Isolation and culture of preantral follicles for retrieving oocytes for the embryo production: present status in domestic animals

The development of efficient ovarian preantral follicle (PF) isolation and culture systems provide a large number of oocytes for the manipulation and embryo production. It also helps for understanding the mechanisms of follicle and oocyte development. Isolation and culture protocols for PFs were developed for many domestic species like cattle, buffalo, sheep, goat, pig, horse, camel, dog and cats; however, embryo production from oocytes derived from in vitro grown PFs was reported only in pigs, buffalo, sheep and goat. The rate of oocyte maturation from PFs grown in vitro is low and requires considerable research. This paper presents an overview of isolation and culture systems of PFs that have been developed for domestic species (cattle, buffalo, sheep, goat, pigs, horse, camel, dog and cat) along with the current status of progress achieved in the direction of producing embryos using PFs as the source of oocyte in these species.     

Production and quality of bovine oocytes and embryos

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.     

NAD+/SIRT2途径调节衰老卵母细胞成熟质量的分子机制

卵母细胞成熟质量不仅是哺乳动物繁殖能力的基础，更能直接决定后代的优劣。卵巢早衰通常引起卵子数量和质量下降，如何提高其成熟质量成为生殖衰老领域的研究热点。近年来，烟酰胺腺嘌呤二核苷酸(NAD+)依赖性去乙酰化酶家族Sirtuins(SIRT1-7)在生殖衰老中的功能愈发受到关注，尤其抗衰老因子SIRT2的乙酰化底物直接与卵母细胞成熟事件相关。本文从乙酰化修饰调控卵母细胞衰老与成熟的全新视角，重点综述了NAD+/SIRT2通过减数分裂、能量代谢、线粒体氧化应激、线粒体质量控制等重要生理环节改善衰老卵母细胞成熟质量的最新研究进展，以期为提高老龄母畜的卵母细胞质量及延长繁殖利用年限提供新思路。     

Production and Quality of Bovine Oocytes and Embryos

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.     

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.     

Heterologous gap junctions between granulosa cells and oocytes in ayu (Plecoglossus altivelis): formation and role during luteinizing hormone-dependent acquisition of oocyte maturational competence

Gap junctions (GJs) are intercellular channels. Molecules with a molecular weight of 1 kDa or less can pass back and forth between adjacent cells through GJs. Communication between oocytes and the somatic cells that surround them via GJs is known to play key roles to initiate oocyte maturation in many vertebrates. However, little is known of the detailed functions of ovarian GJs during oocyte maturation in fish. The oocyte maturation of fish is induced by a maturation-inducing steroid (MIS). The sensitivity of oocytes to the MIS is known as oocyte maturational competence (OMC) and is induced by luteinizing hormone (LH). However, LH receptors are found on the surface of granulosa cells rather than oocytes. We therefore proposed that the LH signals received by granulosa cells were passed to oocytes via GJs. This review describes current knowledge of the role of GJs between granulosa cells and oocytes during the LH-induced acquisition of OMC in fish.     

Effects of maternal environment during gestation on ovarian folliculogenesis and consequences for fertility in bovine offspring

Mammals such as cattle, swine, sheep and humans are born with a highly variable number of ovarian follicles and oocytes in the ovaries that dwindle during ageing and are never replenished. This variation in the ovarian reserve is reflected in the numbers of antral follicles in the ovaries at all ages after birth. As numbers of follicles in ovaries are determined during gestation, the role of maternal nutrition and health during gestation (at time of ovarian development in their foetuses) has been investigated as factors that may impact oogonia proliferation and thus follicle numbers post-natally. These studies have found that both nutrition and health impact numbers of follicles in their offspring. The idea that numbers of follicles and oocytes in ovaries impact fertility is a long-held belief in reproductive biology. This has recently been tested in cattle, and it has been shown that cows with a relatively high number of antral follicles in ovaries have higher pregnancy rates, shorter calving to conception intervals and fewer artificial inseminations during the breeding season compared with cows with a lower number of follicles, and similarly, heifers with many follicles had higher pregnancy rates than those with fewer follicles. Studies summarized in this review highlight the importance of the maternal environment during gestation in determining the size of the ovarian reserve in their offspring and also the contribution of the ovarian reserve to subsequent fertility in cattle.     

