PCV2 induces apoptosis and modulates calcium homeostasis in piglet lymphocytes in vitro

This study investigated the process of PCV2-induced apoptosis and the effect of PCV2 inoculation on calcium homeostasis in piglet lymphocytes in vitro. PCV2-inoculated lymphocytes exhibited chromatin condensation, chromatin segregation, the appearance of membrane-enclosed apoptotic bodies, and DNA fragmentation. Moreover, the proportion of apoptotic cells increased significantly in PCV2-inoculated lymphocytes compared with controls. These results demonstrate that PCV2 induces lymphocyte apoptosis. Some evidence suggests that an alteration in the intracellular free Ca(2+) concentration ([Ca(2+)]i) could cause apoptosis. We measured elevated [Ca(2+)]i in PCV2-inoculated lymphocytes for 12 or 24h compared with controls. Our results support that PCV2-induced apoptosis may be relative to [Ca(2+)]i. In addition, calmodulin (CaM) was increased in PCV2-inoculated lymphocytes for 12h compared with controls. The amount of CaM-dependent protein kinase II (CaMKII) did not change with PCV2 inoculation. We infer that the increased [Ca(2+)]i can bind CaM protein, but functions independently of CaMKII. Inositol 1,4,5-trisphosphate receptor (IP3R)-1 mRNA expression increased with PCV2 inoculation, whereas plasma Ca(2+)-ATP4 mRNA expression decreased. A decreased Ca(2+)-ATP4 level may inhibit Ca(2+) efflux, and the increased IP3R-1 may trigger Ca(2+) release from the endoplasmic reticulum. Both of these changes may contribute to increased [Ca(2+)]i.     

Glycine ameliorates mitochondrial dysfunction caused by ABT-199 in porcine oocytes

Mitochondria play an important role in controlling oocyte developmental competence. Our previous studies showed that glycine (Gly) can regulate mitochondrial function and improve oocyte maturation in vitro. However, the mechanisms by which Gly affects mitochondrial function during oocyte maturation in vitro have not been fully investigated. In this study, we induced a mitochondrial damage model in oocytes with the Bcl-2-specific antagonist ABT-199. We investigated whether Gly could reverse the mitochondrial dysfunction caused by ABT-199 exposure and whether it is related to calcium regulation. Our results showed that ABT-199 inhibited cumulus expansion, decreased the oocyte maturation rate and the intracellular glutathione (GSH) level, caused mitochondrial dysfunction, which was confirmed by decreased mitochondrial membrane potential (ΔΨm) and the expression of mitochondrial function-related genes PGC-1α, and increased reactiveoxygenspecies (ROS) levelsand the expression of apoptosis-associated genes Bax, Caspase-3, and Cyto C.More importantly, ABT-199-treated oocytes showed an increase in the intracellular free calcium concentration ([Ca²⁺]i) and had impaired cortical type 1 inositol 1,4,5-trisphosphate receptors (IP₃R1) distribution. Nevertheless, treatment with Gly significantly ameliorated mitochondrial dysfunction, oxidative stress, and apoptosis, and Gly also regulated [Ca²⁺]i levels and IP₃R1 cellular distribution, which further protects oocyte maturation in ABT-199-induced porcine oocytes.Taken together, our results indicate that Gly has a protective action against ABT-199-induced mitochondrial dysfunction in porcine oocytes.     

Meiotic arrest with roscovitine and sexual maturity improve competence of mouse oocytes by regulating expression of competence-related genes

We have studied the mechanisms by which meiotic arrest maintenance (MAM) with roscovitine, female sexual maturity, and the surrounded nucleoli (SN) chromatin configuration improve the competence of mouse oocytes by observing the expression of oocyte competence-related genes in non-surrounded nucleoli (NSN) and SN oocytes from prepubertal and adult mice following maturation with or without MAM. The results demonstrated that MAM with roscovitine significantly improved the developmental potential of adult SN and prepubertal NSN oocytes, but had no effect on that of prepubertal SN oocytes. Without MAM, while 40% of the 2-cell embryos derived from prepubertal SN oocytes developed into 4-cell embryos, none of the 2-cell embryos derived from prepubertal NSN oocytes did, and while 42% of the 4-cell embryos derived from adult SN oocytes developed into blastocysts, only 1% of the 4-cell embryos derived from prepubertal SN oocytes developed into blastocysts. Furthermore, MAM with roscovitine, SN configuration, and female sexual maturity significantly increased the mRNA levels of competence-beneficial genes and decreased those of competence-detrimental genes. In conclusion, our results suggest that MAM with roscovitine, SN chromatin configuration, and female sexual maturity improve oocyte competence by regulating the expression of competence-related genes, suggesting that Oct4, Stella, Mater, Zar1, Mapk8, and Bcl2 are oocyte competence-beneficial genes, whereas Foxj2, Ship1, and Bax are competence-detrimental genes.     

