卵泡内环境对猪卵泡卵体外成熟和发育的影响

研究卵泡内环境对猪卵母细胞体外成熟、受精及受精卵体外发育的影响。主要结果如下：直径≥5mm、4－4.9mm、3－3.9mm和2－2.9mm的卵泡卵母细胞体外成熟率分别为90.5%、89.7%、85.4%和67.4%,体外受精后,卵母细胞的发育能力随卵泡直径的增大而增强,直径≥5mm和4－4.9mm卵泡卵的2－细胞、3－4－细胞发育率显著高于直径2－2.9mm的卵泡卵（P＜0.05或0.01）。体外成熟培养36h、42h和48h,直径2－2.9mm卵泡卵的体外成熟率,体外受精后的卵裂率差异不显著（P＞0.05）。在体外成熟培养液中添加5％或15％的不同直径卵泡的卵泡液,各组间卵母细胞的体外成熟率,受精卵的体外发育率均无显著差异,结果表明：卵泡大小对猪卵母细胞体外成熟、受精及受精卵体外发育有重要影响。     

Meiotically incompetent and competent goat oocytes: timing of nuclear events and protein phosphorylation

The ability of mammalian oocytes to resume meiosis and to complete the first meiotic division is acquired sequentially during their growth phase. The acquisition of meiotic competence in goat oocytes has been previously correlated with follicular size (9). Since protein phosphorylation/dephosphorylation play a key role in oocyte maturation, it could be that in meiotically incompetent oocytes, such post-translational modifications are inadequate. The aim of this study was to analyze whether changes in oocyte proteins phosphorylation occurred during the acquisition of meiotic competence. For this propose, goat oocytes were divided into 4 classes according to follicular size and meiotic competence: Class A oocytes from follicles < 0.5 mm in diameter: Class B oocytes from follicles 0.5-0.8 mm; Class C oocytes from follicles 1-1.8 mm and class D oocytes from follicles > 3 mm. The protein phosphorylation patterns of these classes of oocytes were studied at different times of in vitro maturation. After 4h of culture, when all oocytes were in the germinal vesicle stage, only the oocytes from Class D displayed the phosphoproteins at 110 kD, 31 kD and around 63 kD. In contrast to Class D oocytes Classes B and C oocytes were partially competent to mature, they underwent germinal vesicle breakdown later than fully competent Class D oocytes and remained in early prometaphase I or in metaphase I, respectively. They exhibited the phosphoprotein changes that are associated with commitment to resume meiosis; but the changes occurred later than in Class D oocytes, which were fully competent to reach metaphase II. After 27 h of culture, the phosphorylation patterns of Class B, C and D oocytes were identical, whereas the meiotic stages reached were quite different. The phosphoprotein changes associated with oocyte maturation did not occur in meiotically incompetent Class A oocytes, which were blocked at the germinal vesicle stage. From these results it can be concluded that, at the GV stage, meiotically incompetent and competent goat oocytes display different patterns of protein phosphorylation. Once oocytes are able to resume meiosis they undergo specific phosphorylation changes, but whether these changes are markers or regulators of maturation events remains to be determined.     