Effects of melatonin on oocyte maturation in PCOS mouse model

The purpose of oocyte in vitro maturation is generation of mature oocytes that could support future development. Efforts have been made to enhance oocyte developmental competence by developing optimal culture conditions. The present study is conducted to determine melatonin effects on quality of polycystic ovarian syndrome (PCOS) oocytes when it has been added during in vitro maturation, and immature oocytes were cultured in defined conditioned medium with and without different melatonin concentrations. Melatonin could significantly improve nuclear maturation of PCOS oocytes (81.1% vs. 56.3%, P < 0.05 were achieved with 10^?6 mol/L concentration). Cleavage rate was significantly higher in 10^?5 mol/L concentration compared to untreated oocytes in PCOS (54% vs. 35%, respectively) and it was significantly higher with 10^?6 mol/L concentration in the control group, 55% versus 38%, compared to untreated oocytes. This study showed that melatonin has the potential to induce oocyte nuclear maturation and guarantee fertilization potential. © 2016 Japanese Society of Animal Science     

谷胱甘肽和活性氧对哺乳动物配子作用的研究进展

文章综述了近年来关于谷胱甘肽对哺乳动物精子、卵母细胞和胚胎早期发育的作用及活性氧对精子功能的影响。在雄性和雌性配子中,谷胱甘肽起着保护精子和卵母细胞免受氧化损害的作用。在卵母细胞减数分裂中谷胱甘肽具有维持纺锤体形态的作用,受精后,巯基在精核的形成中起着积极的作用,并促进早期胚胎发育到囊胚阶段。在卵母细胞成熟过程中,谷胱甘肽的浓度发生着变化。它的合成受促性腺激素的调节,在精子的发生过程中随着精子的成熟,谷胱甘肽的浓度逐渐降低,但活性氧对精子功能也有多方面的作用。文章对卵丘细胞中小分子巯基化合物在谷胱甘肽合成中的重要作用也进行了综述。     

Factors affecting cryopreservation of porcine oocytes

Recent improvements in cryopreservation of mammalian eggs enable the long-term preservation of female germ cells in several mammalian species. Nevertheless, cryopreservation of porcine oocytes is still considered as a challenge. Although the use of vitrification techniques result in reasonable survival rates, developmental competence of vitrified oocytes has been compromised. Alterations of zona characteristics, cytoskeleton, mitochondrial functions and antioxidant-defense ability caused by vitrification are among the most frequently observed malformations which may be responsible for the low developmental competence of cryopreserved porcine oocytes. Furthermore, in vitro maturation, fertilization and embryo culture technologies, which are indispensable for generating embryos from cryopreserved oocytes, generate high rates of abnormal fertilization (polyspermy) and additional stress in resultant embryos further compromising their developmental competence. As a result, embryo development of porcine cryopreserved oocytes is still at low level and to date no piglet has been produced from such oocytes. The aim of the present review is to summarize knowledge on viability and developmental competence of vitrified porcine oocytes and to give ideas for future perspectives for the improvement of porcine oocyte cryopreservation technology.     

谷胱甘肽对哺乳动物卵母细胞的影响

谷胱甘肽与哺乳动物卵母细胞之间关系密切,谷胱甘肽具有一种还原能力。谷胱甘肽参与保护雌性配子细胞免受氧化物的伤害,还参与维持卵母细胞减数分裂纺锤体形态。卵母细胞中谷胱甘肽浓度可以作为衡量哺乳动物卵母细胞质和核成熟程度的标志。它对于受精后雄原核形成起了积极的作用,并且还有助于早期胚胎发育。卵丘细胞对卵母细胞内的谷胱甘肽合成起重要作用。不同种类的低分子量硫醇复合物可以诱导卵母细胞中谷胱甘肽的合成。     

miRNA在哺乳动物配子发生中的作用研究进展

哺乳动物配子发生的基因表达调控包括编码基因的阶段特异性表达调控及非编码基因的转录和转录后水平调控。微小RNA(microRNA, miRNA)作为一类小的非编码RNA, 通过识别靶基因非翻译区的结合位点, 导致mRNA降解或者蛋白质翻译抑制, 从而在转录后水平发挥调控作用。近年来, miRNA在哺乳动物生殖活动中的作用逐渐被揭示, 越来越多的研究表明, miRNA在哺乳动物精子发生、精子成熟、卵母细胞成熟、卵泡发育及早期胚胎发育等过程中都发挥着重要的调节作用, 其可通过调节支持细胞的增殖和凋亡或精原细胞、精母细胞及精细胞的细胞周期进程, 在精子发生的不同阶段发挥间接或直接调控作用, 也可通过调节卵母细胞、卵丘细胞以及颗粒细胞的增殖、凋亡、激素合成和细胞间作用, 对卵母细胞的发育和成熟过程进行调控。作者主要介绍了在哺乳动物配子发生过程中, miRNA的细胞和阶段特异性表达及其对靶基因的调节作用, 以期为深入研究哺乳动物配子发生的调控机制提供参考。     

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.     

