Kisspeptin, a neuropeptide and the master controller of reproductive axis upstream to GnRH neurons, and its receptor are also expressed in extrahypothalamic tissues, such as ovaries. As systemic kisspeptin has been shown to modulate follicular dynamics in cattle, we hypothesized that kisspeptin has direct actions on the ovarian follicular development. We also hypothesized that kisspeptin regulation of primordial follicle development is via modulation of VEGF expression. In order to test these hypotheses, we cultured caprine ovarian cortical strips in vitro for 7 days with supplementation of kisspeptin at 1, 10 and 100 μM concentration and observed the development of primordial follicles into intermediate, primary and secondary follicles. We also studied the alteration in the expression profile of VEGF and VEGF transcript variant 2 mRNA during follicular development in the presence of kisspeptin. We confirmed the presence of GPR54 in goat ovaries in our preliminary studies. Supplementation of kisspeptin at 1 and 10 μM concentration facilitated the development of primordial follicles into intermediate, primary and secondary follicles with less number of degenerated follicles while the same at 100 μM resulted in degeneration of follicles. We observed a drastic increase in the expression profile of VEGF and VEGF transcript variant 2 mRNA upon culture which was independent of kisspeptin treatment. In conclusion, our studies show that kisspeptin facilitates ovarian primordial development in vitro.

Kisspeptin-10 (KP-10) has been reported to act as a tumor metastasis suppressor via its receptor, G protein-coupled receptor 54 (GRP54). The KP-10/GPR54/BMPs signaling pathway plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. The aim of this study was to confirm the molecular mechanism for the action of KP-10 on osteoblast differentiation. Expression of the Bone morphogenetic protein-2 (BMP2) and osteogenic genes were determined by RT-PCR and real-time PCR analysis in C3H10T1/2 cells. Transient transfection assays were performed to confirm the effects of KP-10 on BMP2-Luc activity. BMP2 and phospho-Smad1/5/9 protein levels were determined by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. To further confirm the effect of KP-10-induced GPR54, we used GPR54 Knock out (KO) C3H10T1/2 cells. KP-10 significantly increased osteogenic gene such as Runx2, ALP and Dlx5 in C3H10T1/2 cells. The ALP staining levels were also increased by KP-10. Interestingly, BMP2 mRNA, protein expression and promoter activity were also increased by KP-10. However, KP-10-induced BMP2 expressions were not increased in GPR54 KO cells. These results suggest that KP-10 increases BMP2 expression through GPR54. Next, Western blot analysis shown Smad1/5/9 phosphorylation were enhanced in a time-dependent manner by KP-10 treatment. It is well known that BMP2 increased phosphorylation of Smad1/5/9 via BMP2 receptor. In addition, KP-10 increased NFATc4 mRNA levels and NFATc4 overexpression enhance BMP2 mRNA levels. To confirm the KP-10-induced BMP2 action, we used KP-10-treated medium in wild type cells and GPR54 KO cells. The osteogenic genes were not elevated by KP-10-treated medium (GPR54 KO cells) whereas increased expression levels by KP-10 medium (wild type cells). These data indicate that KP-10 induced osteoblast differentiation through NFATc4-mediated BMP2 signaling.

최근 여러 포유류 종에서 Kisspeptin이 그 수용체인 GPR54와 함께 GnRH 분비를 자극하여 성 성숙과 최종배란에 영향을 미친다고 보고되고 있다. 이러한 보고들은 KiSS-GPR54 system이 번식과 밀접한 연관이 있음을 제시하고 있지만, 어류에 관한 연구는 미비한 실정이다. 이 연구는 어류에서의 KiSS-GPR54 system의 생리적 기능과 번식내분비적 기능을 탐색하기 위해 홍바리 Epinephelus fasciatus 뇌에서 KiSS1

Kisspeptin has been implicated in the process of puberty onset in various animal groups. This peptide is encoded by a gene, Kiss1 in avian and mammalian species. Contrary to these higher vertebrates, however, fish appeared to have another gene, Kiss2 that also codes for the precursor peptide of kisspeptin. To figure out biological significance of this gene during the puberty onset in fish, the expression profile of Kiss2 gene was investigated in the brain of Nile tilapia together with genes of GPR54, GnRH receptorI (rGnRHI) and GTH subunits ( and ). Expression of Kiss2 mRNA significantly increased at 2 weeks post hatch (wph) and 13 wph (<0.05). This increase coincided with the increases of GPR54 and rGnRH I gene expression. Detection of and subunit gene expression was possible later than 13 wph, indicating the activation of gonadotrophs in the pituitary. Data obtained from this study strongly suggest that, in addition to Kiss1 gene, Kiss2 gene is deeply associated with the onset of puberty by the activation of hypothalamus pituitary gonadal axis in Nile tilapia.

GPR 54 (G protein-coupled receptor 54)는 kisspeptin receptor로 알려져 있으며 KISS1 receptor (KISS1r)로 불리기도 한다. GPR 54는 kisspeptin과의 연관성이 알려지기 이전부터 kisspeptin과는 별개로 그 구조와 기능이 연구되었다. 틸라피아 (Nile tilapia, Oreochromis niloticus)에서는 2004년도에 뇌에서 분리 동정되었으며, 이 어종의 뇌에 있는 GnRH 분비세포와 같은 위치에서 GPR 54 유전자의 발현이 관찰되었다 (Parhar et al., 2004). KISS/GPR 54 system은 포유류의 성 성숙 및 번식과 연관되어 있는 것으로 밝혀졌으며, 어류에서도 번식 조절 체계에서 외부의 환경신호와 체내의 대사적 신호를 중개하는 연결고리로 작용할 가능성이 높다. 그러나 어류에서 KISS/GPR 54 system에 관한 연구는 아직 많지 않아 역할구명을 위한 연구가 필요하다. 본 연구에서는 KISS/GPR 54 system역할 구명에 대한 기초자료를 얻고자 틸라피아를 대상으로 개체크기별 및 조직별 GPR 54유전자의 발현 양상을 조사하였다. 선문대학교 수산생명의학과 수조실에서 사육중인 틸라피아를 아래의 표와 같이 크기별로 나누어 실험을 진행하였다. 자․치어를 제외한 나머지 실험어에서는 뇌를 적출하였으며, 아가미, 간, 근육, 생식소를 샘플하여 GPR 54유전자의 발현 양상을 비교하였다. 부화 후 1일, 부화 후 7일, 부화 후 30일, 부화 후 60일의 모든 그룹에서 GPR 54의 발현을 확인할 수 있었고 특히, 난황을 달고 있는 부화 후 1일 자어그룹에서 발현양이 가장 많은 것으로 확인되었다. 조직 내 GPR 54의 발현은 소형어, 중형어, 그리고 대형어 그룹 모두에서 뇌와 난소에서만 발현되는 특징을 보였다. 소형어에서의 뇌와 난소에서의 발현이 중형어와 대형어와 비교해 높은 GPR 54유전자 발현을 나타냈다. 뇌와 난소에서의 높은 발현은 개체의 크기가 커질수록 낮아지는 경향을 보였다. 자․치어를 제외한 모든 실험어는 puberty 단계에 도달한 개체를 사용하였지만 개체의 크기에 따라 발현양에 차이가 나타나는 것을 확인할 수 있었다. 이러한 결과는 GPR 54가 틸라피아 성적 발달 과정과 연관되어 있음을 시사한다.