SRT1720 enhances maturity and quality of oocytes in aged mice

This study aims to investigate the morphology and distribution of mitochondria, spindles, and chromosomes in oocytes of aged mice and examine the effects of SRT1720 on oocyte maturation. C57BL/6J mice were divided into young (4–8 weeks) and aged groups (48–52 weeks). In vitro maturation media contained (0.05, 0.1, and 1.0 μM) SRT1720 and 0.1-μM dimethyl sulfoxide (DMSO control). The rate of chromosome misalignment and spindle misorientation in oocytes of aged mice were significantly higher than that of young mice (P < 0.01). Fluorescence intensity of mitochondria from oocytes of aged mice was significantly lower than that of young mice (P < 0.01). SRT1720 at 0.1 μM significantly improved oocyte maturation, fertilization, and blastocyst formation in aged mice compared with young mice (P < 0.01). Additionally, immunofluorescence intensity of mitochondria, normal spindle morphology, and chromosome alignment were notably enhanced with SRT1720 when compared with the DSMO control group for metaphase II (MII)-stage oocytes matured in vitro (P < 0.01); 0.1-μM SRT1720 enhanced the expression level of SRIT1 in oocytes from aged mice. In summary, the aged mice oocytes showed increased nuclear and cytoplasmic defects, whereas SRT1720 enhanced oocyte maturation and quality. We concluded that 0.1-μM SRT1720 was an appropriate concentration for in vitro maturation media.     

Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes

Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.     

Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytes

During fertilization in mammalian species, a sperm-induced intracellular Ca²⁺ signal ([Ca²⁺]_i) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP₃R1), the channel responsible for Ca²⁺ release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP₃R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP₃R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP₃R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34^cdc2 kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP₃R1 phosphorylation, although inactivation of p34^cdc2 kinase by roscovitine dramatically reduced IP₃R1 phosphorylation. Neither inhibitor affected total expression of IP₃R1. Altogether, our results show that IP₃R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization.     

The use of transmission electron microscopy and oocyte transfer to evaluate in vitro maturation of equine oocytes in different culture conditions

The current study evaluates the ability of equine oocytes matured in different conditions to undergo nuclear and cytoplasmic maturation. After oocyte transfer, embryonic development was diagnosed at 15 and 90 days of gestation. For each group, immature oocytes obtained from slaughterhouse ovaries were matured in vitro (5 replicates). In experiment I, three different media were tested, HTF:BME, SOFaa, and TCM 199. In experiment II, the HTF:BME was chosen as maturation medium containing pFSH, eFSH, or eFSH + eGH. Nuclear maturation was estimated after stripping the oocytes and staining with Hoechst 33342. The evaluation of cytoplasmic maturation was performed by transmission electron microscopy. For oocyte transfer, six non-cycling recipient mares were used, and 8 to 15 oocytes were transferred in each mare. In experiment I, the results showed no differences (P > .05) in nuclear maturation (MII) among experimental groups. The percentage of MII was 29.3 (±9.6), 23.4 (±8.4), and 13.5 (±12.4) for HTF:BME, SOF, and TCM, respectively. In experiment II, all media tested were efficient in inducing metaphase II. Also, no statistical differences (P > .05) were observed in percentages of nuclear maturation rates when porcine (37.1 ± 22.4) or equine (25.8 ± 8.2) FSH were used, or when eFSH + eGH was added to HTF:BME (29.4 ± 12.3). The analysis of cytoplasmic morphology of oocytes cultured in TCM 199 and SOFaa showed signs of incomplete cytoplasmic maturation and premature cortical reaction. Meanwhile, oocytes cultured in HTF:BME medium presented cytoplasmic characteristics similar to those described by others for in vivo-matured oocytes. The addition of eFSH to the HTF:BME medium resulted in an improvement of cytoplasmic morphology. After oocyte transfer, two mares became pregnant, one from pFSH group and one from eFSH+eGH group. These results indicate that although in vitro matured equine oocytes are capable of fertilization and embryonic development, the percentage of competent oocytes is still low.     

