BrdU labelling of oocyte in the rat fetus ovary

为了动态分析卵泡的发育，通过标记部分卵泡并跟踪其生长来建立队列是有效的。众所周知，大鼠卵母细胞在胚胎期DNA倍增，其有丝分裂一直停滞到排卵前的第二次减数分裂。我们注意到这一现象可以用来确定在胚胎阶段标记某一组卵母细胞核的可能性。材料和方法将妊娠大鼠分为A、B、C和D组，每组4只，在怀孕第17天一次性经腹注射溴脱氧尿苷(BrdU)5g/体重(G)。他们被抚养长大，直到分娩。分别于1周龄(A组)、2周龄(B组)、3周龄(C组)、4周龄(D周)处死雌性新生动物，摘除双侧卵巢和肝脏。这些器官在福尔马林中固定，石蜡包埋，然后在5米厚的…中连续切片更多的纯净。每七个部分都安装在滑动玻璃上。抗BrdU单抗(Boehringer-Mannheim BioChemica)免疫组织化学染色采用生物素-链霉亲和素方法。结果与讨论1)部分胚胎卵母细胞在妊娠第17天开始被BrdU标记，并在出生后4周一直维持在细胞核内(A、B、C、D组)。A组肝细胞BrdU染色阳性，B、C、D组未检测到BrdU标记，说明肝细胞对BrdU的摄取在每次有丝分裂时被稀释到无法证实的程度。2)BrdU标记主要见于不含颗粒细胞的原始卵泡。出生后1周(A组)，BrdU标记的卵泡为原始卵泡的100%。出生2周原始卵泡占标记卵泡的96.3%(B组)，3周原始卵泡占标记卵泡的46.7%(C组)。BrdU标记的原始卵泡发生率的降低表明，在给定的BrdU标记的队列中，一些卵泡发育成下一类初级卵泡，并伴有几层颗粒细胞。出生后1~4周，仅有1~2个囊泡卵泡显示BrdU掺入。较少

INTRODUCTIONIn order to analyze the follicular development dynamically, it is effective to set up a cohort by labelling a part of follicles and to follow up their growth. It is widely known that rat's oocyte doubles its DNA in the embryonal stage and its mitosis has been arrested until the second meiosis just before ovulation. We noticed this phenomenon is available to determine the possibility of labelling a certain group of oocyte's nuclei in the embryonal stage.MATERIALS AND METHODSThe pregnant rats were divided into four groups (A, B, C and D group, each consisted of 4 animals) and transabdominally injected with bromodeoxyuridine (BrdU) 5 g/body weight (g) at one time in the 17th day of pregnancy. They were reared to delivery. Thereafter, the female newborn animals were reared and sacrificed at 1week (group A), 2week (B), 3week (C) and 4week (D) to excise bilateral ovaries and liver. These organs were fixed in formalin, embedded in paraffin, and then serially sliced at 5 m in thick … More ness. Every seventh section was mounted on slide glass. Immunohistochemical staining was employed with anti-BrdU monoclonal antibody (Boehringer-Mannheim Biochemica) by using Biotin-streptAvidin's method. We investigated the localization of BrdU which was incorporated into the cell.RESULTS AND DISCUSSION1) A part of fetal oocytes were labelled by BrdU in the 17th day of pregnancy and their labelling in the nuclei were maintained up to 4th week after delivery (group A, B, C, D). On the contrary, liver cells showed positive staining of BrdU in group A, however, BrdU labelling was not detectable in group B, C, and D. Therefore, it was clarified that BrdU-uptake in the liver cell was so diluted at its every mitosis to the degree which could not be demonstrated.2) BrdU labelling was evident mainly in the primordial follicle which did not have granulosa cells. At 1 week of delivery (group A), the follicles labelled by BrdU were 100% of the primordial follicles. The primordial follicles occupied 96.3% of the tagged follicles at 2 week of delivery (group B) and 46.7% at 3 week (group C). This decreased incidence of BrdU-labelled primordial follicles presented that some follicles in given BrdU-labelled cohort were developed into the next category of primary follicles accompanied with a few layers of granulosa cells. However, there were only 1 or 2 vesicular follicles showing BrdU incorporation through 1 to 4 week of delivery. Less