Dose-dependent effects of gonadotropin on oocyte developmental competence and apoptosis

The aim of the present study was to evaluate the effect of gonadotropins (Gn) on oocyte maturation, developmental competence and apoptosis in an animal model. Bovine cumulus-oocyte complexes (COCs) were matured for 24 h in media supplemented with varying concentrations of Bravelle (B), B + Menopur (B+M) or B + Repronex (B + R) (Ferring Pharmaceuticals, Parsiappany, NJ, USA). Then, nuclear maturation, embryo development, and apoptosis in cumulus cells and oocytes were evaluated. Low to moderate Gn concentrations (75-75 00 mIUmL(-1)) effectively improved nuclear maturation and in vitro development. Higher concentrations of Gn (75 000 mIUmL(-1)) did not have any added beneficial effects and nuclear maturation and blastocyst rates in the presence of these concentrations were comparable to control (P>0.05). Most COCs showed slight apoptosis when exposed to 75, 750 and 75 00 mIUmL(-1) Gn; however, when the concentration was increased to 75 000 mIUmL(-1), the proportion of moderately apoptotic COCs increased. In conclusion, extremely high concentrations of Gn have detrimental effects on oocyte nuclear maturation and embryo development and increase apoptosis in cumulus cells, suggesting the importance of judicious use of Gn in assisted reproductive technologies (ART).     

In vitro maturation of oocytes

In vitro maturation (IVM) of human oocytes is an emerging assisted reproductive technology with great promise. To be successful, this process must entail both nuclear and cytoplasmic maturation. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by endocrine parameters, oocyte/follicular cross-talk, and intra-oocyte kinase/phosphatase interactions. Although nuclear maturation during human oocyte IVM progresses normally, cytoplasmic maturation is significantly lacking, as exemplified by poor embryonic developmental competence and pregnancy rates. Advances made in immature oocyte isolation and oocyte and embryo culture conditions have increased the clinical feasibility of IVM. However, in order to achieve acceptable birth rates, future studies should focus on characterization and regulation of oocyte cytoplasmic maturation, and how oocyte-derived factors influence zygotic genome activation and embryonic developmental competence.     

Follicle size and oocyte diameter in relation to developmental competence of buffalo oocytes in vitro

Follicular size, oocyte morphology and diameter were investigated for their possible relationship with in vitro developmental competence of buffalo oocytes. Cumulus oocytes complexes (COCs), aspirated from small (<3 mm), medium (3-8 mm) and large (>8 mm) follicles of normal ovaries and cystic ovarian follicles of abattoir-derived ovaries, were graded for their morphological appearance and were cultured to assess their developmental competence. The influence of cystic follicles on maturational competence of COCs recovered from co-existing follicles of cystic ovaries was studied. The mean diameter of oocytes from follicles of different size were examined, and the influence of oocyte diameter--(i) <126 microm; (ii) 127-144 microm; (iii) 145-162 microm; and (iv) >163 microm--on in vitro maturation, cleavage and embryo yield was studied. Results suggested that increased fertilization, cleavage and embryo development were significantly (P<0.05) higher in COCs aspirated from large follicles, followed by medium and small-sized normal follicles, and the presence of cystic follicles had no significant (P<0.05) effect on the maturation competence of the COCs recovered from co-existing follicles. The mean diameter of the buffalo oocyte obtained from normal ovaries was found to be 146.4 microm and the rate of blastocyst production in vitro was significantly higher (P<0.05) in oocytes with diameters greater than 145 microm. In conclusion, the larger the size of the follicles and oocytes, the greater the developmental competence in vitro of buffalo oocytes.     

Paradoxical effects of kisspeptin: it enhances oocyte in vitro maturation but has an adverse impact on hatched blastocysts during in vitro culture

Kisspeptin (Kp) is best known as a multifunctional peptide with roles in reproduction, the cardiovascular system and cancer. In the present study the expression of kisspeptin hierarchy elements (KISS1, GNRH1 and LHB) and their receptors (KISS1R, GNRHR and LHCGR, respectively) in porcine ovary and in cumulus-oocyte complexes (COCs) were investigated, as were its effects on the in vitro maturation (IVM) of oocytes and their subsequent ability to sustain preimplantation embryo competence after parthenogenetic electrical activation. Kp system elements were expressed and affected IVM of oocytes when maturation medium was supplemented with 10(-6)M Kp. Oocyte maturation, maternal gene expression (MOS, GDF9 and BMP15), blastocyst formation rate, blastocyst hatching and blastocyst total cell count were all significantly increased when oocytes were matured in medium containing Kp compared with the control group (without Kp). A Kp antagonist (p234) at 4×10(-6)M interfered with this hierarchy but did not influence the threshold effect of gonadotrophins on oocyte maturation. FSH was critical and permissive to Kp action on COCs by increasing the relative expression of KISS1R. In contrast, Kp significantly increased apoptosis, the expression of pro-apoptotic gene, BAK1, and suppressed trophoblast outgrowths from hatched blastocysts cultured on feeder cells. The present study provides the first functional evidence of the Kp hierarchy in porcine COCs and its role in enhancing oocyte maturation and subsequent developmental competence in an autocrine-paracrine manner. However, Kp supplementation may have a harmful impact on cultured hatched blastocysts reflecting systemic or local regulation during the critical early period of embryonic development.     