玻璃化冷冻对哺乳动物卵母细胞Ca2+的影响机制

卵母细胞冷冻保存是胚胎生物技术(如体外受精、胞浆内单精子注射、体细胞克隆)的重要组成部分,对优良种畜和濒危动物种质资源保存,加速家畜品种改良进程都具有重要意义。与常规冷冻法相比,玻璃化冷冻具有操作简单、降温速率快、耗时短、冷冻效率高等优点,被越来越广泛地应用于家畜卵母细胞的冷冻保存。然而,与新鲜卵母细胞相比,玻璃化冷冻卵母细胞的受精率及发育能力仍不理想,这严重影响了玻璃化冷冻卵母细胞的应用潜力。玻璃化冷冻会引起卵母细胞Ca²⁺浓度升高及钙振荡模式异常,导致其透明带硬化、受精信号紊乱等问题。综合前人研究进展,作者分析了玻璃化冷冻对胞内Ca²⁺浓度、钙振荡模式的影响和作用机制,并指出胞外Ca²⁺内流和胞内钙库Ca²⁺释放是导致冷冻卵母细胞胞内Ca²⁺浓度升高的主要原因,1,4,5-三磷酸肌醇(IP3)Ⅰ型受体分布异常和线粒体损伤可能是导致冷冻卵母细胞钙振荡模式异常的重要原因,以期为正向调控冷冻卵母细胞Ca²⁺浓度及钙振荡模式提供技术参考,从而进一步提高玻璃化冷冻卵母细胞的受精和后续的发育能力。     

水牛卵母细胞在不同培养液中成熟与凋亡的比较研究

为了解卵母细胞体外成熟与凋亡过程,进而提高卵母细胞体外成熟率,本试验研究了在培养液中添加不同浓度的表皮生长因子(EGF)、胰岛素样生长因子-1(IGF-1)对水牛卵母细胞体外成熟和凋亡的影响。结果表明:(1)添加各种浓度的EGF(10,20,30,50,100 ng/mL)均可以提高水牛卵母细胞的成熟率,降低卵母细胞的凋亡率,其中50 ng/mL EGF有显著影响(P<0.05);(2)添加各种浓度的IGF-1(10,30,50,100 ng/mL)均能提高水牛卵母细胞体外成熟率,降低卵母细胞的凋亡率,以30 ng/mL效果明显(P<0.05);(3)添加20 ng/mL EGF+30ng/mL IGF-1组卵母细胞体外成熟率和凋亡率分别高于和低于单独添加IGF-1和EGF组。     

Vitrification procedure decreases inositol 1,4,5‐trisphophate receptor expression,resulting in low fertility of pig oocytes

Although cryopreservation of mammalian oocytes is an important technology, it is well known that unfertilized oocytes, especially in pigs, are highly sensitive to low temperature and that cryopreserved oocytes show low fertility and developmental ability. The aim of the present study was to clarify why porcine in vitro matured (IVM) oocytes at the metaphase II (MII) stage showed low fertility and developmental ability after vitrification. In vitro matured cumulus oocyte complexes (COCs) were vitrified with Cryotop and then evaluated for fertility through in vitro fertilization (IVF). Although sperm‐penetrated oocytes were observed to some extent (30–40%), the rate of pronuclear formation was low (9%) and none of them progressed to the two‐cell stage. The results suggest that activation ability of cryopreserved oocytes was decreased by vitrification. We examined the localization and expression level of the type 1 inositol 1,4,5 trisphosphate receptor (IP₃R1), the channel responsible for Ca²⁺ release during IVF in porcine oocytes. Localization of IP₃R1 close to the plasma membrane and total expression level of IP₃R1 protein were both decreased by vitrification. In conclusion, our present study indicates that vitrified‐warmed porcine COCs showed a high survival rate but low fertility after IVF. This low fertility seems to be due to the decrease in IP₃R1 by the vitrification procedure.     

