Relationship between DNA fragmentation and nuclear status of in vitro-matured porcine oocytes: role of cumulus cells

The present study was conducted to investigate the effects of the attachment of cumulus cells to oocytes and coculture with cumulus cells during maturation culture on the nuclear status and DNA fragmentation of porcine denuded oocytes (DOs). In the first experiment, cumulus cells were removed from cumulus-oocyte complexes (COCs) at 0, 8, 16, 24 or 32 h after the onset of maturation culture and the DOs were then cultured in their original droplets until 42 h of culture was reached. In the second experiment, all COCs were denuded before the onset of culture and the DOs were cocultured with their removed cumulus cells. The DOs were transferred into fresh medium at 0, 8, 16, 24 or 32 h after the onset of coculture with cumulus cells and then cultured until 42 h of culture was reached. After culture, DNA fragmentation and the nuclear status of oocytes were examined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) method. When the DOs were returned to the same droplets after removal of the cumulus cells, the removal of the cumulus cells after 16 h of culture significantly decreased the proportion of oocytes remaining at the germinal vesicle (GV) stage. However, coculture treatment of DOs in the presence of their removed cumulus cells had no significant effects on the GV breakdown (GVBD) of oocytes. There were no significant differences in the proportion maturing to MII oocytes among the groups following removal of cumulus cells after the onset of maturation culture; however, DOs cocultured with cumulus cells until the end of maturation culture exhibited an increased maturation rate compared with DOs cocultured for 8 and 16 h. The total proportion of TUNEL-positive oocytes of oocytes remaining at the GV stage was higher than that of oocytes reaching other stages, irrespective of the removal of cumulus cells and coculture treatments. However, coculture for more than 16 h decreased the total proportion of TUNEL-positive oocytes. Our results indicate that the attachment of cumulus cells to oocytes may have a critical role for oocytes undergoing GVBD and that coculture with cumulus cells promotes the ability of oocytes to complete maturation. Moreover, coculture with cumulus cells may assist the oocyte to avoid undergoing DNA fragmentation.     

Bovine in vitro oocyte maturation as a model for manipulation of the gamma-glutamyl cycle and intraoocyte glutathione

Glutathione (GSH) is the main non-enzymatic defence against oxidative stress and is a critical intracellular component required for oocyte maturation. In the present study, several modulators of intracellular GSH were assessed for their effect on the in vitro maturation (IVM) and intracellular GSH content of bovine metaphase (MII) oocytes. Of the five GSH modulators tested, only the cell-permeable GSH donor glutathione ethyl ester (GSH-OEt) significantly increased the GSH content of IVM MII oocytes in a concentration-dependent manner without adversely affecting oocyte maturation rate. The GSH level in IVM MII oocytes was greatly influenced by the presence or absence of cumulus cells and severely restricted when oocytes were cultured in the presence of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. The addition of GSH-OEt to cumulus-denuded or BSO-treated oocytes increased the GSH content of bovine MII oocytes. Supplementation of the maturation medium with bovine serum albumin (BSA) or fetal calf serum (FCS) affected the GSH content of IVM MII oocytes, with greater levels attained under BSA culture conditions. The addition of GSH-OEt to the maturation medium increased the GSH content of IVM MII oocytes, irrespective of protein source. Spindle morphology, as assessed by immunocytochemistry and confocal microscopy, displayed distinct alterations in response to changes in oocyte GSH levels. GSH depletion caused by BSO treatment tended to widen spindle poles and significantly increased spindle area. Supplementation of the IVM medium with GSH-OEt increased spindle length, but did not significantly alter spindle area or spindle morphology. GSH-OEt represents a novel oocyte-permeable and cumulus cell-independent approach for effective elevation of mammalian oocyte GSH levels.     

