In vitro and in vivo developmental ability of oocytes derived from porcine primordial follicles xenografted into nude mice

We evaluated the developmental ability of oocytes in porcine primordial follicles xenografted into nude mice. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the kidney capsules of ovariectomized nude mice. Forty-nine to 89 days after grafting (mean +/- SEM, 66.9 +/- 1.9 days; n = 64), the host mice showed the presence of cornified epithelial cells in their vaginal smears for the first time. The mice were then treated with 4 IU of equine chorionic gonadotropin (eCG) 60 days after first detection of vaginal cornification. Oocytes were collected from the host mice 48 h after treatment with eCG, and then matured. The maturation rates, based on the incidence of first polar body, ranged from 25.1% to 42.5%. They were then fertilized in vitro and cultured in vitro for 6 days, or transferred into estrous-synchronized recipients and recovered after 6 days. On Day 6 of culture, 15.4% of the matured oocytes had cleaved to the 2- to 8-cell stage. However, neither the embryos cultured in vitro nor those transferred and recovered developed to advanced embryonic stages, such as morulae or blastocysts. This result suggests that the developmental ability of xenografted oocytes is insufficient, even after in vitro maturation. Further strategies, such as improvement of hormonal treatment for host mice, are required to enable oocytes in xenografted ovarian tissues to acquire the cytoplasmic maturation necessary for embryonic development.     

Growth and maturation of follicles and oocytes following xenotransplantation of porcine ovarian tissues and in vitro maturation

This is the first report to show morphological evidence of in vitro maturation of oocytes recovered from xenotransplanted antral follicles. To develop a suitable tool for studing the growth and maturation of follicles and oocytes, we xenotransplanted small pieces of ovarian cortical tissue from sows, which contained small preantral follicles (primordial, primary, and secondary follicles; less than 0.05, 0.1 and 0.3 mm in diameter, respectively), under the capsules of kidneys of adult female severe combined immunodeficient (SCID) mice for 2 and 8 weeks, and then recovered cumulus-oocyte complexes from the growing tertiary follicles in xenografted tissues. The distribution of processes from cumulus cells to oocytes and the follicular growth, development, and maturation during xenotransplantation were histochemically analyzed. Tertiary follicles, 0.5 to 3.0 mm in diameter, were obtained from grafted tissues 2 (85%: 52 follicles/61 grafted tissues) and 8 (50%: 15/30) weeks after xenotransplantation, and then oocytes, which were tightly attached to cumulus cells, were collected from each tertiary follicle and cultured to assess their quality. At 2 weeks after grafting, 17.6% of the oocytes had matured to the metaphase II stage, but no such maturation was observed 8 weeks after grafting. Thus, in the 2 weeks group, preantral follicles rapidly grew in xenotransplanted porcine ovarian tissues to the tertiary stage, and oocytes could be recovered and matured from them by in vitro culture.     

Oxytocin mediates some effects of insulin-like growth factor-I on porcine ovarian follicles

The aims of the present study were (1) to investigate the influence of insulin-like growth factor-I (IGF-I) on follicular size, on the secretion of oxytocin (OT), progesterone (P), estradiol (E), IGF binding protein-3 (IGFBP-3), inhibin A, inhibin B and cAMP and on the expression of proliferation-associated peptide PCNA, ERK-related mitogen activated protein kinase (MAPK/ERK1, 2) and protein kinase A (PKA) in cultured porcine ovarian follicles; (2) to examine the effects of OT on IGF-I and on these functions; and (3) to determine whether the effects of IGF-I can be mediated by OT. To define the involvement of OT in mediating IGF-I action, we compared responses of porcine ovarian follicles to IGF-I and OT and examined whether blockade of endogenous OT by specific antiserum can affect IGF-I action. It was observed that IGF-I (1, 10 or 100 ng/ml) was able to prevent a decrease in the size of ovarian follicles during culture and caused an increase in the diameter of some follicles. It also stimulated the secretion of OT, P, IGFBP-3, inhibin A and cAMP, decreased the secretion of E and inhibin B (RIA/EIA/ELISA), and induced the expression of PCNA, PKA, MAPK/ERK1, but not MAPK/ERK2 (Western blotting). Like IGF-1, OT (100 ng/ml) prevented decrease in follicular size and increased the diameter of some follicles. It also stimulated the secretion of P and IGF-I, but not E. Antiserum against OT (1%), when given alone, did not affect the reduction of follicular size but slightly increased the percentage of follicles increasing their diameter during culture. The antiserum also inhibited secretion of OT and cAMP but not the secretion of P, E, IGFBP-3 or the expression of PKA, MAPK/ERK1 or 2. When given together with IGF-I, the antiserum prevented the stimulatory action of IGF-I on the proportion of enlarged follicles and on OT, IGFBP-3 and MAPK/ERK1. It augmented the effect of IGF-I on P, but not the effect on E, cAMP, PKA or MAPK/ERK2. These observations demonstrate the involvement of IGF-I and OT in the control of ovarian follicular size and follicular cell proliferation, progestagen, estrogen, IGFBP-3, inhibin A and B secretion and in cAMP/PKA- and MAPK/ERK1-dependent intracellular mechanisms. Furthermore, the reciprocal stimulation of IGF-I and OT and the similarity of some their effects, together with the prevention or augmentation of some IGF-I effects after OT blockade, suggest that IGF-I action can be mediated by OT.     