钙稳态失衡在锰致体外培养鸡胚脑神经细胞凋亡中的作用

旨在探讨钙稳态失衡在锰致体外培养鸡胚脑神经细胞凋亡中的作用。以体外培养鸡胚脑神经元为研究对象,在含终浓度为0、1.5、22、.5 mmol.L-1MnCl2的DMEM培养液中培养24 h,应用流式细胞仪检测磷脂酰丝氨酸(PS)和线粒体膜电位(ΔΨm),琼脂糖凝胶电泳法(DNA Ladder)检测细胞染色质的断裂,以Fura-3/AM为探针检测细胞内游离钙离子浓度([Ca2+]i),用实时定量PCR法检测细胞内CaMmRNA的表达水平。结果表明,随着MnCl2浓度的升高,ΔΨm呈降低趋势,PS膜外翻增加,细胞染色质DNA发生损伤,产生明显的梯形条带,细胞内[Ca2+]i呈升高趋势,CaMmRNA的表达水平下降。结果提示,过量锰通过抑制CaM活性,引起神经元内[Ca2+]i升高,膜通透性发生改变,ΔΨm降低,从而导致鸡胚脑神经元发生凋亡。     

Distribution of protein disulfide isomerase during maturation of pig oocytes

Oocyte maturation in mammals is characterized by a dramatic reorganization of the endoplasmic reticulum (ER). In mice, the ER forms accumulations in the germinal vesicle (GV) stage and distinctive cortical clusters in metaphase II (MII) of the oocyte. Multiple evidence suggests that this ER distribution is important in preparing the oocyte for Ca²⁺ oscillations, which trigger oocyte activation at fertilization. In this study, we investigated the time course and illustrated the possible functional role of ER distribution during maturation of porcine oocytes by immunostaining with protein disulfide isomerase (PDI). PDI forms clusters in the cytoplasm of oocytes. After immunostaining, PDI clusters were identified throughout the cytoplasm from the GV to metaphase I (MI) stage; however, at the MII stage, the PDI formed large clusters (1–2 µm) in the animal pole around the first polar body. PDI distribution was prevented by bacitracin, a PDI inhibitor. Our experiments indicated that, during porcine oocyte maturation, PDI undergoes a dramatic reorganization. This characteristic distribution is different from that in the mouse oocyte. Moreover, our study suggested that formation of PDI clusters in the animal pole is a specific characteristic of matured porcine oocytes.     

生长激素对体外培养猪COCs影响的研究

研究了生长激素对猪COCs体外成熟过程中卵丘细胞扩展、卵丘细胞凋亡、卵母成熟及孤雌激活后卵裂的影响。结果表明:生长激素(STH)能够促进卵丘细胞扩展,抑制卵丘细胞凋亡,对卵母细胞的成熟和激活后卵裂呈现双重效应。在猪COCs培养液中添加0.15μg/mL STH成熟率(73.83%±1.80%)和卵裂率(64.76%±2.84%)显著高于对照组(mNCSU-23+PMSG(10IU/mL)+hCG(10 IU/mL))及添加0.01、0.05、0.1、2μg/mL STH组(P<0.05),在本实验中为猪卵母细胞体外成熟的最佳条件。     

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.     

表皮生长因子和胰岛素样生长因子对水牛卵母细胞胞质成熟的影响

以皮层颗粒（CG）单层分布于质膜下做为卵母细胞胞质成熟的标志,利用CG荧光染色法,研究了不同浓度表皮生长因子（EGF）和胰岛素样生长因子（IGF-1）及其组合对水牛卵母细胞胞质成熟的影响。结果发现：（1）添加不同浓度的EGF（10、20、30、50ng／mL）都可以提高胞质的成熟率,但是组间差异不显著（P〉0．05）;皮质颗粒的分布随着EGF浓度的升高逐步由中间分布向皮层分布转变;（2）在成熟液中添加IGF-130ng／mL时效果较好,能显著提高卵母细胞胞质的成熟率;皮质颗粒的分布随着IGF-1浓度的升高逐步由中间分布向皮层分布转变,在添加IGF一130ng／mL时,在皮层分布最好,随着IGF-1量的进一步增加,皮质颗粒又向中间分布转变;（3）添加20ng／mL EGF＋30ng／mL IGF-1组卵母细胞体外成熟率高于添加30ng／mL的IGF-1组,显著高于添加20ng／m LEGF组和对照组（P〈0.05）。由此表明,EGF和IGF-1对水牛卵母细胞体外成熟有协同作用。     

Improvement of cumulus-free oocyte maturation in vitro and its application to microinsemination with primary spermatocytes in mice

