Characterization of Luteinizing Hormone and Luteinizing Hormone Receptor and Their Indispensable Role in the Ovulatory Process of the Medaka
Figure 3
Histological localization analysis of gonadotropin receptors in the medaka ovary.
(A) In situ hybridization analysis for fshra mRNA was conducted using frozen sections of the ovary isolated 1 h after ovulation. Staining with the antisense probe is shown in the left and middle panels. The boxed area in the left panel is shown at higher magnification in the middle panel. As a control, staining with the sense probe is shown in the right panel. Black arrows indicate positive staining associated with the follicle layers of middle-sized follicles, and white arrows indicate positive staining associated with the cytoplasm of small follicles. Bars represent 500 µm (for the right and left panels) and 100 µm (for the middle panel). *, large preovulatory follicle; #, medium-sized follicle; +, postovulatory follicle with no oocyte. (B) In situ hybridization analysis for lhcgrbb mRNA was conducted using paraffin sections of the ovary isolated 1 h after ovulation. The left two panels and right two panels show the staining with the antisense and sense probe, respectively. The boxed areas in the antisense and sense staining are also shown at higher magnification to the right. The white arrows indicate positive staining associated with the cytoplasm of small follicles. The black arrowhead and the white arrowhead in the enlarged antisense staining panel indicate the theca cell layer (TC) and granulosa cell layer (GC), respectively, that surround the egg membrane (EM) of follicles. Bars represent 400 µm (for the low-magnification panels) and 50 µm (for the higher-magnification panels). *, large preovulatory follicle; #, middle-sized follicle; +, postovulatory follicle with no oocyte. (C) Immunohistochemical analysis for Lhcgrbb was conducted using paraffin sections of the ovary isolated 1 h after ovulation. Staining with the antibody for medaka Lhcgrbb is shown in the left and middle panels. The boxed area in the left panel is shown at higher magnification in the middle panel. As a control, staining with the absorbed antibody is shown in the right panel. Black arrows indicate positive staining associated with the follicle layers of large and medium-sized follicles, and white arrows indicate positive staining associated with the cytoplasm of small follicles. The black arrowhead and the white arrowhead indicate the TC and GC layers, respectively. Bars represent 500 µm (for the right and left panels) and 100 µm (for the middle panel). *, large preovulatory follicle; #, middle-sized follicle; +, postovulatory follicle with no oocyte. The reproducibility of all findings was confirmed by conducting four separate experiments. The results of a representative experiment are presented.