AMH蛋白在性成熟小鼠卵泡发育不同阶段及自然衰老过程卵巢组织中的表达变化

目的:通过检测抗苗勒氏管激素(AMH)蛋白在成年小鼠卵泡发育不同阶段和自然衰老过程卵巢中的表达变化,为卵泡的生长发育调控及卵巢衰老发生发展的机制研究奠定理论基础。方法:选用12,24,36,48周龄自然衰老C57BL/6小鼠为模型。收集12周龄C57BL/6小鼠不同发育阶段的卵巢和24,36,48周龄自然衰老C57BL/6小鼠卵巢。采用免疫组织化学方法和蛋白印迹技术检测各组卵巢中AMH蛋白的表达。结果:免疫组织化学显示,AMH蛋白在始基卵泡、初级卵泡,次级卵泡、窦状卵泡的颗粒细胞中均有表达,但在黄体和闭锁卵泡的颗粒细胞中未见表达。始基卵泡颗粒细胞中AMH蛋白表达明显低于初级卵泡、次级卵泡和窦状卵泡(P<0.05);窦状卵泡颗粒细胞中AMH蛋白表达明显低于初级卵泡和次级卵泡(P<0.05);初级卵泡和次级卵泡的AMH蛋白表达未见明显差异(P>0.05)。不同鼠龄的小鼠同级别卵泡颗粒细胞AMH的染色程度未见明显差异;但是24周龄以后间质中开始出现AMH蛋白表达,并且AMH的表达随鼠龄增加而上升;48周龄小鼠卵巢间质中AMH蛋白表达明显高于24周龄和36周龄(P<0.05);24周龄与36周龄比较差异无统计学意义(P>0.05)。蛋白印迹技术结果显示,AMH蛋白在小鼠卵巢组织中的表达随鼠龄增加而降低,差异有统计学意义(P<0.05)。结论:AMH蛋白主要表达于卵泡发育早期阶段的颗粒细胞,并且其在间质中随鼠龄增加表达增强,但是在整个卵巢组织中其表达量随鼠龄增加而降低。提示AMH可能在卵泡发育以及卵巢衰老中起重要的调控作用。     

两种冷冻方法对人类卵巢组织卵泡形态和组织增殖活性的影响

目的:探讨玻璃化冷冻和慢速冷冻何者更适于冻存人卵巢组织。方法:将10例因卵巢良性囊肿剔除术获取的人卵巢皮质组织切成薄片后随机分配到新鲜卵巢组(A组)、玻璃化冷冻组(B组)和慢速冷冻组(C组),通过光学显微镜和透射电子显微镜观察比较卵泡形态变化,免疫组织化学检测组织细胞增殖细胞核抗原(PCNA)表达变化。结果:A、B、C组中形态正常的原始卵泡比例分别占71.4%、70.1%、52.3%;形态正常的初级卵泡比例分别占76.0%、43.5%、31.8%;C组中形态正常的原始卵泡比例和初级卵泡比例均明显低于A、B组(P<0.05);B组形态正常的原始卵泡比例和初级卵泡比例与A组相比无统计学差异(P>0.05)。A、B组中形态正常的原始卵泡超微结构无明显改变,但B组中初级卵泡和C组中原始卵泡和初级卵泡的超微结构存在一定程度的改变。PCNA阳性表达主要见于卵母细胞、颗粒细胞和卵巢组织间质细胞,3组中均有PCNA表达,且表达无统计学差异。结论:玻璃化冷冻较慢速冷冻对人卵巢组织影响小,是一种较适宜的人卵巢组织冷冻保存方法。     