Growth of primordial oocytes in neonatal and adult mammals

Mammalian ovaries are endowed with a huge number of small oocytes (primordial oocytes) in primordial follicles. A small number of primordial oocytes start to grow, while others remain quiescent. Little is known about the mechanism regulating the activation of primordial oocytes. Recently, we found that primordial follicles in mature cows and prepubertal pigs took longer to initiate growth in xenografts compared with those in neonatal animals. We think that primordial oocytes in adult mammals are different from those in neonatal mammals. In this review, we summarize the results regarding the activation of primordial oocytes in neonatal and adult ovaries of different species and propose a model in which ovaries of neonatal mammals contain a mixed population of both quiescent and activated primordial oocytes, while almost all primordial oocytes are quiescent in adult females. The dormancy of primordial oocytes may be required to reserve the non-growing oocyte pool for the long reproductive life in mammals. FOXO3 is considered one of the molecules responsible for the dormancy of primordial oocytes in adult ovaries. These quiescent primordial oocytes are activated, perhaps by certain mechanisms involving the interaction between stimulatory and inhibitory factors, to enter the growth phase.     

FOXO3 knockdown accelerates development of bovine primordial follicles

The objective of the present study was to elucidate the involvement of FOXO3 in the activation of bovine primordial follicles. In immunohistochemistry, FOXO3 was detected in all of the oocytes in primordial and primary follicles. The FOXO3 decreased after treatment with FOXO3 small interfering RNAs (siRNAs). Ovarian tissues containing dominantly primordial follicles were treated with FOXO3 siRNAs and then xenografted to severe combined immune deficiency (SCID) mice. Two months after xenografting, some primordial follicles developed to the secondary and tertiary stages, and the total percentage of these developing follicles (secondary and tertiary follicles: 18 ± 7%) was higher than in the control grafts treated with control siRNA (7 ± 1%). It is thought that bovine primordial follicle activation is regulated by the FOXO3-dependent mechanism and that knockdown of FOXO3 induces the release of primordial follicles from FOXO3 suppression, initiating their growth.     

Effect of estradiol‐17β during in vitro growth culture on the growth,maturation, cumulus expansion and development of porcine oocytes from early antral follicles

Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full‐grown oocytes. We postulated that estradiol‐17β (E₂) supported the acquisition of meiotic and developmental competence as well as cumulus‐expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10^?7, 10^?6, 10^?5 or 10^?4 mol/L E₂; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus‐expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro‐stimulation was applied to the oocytes grown with 10^?5 mol/L E₂. After 6 days of culture, in vitro‐grown oocytes developed to the blastocyst stage at a rate similar to that for full‐grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E₂ during growth culture improves the meiotic and developmental competence of oocytes, cumulus‐expansion ability, and cumulus cell attachment to the oocytes.     

