Nowak R A, Haimovici F, Biggers J D, Erbach G T
Department of Obstetrics and Gynecology, Brigham & Women's Hospital, Boston, MA 02115, USA.
Biol Reprod. 1999 Jan;60(1):85-93. doi: 10.1095/biolreprod60.1.85.
The goals of this study were 1) to compare the effects of transforming growth factor-beta (TGF-beta) and parathyroid hormone-related protein (PTHrP) on mouse blastocyst attachment and outgrowth in vitro, 2) to determine whether TGF-beta acts through a mechanism involving PTHrP, 3) to examine effects of PTHrP on preimplantation mouse embryo development, and 4) to determine the pattern of expression of PTHrP protein in the uterus of the mouse during early gestation. In the first set of experiments, hatched blastocysts were placed in fibronectin-coated wells. Cultures were treated with PTHrP or TGF-beta1 and assessed at 24, 48, and 72 h for attachment and surface area of blastocyst outgrowth. Results showed that both PTHrP and TGF-beta1 increased blastocyst outgrowth significantly. A PTHrP-neutralizing antibody blocked the stimulatory effect of both PTHrP and TGF-beta1, suggesting that TGF-beta1 acts to increase endogenous production of PTHrP by the blastocyst. Immunoassay of conditioned medium from blastocysts treated with either TGF-beta1 or PTHrP 1-34 confirmed a 3- to 4-fold increase in levels of PTHrP 1-141. In the second series of experiments, pronuclear zygotes were cultured in various concentrations of PTHrP for 96 h. Blastocysts then were subjected to differential fluorescent staining of inner cell mass and trophectoderm cells. Treatment of mouse embryos with the various concentrations of PTHrP altered neither the number developing to the blastocyst stage nor the number of inner cell mass or trophectoderm cells in the resulting blastocysts. In the third experiment, pregnant mice were killed at Days 3, 4, 5, 6, and 7 of gestation, and uterine horns were processed for immunohistochemistry. Uterine sections were stained with antibodies to PTHrP, desmin, and laminin. On Days 3, 4, and 5, uterine luminal and glandular epithelial cells stained intensely for PTHrP, while stromal cells were negative. By Days 6 and 7, decidualized stromal cells stained positively for PTHrP, desmin, and laminin. These results support the hypothesis that TGF-beta and PTHrP play an important role in the process of implantation.
1）比较转化生长因子-β（TGF-β）和甲状旁腺激素相关蛋白（PTHrP）对小鼠囊胚体外黏附和孵出的影响；2）确定TGF-β是否通过涉及PTHrP的机制发挥作用；3）研究PTHrP对植入前小鼠胚胎发育的影响；4）确定妊娠早期小鼠子宫中PTHrP蛋白的表达模式。在第一组实验中，将孵化的囊胚置于纤连蛋白包被的孔中。用PTHrP或TGF-β1处理培养物，并在24、48和72小时评估囊胚孵出的黏附情况和表面积。结果显示，PTHrP和TGF-β1均显著增加了囊胚孵出。一种PTHrP中和抗体阻断了PTHrP和TGF-β1的刺激作用，表明TGF-β1通过增加囊胚内源性PTHrP的产生发挥作用。对用TGF-β1或PTHrP 1-34处理的囊胚的条件培养基进行免疫测定，证实PTHrP 1-141水平增加了3至4倍。在第二系列实验中，将原核受精卵在不同浓度的PTHrP中培养96小时。然后对囊胚进行内细胞团和滋养外胚层细胞的差异荧光染色。用不同浓度的PTHrP处理小鼠胚胎，既不改变发育到囊胚阶段的数量，也不改变所得囊胚中内细胞团或滋养外胚层细胞的数量。在第三个实验中，在妊娠第3、4、5、6和7天处死怀孕小鼠，对子宫角进行免疫组织化学处理。子宫切片用抗PTHrP、结蛋白和层粘连蛋白的抗体染色。在第3、4和5天，子宫腔和腺上皮细胞对PTHrP染色强烈，而基质细胞为阴性。到第6和7天，蜕膜化的基质细胞对PTHrP、结蛋白和层粘连蛋白染色呈阳性。这些结果支持了TGF-β和PTHrP在着床过程中起重要作用的假说。