FOXO3a转录因子参与调控新生大鼠卵母细胞凋亡的实验研究

目的:探讨新生大鼠卵巢卵母细胞的凋亡是否与FOXO3a(forkhead box group O)转录因子在细胞核内高表达有关。方法:取新生1d、2d、3d、4d雌性SD大鼠卵巢,用免疫组织化学方法检测FOXO3a与其靶分子促凋亡蛋白BIM的表达变化;TUNEL化学染色观察新生大鼠卵巢中卵母细胞凋亡状况;再用免疫组织荧光联合TUNEL荧光检测技术观察FOXO3a和其靶分子BIM表达水平与卵母细胞凋亡的关系。结果:免疫组织化学结果显示,FOXO3a在一些卵母细胞巢内和原始卵泡的卵母细胞核内高表达,其阳性率在出生后1-2d较高,随后逐渐下降。与FOXO3a相似,BIM也是在一些卵母细胞巢内和原始卵泡的卵母细胞中高表达,阳性率变化规律也与FOXO3a一致。凋亡细胞主要见于卵母细胞巢内和原始卵泡的卵母细胞中,其凋亡率变化与FOXO3a阳性率变化一致。免疫组织荧光联合TUNEL荧光检测结果表明,FOXO3a(核内)与BIM高表达的卵母细胞正是凋亡的卵母细胞。结论:FOXO3a转录因子参与新生大鼠卵巢中卵母细胞巢内卵母细胞和原始卵泡卵母细胞的凋亡调控。     

孕酮和表皮生长因子在体外培养新生大鼠卵巢原始卵泡生长启动中的作用

目的:探讨孕酮(P_4)和表皮生长因子(epidermal growth factor,EGF)在原始卵泡生长启动中的作用。方法:取出生2d的SD大鼠卵巢,分别在含不同浓度P_4、EGF和P_4+EGF的Waymouth培养体系中培养,以单纯Waymouth培养体系培养的大鼠卵巢为对照,于培养8 d后石蜡包埋切片, HE染色,观察各级卵泡形态,并统计分析各级正常卵泡数。结果:P_4各浓度组的原始卵泡数明显多于对照组(P<0.01),50 ng/ml EGF组的初级卵泡和次级卵泡数明显多于对照组(P<0.01),但1 000 ng/ml EGF组的初级卵泡数和次级卵泡数均明显少于对照组(P<0.01);50 ng/ml EGF+10~(-5) mol/L P_4与相同剂量单独作用的P_4和EGF比较各级卵泡数有统计学差异(P<0.05),与对照组比较无明显差异(P<0.05)。结论:在Waymouth培养体系中孕酮对原始卵泡生长有抑制作用,一定浓度的EGF能促进原始卵泡生长,而同时加入P_4和EGF,两者的作用有部分抵消。     

PRXⅡ蛋白及mRNA在小鼠卵巢不同发育阶段的表达

目的:探讨PRXⅡ在小鼠卵泡发育不同阶段的表达。方法:选择不同发育时期的昆明小鼠卵巢,用免疫组化SP染色法,半定量逆转录聚合酶链反应(RT-PCR)检测不同发育期卵巢组织中PRXⅡ蛋白、mRNA的表达水平。结果:PRXⅡ蛋白在各级卵泡的卵母细胞及颗粒细胞中均有表达,其表达水平随卵泡发育逐渐增强。始基卵泡与初级、次级、窦状卵泡相比,其卵母细胞及颗粒细胞中PRXⅡ蛋白表达水平明显较低,差异有统计学意义(P<0.005)。初级卵泡分别与次级、窦状卵泡相比其颗粒细胞中PRXⅡ蛋白表达水平较低,有显著差异(P<0.005)。闭锁卵泡PRXⅡ蛋白呈强阳性表达,明显高于生长卵泡中的表达水平。黄体及间质中亦见少量PRXⅡ表达。PRXⅡmRNA在卵巢组织中的表达水平随卵巢的生长发育呈先增高后降低的趋势。3、6、11周小鼠卵巢中PRXⅡmR-NA表达水平无统计学差异(P>0.05),但其表达水平是8天及15周小鼠卵巢组织的两倍。结论:卵泡发育不同阶段PRXⅡ均有表达,在卵泡发育后期和闭锁卵泡中强表达,提示PRXⅡ在卵泡的生长发育及闭锁过程中可能起重要的调控作用。     