Supplementation with cumulus cell masses improves the in vitro meiotic competence of porcine cumulus–oocytes complexes derived from small follicles

The present study was conducted to examine the supplemented effect of cumulus cell masses (CCMs) derived from middle follicle (MF; 3–6 mm diameter) on the morphology and the meiotic or developmental competence of oocytes from small follicles (SF; 1–2 mm diameter). The number of cumulus cells surrounding oocytes just after collection was also lower in cumulus–oocyte complexes (COCs) from SF than MF. The ooplasmic diameter of oocytes was significantly smaller in SF‐derived oocytes than MF‐derived ones before and after in vitro maturation (IVM), whereas the diameter significantly increased during the culture. Co‐culture of SF‐derived COCs with MF‐derived CCMs during IVM significantly improved the meiotic competence of the oocytes to the metaphase‐II stage. Furthermore, the ooplasmic diameter of SF‐derived COCs during IVM was increased to the similar size of MF‐derived those in the presence of MF‐derived CCMs. The abilities of oocytes to be penetrated, to form male pronuclear formation and to cleave or develop to the blastocyst stage were not affected by the co‐culture with CCMs. Electrophoretic analysis of CCM secretions clearly showed the presence of more protein(s) approximately 27.6 kDa in the conditioned medium when supplemented with MF‐derived CCMs. In conclusion, we demonstrate that supplementation with MF‐derived CCMs improves the ooplasmic diameter and meiotic competence of SF‐derived oocytes.     

Allometric study on the relation between the growth of preantral and antral follicles and that of oocytes in bovine ovaries

We examined the relation between the growth of preantral and antral follicles and that of their oocytes in the ovaries of Holstein cows. We recovered follicles and oocytes (419 pairs) from the ovaries of 61 cows, and examined the relative growth relating the follicle diameter to the oocyte diameter by using six regression models for only healthy oocytes and all the oocytes including degenerated ones with and/or without zona pellucida. The best fitting model was found to be a hyperbolic regression (R(2): 0.999). The differentiated equation for the hyperbolic curve in normal oocytes with zona pellucida and the follicles was found to be y'=41.0/(x+0.253) (2): y and x are diameters of oocytes (microm) and follicles (mm), respectively. When follicles grew more than 4.0 mm in diameter, the growth rate of the oocytes calculated by the differentiation equation was found to be an asymptotic depression around zero. Thus, it is suggested that when the follicles grow more than 4.0 mm in diameter, the oocytes reach full size and cease to grow. Furthermore, it is considered that the equation can be applied to the assessment of normal growth in oocytes and follicles cultured in vitro.     

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte–cumulus cell complexes in vitro

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2–1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte–cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0–100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.     

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.     

猪卵母细胞体外受精条件的优化

猪卵母细胞体外受精(in vitro fertilization,IVF)技术为受精和胚胎早期发育机理等基础理论的研究及生产实践提供了重要的研究手段。虽然猪IVF的研究已经取得了一些进展,但其囊胚发育率仍然较低,亟需建立更为稳定、高效的体外受精方法。本试验通过比较精子浓度,精子获能处理,不同精卵共孵育培养体系以及在卵母细胞成熟过程中添加不同激素等影响猪卵母细胞体外受精胚胎发育能力的因素,以求找到最佳的猪卵母细胞体外受精体系。结果显示:在精子浓度为1×10~5~1×10~7/mL,5×10~6/mL的精子浓度显著提高体外受精效率(P<0.05);以茶碱、咖啡及咖啡因联合使用肝素作为获能物质处理精子,发现2.5 mmol/L茶碱处理组体外受精效率显著提高(P<0.05);通过比较不同的受精体系,发现使用40个卵/500μL体系显著提高体外受精效率(P<0.05);在卵母细胞成熟液中添加不同激素组合,发现添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH激素组合,体外受精效率显著提高(P<0.05)。本试验结果表明,在M199中添加10IU/mL PMSG,10IU/mL HCG和2.5IU/mL FSH体外成熟培养卵母细胞,以5×10~6/mL精子浓度,2.5mmol/L茶碱作为获能物质处理精子,40个卵/500μL共孵育的IVF体系效果最佳,其卵裂率为(61.33±0.77)%,囊胚率为(28.33±1.08)%。本试验将为深入研究猪IVF提供理论参考。     

Activation of goat primordial follicles in vitro: Influence of alginate and ovarian tissue