High-resolution ultrasonography of xenografted domestic cat ovarian cortex

Transplantation of ovarian tissue has high potential for female gamete conservation. However, optimal timing of oocyte recovery for in vitro maturation and fertilization is still critical. Therefore the aim of the present study was to use high-resolution transcutaneous ultrasonography to monitor follicular development within xenografted ovarian tissue. Ovarian cortex fragments (n=44) from domestic cats were transplanted into athymic nude rats (n=12). Graft development in the animals was assessed weekly by high frequency ultrasound (10-22 MHz) under two different FSH regimes. Blood collection for serum estradiol determination and vaginal smears were performed simultaneously. The xenografts were removed at different time points according to the ultrasound findings. The survival rate of the transplants 4 weeks after surgery was 54.5% and antral follicular growth was observed within 10 grafts from 5 different hosts (8.6 +/- 6.43 follicles per graft). Early follicle antrums could be detected from 0.4 mm onwards. The growth rate of the antral cavity was calculated from weekly measurements (0.56 +/- 0.44 mm per week). Although vaginal cells and estradiol levels followed a cyclic pattern, no correlation was found between follicular diameter, estradiol and keratinized vaginal cells. We recovered 5, 1 and 4 cumulus oocyte complexes from three different individuals during weeks 19, 21, and 23 respectively. Extrusion of a polar body (1 oocyte) and germinal vesicle break down (7 oocytes) indicated progression of maturation after in vitro culture. We conclude that ultrasonography und provided a reliable method to examine xenograft survival and follicular development within the grafts. Furthermore, this technique is suitable for assessment of the efficiency of hormonal treatment and narrowing of the optimal time frame for oocyte retrieval. To our knowledge, this is the first report of the in vivo development of early antral follicles in mammalian species.     

Bmp/Smad信号通路及其在哺乳动物卵巢发育中的作用

开展绵羊多羔性状基因的研究对于揭示其分子调控机制和提高绵羊繁殖力具有重要意义。骨形态发生蛋白(BMPs)为转化生长因子-β(TGF-β)超家族成员,与哺乳动物繁殖活动密切相关。研究表明,BMPs可促使原始卵泡向次级卵泡转化,对哺乳动物卵巢颗粒细胞增殖、生殖激素的合成和分泌以及卵母细胞成熟和排卵等方面起重要调节作用,而BMPs发挥功能主要依赖于经典的Bmp/Smad信号通路。本文就Bmp/Smad信号通路成员的表达、对早期胚胎发育的影响以及在哺乳动物卵巢发育中的作用等方面的研究进行总结,以期为进一步研究BMPs及其信号通路的调控机制提供参考。     

Cyclopamine did not affect mouse oocyte maturation in vitro but decreased early embryonic development

Hedgehog (Hh) pathway has been studied in various animal body life procedures and is suggested to be important for the development of multiple organs. The genes involved in the Hh signaling pathway were expressed in the ovary of mice, pigs and cattle. However, the function of Hh signaling pathway on oocyte maturation and early embryonic development is still controversial. We detected the effect of sonic hedgehog (Shh) and cyclopamine on the in vitro maturation of mouse oocytes and embryo development. The results showed that the presence of Shh or cyclopamine resulted in similar oocyte maturation to control groups. Shh did not improve early embryonic development. However, the supplement of cyclopamine depressed early embryo development. The mRNA of shh, ptch1, smo and gli1 were less detected in the denuded oocytes. The expression levels of ptch1 ascended from the uncleaved zygote to blastocyst stage. Smo or gli1 were expressed on a higher level at the two‐cell or four‐cell stage in early embryonic development separately. Therefore, Shh did not affect mouse oocyte maturation and early embryo development, but cyclopamine led to inhibited development of mouse early embryo. The effects of Hh signaling on the oocyte maturation and early embryo development might be species‐specific.     