1000IU hCG在控制性卵巢刺激过程中诱发卵泡成熟作用的初步研究

目的:评估1 000 IU低剂量hCG在控制性卵巢刺激(COS)过程中诱发卵泡成熟的作用。方法:35例接受IVF/ICSI-ET治疗的患者进行35个周期的COS。在诱发卵泡成熟日,肌肉注射hCG1 000 IU;并按照血E2水平分为卵巢正常反应组(E2<3 000 pg/ml,A组,12例)和卵巢高反应组(E2≥3 000 pg/ml,B组,23例),统计分析IVF/ICSI-ET结局。结果:35例患者均获得成熟卵母细胞并进行了胚胎移植,平均获卵数为13.5±7.7枚,平均成熟卵母细胞数为11.6±6.7枚,平均受精卵数为10.8±5.6枚,平均胚胎数为10.0±5.6枚,平均移植胚胎数1.9±0.2枚,平均冷冻保存胚胎数为4.3±3.3枚,临床妊娠率A组为50.00%,显示高于B组(30.43%)(P<0.05),继续妊娠率A组为41.67%,显示高于B组(26.09%)(P<0.05);所有患者均未发生无中/重度卵巢过度刺激综合征。A组与B组相比较,获卵率、受精率、优质胚胎率及早期流产率均无统计学差异(P>0.05)。结论:1 000 IU的低剂量hCG能够在COS过程中诱发卵泡成熟,不仅适应于卵巢反应高者,而且也适应于卵巢反应正常者。     

KL与其受体Kit在不同发育阶段大鼠卵巢卵泡中的表达研究

目的:探讨KL(kitligand)与Kit蛋白在各年龄段大鼠卵巢组织的各种细胞中的表达。方法:分别取1d、2d、4d、8d、16d、3个月、12个月龄大鼠卵巢,制备组织切片,用免疫组织化学技术观察KL及Kit蛋白在各年龄段大鼠卵巢组织各种细胞中的表达状况。结果:在实验观察期,大鼠卵巢中KL在部分早期原始卵泡和原始卵泡的卵母细胞中表达,在初级卵泡以后各级卵泡的卵母细胞中均表达,而各级卵泡的颗粒细胞均不表达。在性成熟前,随着卵巢的发育KL逐渐出现在卵巢膜细胞和间质细胞中;性成熟后卵巢的间质细胞、卵泡膜细胞、卵巢膜上皮细胞以及黄体细胞均表达大量的KL蛋白。在实验观察的各年龄段大鼠卵巢中从早期原始卵泡到窦状卵泡的卵母细胞均表达Kit蛋白,但表达量随卵泡的发育逐渐减弱;随着卵巢的发育,Kit蛋白逐渐出现在卵巢的膜上皮细胞、卵泡膜细胞和间质细胞中;Kit也在黄体细胞中表达。在一些初级卵泡至窦状卵泡的少数颗粒细胞中也观察到Kit蛋白表达。结论:KL蛋白在各级卵泡的卵母细胞中表达,在颗粒细胞中不表达,KL与其受体Kit都在卵母细胞中表达,这些结果提示卵母细胞的生长可能受自身KL-Kit信号的调节。     

βB2晶状体蛋白对小鼠卵巢发育及动情周期的影响

目的:初步探讨βB2晶状体蛋白(Crybb2)参与调节生殖过程的作用机制。方法:选取11-13周龄βB2基因敲除(KO组,n=19)与野生型C57BL/C(WT组,n=23)雌性小鼠,阴道涂片观察动情周期变化;HE染色观察卵巢病理变化;光学显微镜下计数卵巢最大切面原始卵泡、初级卵泡、闭锁卵泡;Western blotting和免疫组织化学确定βB2晶状体蛋白在卵巢组织中的表达与定位。结果:βB2晶状体蛋白主要表达在WT组小鼠卵巢颗粒细胞内。与WT组小鼠相比,KO组小鼠卵巢相对重量减轻,动情周期紊乱,原始卵泡、初级卵泡减少,闭锁卵泡增多。结论:βB2基因敲除小鼠的动情周期及卵巢发育异常,βB2晶状体蛋白对小鼠卵巢的发育有重要影响。     