The present study aimed to evaluate the effect of three culture systems on caprine primordial follicle activation in vitro: follicles cultured either in the isolated form within alginate (Isolated follicles + Alginate treatment), or enclosed in ovarian tissue (in situ), with or without alginate (Fragment + Alginate, and Fragment alone treatments, respectively). After culture, the Isolated follicles + Alginate treatment presented a percentage of morphologically normal follicles (MNF) similar to both the non-cultured control and the Fragment Alone treatments. Nevertheless, Fragment + Alginate treatment showed a significant reduction in the number of MNF when compared to the other treatments. Regarding follicle development, our results showed that regardless of the alginate, the presence of ovarian tissue limited primordial follicle activation during in vitro culture. Remarkably, the Isolated primordial follicle + Alginate treatment was the only one that significantly promoted follicle activation and increased both follicle and oocyte diameters during IVFC, pointing out a higher cell proliferation. In conclusion, the presence of ovarian tissue with or without alginate limited follicle development (activation) after culture. Nevertheless, when primordial follicles were isolated and encapsulated in alginate they presented suitable survival rates, higher rates of follicle activation and continued to grow throughout the culture period.     

In vitro development of primordial follicles after long-term culture of goat ovarian tissue

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.     

In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage.

Two groups of mouse preantral follicles with diameters of 125-150 and 151-175 microm were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rate at the end of culture was higher in the follicles of 151-175 microm (89%) than 125-150 microm (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 microm follicles formed antra earlier than 125-150 microm follicles (days 4 and 5 vs. 5 and 6). However, follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however, showed less cleavage (30 and 35%) than the in vivo derived oocytes (89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization, blastocysts were only obtained from oocytes derived from the 151-175 microm category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 microm to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage.     

Effects of reaggregated granulosa cells and oocytes derived from early antral follicles on the properties of oocytes grown in vitro

In this study, we examined the effects of reconstructed oocyte–granulosa cell complexes (OGCs) on the development of porcine oocytes derived from early antral follicles (EAFs; 0.5–0.7 mm in diameter). When denuded oocytes were cocultured with granulosa cells derived from other EAFs, the oocytes and granulosa cells aggregated to form OGCs after 2 days of culture. After 14 days of culture, we compared cell number, oocyte diameter, and oocyte chromatin configuration in unmanipulated (natural) OGCs, reconstructed OGCs, and OGCs collected from antral follicles (AFs, 3.0–6.0 mm in diameter). The diameters of oocytes from reconstructed OGCs grown in vitro were not different from those of oocytes from natural OGCs, although they were significantly smaller than those of oocytes from antral follicle (AF) OGCs. Oocyte chromatin configuration did not differ among the 3 OGC groups, but the oocyte nuclear maturation rate was lower in the reconstructed OGCs and higher in the AF OGCs. However, when the in vitro culture period for the reconstructed OGCs was extended by 2 days, the nuclear maturation rate of oocytes from reconstructed OGCs was similar to that of oocytes from natural OGCs. In addition, blastocysts were successfully obtained from oocytes from reconstructed OGCs. In conclusion, we established an innovative culture method that allows oocytes and granulosa cells from EAFs to reaggregate as reconstructed OGCs, which yield oocytes with the ability to develop to the blastocyst stage.     

Oocyte ultrastructural characteristics in camel (Camelus dromedarius) primordial to large antral follicles

This study was conducted to describe in detail the ultrastructural features and morphological characteristics of camel oocytes from preantral follicles in relation to the sequential stages of follicular development and also for oocytes from antral follicles in relation to their diameter. Camel oocytes from primordial, primary, secondary and also early to late antral follicles were processed and examined by light and transmission electron microscopy. Primordial follicular oocytes were characterized by a layer of flattened granulosa cells around and also eccentric nucleus and few cytoplasmic organelles in the peripheral region. Up to the secondary follicle stage, flat cells were replaced by cuboidal granulosa cells and their number increased and also an increase in the number of organelles such as vesicles, mitochondria and endoplasmic reticulum was observed. In the early antral stage, the formation of zona pellucida, appearance of microvilli and pleomorphic mitochondria was seen and the nucleus was dislocated to the peripheral region. During final growth phase, the extent of endoplasmic reticulum, vesicles and mitochondria increased, the number of lipid droplets decreased and cumulus cell process endings (CCPE) were observed. In conclusion, the growth of camel oocyte is associated with progressive increase in the number of mitochondria, endoplasmic reticulum, Golgi complexes and cytoplasmic vesicles as well as decrease in the number of lipid droplets and the nucleus migration from an eccentric in preantral to a peripheral location in antral follicles.     