Temporal effects of exogenous oocyte-secreted factors on bovine oocyte developmental competence during IVM

We investigated whether paracrine signalling between the bovine oocyte and cumulus cells is altered during the course of in vitro maturation (IVM). Bovine COCs were cocultured with denuded oocytes or treated with specific oocyte-secreted factors, namely recombinant bone morphogenetic protein (BMP)-15 or growth differentiation factor (GDF)-9, beginning from 0 or 9h IVM. To generate a 9-h denuded oocyte (DO) group, COCs were cultured intact for the first 9h of IVM and then denuded. Coculturing intact COCs with DOs denuded immediately after collection or following 9h of maturation did not affect cleavage rate, but improved blastocyst yield (P<0.05) on Day 8 (51 and 61%, respectively; P<0.05) and cell number compared with COCs cultured alone (41%). Significantly, we observed higher levels of endogenous GDF-9 and BMP-15 protein in oocytes of COCs matured for 9h compared with no incubation. The addition of 175 ng mL(-1) GDF-9 or 10%v/v BMP-15 from partially purified transfected 293H cell supernatant for 24h IVM significantly enhanced development to the blastocyst stage from 40% (control) to 51 and 47%, respectively (P<0.05). However, treatment of COCs with GDF-9 or BMP-15 between 9 and 24h of IVM did not increase blastocyst yield. These results provide evidence of quantitative and possibly qualitative temporal changes in oocyte paracrine factor production during IVM.     

Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells

This paper describes the use of the protein synthesis inhibitor cycloheximide (CHX) to synchronize nuclear progression during meiotic maturation in porcine oocytes, and also the time-dependence of nuclear maturation on exposure of the oocyte to cumulus cells. Prior to culture, the majority of oocytes were at the germinal vesicle (GV) stage (95-100%), but distributed from GVI to GVIV (GVI 56.1 +/- 9.1%, GVII 15.3 +/- 1.4%, GVIII 21.5 +/- 7.1%, GVIV 7.1 +/- 3.5%). During culture of cumulus-enclosed oocytes (COCs) from 12 h to 48 h in a conventional culture system, all meiotic stages were represented at any time point examined, with 63.6 +/- 4.2% of oocytes maturing to metaphase II (MII). Cycloheximide blocked the progression of nuclear development in a dose-dependent manner. Treatment for 12 h with CHX at 1-25 microg mL(-1) resulted in 95-100% oocytes being arrested and synchronized at GVII. With >5 microg mL(-1) CHX, all oocytes were arrested before germinal vesicle breakdown (GVBD) (mostly at GVIII) by 24 h. A 12 h preincubation with 5 microg mL(-1) CHX followed by 24 h of further culture without CHX resulted in >80% of oocytes maturing to MII. The profile of nuclear progression during maturation revealed discrete peaks of occurrence of different meiotic stages, with GVBD at 6-12 h, metaphase I (MI) at 10-18 h and anaphase I/telophase I at 16-20 h. After 12 h preincubation with 5 microg mL(-1) CHX, denuded oocytes (DOs) matured to MI as COCs. However, DOs matured to MII as normal when denuded at MI. In conclusion, CHX not only efficiently blocks and synchronizes the meiotic progression of porcine oocytes at a specific GV stage, but it also effectively synchronizes subsequent meiotic progression to MII, resulting in discrete peaks of occurrence of different meiotic stages. Using this technique, the study showed that cumulus cells are essential for oocytes to mature from MI to MII but exposure to cumulus cells must occur before MI.     

Enhancement of lipid metabolism with L-carnitine during in vitro maturation improves nuclear maturation and cleavage ability of follicular porcine oocytes

