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。     

Role of VEGF in pig preantral follicles

Veterinary Research Communications -     

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.     

Live offspring from cryopreserved embryos following in vitro growth, maturation and fertilization of oocytes derived from preantral follicles in mice

This study was undertaken to examine pre- and postimplantation developmental potency of cryopreserved embryos that had undergone in vitro growth (IVG), maturation (IVM) and fertilization (IVF) of oocytes from the preantral follicle stage. An oocyte culture system for IVG and IVM was used in oocyte-granulosa cell complexes (OGCs) derived from preantral follicles in 12-day-old mice. The rate of oocyte maturation was improved by the addition of gonadotropins (FSH/LH) and cytokines (IGF-I/SCF) to culture medium for IVG. During culture for IVG, estradiol-17β and progesterone concentrations increased progressively to the latter period of culture. This culture system enabled IVG, IVM, IVF and pre- and postimplantation development. From 90 cryopreserved 2-cell stage embryos transferred into recipients after warming, 10 live pups were produced. Cryopreservation of embryos by vitrification at the 2-cell stage showed no harmful effect on development to the blastocyst stage or on the cell numbers of the inner cell mass (ICM) and trophectoderm (TE). This study demonstrated that embryos derived from oocytes grown in vitro have tolerance for vitrification and competence to develop to term after warming. This IVG-IVM-IVF technology combined with embryo cryopreservation might be useful for assisted reproduction in mice.     

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.     

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 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.     

Gene expression and immunolocalization of fibroblast growth factor 2 in the ovary and its effect on the in vitro culture of caprine preantral ovarian follicles

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate 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.     

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.     

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。     

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.     

Influence of nitric oxide on in vitro growth, survival, steroidogenesis, and apoptosis of follicle stimulating hormone stimulated buffalo (Bubalus bubalis) preantral follicles

Effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor, on in vitro survival, growth, steroidogenesis, and apoptosis of buffalo preantral follicles (PFs) was investigated. PFs (200~250 µm) were isolated by micro-dissection and cultured in 0 (control), 10^-3, 10^-5, 10^-7, and 10^-9 M SNP. To examine the reversible effect of SNP, PFs were cultured with 10^-5 M SNP + 1 mM N^ω-nitro-L-arginine methyl ester (L-NAME) or 1.0 µg hemoglobin (Hb). The results showed that greater concentrations of SNP (10^-3, 10^-5, 10^-7 M) inhibited (p < 0.05) FSH-induced survival, growth, antrum formation, estradiol production, and oocyte apoptosis in a dose-dependent manner. However, a lower dose of SNP (10^-9 M) significantly stimulated (p < 0.05) the survival, growth, antrum formation, follicular oocyte maturation, and stimulated progesterone secretion compared to the control. A combination of SNP + L-NAME promoted the inhibitor effect of SNP while a SNP + Hb combination reversed this effect. Nitrate and nitrite concentrations in the culture medium increased (p < 0.05) in a dose-dependent manner according to SNP concentration in the culture medium. At higher concentrations, SNP had a cytotoxic effect leading to follicular oocyte apoptosis whereas lower concentrations have stimulatory effects. In conclusion, NO exerts a dual effect on its development of buffalo PFs depending on the concentration in the culture medium.     

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.     

Sequential medium with GH and IGF‐1 improved in vitro development of bovine preantral follicles enclosed in ovarian tissue

The growth hormone (GH) and growth insulin‐like factor‐1 (IGF‐1) act directly upon the regulation and growth in the different phases of preantral follicles. Thus, it is necessary to define their sequentiality until the in vitro preovulatory development. Therefore, the study aimed to assess the effects of a sequential medium containing GH and/or IGF‐1 in the long‐duration in vitro culture of preantral ovarian follicles. Ovarian fragments were cultivated: first half (days 1–7), second half (days 7–14) or during 14 culture days. Treatments were identified as: αMEM+; GH → IGF‐1; IGF‐1 → GH and GH + IGF‐1. The culture was designed in 24‐well plates, in an incubator at 37°C and 5% CO₂. The parameters of normality, viability, follicles (primordial/in developing) and follicle diameter were evaluated. In addition, the ultrastructure was confirmed with electron transmission microscopy. The results showed that the culture treated with GH → IGF‐1 kept the follicular normality and the viability until the 14th day of culture and increased both in the follicular development until 7th day and in the follicular diameter until 14th day, when compared to the control. The treatments IGF‐1 → GH and GH + IGF‐1 were not effective in the developing and follicular diameter after 7 days of culture, and also reduced the percentage of viability. It is concluded that the bovine preantral follicles cultured in the sequential medium treated with GH → IGF‐1 improved the follicular development until the first half of the culture and kept these parameters with normality, viability and ultrastructure until the second half of the in vitro culture.     

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.