Grazul-Bilska A T, Redmer D A, Reynolds L P
Cell Biology Center, North Dakota State University, 58105, Fargo, ND,
Endocrine. 1996 Oct;5(2):225-33. doi: 10.1007/BF02738710.
Cellular interactions mediated by contact-dependent pathways may be important to maintain luteal function. The objective of the present experiment was to evaluate the role of LH and prostaglandin F(2α) (PGF) in regulation of contact-dependent, gap junctional intercellular communication (GJIC) of ovine luteal cells from several stages of luteal development. Corpora lutea (CL) obtained from superovulated ewes on days 5 (n=7), 10 (n=8), and 15 (n=9) after estrus were dispersed with collagenase and cell types were separated by elutriation. Cells were plated as a mixed population (nonelutriated), or as small or large luteal cell fractions, and incubated in serum-free media containing no hormone, LH (100 ng/mL), PGF (100 ng/mL), LH+PGF, or dibutyryl cAMP (dbcAMP; 2 mM) for 18-24 h. Media were collected for evaluation of progesterone (P4) concentrations and replaced with media containing fluorescent dye. Then the rate of GJIC was evaluated by using the fluorescence recovery after photobleaching technique and laser cytometry. The rate of GJIC was determined for selected cells: small luteal cells in contact only with small luteal (S-S) cells; large luteal cells in contact only with small luteal (L-S) cells; and large luteal cells in contact only with large luteal (L-L) cells. LH increased (p<0.01) GJIC for S-S on d 5 and 10 and for L-S cells across the estrous cycle, but did not affect GJIC for L-L cells. PGF increased (p<0.05) GJIC for L-L cells on d 10 and 15, and decreased (p<0.05) GJIC for S-S cells from d 5 and 10 of the estrous cycle. LH+PGF increased (p<0.05) GJIC for S-S cells on d 5 and 10, and for L-S and L-L cells on d 10 and 15 of the estrous cycle. In addition, PGF diminished (p<0.05) LH-stimulatory effects on GJIC for S-S cells from d 5 and 10, and for L-S cells from d 5 of the estrous cycle. Dibutyryl cAMP stimulated (p<0.05) GJIC between all evaluated cell types across the estrous cycle. LH and dbcAMP stimulated (p<0.05) P4 secretion by mixed and small luteal cell fractions, PGF alone did not affect basal P4 secretion, but LH+PGF stimulated (p<0.05) P4 production by small luteal cells across the estrous cycle. PGF diminished (p<0.05) LH-stimulatory effects on P4 production in mixed populations of luteal cells across the estrous cycle.These data demonstrate that both luteal cell types communicate with each other, and the rate of communication was affected by LH, PGF, and dbcAMP. Modulation of gap junctional contact-dependent intercellular communication may be an important mechanism by which regulatory signals are transduced during luteal growth, differentiation, and regression in sheep.
由接触依赖性途径介导的细胞间相互作用对于维持黄体功能可能很重要。本实验的目的是评估促黄体生成素（LH）和前列腺素F2α（PGF）在调节来自黄体发育几个阶段的绵羊黄体细胞的接触依赖性、缝隙连接细胞间通讯（GJIC）中的作用。从发情后第5天（n = 7）、第10天（n = 8）和第15天（n = 9）的超排母羊获得黄体（CL），用胶原酶分散，通过淘洗分离细胞类型。将细胞接种为混合群体（未淘洗），或作为小黄体细胞或大黄体细胞组分，并在不含激素、LH（100 ng/mL）、PGF（100 ng/mL）、LH + PGF或二丁酰环磷腺苷（dbcAMP；2 mM）的无血清培养基中孵育18 - 24小时。收集培养基以评估孕酮（P4）浓度，并用含荧光染料的培养基替换。然后使用光漂白后荧光恢复技术和激光细胞术评估GJIC的速率。针对选定的细胞确定GJIC的速率：仅与小黄体（S - S）细胞接触的小黄体细胞；仅与小黄体（L - S）细胞接触的大黄体细胞；以及仅与大黄体（L - L）细胞接触的大黄体细胞。LH在发情周期的第5天和第10天增加（p < 0.01）S - S细胞以及整个发情周期中L - S细胞的GJIC，但不影响L - L细胞的GJIC。PGF在发情周期的第10天和第15天增加（p < 0.05）L - L细胞的GJIC，并在发情周期的第5天和第10天降低（p < 0.05）S - S细胞的GJIC。LH + PGF在发情周期的第5天和第10天增加（p < 0.05）S - S细胞的GJIC，并在发情周期的第10天和第15天增加L - S和L - L细胞的GJIC。此外，PGF在发情周期的第5天和第10天减弱（p < 0.05）LH对S - S细胞以及发情周期第5天的L - S细胞GJIC的刺激作用。二丁酰环磷腺苷在整个发情周期中刺激（p < 0.05）所有评估细胞类型之间的GJIC。LH和二丁酰环磷腺苷刺激（p < 0.05）混合和小黄体细胞组分分泌P4，单独的PGF不影响基础P4分泌，但LH + PGF在整个发情周期中刺激（p < 0.05）小黄体细胞产生P4。PGF在整个发情周期中减弱（p < 0.05）LH对黄体细胞混合群体中P4产生的刺激作用。这些数据表明两种黄体细胞类型相互通讯，并且通讯速率受LH、PGF和二丁酰环磷腺苷影响。缝隙连接接触依赖性细胞间通讯的调节可能是绵羊黄体生长、分化和退化过程中调节信号转导的重要机制。