The aim of the present study was to assess the effects of L-carnitine, an enhancer of lipid metabolism and mitochondrial activity, during in vitro maturation (IVM) on nuclear maturation and in vitro fertilisation of porcine follicular oocytes and subsequent embryo development. Mitochondrial functions, intracellular lipid content and reactive oxygen species (ROS) levels in oocytes were also investigated. L-carnitine supplementation in 0.6-5mgmL(-1) concentration during IVM significantly improved (P<0.05) the rates of metaphase-II (MII) stage oocytes compared with the control; however, fertilisation rates and monospermy were not improved. Although supplementation of IVM medium with L-carnitine significantly increased oocyte cleavage (P<0.05), further development to the blastocyst stage was not improved. The density of active mitochondria was significantly higher and the density of lipid droplets was significantly lower (P<0.05) in L-carnitine-treated oocytes compared with the control. Furthermore, the ROS levels in L-carnitine-treated oocytes were significantly lower than those in the control. In conclusion, enhancing mitochondrial functions by L-carnitine improved oocyte maturation and cleavage underlining the importance of lipid metabolism for nuclear and cytoplasmic maturation of porcine oocytes.     

Follicular fluid supplementation during in vitro maturation promotes sperm penetration in bovine oocytes by enhancing cumulus expansion and increasing mitochondrial activity in oocytes

The aim of this study was to examine the effects of bovine follicular fluid (bFF) on mitochondrial activity in in vitro-matured (IVM) oocytes and to assess its importance for fertilisation and embryo development. Bovine follicular oocytes were subjected to IVM in medium supplemented either with polyvinylpyrrolidone, bovine serum albumin, calf serum or bFF. Nuclear maturation, cumulus expansion, mitochondrial distribution and ATP content in oocytes were compared between groups along with subsequent in vitro fertilisation (IVF) and embryo development. Compared with other supplements, bFF generated significantly enhanced re-distribution of active mitochondria in oocytes and this effect was associated with elevated intracellular ATP content. Furthermore, bFF significantly improved cumulus expansion, which was associated with improved fertilisation rates when cumulus-enclosed oocytes were subjected to IVF; however, its promoting effect was neutralised when denuded oocytes were inseminated. Elevating ATP content in oocytes by bFF did not affect maturation or embryo development but promoted fertilisation when mitochondrial electron transport was blocked in oocytes before IVF by Rotenone. In conclusion, supplementation of IVM medium with bFF promotes sperm penetration both by the improvement of cumulus expansion and by enhancing ATP levels in oocytes, which maintains their ability to be fertilised after mitochondrial stress.     

Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development

During cumulus-oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-L-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.     

添加两种不同培养成分对人未成熟卵体外成熟培养的影响

目的:研究添加两种不同培养成分对人未成熟卵母细胞体外成熟培养(IVM)的效果。方法:选择接受ICSI治疗的77名不孕患者共收集未成熟卵177枚,随机分A组(激素组)33例,添加0.075 IU/ml FSH及0.075 IU/ml LH至基础IVM培养液中;B组(成熟卵泡液组)44例,50%基础IVM培养液与50%人成熟卵泡液配成。将两组收集的未成熟卵母细胞放入不同培养液中培养48 h,每24 h倒置显微镜下观察卵母细胞的形态;对MII卵进行ICSI,ICSI操作后继续培养,ICSI后16～20 h进行受精观察,24及48 h分别进行卵裂观察及胚胎评分。结果:①B组未成熟卵母细胞48 h成熟率和MI期卵母细胞48 h成熟率显著高于A组,差异有统计学意义(P<0.05),两组培养液GV期卵母细胞24和48 h成熟率比较差异无统计学意义(P>0.05)。②B组2PN卵裂率显著高于A组,差异有统计学意义(P<0.05),两组2PN受精率及可利用胚胎率差异无统计学意义(P>0.05)。结论:人成熟卵泡液内可能含有一些未知的有利于卵母细胞成熟的因子,深入研究卵泡液的成分,进一步明确其促卵母细胞成熟机制对开发新的IVM培养液、改善IVM的临床结局有重要意义。     

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.     