不同浓度血管活性肠肽(VIP)对新生大鼠原始卵泡体外发育影响的实验研究

目的:观察不同浓度血管活性肠肽(VIP)对新生4 d大鼠卵巢体外培养过程中原始卵泡存活和生长发育的影响。方法:取新生4 d大鼠的卵巢72个,随机分为6组:新鲜组、基础培养组和不同浓度VIP组(10-9~10-6 mol/L)。新鲜组不经培养、其余各组体外培养14 d行组织形态学检查、增殖细胞核抗原(PCNA)免疫组织化学和TUNEL凋亡分析。结果:①各培养组的1级(早期初级)卵泡百分比明显高于新鲜组(P<0.05),且VIP 10-7 mol/L组最高。②各培养组PCNA阳性率与新鲜组比较均有统计学差异(P<0.001);不同浓度VIP组与基础培养组比较有统计学差异(P<0.05);VIP 10-7 mol/L组卵泡PCNA阳性率最高,与其他VIP浓度组比有显著差异(P<0.05),③各培养组卵泡凋亡率均显著高于新鲜组(P<0.001),培养组中以VIP 10-7 mol/L组较低,与VIP 10-8 mol/L组和VIP 10-9 mol/L组间有统计学差异(P<0.05)。结论:在卵巢体外培养过程中,VIP可以促进新生4 d大鼠的原始卵泡向初级卵泡转化;不同浓度的VIP均可降低卵巢组织卵泡细胞的凋亡,提高卵泡体外存活能力,VIP10-7 mol/L较其他浓度更明显地促进卵泡细胞的增殖,降低卵泡细胞的凋亡。     

新生小鼠原始卵泡成熟的诱导

目的:建立人工诱导新生小鼠原始卵泡成熟的方法。方法:30只新生小鼠卵巢同种异体移植于去势的10只成年雌小鼠肾被膜下,14d后分离窦前卵泡进行体外成熟培养。结果:卵泡成活率为50.6%,窦腔形成率为22.3%,卵母细胞成熟率为10.3%,MⅡ期卵母细胞平均直径为71.8±5.6μm。结论:本方法可以诱导新生小鼠原始卵泡发育,从而得到第二次减数分裂中期(MⅡ期)的卵母细胞。     

茶多酚对新生大鼠卵巢发育的影响

目的:探讨茶多酚对新生大鼠卵泡发育和卵母细胞凋亡的影响及相关调控机制。方法:受孕11.5dSD大鼠灌胃茶多酚(100mg/kg,qd)直至分娩,分别用多聚甲醛固定出生后1d、2d、4d、8d龄雌仔鼠卵巢(A组),同时将正常出生后1d的雌幼SD大鼠腹腔注射茶多酚(50mg/kg×1-8d),分别固定出生后2d、4d、8d龄卵巢(B组);所有卵巢经HE染色,观察不同发育阶段的卵泡比例,TUNEL(TdT-mediated dUTP Nick-End Labeling)荧光染色检测卵巢内卵母细胞凋亡变化,免疫组化方法观察叉头转录因子(FOXO3a)、促凋亡因子(Bim)和抗凋亡因子(MnSOD2)在卵巢组织中的表达水平,并以母、幼均未处理的正常同龄大鼠卵巢为对照(C组)。结果:在茶多酚干预组(包括A组和B组)卵巢内,1d、2d龄未装配卵泡比例及4d、8d龄原始卵泡比例均高于C组;A组、B组中卵母细胞TUNEL阳性率显著低于C组;FOXO3a、MnSOD2在A组的1d、2d、4d龄卵巢中的表达高于C组,但二组间的Bim表达水平相一致。结论:茶多酚能延缓新生大鼠卵母细胞巢破裂,抑制原始卵泡的发育启动,减少卵泡的消耗,可能有益于延长卵巢的生殖寿命;茶多酚可能通过上调FOXO3a的活性从而抑制原始卵泡的发育启动,同时通过激活其下游靶分子MnSOD2抑制卵母细胞的凋亡,促进卵母细胞的存活。     

Morphometric characteristics of preantral and antral follicles and expression of factors involved in folliculogenesis in ovaries of adult baboons (<Emphasis Type="Italic">Papio anubis</Emphasis>)

Purpose

Baboons are commonly utilized as an animal model for studies of human reproduction. However, folliculogenesis in this species has not been fully documented. The aim of this study was to assess follicle morphometry and expression of essential proteins involved in folliculogenesis in baboons.