人卵丘细胞内基因表达与胚胎发育潜能的关系

缺乏准确、客观、非侵入性的胚胎评估标准是目前体外受精-胚胎移植技术所面临的主要挑战之一。本文对卵丘细胞内基因表达与胚胎及卵子发育潜能之间的相关研究进行了总结,发现卵丘细胞内特定基因的表达与胚胎发育、成熟及发育潜能之间存在紧密的联系。近年来,通过实时定量逆转录聚合酶链反应（qRT-PCR）及微阵列技术筛选出一系列颗粒细胞中能够预测受精结局、胚胎形态学评分、妊娠结局等胚胎发育潜能的候选标记基因,这些基因主要涉及卵丘扩展、脂类代谢、细胞凋亡等过程。然而,卵母细胞及胚胎的发育受卵泡内、外多种因素的影响。欲确立一种全面、准确、非侵入性的胚胎发育潜能评估方法仍需更深入的研究。     

Evaluation of oocyte quality: morphological, cellular and molecular predictors

Mounting evidence that oocyte quality profoundly affects fertilisation an d subsequent embryo development drives the continued search for reliable predictors of oocyte developmental competence. In the present review, we provide an overall summary and analysis of potential criteria that can be used to evaluate oocyte quality. These criteria are specifically classified as morphological and cellular/molecular predictors. Traditional methods for the evaluation of oocyte quality are based on morphological classification of thefollicle, cumulus-oocytecomplex, polar body and/or meiotic spindle. Although the use of morphological characteristics as predictors of oocyte quality is controversial, such a grading system can provide valuable information for the preselection of oocytes with higher developmental competence and, therefore, may maximise embryo developmental outcome. Several intrinsic markers (such as mitochondrial status and glucose-6-phosphate dehydrogenase 1 activity) and extrinsic markers (such as apoptosis of follicular cells and levels of the transforming growth factor-beta superfamily in follicular fluid or serum) have also been reported as useful indicators of oocyte competence and embryo quality. Compared with the morphological parameters, these cellular and molecular predictors of oocyte quality may prove to be more precise and objective, although further studies and refinement of techniques are needed.     

人卵母细胞玻璃化冷冻的分子表达变化

卵母细胞冷冻技术是体外受精助孕的重要组成部分,是保存女性生育力的主要途径,卵母细胞冷冻保存易对卵母细胞造成不可逆损伤。卵丘细胞对卵母细胞体外成熟过程具有重要作用,在冷冻过程也可能受损伤。目前已知玻璃化冷冻/解冻对卵母细胞（包括成熟与未成熟卵母细胞）的影响,可改变卵母细胞正常发育必需基因的表达,如卵母细胞成熟（GDF9、BMP15）、凋亡（Bax、Bcl-2）、细胞骨架（CK8、ACTB）、细胞周期（CCNA1、MCM10）、缝隙连接（GJA1）、发育能力（HDAC1、STAT3、SMARCAL1和DNMT3B）、脂质代谢（cGMP）等方面的改变。就卵母细胞玻璃化冷冻前后的分子及基因变化进行阐述,为卵母细胞玻璃化冷冻提供更好的理论基础和实验依据。     

BDNF对小鼠卵母细胞体外成熟及发育能力的影响

目的:观察脑源性神经营养因子(BDNF)对小鼠未成熟卵的体外成熟及发育能力的影响。方法:以α-MEM为基础培养液,添加不同浓度(0、1、5、10 ng/ml)的BDNF以及FSH、FBS培养小鼠未成熟卵,并进行体外受精,观察卵母细胞成熟率、受精率和胚胎发育至囊胚的能力,了解不同培养条件下BDNF对卵母细胞发育能力的影响。结果:当体外成熟培养液中含有FSH和10%的FBS时,与体外成熟对照组比较,BDNF虽然不影响卵母细胞的成熟率和受精率,但BDNF 5 ng/ml组的囊胚形成率(75.00%)显著高于体外成熟对照组(56.63%),而接近体内成熟组(76.92%);当培养液中仅含FBS时,各组间卵母细胞成熟率和受精率没有差异,但与对照组比较,BDNF显著提高囊胚形成率;当培养液中不含FBS、FSH时,虽然无囊胚形成,但BDNF显著提高了卵母细胞的受精率。结论:BDNF能促进小鼠未成熟卵胞质的发育,提高卵母细胞的发育能力。     