山羊卵母细胞的减数分裂进程

The meiotic progression of goat oocytes from follicles of different diameters was investigated in this study. The results were summarized as follows: (1) The in vitro meiotic maturation capacity was different among oocytes from follicles of different diameters. And thus oocytes from < or = 0.5 mm follicles were unable to resume meiosis; oocytes from 0.8-1.2 mm follicles were capable to resume meiosis, but could develop only to MI stage (60% at 24 h); oocytes from 1.5-5 mm follicles had acquired full-meiotic maturation capacity and 91% of them developed to M II stage at 24 h of culture. (2) The percentage of oocytes with intact-germinal vesicles from 1.5-5 mm follicles decreased significantly during 2-8 h of in vitro maturation and the decrease was even more rapid during 4-6 h of culture (from 60% to 19%, p < 0.0005). The percentage of oocytes at M I-stage increased from 24% to 61% during 6-12 h of in vitro maturation, and it then decreased. By 24 h of culture, only 2% oocytes remained at M I-stage. Twenty one percent of the oocytes in this group developed to M II-stage at 16 h of culture, and by 24 h of culture, 91% were at M II-stage. (3) Statistic analysis of the meiotic progression (the duration of each cell cycle stage) of oocytes from 1.5-5 mm follicles showed that GV stage lasted from 0 to 3 h of culture, prometaphase-I stage was from 3.0 to 7.0 h, metaphase-I stage was from 7.0 to 14.6 h, anaphase-I/telophase-I was from 14.6 to 18.4 h and metaphase-II stage lasted from 18.4 to 24 h. (4) Whether the oocytes capable of GVBD and entrance of M I developed to M II, the timing of meiotic progression prior to M I was similar. In summary, our results provided necessary data for studies on the mechanisms and control of meiosis in mammalian oocytes.     

不同直径山羊卵泡卵母细胞的发育特征及体外发育能力

目的研究山羊卵巢表面不同直径卵泡卵母细胞的发育特征及体外发育能力,优化山羊早期胚胎体外生产系统。方法收集繁殖期和非繁殖期山羊卵巢,采集表面直径小于1.5 mm、1.5-2.5 mm、2.5-3.5 mm和大于3.5 mm4种卵泡卵母细胞,以Hoechst33342染色检查核发育阶段;同时,利用体外培养方法观察不同直径卵泡卵母细胞的成熟、受精和早期胚胎发育能力。结果直径小于1.5 mm卵泡卵母细胞主要处于GVI期;1.5-2.5 mm卵泡卵母细胞以GVⅠ、GVⅡ和GVⅢ期为主;2.5-3.5 mm卵泡卵母细胞在GVⅡ到GVⅣ期间平均分布;大于3.5mm卵泡卵母细胞主要为GVⅢ到GVBD期。体外培养实验发现,直径小于1.5 mm卵泡卵母细胞仅有个别能完成成熟和卵裂;大于1.5 mm卵泡卵母细胞具有核成熟能力,能完成成熟和受精,但1.5-2.5 mm卵泡卵母细胞的受精卵通常阻滞于4-8细胞期;当卵泡直径大于2.5 mm时,卵母细胞才能较好地支持胚胎继续发育,其桑/囊胚的比例达到30%以上。卵泡卵母细胞的发育特征和体外发育能力与动物所处的繁殖季节无关。结论山羊卵巢上直径大于1.5 mm卵泡卵母细胞具有核成熟能力,大于2.5 mm卵泡卵母细胞能支持早期胚胎继续发育。     

Germinal vesicle configurations and patterns of polypeptide synthesis of procine oocytes from antral follicles of different size, as related to their competency for spontaneous maturation

The cytogenetic configurations of germinal vesicle (gv) chromatin were analyzed for pools of porcine oocytes harvested from small (1.0-2.0 mm), medium (3.0-5.0 mm), and large (6.0-10.0 mm) antral follicles. Groups of oocytes from these follicular classes also were examined by high-resolution, two-dimensional, polyacrylamide gel electrophoresis to compare their patterns of polypeptide synthesis. The results show a high incidence of gross and cytogenetic degeneration among oocytes from small antral follicles as compared with those from medium or lage follicles. Pools of oocytes could be separated, on the basis of gross morphology and integrity of adherent granulosa cells, into two classes: "Type A" which appeared normal, and "type B" which appeared to be atretic. Among selected "type A" oocytes a particular chromatin configuration, termed "fibrous" characterizes the gv of oocytes from small follicles; whereas a different configuration, termed "diffuse," characterizes the gv of oocytes from large follicles. The patterns of polypeptide synthesis were markedly different for samples of "type A" oocytes of the three follicular classes; and the patterns for oocytes from medium and large follicles were more similar to each other than to patterns for oocytes from slall follicles. The incidences of maturational development beyond the gv stage in vitro were similar for "type A" oocytes from the three follicular classes (i.e., 66% to 82% maturation); although "type B" oocytes underwent maturation beyond the gv at a significantly reduced incidence (i.e., 20% to 29% maturation). "Type A" oocytes from large follicles completed maturation in vitro (i.e., underwent the first meiotic division) at a significantly higher incidence (55%) than did oocytes from small (11% to 20%) or medium (16%) follicles. Our findings are consistent with the hypotheses that a high proportion of oocytes from small antral follicles are atretic, and that a developmental program controls the molecular and cytogenetic changes occurring in porcine oocytes during follicular growth. These changes appear to be highly correlated with the acquisition of competency to complete maturation in vitro, and possibly also are required for normal fertilization and embryogenesis.     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