不同方法冻融小鼠未成熟与成熟卵母细胞的研究

目的:探讨不同冷冻方法对小鼠生殖泡(GV)期与成熟期(MⅡ)卵母细胞体外成熟、体外受精及胚胎发育能力的影响。方法:收集ICR小鼠GV期与MⅡ期卵母细胞,分别行程序化冷冻(慢速冷冻-快速解冻)与玻璃化冷冻。解冻后比较各组成熟率、受精率、卵裂率及8细胞胚胎形成率。结果:采用玻璃化冷冻法冷冻GV期卵母细胞(Ⅰ组),解冻后存活率达84.2%,显著高于MⅡ期卵母细胞(Ⅱ组)60.5%及程序化冷冻组(Ⅲ组)56.9%、(Ⅳ组)55.7%(均P<0.01)。各组成熟率、受精率、卵裂率及8细胞胚胎形成率比较,差异无统计学意义(P>0.05)。结论:在进行卵母细胞冷冻保存时,宜选择玻璃化冷冻法,且GV期卵母细胞冷冻效果好于MⅡ期卵母细胞。     

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 microm cysteamine. Supplementation of IVM medium with 50 microm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 microm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 microm (P < 0.05) cysteamine, whereas 200 microm cysteamine was ineffective. Supplementation of both IVM medium with 50 microm cysteamine and of IVC medium with 100 microm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.     

Effect of hexoses and gonadotrophin supplementation on bovine oocyte nuclear maturation during in vitro maturation in a synthetic follicle fluid medium

In vitro oocyte maturation (IVM) culture conditions have been relatively unchanged over the past few decades and remain suboptimal. In contrast, studies of the in vivo environment have led to significant improvements to in vitro embryo culture technologies. The aim of the present study was to determine the effect of maturing bovine cumulus-oocyte complexes (COCs) in medium based on the composition of bovine follicular fluid (Bovine VitroMat; Cook Australia, Eight Mile Plain, Qld, Australia). In particular, the effect of different glucose concentrations and glucosamine supplementation on meiotic maturation was determined. Culturing COCs in the presence of gonadotrophins in Bovine VitroMat, containing either physiological glucose concentrations (2.3 mM) or 5.6 mM (equivalent to levels in Tissue Culture Medium 199 (TCM199)) supplemented with glucosamine resulted in comparable cumulus expansion to COCs cultured in TCM199 plus gonadotrophins. However, nuclear maturation was 1.3-fold lower in Bovine VitroMat cultures containing 2.3 mM glucose compared with 5.6 mM glucose and this effect was independent of glucosamine supplementation. Investigations into the effects of different glucose concentrations and gonadotrophin supplementation during the initial 6 h of maturation demonstrated that COCs cultured in Bovine VitroMat with 5.6 mM glucose without gonadotrophins had a twofold acceleration of the rate of meiotic resumption, yet the rate of polar body formation was decreased by approximately 20% compared with cultures in 2.3 mM glucose and TCM199. However, this effect was not seen when COCs were cultured for the initial 16 h in Bovine VitroMat + 5.6 mM minus gonadotrophins or in Bovine VitroMat + 2.3 mM glucose +/- gonadotrophins. These data demonstrate that glucose concentrations and the timing of the introduction of gonadotrophin during IVM have variable effects on nuclear maturation. Manipulation of glucose concentrations may be a mechanism to influence oocyte meiotic progression and may lead to the development of improved IVM systems, allowing for an increased developmental capacity of bovine oocytes.     

Effect of homologous follicular fluid from medium-sized and large follicles on in vitro maturation of equine cumulus-oocyte complexes

