Johnson Mary Lynn, Redmer Dale A, Reynolds Lawrence P, Bilski Jerzy J, Grazul-Bilska Anna T
Endocrine. 2002 Aug;18(3):261-70. doi: 10.1385/ENDO:18:3:261.
Throughout each estrous cycle, the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are involved in regulation of folliculogenesis. We have shown that LH or FSH affect cellular interactions mediated by gap junctions in bovine granulosa and thecal cells in vitro. To evaluate further the hypothesis that gonadotropins influence gap junctional intercellular communication (GJIC) and expression of gap junctional proteins known as connexins (Cx), throughout antral follicle development, granulosa and thecal cells from large (>10 mm; n = 13), medium (5-10 mm; n = 20), and small (<5 mm; n = 27) follicles were cultured (n = 4 cultures per size) with or without LH, FSH, or LH + FSH for 24 h. GJIC was evaluated (n = 125-150 cells/treatment group) by using the fluorescent recovery after photobleaching technique and laser cytometry. Additionally, Cx43, Cx32, and Cx26 were detected in cultured cells by immunocytochemistry and Cx43 by Western immunoblot analysis. Finally, progesterone production by cultured cells was evaluated by radioimmunoassay. Across all follicles and treatments, GJIC was greater (p < 0.01) for granulosa than thecal cells (4.9 +/- 0.05 vs 3.8 +/- 0.04%/min). For granulosa cells of large and medium follicles, LH and/or FSH did not affect GJIC. For granulosa cells of small follicles, FSH increased (p < 0.05), but LH or LH + FSH had no effect on GJIC. For thecal cells of large follicles, LH increased (p < 0.01) GJIC, whereas FSH or LH + FSH had no effects. For thecal cells of medium and small follicles, LH and/or FSH did not affect GJIC. These results demonstrate that FSH influenced GJIC of granulosa cells from small, but not from medium or large, follicles, and LH influenced GJIC of thecal cells from large, but not from medium or small, follicles. Cx43 was present as punctate staining between granulosa or thecal cells from all cultures, indicating assembled gap junctions. LH + FSH increased (p < 0.05) expression of Cx43 only by thecal cells from large follicles. Cx32 was detected in the perinuclear cytoplasm of cultured granulosa or thecal cells, and in the cytoskeleton of a few cells per culture dish in all sizes of follicles. Cx26 was present in a regular pattern throughout the cytoplasm of granulosa or thecal cells in all sizes of follicles. For granulosa cells from large follicles, progesterone production was stimulated (p < 0.05) with LH or FSH alone but was unaffected by LH + FSH. For granulosa cells from medium and small follicles, progesterone production was unaffected by LH and/or FSH. For thecal cells from all sizes of follicles, LH, FSH, and LH + FSH stimulated (p < 0.05) production of progesterone. These data indicate that LH and FSH influence gap junction function and expression, which likely contributes to the development and maintenance of ovarian follicles.
在每个发情周期中，促性腺激素，即黄体生成素（LH）和卵泡刺激素（FSH），都参与卵泡生成的调节。我们已经表明，LH或FSH在体外会影响牛颗粒细胞和卵泡膜细胞中由缝隙连接介导的细胞间相互作用。为了进一步评估促性腺激素在整个窦状卵泡发育过程中影响缝隙连接介导的细胞间通讯（GJIC）以及缝隙连接蛋白（连接蛋白，Cx）表达的假说，将来自大卵泡（>10 mm；n = 13）、中卵泡（5 - 10 mm；n = 20）和小卵泡（<5 mm；n = 27）的颗粒细胞和卵泡膜细胞（每种大小n = 4个培养物）在添加或不添加LH、FSH或LH + FSH的情况下培养24小时。通过光漂白后荧光恢复技术和激光细胞术评估GJIC（每个处理组n = 125 - 150个细胞）。此外，通过免疫细胞化学检测培养细胞中的Cx43、Cx32和Cx26，并通过Western免疫印迹分析检测Cx43。最后，通过放射免疫测定法评估培养细胞的孕酮生成。在所有卵泡和处理组中，颗粒细胞的GJIC均高于卵泡膜细胞（4.9±0.05 vs 3.8±0.04%/分钟，p < 0.01）。对于大卵泡和中卵泡的颗粒细胞，LH和/或FSH不影响GJIC。对于小卵泡的颗粒细胞，FSH可增加GJIC（p < 0.05），但LH或LH + FSH对GJIC无影响。对于大卵泡的卵泡膜细胞，LH可增加GJIC（p < 0.01），而FSH或LH + FSH无影响。对于中卵泡和小卵泡的卵泡膜细胞，LH和/或FSH不影响GJIC。这些结果表明，FSH影响小卵泡而非中、大卵泡颗粒细胞的GJIC，LH影响大卵泡而非中、小卵泡卵泡膜细胞的GJIC。Cx43在所有培养物的颗粒细胞或卵泡膜细胞之间呈点状染色，表明存在组装好的缝隙连接。LH + FSH仅增加大卵泡卵泡膜细胞中Cx43的表达（p < 0.05）。在所有大小卵泡的培养颗粒细胞或卵泡膜细胞的核周细胞质中以及每个培养皿中少数细胞的细胞骨架中检测到Cx32。Cx26以规则模式存在于所有大小卵泡的颗粒细胞或卵泡膜细胞的整个细胞质中。对于大卵泡的颗粒细胞，单独使用LH或FSH可刺激孕酮生成（p < 0.05），但LH + FSH对其无影响。对于中卵泡和小卵泡的颗粒细胞，LH和/或FSH对孕酮生成无影响。对于所有大小卵泡的卵泡膜细胞，LH、FSH和LH + FSH均可刺激孕酮生成（p < 0.05）。这些数据表明，LH和FSH影响缝隙连接功能和表达，这可能有助于卵巢卵泡的发育和维持。