Effect of follicular size on in vitro developmental competence of oocytes and viability of embryos after transfer in the dromedary (Camelus dromedarius)

The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3-6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 microM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs (n=30) denuded, fixed and stained with aceto-orcein. In vitro fertilization was performed using fresh semen (0.5 x 10(6)spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 5% O(2) and 90% N(2). The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively (P>0.1). The rate of total cleavage (two cells to blastocyst stage) was greater (P<0.05) for oocytes originating from large follicles (72%; 116/162) than for those derived from small follicles (59%; 140/237). The percentage of fertilized oocytes reaching the blastocyst stage was 35% (57/162) and 20% (48/237) for oocytes collected from large and small follicles, respectively (P<0.05). The viability of in vitro-produced hatched blastocyst from the two groups (15 from 3 to 6mm follicle size and 22 from follicles >6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% (n=8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular development and this developmental ability translates into greater pregnancy rates after transfer of in vitro produced hatched blastocysts.     

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.     

Origin of bovine follicular fluid and its effect during in vitro maturation on the developmental competence of bovine oocytes

Protein supplementation during in vitro maturation can profoundly affect both the rate and overall efficiency of the maturation procedure. The present study was conducted to assess the ability of different concentrations (1, 5, and 10%) of bovine follicular fluid (bFF) to support in vitro maturation of oocytes and subsequent developmental capacity. The bFF was derived either from competent follicles ( > 8 mm) obtained by transvaginal recovery following superovulation or from a pool of small follicles (2-5 mm) from abbatoir-derived ovaries. Bovine oocytes were cultured for 24 h in synthetic oviduct fluid medium (m-SOF) supplemented with polyvinylpyrrolidone. Following fertilization and embryo culture, more oocytes (P < 0.05) reached the blastocyst stage when oocytes were cultured with 5% bFF from competent follicles (41 +/- 3.7%) compared with bFF derived from small follicles (16 +/- 2.9%). Estradiol and recombinant human follicle stimulating hormone added to the competent bFF during maturation acted in synergy to increase blastocyst production rate (P < 0.05); this blastocyst production rate (57 +/- 1.2%) was higher than those obtained with the addition of these two hormones to bFF derived from small follicles (26 +/- 2.9%). The quality of blastocysts obtained was reflected by inner cell mass (51.30 +/- 3.5 and 25.50 +/- 3.7) and trophectoderm cell numbers (99.72 +/- 2.5 and 94.80 +/- 4.7) for bFF from competent and small follicles, respectively. In conclusion, follicular fluid originating from competent follicles increased the developmental competence of abbatoir-derived oocytes.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Dynamics of meiosis and protein kinase activities in bovine oocytes correlated to prolactin treatment and follicle size