The in vitro maturation (IVM) of equine oocytes is typically performed using various synthetic media; however, an optimal IVM system for equine oocytes has not been developed. The aim of the present study was to evaluate the effects of two types of follicular fluid (FF) obtained from cyclic mares and two incubation intervals for the IVM of equine cumulus-oocyte complexes (COCs). Follicular fluid was collected from medium-sized (20-29 mm diameter) and large (e30 mm; post-human chorionic gonadotrophin administration) follicles using transvaginal ultrasound-guided follicle aspiration. Compact (n = 232) and non-compact (n = 183) COCs obtained from a slaughterhouse were incubated separately in the following groups: (1) FF from medium follicles for 24 h; (2) FF from large follicles for 24 h; (3) control (synthetic) medium for 24 h; (4) FF from medium follicles for 24 h then FF from large follicles for an additional 24 h; (5) FF from large follicles for 48 h; and (6) control medium for 48 h. For compact COCs, there was a tendency (P = 0.06) for more COCs incubated in FF from large follicles for 24 h to reach metaphase II compared with those incubated in control medium for 24 h (58% v. 35%, respectively). More (P < 0.05) compact COCs had degenerated after incubation in control medium for 48 h compared with all other groups (51% v. 14-24%, respectively). For non-compact COCs, incubation in FF from medium follicles for 24 h resulted in more (P = 0.05) COCs at metaphase II compared with control medium for 48 h (58% v. 29%, respectively). These results indicate that homologous FF from cyclic mares is a suitable alternative for the IVM of equine COCs and that it may be superior to conventional media for longer (i.e. >24 h) incubation intervals.     

Effect of serum on the in vitro maturation of canine oocytes

The meiotic competence of canine oocytes cultured for 72 h in medium supplemented with three different concentrations (5, 10 and 20%) of anoestrous, oestrous or metoestrous bitch serum, or with 0.3% bovine serum albumin (BSA), was examined. The oestrous serum supplement had a positive effect on the resumption of meiosis, compared with the other supplements (P<0.05). The number of oocytes that reached metaphase I (MI) and metaphase II (MII) was significantly higher (P<0.05) with the oestrous serum supplement than with the anoestrous serum supplement. There were no significant differences among the three different concentrations in each serum type with respect to the proportion of oocytes that completed meiosis (MI to MII). The number of oocytes that resumed meiosis in the 10% oestrous serum supplement was significantly higher (P<0.05) than those of each concentration of the anoestrous and metoestrous serum supplements, and of the 0.3% BSA supplement. Moreover, a higher number of oocytes reached MII in the presence of the 10% oestrous serum supplement than with the 10% anoestrous serum supplement. These results suggest that supplementation of the culture medium with 10% oestrous serum is the optimal treatment for in vitro maturation of canine oocytes.     

OPS法玻璃化冷冻未成熟卵母细胞的效果观察

目的:探讨OPS(open pu lled straw,开放式拉细麦管)法玻璃化冷冻技术冻存未成熟卵母细胞的效果。方法:小鼠GV期卵母细胞用OPS法玻璃化冷冻复苏后行体外成熟培养(in-vitro m aturation,IVM)或直接行IVM,所获成熟卵行体外受精(IVF)和胚胎的体外培养(IVC)。以体内成熟卵行IVF/IVC作为in-vivo对照组。结果:GV期卵冻融后65.11%存活,IVM后成熟率为52.86%,其成熟率显著低于IVM对照组。冻融卵成熟后受精、卵裂率(33.78%、76.00%)低于IVM对照组和in-vivo对照组,培养后未见囊胚形成。结论:OPS法玻璃化冷冻技术可有效冻存GV期卵母细胞,复苏后可发育成熟,但卵的受精和继续发育能力欠佳。     

Timing and regulatory aspects of oocyte maturation in vitro in the tammar wallaby (Macropus eugenii)