In micromanipulation experiments using immature oocytes, final ooplasmic maturation is often compromised because the oocytes are usually first freed from their nurturing cumulus cells. This study was undertaken to determine whether cumulus-free in vitro maturation (IVM) in mice could be improved by modifying IVM medium having defined components. Cumulus-free germinal vesicle (GV) stage oocytes were subjected to IVM in either alphaMEM medium, TYH medium, or a 1:1 mixture of the two (termed TaM). TYH medium produced a better maturation rate (181/196; 92.3%) than alphaMEM (184/257; 71.6%). However, alphaMEM supported better embryo development to the morula/blastocyst stage than TYH following in vitro fertilization (93.3% vs. 76.5%) or parthenogenetic activation (82.4% vs. 60.4%). Mitochondrial distribution in MII oocytes was diffuse following IVM in alphaMEM, but was aggregated with TYH. The maturation promoting factor (MPF) activity in MII oocytes was significantly higher in TYH than in alphaMEM (P<0.05). Oocytes cultured in TaM had intermediate characteristics and essentially resembled in vivo matured oocytes, with the mitochondrial distribution pattern being most typical of that condition. The highest rate of development from GV oocytes to full-term fetuses following in vitro fertilization and embryo transfer to foster mothers (23.8%) was obtained using TaM. When this IVM system was applied to MI oocytes injected with spermatocytes, offspring were first obtained without cytoplasmic replacement at MII. Thus, optimization of the culture medium can considerably improve the quality of cumulus-free oocyte IVM in mice.     

Proteomic-based identification of oocyte maturation-related proteins in mouse germinal vesicle oocytes

Oocyte proteins play an important role in oocyte maturation, fertilization and embryonic development. However, the protein composition of mouse germinal vesicle (GV) oocytes is still unclear. Using one-dimensional Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (1D SDS-PAGE) and Reverse-phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS), we constructed a protein profile of mouse GV oocytes. First, our proteomics profile identified 1,405 different proteins from 11,000 mouse GV oocytes lacking zona pellucida. Second, with detailed bioinformatics analysis, a group of proteins that play an essential role in oocyte maturation was screened. In addition, the expression and localization of suppressor of G2 allele of skp1(SUGT1, also called SGT1), heterogeneous nuclear ribonucleoprotein K (Hnrpk), Seruin, Cullin1(Clu1) and nuclear distribution protein C (Nudc) in mouse ovaries and early embryos were also captured and investigated in this study. Moreover, the protein profile was submitted to the Proteomics Identifications Database (PRIDE) and is available via ProteomeXchange with the identifier PXD014314. Our research provides valuable resources for the study of oocyte proteins and oocyte maturation and helps to clarify the mechanisms of oocyte maturation.     

Insights into the NAD+ biosynthesis pathways involved during meiotic maturation and spindle formation in porcine oocytes

Treatments that elevate NAD⁺ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD⁺ is vital during oocyte maturation. This study aimed to examine the influence of different NAD⁺ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid [2-HNA, nicotinic acid phosphoribosyltransferase (NAPRT) inhibitor], FK866 [nicotinamide phosphoribosyltransferase (NAMPT) inhibitor], or gallotannin [nicotinamide mononucleotide adenylyltransferase (NMNAT) inhibitor] and their respective NAD⁺ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 h of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD⁺ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with gallotannin and nicotinamide mononucleotide together showed improvements in spindle width, while treating oocytes with 2-HNA and nicotinic acid combined showed an improvement in both spindle length and width. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.     

Ultrastructural Characteristics of Non-matured and In Vitro Matured Oocytes Collected from Pre-pubertal and Adult Domestic Cat Ovaries

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4°C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38°C in a humidified environment of 5% O₂, 5% CO₂ and 90% N₂. Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.     

The roles of vitamin A for cytoplasmic maturation of bovine oocytes

Vitamin A is one of the micronutrients which have been implicated in cattle reproduction. In cattle, ingested vitamin A, mainly as beta-carotene (BC) from forages and retinol ester from formula feed, is metabolized and transported to the oocytes and cumulus-granulosa cells in ovarian follicles through binding to various interacting molecules. The active form of vitamin A, retinoic acid (RA), functions as a regulator of gene expression in these targets. Early research showed the positive effects of vitamin A supplementation on bovine fertility in artificial insemination, and several studies on effects of vitamin A metabolites used in other artificial reproductive techniques (ART), including superovulation, ovum pick up, and in vitro maturation culture have provided evidence for the specific roles of vitamin A in oocyte cytoplasmic maturation (acquisition of developmental competence of oocytes during their meiotic maturation period for the embryonic development after fertilization). BC may enhance cytoplasmic maturation by its antioxidant properties which cannot be replaced by RA. Furthermore, RA may promote cytoplasmic maturation of bovine oocytes via its modulatory effects on the gene expression of gonadotrophin receptors, midkine, cyclooxygenase-2, and nitric oxide synthase in cumulus-granulosa cells.