Oocyte developmental competence depends on the size of the original follicle and is affected by compounds like prolactin. We wished to investigate nuclear and cytoplasmic maturation of bovine oocytes correlated to their origin and response to prolactin treatment, by monitoring at frequent intervals meiotic configuration of chromosomes and activity of histone H1 and MAP-kinase. Bovine ovaries were obtained from a slaughterhouse and oocytes were recovered by follicle isolation. Oocytes (n = 1,397) with a compact cumulus were selected from small (2 to 3 mm) and large (4 to 5 mm in diameter) follicles and cultured up to 28 h in TCM 199+20% bull serum with or without 50 ng/mL bovine prolactin. Four groups of oocytes were formed: originating from small or large follicles, and treated or not treated with prolactin. At the scheduled time intervals for in vitro maturation, cumulus oocyte complexes from the 4 groups were randomly selected and the oocytes were analyzed for histone H1 and MAP-kinase, and for chromatin configuration. The first meiotic division took longer to complete in oocytes from large follicles (P < 0.01). Under the influence of prolactin the meiosis was prolonged in oocytes both from small and large follicles (P < 0.05). Histone H1 and MAP-kinases started to be activated at approximately the same time, around 6 h after beginning maturation. But after this time, significantly lower levels of both kinase activities were found in oocytes treated with prolactin, especially those treated during Meiosis I (P < 0.05). Our results indicate a correlation of chromatin configuration and histone H1/MAP-kinase activities.     

Meiotic competence of in vitro grown goat oocytes

The objective of the present study was to grow meiotically incompetent goat oocytes from early antral follicles in vitro and to render them competent to undergo germinal vesicle breakdown. Cumulus-oocyte complexes with pieces of parietal granulosa cells were isolated from follicles 0.35-0.45 mm in diameter using both mechanical and enzymatic methods. The cumulus-oocyte complexes were divided into two groups according to oocyte diameter (group A: < 95 microm; group B: > 95 microm) and cultured for 8 or 9 days on granulosa cell monolayers. Within 8 days of culture, the mean oocyte diameter increased from 86 +/- 0.4 microm to 95 +/- 0.7 microm in group Aand from 106 +/- 0.2 microm to 109 +/- 0.5 microm in group B. After 9 days of culture, the mean diameter of oocytes from groups A and B were 99 +/- 0.5 microm and 112 +/- 0.4 microm, respectively. The meiotic competence of oocytes grown in vitro was evaluated by in vitro maturation. Within 8 days of culture, only 3% of oocytes from group A and 6% of oocytes from group B acquired the ability to undergo germinal vesicle breakdown. After 9 days of culture, 7% of group A oocytes and 42% of group B oocytes were competent to resume meiosis. The expression of p34(cdc2) in oocytes grown in vitro was analysed by the western blot technique. During 9 days of culture, p34(cdc2) accumulated in both groups of growing oocytes, but its concentration was lower than in fully grown oocytes used as controls. The results showed for the first time that goat oocytes from early antral follicles can grow, accumulate p34(cdc2) and acquire the ability to resume meiosis, when cultured for 9 days on granulosa cell monolayers.     

Acquisition of meiotic competence in growing pig oocytes correlates with their ability to activate Cdc2 kinase and MAP kinase

Meiotic maturation of mammalian oocytes is under the control of cell cycle molecules Cdc2 kinase and MAP kinase (mitogen-activated protein kinase). In the present study, we investigated the relationship between the ability to activate Cdc2 kinase and MAP kinase and the acquisition of meiotic competence during pig oocyte growth. Growing and fully grown pig oocytes were collected from four groups of antral follicles of various diameters (A, 0.5-0.7 mm; B, 1.0-1.5 mm; C, 2.0-2.5 mm; D, 4.0-6.0 mm) and cultured in vitro. Fully grown oocytes from class D follicles, which have full competence to mature to metaphase II, had the ability to activate both Cdc2 kinase and MAP kinase. In contrast, growing oocytes from class A follicles, which have limited competence to resume meiosis, had no such ability. Cyclin B1 molecules did accumulate, however, with phosphorylated 35 and 36 kDa bands of p34cdc2 appearing in the cultured oocytes. Of the growing oocytes from class B follicles, 60% resumed meiosis but arrested at metaphase I. Some of the oocytes in this class were capable of activating Cdc2 kinase, although they did not appear to have established a MAP kinase-activating pathway or the ability to activate MEK. These results suggest that limited meiotic competence in growing oocytes from class A follicles is due to their inability to activate Cdc2 kinase and their incomplete MEK-MAP-kinase pathway, although the oocytes are capable of accumulating cyclin B1 molecules. During the final growth phase, pig oocytes acquire the ability to activate Cdc2 kinase and then establish the MEK-MAP-kinase pathway for full meiotic competence.     

