哺乳动物腔前卵泡体外培养研究进展

哺乳动物腔前卵泡的体外培养可充分挖掘优良母畜潜在的遗传和繁殖潜力,为体外胚胎生产提供大量的卵母细胞来源,因而正日益受到人们的重视。本文就哺乳动物腔前卵泡的体外发育中的培养系统、培养方法,培养形式以及影响其体外发育的因素等几个方面进行了阐述。     

Regulation of in vitro growth of preantral follicles by growth factors in goats

Goat preantral follicles were cultured to investigate the effects of insulin-like growth factor-I (IGF-I), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the in vitro growth and viability of oocytes. Preantral follicles were isolated mechanically and enzymatically (using collagenase and DNase) from prepuberal goat ovaries. The working medium was composed of Defined Eagle's Minimum Essential Medium (DMEM) supplemented with HEPES (20 mM), 10% fetal calf serum (FCS), hypoxanthine (2 mM), dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP) (2 mM), penicillin (75 ng/ml) and streptomycin (50 ng/ml). The culture medium consisted of the working medium with follicle stimulating hormone (FSH) (100 ng/ml) and hydrocortisone (40 ng/ml) added. In the experiment, goat preantral follicles were cultured for 9 days in the culture medium and in the culture medium supplemented with either IGF-I (100 ng/ml), EGF (50 ng/ml), bFGF (50 ng/ml) or IGF-I (100 ng/ml)+EGF (50 ng/ml). The results indicated that IGF-I (100 ng/ml) effectively maintained the survival of oocytes and promoted their growth; EGF (50 ng/ml) enhanced the survival rate of oocytes but had a negative effect on oocyte growth; bFGF (50 ng/ml) stimulated oocyte survival but had no obvious effect on their growth while IGF-I (100 ng/ml) and EGF (50 ng/ml) in combination had a greater effect on both survival and growth rate of oocytes than IGF-I or EGF alone. The supplementation of IGF-1 and EGF to the culture medium is recommended in the culture of goat preantral follicles.     

水牛腔前卵泡的分布及形态学研究

用常规石蜡切片法研究水牛卵泡在卵巢中的分布规律与结构特征。结果表明各级卵泡的分布具有明显的区域性;原始卵泡位于卵巢皮质的最外层,形成原始卵泡层,初级卵泡位于皮质的中层,次级卵泡位于富含血管的皮质最内层;原始卵泡、初级卵泡和次级卵泡的直径分别为(36.9±7.7)μm、(48.7±8.9)μm和(91.9±27.3)μm,与之对应卵母细胞的直径分别为(19.6±6.0)μm、(27.3±7.1)μm和(49.1±17.4)μm;原始卵泡和初级卵泡的颗粒细胞数为(10.8±2.3)个和(18.1±3.1)个;次级卵泡含有2~6层颗粒细胞,2~4层的颗粒细胞分布不均。     

Catalase addition to vitrification solutions maintains goat ovarian preantral follicles stability

The aim of this study was to verify whether the addition of catalase (20 IU/mL) at different steps of goat ovarian tissue vitrification affects ROS levels, follicular morphology and viability, stromal cell density, apoptosis and the expression of proteins related to DNA–damage signaling (γH2AX) and repair (53BP1). Goat ovarian tissues were analyzed fresh (control) or after vitrification: without catalase (VS–/WS–), with catalase in vitrification solutions (VS+/WS–), with catalase in washing solutions (VS–/WS+) or with catalase in both solutions (VS+/WS+). The vitrification without catalase had higher ROS levels than the control. The catalase, regardless the step of addition, maintained ROS levels similar to the control. There were no difference between treatments regarding follicular viability, stromal cell density and detection of γH2AX and 53BP1. There was no difference in follicular morphology and DNA fragmentation between groups vitrified. In conclusion, catalase addition to vitrification solutions prevents ROS formation in cryopreserved goat ovarian tissues.     