Oocytes from a marsupial, the tammar wallaby (Macropus eugenii), resemble those of eutherian mammals in their ability to resume meiosis in vitro when cultured under suitable conditions. Culture for 42-48 h in Eagle's minimum essential medium (EMEM) supplemented with 10% fetal calf serum, and 10 microg mL(-1) porcine luteinizing hormone (pLH) was required in order for oocytes, collected from the large antral follicles (> 2 mm diameter) of tammar wallabies (primed with 6 mg of porcine follicle stimulating hormone twice daily for four days), to proceed to metaphase II (MII) of meiosis. Under these conditions, chromatin condensation was observed within 4-8 h of culture in 61% of oocytes; metaphase I (MI) chromosomes were observed from 18-30 h of culture (66%); and most oocytes (76%) progressed to MII by 42 h in vitro. The addition of cycloheximide, a protein synthesis inhibitor, at concentrations of 1-100 microg mL(-1), prevented maturation of tammar wallaby oocytes in vitro. This effect was reversible, as oocytes washed free of cycloheximide after 4 h of incubation were able to progress to MII. The addition of cycloheximide to wallaby oocytes at MI of meiosis prevented normal progression to MII suggesting that proteins critical for nuclear maturation are synthesized throughout the maturation process. Genistein, a protein kinase inhibitor decreased maturation of wallaby oocytes in a dose dependent manner. However, the concentration required to significantly inhibit maturation of wallaby oocytes (60 microg mL(-1)) was greater than that required for eutherian species. Most wallaby oocytes were able to undergo germinal vesicle breakdown (GVBD) in the presence of high concentrations of genistein but produced abnormal chromatin configurations and were unable to progress to MII. Future studies will examine whether cytoplasmic changes occur in marsupial oocytes in vitro and their temporal relationship to nuclear maturation.     

玻璃化冷冻法在未成熟卵子冷冻技术中的应用

目的:探讨玻璃化冷冻法在未成熟卵子冷冻方面的应用价值。方法:选取GV期卵母细胞744枚,随机分为5组,A组(128枚),GV期卵子直接进行未成熟卵母细胞体外培养(IVM);B组(220枚),GV期卵子直接行玻璃化冷冻;C组(155枚),GV期卵子直接行IVM,待培养至MⅡ期,再行玻璃化冷冻;D组(112枚),GV期卵子去除冠-丘细胞,行玻璃化冷冻;E组(129枚),GV期卵子保留除冠-丘细胞行玻璃化冷冻。比较各组卵母细胞的存活率、成熟率、受精率、卵裂率及囊胚形成率。结果:与A组相比,B组和C组的成熟率、受精率、卵裂率和囊胚形成率均降低(P<0.05);B组与C组间存活率、成熟率、受精率、卵裂率和囊胚形成率无差异(P>0.05)。E组的成熟卵子率较D组提高(P<0.05),但两组在解冻卵子存活率、受精率、2细胞数和>2细胞数方面无差异(P>0.05)。结论:未成熟卵子的玻璃化冷冻保留冠-丘细胞可显著提高冻融效果。     

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.     

Effect of commercially available PMSG on maturation, fertilization and embryo development of buffalo oocytes in vitro

In vitro fertilization (IVF) technology provides an opportunity to produce embryos for genetic manipulation, embryo transfer and basic research in developmental physiology, and can be exploited for emerging biotechnologies such as transgenesis and cloning. In the present study, the effects of different concentrations of commercially available pregnant mare serum gonadotrophin (PMSG) (Folligon; Intervet, International B.V, Boxmeer, Holland) in oocyte culture media, on maturation, fertilization and embryonic development of buffalo oocytes in vitro were investigated. Oocytes aspirated from abattoir-derived ovaries were cultured in media containing TCM-199 + PMSG at 0, 2.5, 20, 30, 40 and 50 IU mL(-1) in presence or absence of steer serum (10%) for 24 h in a CO2 incubator. The maturation rate was assessed on the basis of degree of expansion of cumulus cells. The matured oocytes were inseminated with 9-10 x 10(6) spermatozoa mL(-1) in Brackett and Oliphant medium and the cleavage rate was recorded 40-42 h after insemination. Uncleaved oocytes were stained with aceto-orcein for evaluation of fertilization rates. The cleaved embryos were further cultured in TCM-199 + 10% steer serum on buffalo oviducal cell monolayer for 7 days. Maturation, fertilization, cleavage and embryonic development were significantly higher (P < 0.05) in oocytes cultured in TCM-199 + 10% steer serum supplemented with 40 and 50 IU PMSG mL(-1). It is concluded that commercially available PMSG can effectively be used in place of pure follicle-stimulating hormone for in vitro maturation of buffalo oocytes, making it cost effective for IVF studies.