Immunohistochemical localization of kisspeptin and kisspeptin receptor in the equine ovary: Effect of follicle size and reproductive state

Kisspeptin is considered a regulator of reproduction by stimulating GnRH; however, extra-hypothalamic actions of kisspeptin have been described. The aim of this study was to localize kisspeptin-10 (Kp10) and kisspeptin receptor (Kiss1r) in the ovary and compare staining intensity between follicles in follicular-phase (FP) and luteal-phase (LP) ovaries, as well as between seasonally anovulatory (SA) ovaries and ovaries from mares induced to cycle in winter with estradiol cypionate (ECP) and sulpiride. Eight ovaries were collected from light-horse mares during the breeding and non-breeding seasons and characterized as FP (n = 3), LP (n = 2), or SA (n = 3). Additionally, 3 ovaries were collected from SA mares treated with ECP and sulpiride upon detection of the first 30-mm follicle after treatment. Immunohistochemistry was carried out on whole-ovarian, paraffin-embedded sections using anti-Kp10 and anti-Kiss1r antibodies validated on equine hypothalamus. Staining intensity was evaluated in primordial, preantral, and antral follicles using a scale of 0-3. Given a normal distribution, two-way ANOVA was used to compare staining between follicle sizes and reproductive states. Staining for Kp10 and Kiss1r was detected in all follicle sizes throughout all reproductive states, as well as in oocytes and corpora lutea. Staining intensity for both differed between all follicle sizes and was greatest (P < 0.01)in antral follicles. Staining for Kp10 and Kiss1r was similar in preantral follicles of all reproductive states but was greater (P < 0.01) than primordial follicles. In primordial follicles, staining for Kp10 and Kiss1r was greatest (P < 0.01) in ECP-sulpiride stimulated ovaries, but similar between all other states. Kisspeptin-10 and Kiss1r staining was greatest (P < 0.01) in antral follicles of FP and ECP-sulpiride stimulated ovaries. Additional quantification of Kp10 and Kiss1r immunostaining in granulosa cells (GC) of antral follicles was achieved by evaluating three random, 625 μm2 fields in 2-3 follicles per animal in each reproductive state. Color deconvolution of standardized images was performed, and units of intensity converted to optical density and compared by ANOVA. Staining for both Kp10 and Kiss1r was most intense in GC of FP and ECP-sulpiride-stimulated ovaries and was greater (P < 0.0001) than LP and SA ovaries. No differences were detected between LP and SA. Here we characterize, for the first time, Kp10 and Kiss1r in the equine ovary. Staining intensity for both increased as follicle size increased. Staining was greatest in GC of FP ovaries, indicating a potential mechanistic role for kisspeptin during late-stage follicle growth and perhaps ovulation. Furthermore, staining in ECP-sulpiride stimulated ovaries was similar to FP ovaries, providing evidence that treatment with ECP-sulpiride during seasonal anovulation restored ovarian kisspeptin activity to a FP-like state.