Oocyte developmental competence is reduced in sows during the seasonal infertility period

The modern domestic sow exhibits a period of impaired reproductive performance during the late summer and early autumn months, known as 'seasonal infertility'. A reduction in farrowing rate due to pregnancy loss is the most economically important manifestation of seasonal infertility. The aim of the present study was to determine whether there are changes in oocyte developmental competence associated with season. Ovaries were collected in pairs from sows sourced from commercial piggeries and slaughtered 4 days after weaning during winter and summer-autumn. Following oocyte IVM and parthenogenetic activation, the ability of oocytes from large follicles to form blastocysts was greater in winter (54.94 ± 6.11%) than in summer (21.09 ± 5.59%). During winter, the proportion of oocytes developing to the blastocyst stage from large follicles was significantly higher (54.94 ± 6.11%) than those oocytes from small follicles (23.17 ± 6.02%). There was no effect of season on the proportion of oocytes developing to the blastocyst stage from small follicles. There was no effect of follicle size on blastocyst formation from those oocytes recovered during summer. Blastocysts derived from small follicles during summer had the lowest number of cells (24.25 ± 1.48) compared with blastocysts derived from large follicles during winter (37.5 ± 1.3; P < 0.05). The mean progesterone concentration in follicular fluid collected from small follicles was greater in winter than summer (1235.55 ± 164.47 v. 701.3 ± 115.5 nmol L(-1), respectively; P < 0.001). The mean progesterone concentration in the follicular fluid of large follicles was also greater in winter than in summer (2470.9 ± 169.1 v. 1469.2 ± 156.5 nmol L(-1), respectively; P < 0.001). Regression analysis revealed a positive correlation between progesterone concentration and oocyte developmental competence. The results indicate that porcine oocytes fail to reach their full developmental potential during the period of seasonal infertility, suggesting that the pregnancy losses observed at this time of year may be due to reduced oocyte developmental competence.     

Recent insights into oocyte-follicle cell interactions provide opportunities for the development of new approaches to in vitro maturation

The last 5-10 years of research in ovarian and oocyte biology has delivered some major new advances in knowledge of the molecular and cellular processes regulating oocyte maturation and oocyte developmental competence. These new insights include, among others: (1) the knowledge that oocytes regulate granulosa and cumulus cell differentiation, ovulation rate and fertility via the secretion of soluble paracrine growth factors; (2) new perspectives on the participation of cyclic nucleotides, phosphodiesterases and gap junctions in the regulation of oocyte meiotic arrest and resumption; and (3) the new appreciation of the mechanisms of LH-induced oocyte maturation and ovulation mediated by the follicular cascade of epidermal growth factor (EGF)-like peptides, the EGF receptor and their intracellular second messengers. These recent insights into oocyte-follicle cell interactions provide opportunities for the development of new approaches to oocyte in vitro maturation (IVM). Laboratory IVM methodologies have changed little over the past 20-30 years and IVM remains notably less efficient than hormone-stimulated IVF, limiting its wider application in reproductive medicine and animal breeding. The challenge for oocyte biologists and clinicians practicing IVM is to modernise clinical IVM systems to benefit from these new insights into oocyte-follicle cell interactions in vivo.     

Relationship between donor animal age,follicular fluid steroid content and oocyte developmental competence in the pig

The developmental competence of oocytes recovered from the ovaries of slaughtered prepubertal and adult pigs was evaluated after in vitro maturation, parthenogenetic activation and culture in vitro. In addition, the effect of prepubertal and adult follicular fluid (FF) on the developmental competence of prepubertal and adult oocytes was investigated. When matured in adult FF, the rates of cleavage (92 v. 73% P < 0.01) and blastocyst formation (57 v. 38%; P < 0.05) were greater for adult oocytes than for prepubertal oocytes. Blastocysts derived from adult oocytes had more trophectoderm cells (43 v. 30; P < 0.05) and total cells (51 v. 36; P < 0.05) than blastocysts derived from prepubertal oocytes. The developmental competence of prepubertal oocytes was not affected by the FF donor age, whereas the developmental competence of adult oocytes was. Blastocysts derived from adult oocytes matured in adult FF had more trophectoderm cells (38 v. 24; P < 0.005), inner cell mass cells (7 v. 3; P < 0.01) and total cells (45 v. 27; P < 0.001) than blastocysts derived from adult oocytes matured in prepubertal FF. Characterization of the steroid content of the FF used to supplement the maturation medium revealed that adult FF contained more progesterone (42 v. 23 ng mL(-1); P < 0.005) and androstenedione (70 v. 16 ng mL(-1); P < 0.05) than prepubertal FF. In addition, the molar ratios of progesterone to androstenedione, androstenedione to 17beta-oestradiol and androstenedione to testosterone differed (P < 0.05) between prepubertal and adult FF. The results support the hypothesis that a greater proportion of adult oocytes than of prepubertal oocytes has completed 'oocyte capacitation'. The differences in FF steroid content are indicative of the different follicular environments from which the prepubertal and adult oocytes were isolated, and may be attributed to the observed effects on oocyte developmental competence.     