Regulation of mitogen-activated protein kinase 3/1 activity during meiosis resumption in mammals

In vivo, resumption of oocyte meiosis occurs in large ovarian follicles after the preovulatory surge of luteinizing hormone (LH). The LH surge leads to the activation of a broad signaling network in mural granulosa cells equipped with LH receptors. The signals generated in the mural granulosa cells are further augmented by locally produced peptides or steroids and transferred to the cumulus cell compartment and the oocyte itself. Over the last decade, essential progress has been made in the identification of molecular events associated with the final maturation and ovulation of mammalian oocytes. All new evidence argues for a multiple roles of mitogen-activated protein kinase 3/1 (MAPK3/1) in the gonadotropin-induced ovulation processes. However, the knowledge of gonadotropin-induced signaling pathways leading to MAPK3/1 activation in follicular cells seems limited. To date, only the LH-induced transactivation of the epidermal growth factor receptor/MAPK3/1 pathway has been described in granulosa/cumulus cells even though other mechanisms of MAPK3/1 activation have been detected in other types of cells. In this review, we aimed to summarize recent advances in the elucidation of gonadotropin-induced mechanisms leading to the activation of MAPK3/1 in preovulatory follicles and cultured cumulus-oocyte complexes and to point out a specific role of this kinase in the processes accompanying final maturation of the mammalian oocyte.     

血清和促性腺激素对山羊卵母细胞核体外成熟的影响

试验研究了清和促性腺激素对山羊卵丘扩展和卵母细胞核成熟的,卵母细胞与卵丘扩展的关系以及卵丘扩展与卵母细胞核成熟的关系。结果表明：（1）培养24h，添加PMSG组卵母细胞核成熟率显著高于不加激素组（P＜0．05），而培养到27h，2者成熟率之间差异变得不显著，说明添加PMSG卵母细胞核的最终成熟率没有明显影响，但加速了卵母细胞核的成熟进程；（2）M199＋BSA培养27h，卵母细胞核成熟率显著高于培养24h，而M199＋FCS培养27h与培养24h的成熟差异不显著，说明添加BSA时，卵母细胞核体外成熟速度比添加FCS的慢；（3）卵丘扩展良好与扩展不好的卵母细胞核成熟率以及第1极体形态无统计学差异，说明山羊卵母细胞的核成熟可能不依赖于卵丘扩展；（4）山羊的卵丘扩展产不依赖于卵母细胞；（5）将带壁颗粒细胞与不带壁颗粒细胞的COC分开培养发现，2者卵母细胞核成熟度无明显差异，带有壁颗粒细胞的COC卵丘扩展情况明显优于一般COC。     

Cumulus–Oocyte Communications in the Horse: Role of the Breeding Season and of the Maturation Medium

Horse is a seasonal breeder and information on oocyte quality outside the breeding season is very limited. Ovaries obtained at the slaughterhouse are a convenient but often limited source of oocytes in this species. As the low quantity of ovaries leads to an intensive use of all available material, it would be useful to know whether ovaries collected during the non‐breeding season are suitable for in vitro maturation (IVM). In an attempt to characterize the effect of season on oocyte quality, we investigated the permeability of the gap junctions (GJ) present between cumulus cells and oocytes because of their important role in oocyte growth and maturation. We also compared the effect of supplementing the maturation medium with bovine serum albumin (BSA) or oestrus mare serum (EMS). A total of 645 oocytes isolated from 158 and 154 ovaries collected during the breeding and the non‐breeding season, respectively, were used in this study. Oocytes were matured for 30 h in TCM 199 supplemented either with 10% EMS or with 4 mg/ml BSA. The presence of permeable GJs between cumulus cells and oocytes was investigated with the injection of a 3% solution of the fluorescent dye Lucifer yellow into the ooplasm. No differences in efficiency of oocyte retrieval or oocyte meiotic competence were detected between oocytes collected during the breeding and non‐breeding season. The vast majority (90%) of the oocytes collected during the breeding season had fully functional communications with their surrounding cumulus cells but such communications were completely interrupted in 55.3% of the oocytes collected during the non‐breeding season. During the non‐breeding season, the proportion of oocytes whose communications with cumulus cells were classified as closed or intermediate at the end of maturation was lower in the group matured with BSA than with EMS (71.4 vs 97.7, p < 0.05). The same trend, although not statistically significant, was observed during the breeding season also. The presence of BSA caused an incomplete cumulus expansion during both seasons. Our data indicate that oocytes collected during the non‐breeding season do not show any meiotic deficiency but lack active communication with the surrounding cumulus cells at the time of their isolation from the ovary. No data are available at present for determining the consequences on the developmental competence even if data from other species suggest that this is likely.     

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.