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM⁺) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM⁺ alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM⁺ alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.     

Vitrification of mouse preantral follicles versus slow freezing: Morphological and apoptosis evaluation

The aim of this study was evaluation of survivability, maturation rate and apoptotic gene expression of preantral follicles after vitrification and slow freezing technique. Normal mouse preantral follicles were randomly divided into three experimental groups. In the control group, follicles were cultured immediately; in the vitrification and slow freezing groups, follicles were cultured after vitrification‐warming and slow freezing‐thawing procedures. Follicular viability was assessed by using 0.4% trypan blue, and molecular evaluation of messenger RNA levels of apoptosis‐related genes was performed by the semi‐quantitative RT‐PCR method after 3 h of culture. Oocyte maturation rates were also evaluated on day 14 of culture. Survival and maturation rate in the slow freezing group were significantly lower than those in control and vitrification groups (P ≤ 0.05). Although there was no difference in Survivin expression among the three experimental groups, Bcl‐2 expression was significantly lower in the slow freezing group compared to the other groups (P ≤ 0.05). The expression of Bax, P53, Fas and Bax/Bcl‐2 ratio in the slow freezing group was significantly higher than control and vitrification groups (P ≤ 0.05). Preantral follicle vitrification seems to be better than slow freezing as seen in the survival, maturation and expression rates of apoptotic gene variants.     

牛腔前卵泡的简易机械分离方法

采用两种机械法分离牛腔前卵泡。方法一 (M -1) ,皮肤移植刀切割、剪碎、过滤镜下直接捡卵法。方法二 (M -2 ) ,皮质片剪碎、离心、悬浮、过滤镜下捡卵法。M -1平均每卵巢采卵数为 46 9个± 18 2个 ,M -2为 6 8 4个± 12 5个 ;处理时间M -1为 2 5 4min± 6 7min ,M -2为 46 3min± 16 8min ,两种方法采集腔前卵泡数量及处理时间均存在显著差异。平均每小时采集卵泡数分别为 99 5和 87 2个 ,M -1多于M -2。两种方法获得腔前卵泡大小分布规律也有明显差异 ,M -1采集腔前卵泡直径为 6 0～ 15 0 μm ,绝大部分是次级卵泡 ,而M -2获取腔前卵泡则偏小。总的看来 ,M -1处理时间短 ,回收效率高 ,方法简便 ,且分离卵泡直径较大 ,适于体外培养 ,是机械分离牛腔前卵泡的一种理想方法。     

Effect of different mechanical isolation techniques on developmental competence and survival of buffalo ovarian preantral follicles

The present study aimed to analyze different methods of mechanical isolation of buffalo preantral follicles (Experiment I), in vitro culture of isolated follicles in different groups of culture medium over collagen gel matrix (Experiment II) and subsequent in vitro development and survival of isolated preantral follicles (PFs) (Experiment III). In Experiment I, ovarian cortical pieces were separated and PFs isolated by different mechanical methods. In Experiment II, isolated follicles were divided into three groups and cultured in TCM-199 having 10% FBS, 1% ITS, and 20 ng/ml EGF (Group A, control), addition of 0.5 μg/ml FSH (Group B) or FSH + 100 ng/ml IGF-I (Group C). Follicles were incubated at 38.5 °C in 5% CO₂ with maximum humidity. In Experiment III, based on the outcome of Experiment I and II, PFs were cultured from those isolation method and treatment group, showing better growth and developmental pattern to analyze the impact of growth factors on in vitro growth of follicles in long term culture. It was found that micro-dissected PFs showed higher survival rate and growth after 15 days of culture compared to PFs isolated by other methods. Follicles cultured with FSH + IGF-1 on collagen gel matrix, showed significantly (P < 0.05) higher survival rate and mean diameter of follicles on day 15 of culture compared to control. In summary, it has been shown that isolation of follicles by micro-dissection has advantages over other methods, being relatively simple, inexpensive and less harmful to follicles. Micro-dissected buffalo PFs maintained the architecture, showed antrum formation in presence of FSH and IGF-I over the collagen gel matrix.     

Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles

The aim of the present study was to investigate the effects of fibroblast growth factor-10 (FGF-10) on the survival, activation (transition from primordial to primary follicles), and growth of goat preantral follicles cultured in vitro. Pieces of ovarian cortex were cultured for 1 and 7 d in the absence or presence of FGF-10 (0, 1, 10, 50, 100, and 200 ng/mL). Noncultured and cultured tissues were processed and analyzed by histology, transmission electron microscopy, and viability testing. Results showed that after 7 d, a greater percentage (79.9%) of morphologically normal follicles (containing an oocyte with regular shape and uniform cytoplasm, and organized layers of granulosa cells without a pyknotic nucleus) was observed when cultured with 50 ng/mL of FGF-10 when compared with other concentrations of FGF-10 (0 ng/mL, 67.3%; 1 ng/mL, 68.2%; 10 ng/mL, 63.3%; 100 ng/mL, 64.4%; 200 ng/mL, 52.7%). Ultrastructural analyses and viability testing using fluorescent markers confirmed the follicular integrity of FGF-10 (50 ng/mL)-treated fragments after 7 d of culture. After 7 d, all FGF-10 concentrations reduced the percentage of primordial follicles and increased the percentage of developing follicles. In the presence of 50 ng/mL of FGF-10, follicles increased in diameter after 7 d of culture when compared with other concentrations tested. In conclusion, this study demonstrates that FGF-10 maintains the morphological integrity of goat preantral follicles and stimulates the growth of activated follicles in culture. The culture conditions identified here contribute to the understanding of the factors involved in goat early follicular development.     

In vitro culture of mouse preantral follicles using membrane inserts and developmental competence of in vitro ovulated oocytes

To develop a reliable follicle culture system, mouse preantral follicles 150-200 microm in diameter were cultured individually for 5 or 6 days in membrane inserts or in droplets, and then induced to ovulate with hCG (Experiment 1). The nuclear maturation and developmental competence of the oocytes that ovulated from the follicles cultured in inserts were determined (Experiment 2). There was no significant difference between the two culture systems in the survival rate (83 and 77%). However, follicles cultured in inserts showed a higher ovulation rate (63%) than those cultured in droplets (39%, P<0.05). About 80% of the oocytes that ovulated from the follicles cultured in inserts were at the metaphase II stage. After in vitro fertilization, 75 and 48% of in vitro ovulated oocytes cleaved and developed into blastocysts, respectively. These results demonstrate that the insert culture system is superior to the droplet culture system in terms of follicular growth and ovulation, and can be used to investigate the growth and ovulation of follicles in vitro.     

促性腺激素(FSH、LH)对牛腔前卵泡体外发育及E_2分泌的影响

利用McCoy's 5a基础无血清培养系统研究了促卵泡素(FSH)、促黄体素(LH)2种促性腺激素对牛腔前卵泡的体外生长、存活及雌二醇(E2)分泌的影响.结果显示,培养液中单独添加LH对牛腔前卵泡生长影响不大,而单独添加50、100μg/L以上FSH对牛腔前卵泡直径增长和培养后期体外存活有明显的促进作用;当培养液中有10 μg/L LH存在时,2种促性腺激素协同刺激卵泡生长的作用更加明显.添加10 μg/L LH 50μg/L FSH对腔前卵泡体外生长、发育、存活及E2分泌均具有显著的促进作用(P<0.05).     