Methods

Ovaries were recovered from four adult baboons and processed for histological evaluation and immunohistochemical analyses. Follicle proportion, follicle and oocyte diameter, theca layer thickness, number of granulosa cells, and follicle density were calculated. Immunohistochemical staining was also carried out for connexin 43 (Cx43), aromatase, and zona pellucida 3 (ZP3).

Results

A total of 2221 follicles were counted and measured. Proportions of primordial, primary, secondary, small antral, and large antral follicles were 49, 26, 23, 1, and 1 %, respectively. The increase in follicle diameter was due not only to the increase in oocyte diameter but also to granulosa cell proliferation. Almost all antral follicles were positive for Cx43 (89.8 %), aromatase (84.8 %), and ZP3 (100 %). Most secondary follicles were positive for Cx43 (65 %) and ZP3 (64.5 %), and some primary follicles were positive only for Cx43. No primordial follicles stained positive in any of these immunohistochemical analyses. Only antral follicles showed aromatase activity.

Conclusions

On the basis of these results, we can conclude that folliculogenesis in baboons appears to be similar to that in humans, and this animal therefore constitutes a valuable model.

     

Effect of mouse ovarian tissue cryopreservation by vitrification with Rapid-i closed system

Purpose

Currently, open systems are mainly used for cryopreservation of ovarian tissue, oocytes, and embryos, but there is a potential risk of contamination. This study was performed to assess ovarian tissue cryopreservation by a closed vitrification system (Rapid-i vitrification system?), which is already used clinically for oocyte/embryo cryopreservation.

Methods

Ovaries of C57BL/6J mice were frozen and thawed by using the Rapid-i vitrification system? (Rapid-i) followed by implantation into recipient mice. Hematoxylin-eosin staining was performed for histological examination of the frozen-thawed ovaries to assess follicle grade. Fertility after implantation of the ovaries was assessed from the live birth rate and the number of live pups.

Results

There was no significant difference in grade 1 primary follicles between fresh ovaries (control group, 94.2?±?2.9%) and frozen-thawed ovaries (Rapid-i group, 87.1?±?1.8%). However, there was a significant decrease in grade 1 early and late secondary follicles in the Rapid-i group compared with the control group. The live-birth rate was significantly lower in the Rapid-i group compared with the control group (29.2 vs. 83.3%, p?<?0.05). On the other hand, there was no significant difference in the average number of live pups between the control group and the Rapid-i group (3?±?0.4 vs. 2.7?±?0.3).

Conclusions

The Rapid-i seems to be effective for cryopreservation of mouse ovarian tissue. Under appropriate conditions, the Rapid-i could be employed for ovarian tissue cryopreservation and preservation of fertility in humans.

     

Reduced and delayed expression of GDF9 and BMP15 in ovarian tissues from women with polycystic ovary syndrome

PurposeGrowth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play crucial roles in follicular development and oocyte maturation. This study aimed to investigate and compare the expression of these proteins in ovarian tissues of women with and without polycystic ovary syndrome (PCOS).MethodsOvarian tissues from 28 patients with PCOS and 26 normal ovulatory women were collected, and the expression of GDF9 and BMP15 in oocytes and granulosa cells was evaluated via immunohistochemical staining.ResultsGDF9 and BMP15 were first expressed in primordial follicles at very low levels, and their expression increased gradually with follicular development, reaching the highest levels in Graafian follicles. However, less GDF9 and BMP15 expression was observed in primordial, primary, and secondary follicles in ovarian tissues of PCOS patients compared with levels in the control tissues (P < 0.05). In Graafian follicles, GDF9 and BMP15 expression reached comparable levels in the PCOS and control groups (P > 0.05).ConclusionsThe expression of GDF9 and BMP15 in ovarian tissues varies among the developmental stages in both oocytes and granulosa cells in human ovarian tissues. The expression of these proteins is reduced and delayed in the early follicular stage in PCOS ovarian tissues, and these differences in expression may be associated with aberrant follicular development in patients with PCOS.     