A combination of EGF and IGF-I accelerates the progression of meiosis in bovine follicular oocytes in vitro and fetal calf serum neutralizes the acceleration effect

The effects of a combination of EGF and IGF-I (GFs) on the progress of meiosis and on their developmental competence were examined in cumulus-enclosed bovine oocytes. Exposure to GFs in serum-free, 0.3% PVP-containing maturation medium significantly (P<0.05) increased the frequency of oocytes with the first polar body (PB) at 16 h of culture and decreased those with PB at 20 h. The cleavage rates of PB-extruded oocytes after fertilization were not affected by treatment of GFs during maturation culture, and blastocyst yield was not improved by GFs treatment. Although replacement of PVP from GFs-containing medium with fatty acid-free BSA did not affect the timing of PB extrusion, replacement with 10% FCS neutralized the acceleration effects of GFs. Replacement for macromolecule in maturation medium did not improve blastocyst yield of PB-extruded oocytes after fertilization. These results indicate that the progression of meiosis in bovine oocytes with cumulus cells is accelerated by exposure to GFs in serum-free maturation medium but their developmental competence is not improved, and that the acceleration effects on the progress of meiosis is neutralized by the presence of FCS in maturation medium with no improvement of developmental competence after in vitro fertilization.     

Cultivation of mouse oocytes in vitro: the ability to resume meiosis

It is well established that fully grown oocytes from sexually mature mice are capable of resuming meiosis when cultured in vitro. However it has not been established at what stage during the growth the oocyte acquires the ability to mature. The present study was undertaken to determine the precise stage of growth at which the ability to resume meiosis is acquired in ICR strain mice. Oocytes of various sizes were isolated from the ovary by mechanical dissection. This method yielded about 30% growing and fully-grown oocytes from the ovary. Cumulus-free oocytes dissociated from the ovaries of 5-week-old mice were cultured in vitro in a chemically defined medium for 3 hr. None of the 61- to 65-microns-diameter oocytes resumed meiosis; however 81.8% of the 66- to 70-microns-diameter oocytes were able to resume meiosis. Fifty- to 60-microns-diameter oocytes recovered by enzymatic dispersal of the basement membrane of isolated growing follicles were unable to resume meiosis. Follicle-enclosed 50- to 60-microns-diameter oocytes did not resume meiosis even when cultured in a medium containing Ca-ionophore A 23187 or 8-bromoadenosine 3', 5'-cyclic monophosphate which are known to induce resumption of meiosis of oocytes in Graafian follicles. These results indicate that the ability to resume meiosis is acquired dramatically at a specific stage of oocyte growth.     

The ability to achieve meiotic maturation in the dog oocyte is linked to glycolysis and glutamine oxidation

We tested the hypothesis that meiotic competence of dog oocytes is tightly linked with donor follicle size and energy metabolism. Oocytes were recovered from small (<1 mm diameter, n = 327), medium (1–<2 mm, n = 292) or large (≥2 mm, n = 102) follicles, cultured for 0, 24, or 48 hr, and then assessed for glycolysis, glucose oxidation, pyruvate uptake, glutamine oxidation, and nuclear status. More oocytes (P < 0.05) from large follicles (37%) reached the metaphase‐II (MII) stage than from the small group (11%), with the medium‐sized class being intermediate (18%; P > 0.05). Glycolytic rate increased (P < 0.05) as oocytes progressed from the germinal vesicle (GV) to MII stage. After 48 hr of culture, oocytes completing nuclear maturation had higher (P < 0.05) glycolytic rates than those arrested at earlier stages. GV oocytes recovered from large follicle oocytes had higher (P < 0.05) metabolism than those from smaller counterparts at culture onset. MII oocytes from large follicles oxidized more (P < 0.05) glutamine than the same stage gametes recovered from smaller counterparts. In summary, larger‐sized dog follicles contain a more metabolically active oocyte with a greater chance of achieving nuclear maturation in vitro. These findings demonstrate a significant role for energy metabolism in promoting dog oocyte maturation, information that will be useful for improving culture systems for rescuing intraovarian genetic material. Mol. Reprod. Dev. 79: 186–196, 2012. Published 2011. This article is a U.S. Government work and is in the public domain in the USA.     