Interaction between ascorbic acid and follicle-stimulating hormone maintains follicular viability after long-term in vitro culture of caprine preantral follicles

This study evaluates the effects of ascorbic acid and its interaction with follicle-stimulating hormone (FSH) on the morphology, activation, and in vitro growth of caprine preantral follicles. Ovarian fragments were cultured for 1, 7, or 14 d in minimum essential medium (MEM) containing ascorbic acid (50 or 100 μg/mL), FSH (50 ng/mL), or both of these substances. Ovarian tissue that was either fresh (control) or cultured for 1, 7, or 14 d was processed for histological and ultrastructural evaluation. The results showed that after 14 d of culture, medium supplemented with 50 μg/mL of ascorbic acid alone or combined with FSH showed higher rates of follicular survival compared with MEM. After 7 d of culture, FSH, ascorbic acid at 50 μg/mL with or without FSH, and ascorbic acid at 100 μg/mL increased the percentage of follicular activation compared to fresh control. In addition, FSH alone significantly increased the percentage of growing follicles after 14 d. The combination of 50 μg/mL of ascorbic acid and FSH promoted a significant increase in oocyte and follicular diameter after 7 d of culture. Ultrastructural and fluorescent analysis confirmed the integrity of follicles cultured with 50 μg/mL of ascorbic acid and FSH after 14 d. In conclusion, the combination of 50 μg/mL of ascorbic acid and FSH maintained follicular integrity and promoted follicular activation and growth after long-term in vitro culture of caprine preantral follicles.     

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

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

BMP-15 activity on in vitro development of collared peccary (Pecari tajacu Linnaeus, 1758) preantral follicles

This study investigated the effects of BMP-15 on the in vitro development of preantral follicles of collared peccaries. Ovarian fragments were cultured for 1 or 6 days in Tissue Culture Medium 199 (TCM199⁺) supplemented with BMP-15 at rates of 0, 1, 25 or 50 ng/ml. The fragments were analysed histologically by evaluating follicular morphology, activation and growth as well as the potential for proliferation of granulosa cells. Our results show the addition of 25 ng/ml BMP-15 in the medium provided the greatest percentage of normal follicles (79.67% ± 0.69) when compared to other treatments (p < .05); however, this result is similar to 1 ng/ml BMP-15 (74.00% ± 1.90, p > .05). Moreover, 25 and 50 ng/ml of BMP-15 promoted follicular activation. BMP-15 supplements did not affect oocyte and follicular growth. All concentrations of BMP-15 increased the number of nucleolus organizer regions (NORs) after 1 day of culture when compared to fresh fragments or the control samples (p < .05). However, at the end of the experiment, the number of NORs in follicles cultured in all treatments was higher than that observed in the fresh control (sample taken prior to culturing) (p > .05). In summary, the addition of 25 ng/ml BMP-15 to the culture medium of collared peccary preantral follicles maintained a high number of morphologically healthy follicles and stimulated the activation of primordial follicles after 6 days in culture.     

The effect of LIF in the absence or presence of FSH on the in vitro development of isolated caprine preantral follicles

We investigated the effect of the leukaemia inhibitory factor (LIF) alone or in association with FSH on the in vitro culture (IVC) of caprine preantral follicles. Preantral follicles >200 μm in size were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/ml) in the absence or presence of FSH. Every 6 days, follicular survival, growth and antrum formation were evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM), and their viability and chromatin configuration were assessed. Follicles of the control group and those cultured in 10 ng/ml LIF maintained the structural integrity (particularly the preservation of the basement membrane) when compared to the oocytes cultured in 50 ng/ml LIF, regardless the presence of FSH. In the absence of FSH, the percentage of antrum formation after 18 days of culture in the 50 ng/ml LIF group was significantly lower than in either the control group or the 10 ng/ml LIF group. However, this effect was not observed in the presence of FSH. The rate of resumption of meiosis was significantly higher in the 50 ng/ml LIF group in the absence of FSH in comparison with the control and 10 ng/ml LIF groups. Metaphase II was observed only when follicles were cultured in a combination of FSH and 50 ng/ml LIF. In conclusion, LIF alone does not interfere with antral formation and oocyte growth, but at concentration of 50 ng/ml and combined with FSH, it promotes oocyte maturation.