Effects of luteinizing hormone and follicle stimulating hormone on the developmental competence of porcine preantral follicle oocytes grown in vitro

Background There has been controversy over the role of FSH in the regulation of preantral follicle development. LH is a survival and differentiation factor that increases oocyte maturation in FSH-supplemented cultures of mouse preantral follicles. However, little information exists on the action of LH and FSH in the developmental competence of porcine preantral follicle oocytes in vitro. Materials and methods Porcine preantral follicles were cultured for 3 days in the presence or absence of FSH or LH. Oocytes from these follicles were then matured, fertilized in vitro, and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. Results FSH or combined LH and FSH significantly enhanced follicular growth compared to LH alone or the controls. Combined LH and FSH treatment of preantral follicles significantly increased the percentage (59 ± 5%) of oocytes competent to undergo cleavage to the two-cell stage after fertilization. A significant effect was seen on oocyte competence to develop from the two-cell to the blastocyst stage (30 ± 6%) compared to FSH alone treatment (45 ± 7 and 14 ± 5%, respectively). The amount of estradiol on days 2 and 3 of culture was significantly higher in follicles cultured with FSH (48.75 ± 17, 70.5 ± 14 pg/ml) or combined LH and FSH (63.25 ± 16, 72.5 ± 12 pg/ml) than that cultured with the untreated controls (16 ± 10, 5.66 ± 4 pg/ml). Conclusions The results indicated that FSH is essential for the in vitro growth of porcine preantral follicles, estradiol secretion, and for oocytes to acquire competence to resume meiosis and undergo fertilization and embryonic development. LH with FSH treatment of porcine preantral follicles can improve the quality of oocytes by promoting growth and a higher frequency of embryonic development.     

Evaluation of oxygen consumption in human vitrified and warmed pre-antral follicles after prolonged low temperatures

Purpose

The purpose of this study was to evaluate the effect of transportation at prolonged low temperatures on the survival of pre-antral follicles.

Methods

Ovarian tissue was removed from six women with gender identity disorder. Tissues were stored in an icebox at 4 °C for 6 or 18 h prior to vitrification. After warming, ovarian tissues were cultured for 24 h and follicle survival was assessed via a viability/cytotoxicity kit. Morphological features and oxygen consumption rate (OCR) were evaluated by scanning electrochemical microscopy (SECM).

Results

Survival rate of isolated primordial follicles was 95.7 and 100 %, and that of primary follicles was 91.7 and 81.8 % in the 6- and 18-h groups respectively. There was no difference in morphology between the 6- and 18-h storage groups. In comparison with OCR of vitrified-warmed follicles and OCR of 24-h culture after vitrified-warmed follicles, OCR of 24-h culture after vitrified-warmed primordial follicles was significantly higher in both 6-hour (0.02 ± 0.02 vs 0.07 ± 0.04, P < 0.05) and 18-h groups (0.02 ± 0.02 vs 0.11 ± 0.10, P < 0.05).

Conclusions

This strongly suggests that prolonged transportation of ovarian tissue at low temperatures is useful when there are no available local systems for fertility preservation.     

Variation in ovarian follicle density during human fetal development

Purpose

To obtain a precise estimate of ovarian follicle density and variation in the number of follicles at several gestational ages during human fetal development.

Methods

Twelve necropsied ovaries from 9 fetuses (gestational age: 24 to 36?weeks) and 3 neonates (who died within the first hours of life) were studied. Ovaries were fixed with 4?% formaldehyde and embedded in paraffin. Serial, 7?mm thick sections of the ovaries were cut and evaluated at every 50 cuts. Follicles were counted in 10 regions (each measuring 625 μm²) of the ovarian cortex and the number of follicles per mm3 was calculated.

Results

The number of follicles per 0.25?mm2 ranged from 10.9 (± 4.8) in a neonate to 34.7 (± 10.6) also in a neonate. Among fetuses, follicle density was lowest at 36?weeks of gestation (11.1?±?6.2) and highest at 26?weeks (32?±?8.9). The total number of follicles ranged from 500,000 at the age of 22?weeks to?>?1,000,000 at the age of 39?weeks.

Conclusion

Our results show a peak in the number of follicles during intrauterine life at approximately 26?weeks, followed by a rapid reduction in this number before birth, providing a step forward towards the understanding of primordial follicular assembly in humans and, ultimately, the identification of the determinants of reproductive capacity.