牛有腔卵泡卵母细胞体外成熟的研究

试验从屠宰场收集了 4 8头青年黄牛、34头青年水牛的卵巢共 16 4个 ,共回收可用卵母细胞 86 4枚。水牛平均每个卵巢可回收 3.2 2枚可用卵母细胞 ,相当于黄牛 (6 .72枚 )的一半。试验结果表明 :水牛卵巢生长卵泡少 ,卵母细胞产量少、质量差 ;3种采集黄牛卵泡卵母细胞的方法 ,用抽吸加切割法平均从每个卵巢回收的可用卵数 (7.71枚 )都极显著高于抽吸法 (6 .19枚 )和切割法 (6 .4 4枚 ) (P <0 .0 1) ,但是该法手续繁杂 ,适于一次且只能收集少量卵巢时使用 ;在黄牛和水牛卵母细胞成熟培养液中用 0 .3%PVP代替 10 %FBS ,牛卵母细胞的体外成熟率无显著差异 (P >0 .0 5 )。     

p27(Kip1) negatively regulates the activation of murine primordial oocytes

In mice, small oocytes (primordial oocytes) are enclosed within flattened granulosa cells to form primordial follicles around birth. A small number of primordial oocytes enter the growth phase, whereas others are quiescent. The mechanism regulating this selection of primordial oocytes is not well understood. The objective of the present study was to understand the role of p27(Kip1), which regulates cell cycle progression in somatic cells, in the growth initiation of primordial oocytes in neonatal mice. We studied the localization of p27(Kip1) in 0-, 3-, 5-, 7- and 21-day-old mouse ovaries by immunohistochemistry. Ovaries from 3-day-old mice were treated with p27(Kip1) siRNAs (small interfering RNAs), and knockdown of p27(Kip1) was determined by immunohistochemistry and Western blotting. Ovaries treated with siRNAs were organ-cultured for 6 days, and oocyte growth was estimated histologically. Expression of p27(Kip1) was undetectable in the primordial oocytes of newborn mice. In the 3-day-old ovaries (n=3), p27(Kip1) was demonstrated in the nucleus of 36 ± 6% primordial oocytes. The percentage of p27(Kip1)-positive primordial oocytes increased to 72 ± 8 (n=3), 85 ± 7 (n=3) and 93 ± 5 (n=3) in the 5-, 7- and 21-day-old mouse ovaries, respectively. After knockdown of the p27(Kip1) protein by siRNAs, a higher proportion of oocytes entered the growth phase in cultured ovaries than those in the control. These results suggest that p27(Kip1) negatively regulates primordial oocyte growth and that knockdown of p27(Kip1) leads primordial oocytes to enter the growth phase in vitro.     

玻璃化冷冻对猪卵母细胞超微结构的影响

本研究以GV期和MII期卵母细胞作为试验材料,通过透射电镜方法观察卵母细胞冷冻前后的超微结构变化,结果发现,透射电镜下观察卵母细胞发现,GV期卵母细胞与透明带连接紧密,微绒毛伸入透明带中,皮质区分布大量的线粒体。脂滴分为两种,一种为灰色脂滴,一种为深色脂滴。MII期卵母细胞排出第一极体,质膜下分布大量的皮质颗粒。微绒毛缩短成矮柱状,线粒体常聚集存在,卵周隙出现。冻后GV期卵母细胞微绒毛消失,线粒体肿胀,线粒体内有囊泡样结构,包围脂滴的内质网不完整,胞质内溶解为絮状,未见高尔基体。冻后MII期卵母细胞透明带损伤、微绒毛、细胞膜损伤甚至消失、极少量皮质颗粒分布于皮质区。脂滴部分溶解,相互连接,脂滴周围伴随大量肿胀呈圆形的线粒体,嵴不明显,内呈囊泡样,未见到高尔基复合体结构。