and after TSA or caffeine treatment; (2) in vivo, oocytes became spontaneously parthenogenetic 25h post-hCG treatment; (3) in vitro, cumulus cells did not significantly increase the parthenogenetic activation rate of cultured hamster oocytes; and (4) TSA or caffeine could delay spontaneous oocyte parthenogenetic activation and the aging processes by at least 5h, but also accelerated the hardening of the ZP. These results define the conditions for the aging and anti-aging processes in golden hamster oocytes. TSA and caffeine play roles in controlling spontaneous activation, which could facilitate the storage and use of golden hamster oocytes for studying processes relevant to human reproduction.

Effect of harvesting technique and presence or absence of corpus luteum on in vitro development after parthenogenetic activation of oocytes recovered from buffalo ovaries

Growing human population, increasing urbanization and rising per capital income are predicted to double the demand for the supply of livestock products in developing countries over the next two decades [1]. Livestock production is growing faster than any other sub-sector and it is predicted that by 2020, livestock will produce more than half of the total global agricultural output in terms of value [2]. The buffalo occupies an integral part in the livestock economy of Asia as well as of India and India has highest buffalo population in the world [3]. Buffaloes are major contributors for milk, meat and draught power and the buffalo is considered as the animal of the future in India due to their ability to survive inharshclimaticconditions, in comparison with cattle. However, reproductive efficiency of this species is low and hampered due to delayed puberty, silent estrus, prolonged inter calving intervals, poor conception rates, seasonal anoestrus and poor super ovulatory response [4]. Reproduction lies at the heart of any livestock breeding enterprise and is vital to maintain or increase the number of animals required for production. In order to improve reproductive efficiency of buffalo; assisted reproductive technologies such as artificial insemination (AI), multiple ovulation and embryo transfer (MOET) and production of embryos has been introduced [4]. Progress in the use of embryo transfer in the buffalo is slow due to poor super ovulatory response. embryo production (IVEP) would be an effective technique to improve the efficacy of transferable embryo production [5]. However, embryo production systems in buffalo are still sub optimal when compared with bovine and requires substantial improvement. in vitro