Effects of follicular size of cytoplast donor on the efficiency of cloning in cattle

In cattle, oocytes obtained from follicles smaller than 3 mm in diameter can undergo maturation in vitro, progressing to MII and undergoing fertilization, but are developmentally incompetent. Cytoplasts were prepared from in vitro matured oocytes aspirated from small (1-3 mm) or large (6-12 mm) follicles and fused to serum starved mural granulosa cells. Following activation, reconstructed embryos were cultured for 7 days and classified G1 to G4, before being processed for nuclei counting or transferred to synchronized recipients. Oocytes from small follicles had lower rates of polar body extrusion (59.6 vs. 69%; 731/1230 vs. 608/857) and fusion (71.4 vs. 78.8%; 360/497 vs. 364/465; P < 0.06). There were no differences in total rate of blastocysts development (60 vs. 59.8%; small vs. large), or any grade classification. A significant interaction was detected between follicle size and embryo grade with G3 embryos from small follicles having a greater cell number. Developmental competence of G1 and G2 embryos did not differ at day 27 (48 vs. 46%; 16/33 vs. 17/37; small vs. large). Although there were no differences in fetal size between the two groups, differences in allantois length (53 vs. 86 mm; small vs. large; P < 0.002) and allantois width (9.5 vs. 13 mm; small vs. large; P < 0.06) were seen. No differences in survival to term (2/13 in each group) were observed. These results indicate that cytoplasts from follicles of 1-3 and 6-12 mm in diameter are equally developmentally competent when used in a nuclear transfer procedure.     

Effect of follicular fluid collected from various diameter follicles on the progression of nuclear maturation and developmental competence of pig oocytes

Supplementing in vitro maturation medium with porcine follicular fluid (FF) improves maturation rate, male pronucleus formation, and monospermic fertilization of pig oocytes. This study examined, (1) if there are differences in FF derived from large follicles (LF, 5–6 mm in diameter) and small follicles (SF, 3–4 mm in diameter) on the effect of supplementing the maturation medium with FF on the progression of nuclear maturation, fertilization rate, and developmental competence of porcine oocytes; (2) whether the FF source influences the effect of the FF on the maturation medium on the survival rate and proliferation rate of cumulus cells (CCs) and the expansion of cumulus-oocyte-complexes (COCs); (3) whether the oocyte source (oocytes collected from LFs or SFs) influences the effect of FF on the progression of the nuclear maturation of oocytes; (4) whether the factors in the FF that affect the kinetics of nuclear maturation are proteins, and the range of the molecular weight of the FF factors.

In experiment 1, adding FF from LFs (LFF) significantly accelerated nuclear maturation and improved the fertilization rate; the developmental ratio was comparable with those of adding FF from SFs (SFF). In experiment 2, adding LFF, but not SFF, improved the CC survival rate, although the FF source did not affect the proliferation rate. Expansion of COCs was greater with SFF than LFF. In experiment 3, LFF promoted nuclear maturation of oocytes collected from only LFs. There was a significant interaction between the FF source and the oocyte source in the effect on nuclear maturation stages at 36 h of maturation. In experiment 4, treatment of FF with heat or trypsin diminished the difference between the effect of LFF and SFF on the progression of nuclear maturation. In addition, the predominant effect of LFF compared to that of SFF on nuclear maturation was not affected by ultrafiltration of the FF with a 30-kDa filter, but was diminished by ultrafiltration with a 100-kDa filter. The present study suggests that some proteins present in LFF that range in molecular weight from 30 to 100 kDa improve the developmental competence of oocytes probably via progression of nuclear maturation and cumulus cells viability.