Ceramide and ceramide 1-phosphate in health and disease

Sphingolipids are essential components of cell membranes, and many of them regulate vital cell functions. In particular, ceramide plays crucial roles in cell signaling processes. Two major actions of ceramides are the promotion of cell cycle arrest and the induction of apoptosis. Phosphorylation of ceramide produces ceramide 1-phosphate (C1P), which has opposite effects to ceramide. C1P is mitogenic and has prosurvival properties. In addition, C1P is an important mediator of inflammatory responses, an action that takes place through stimulation of cytosolic phospholipase A2, and the subsequent release of arachidonic acid and prostaglandin formation. All of the former actions are thought to be mediated by intracellularly generated C1P. However, the recent observation that C1P stimulates macrophage chemotaxis implicates specific plasma membrane receptors that are coupled to Gi proteins. Hence, it can be concluded that C1P has dual actions in cells, as it can act as an intracellular second messenger to promote cell survival, or as an extracellular receptor agonist to stimulate cell migration.     

In vitro maturation of porcine oocytes with retinoids improves embryonic development

In the present study, the effects of retinoid metabolite administration during in vitro maturation (IVM) on oocyte maturation, parameters of in vitro fertilisation (IVF) and embryo development were examined. Varying concentrations of 9-cis retinoic acid (RA; 0, 5, 50 and 500 nm; Experiment 1) and all-trans retinol (ROH; 0, 125, 1250 and 12 500 nm; Experiment 2) were included in the maturation medium. Cumulus-oocyte complexes were matured in vitro and inseminated with frozen-thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess IVF parameters or for 7 days to assess embryo development and quality. In Experiment 1, the oocyte maturation rate to metaphase II was significantly decreased (P < 0.001), with values below 5%, in the presence of the highest concentration of RA (500 nm). However, 5 and 50 nm RA had no effect compared with control. Treatment with 5 nm RA improved the blastocyst development rate (P < 0.001). In Experiment 2, the oocyte maturation rate did not differ between 125 and 1250 nm ROH treatment groups and control. However, treatment with 12 500 nm ROH was deleterious because no matured oocytes were observed following the treatment. The penetration rate was lower in the group treated with 1250 nm ROH compared with the 125 nm ROH-treated and control groups, but the blastocyst formation rate did not differ among the three groups. In conclusion, 5 nm RA in the IVM medium significantly increased the blastocyst formation rate, suggesting that RA may play an important role during IVM.     

辅助生殖技术中卵母细胞线粒体功能对胚胎发育的影响

提高辅助生殖技术（ART）成功率和安全性是目前ART面临的重要课题。线粒体是卵母细胞/胚胎中数量最多的细胞器,不仅为卵母细胞的成熟、受精以及随后的胚胎发育过程中的转录与翻译提供能量,还与卵母细胞激活、钙振荡、凋亡程序启动等生命过程密切相关,而且胚胎中的线粒体全部来源于卵母细胞。线粒体功能的损伤也与卵母细胞老化、受精率及胚胎质量降低等密切相关。近年关于人和动物卵母细胞线粒体与ART的研究阐述了线粒体在卵母细胞成熟、受精以及胚胎发育过程中的作用、各种ART手段对卵母细胞线粒体功能的影响以及可能的线粒体保护手段。     

Caffeine and dithiothreitol delay ovine oocyte ageing

The intracellular glutathione levels and developmental competence of aged oocytes after parthenogenetic activation, somatic cell nuclear transfer and intracytoplasmic sperm injection in the presence or absence of caffeine or dithiothreitol (DTT) were examined. The following results were found: (1) ovine oocytes were fully aged 30 h post-onset of maturation culture; (2) the appropriate concentrations of caffeine and DTT for oocyte culture were 5 mM and 1 mM, respectively; (3) when nuclear transfer-reconstructed embryos were treated with caffeine or DTT following fusion, no increase in the frequency of development to blastocyst was observed (P > 0.05), but the cell numbers of blastocysts increased (P < 0.05); (4) both caffeine and DTT increased the blastocyst formation rates of intracytoplasmic sperm-injected embryos (P < 0.05); (5) caffeine increased the glutathione content of aged oocytes (P < 0.05). The glutathione content of DTT-treated aged oocytes was higher than that of oocytes matured for 36 h (P < 0.05). In conclusion, caffeine and dithiothreitol delay oocyte ageing but only to a limited extent.