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 is a key player in the central control of reproductive axis. Central administration of kisspeptin has been shown to advance puberty in rats. Stimulation of hypothalamic GnRH pulse generating mechanism by kisspeptin has been proposed to be the mechanism behind the onset of puberty. We hypothesized that chronic high doses of kisspeptin administration suppresses the reproductive axis and hence delays the pubertal onset. Hence, we investigated the effect of peripheral administration of chronic high doses of kisspeptin on pubertal onset, feed intake and body weight in female rats. Rats were treated with saline or kisspeptin (100 nmoles per day; intraperitoneal) for 26 days (day 25 to day 50 postnatal) and the day of vaginal opening was marked as day of puberty. Kisspeptin treated rats had delayed pubertal onset and reduced feed intake and body weight. Gonadal GPR54 mRNA was reduced suggesting that chronic high doses of kisspeptin may suppress the reproductive functions possibly by downregulation of GPR54 receptor. However, delay in puberty due to reduction in feed intake and body weight could not be ruled out in this study. Further, our study emphasizes the importance of dosage and duration of kisspeptin administration in the manipulation of reproductive axis. Our study, for the first time, suggests that kisspeptin and its analogues, if proven beneficial, could be used to treat precocious puberty in children. It appears that, though a promising tool for enhancing fertility, kisspeptin acts as a double-edged sword and has to be cautiously used to manipulate reproduction.

Chronic and unpredictable stress can disrupt the female reproductive system by suppression for secretion of gonadotrophin-releasing hormone (GnRH) and gonadotrophin, resulted in ovarian malfunction and infertility. In the recent days, kisspeptin has been highly highlighted as a hypothalamic peptide which directly stimulates synthesis and release for GnRH. However, in spite of the key role of kisspeptin in the female reproductive system, little information is still available on the changes of its expression during ovarian cycle under stressed condition. Therefore, we induced chronic and unpredictable stress series to the female mice to analyze kisspeptin expression in the brain and ovary. Stressed mice exhibited changes of behavior and body weight gain during the stress assessment, which suggested that the present stress model in mice was successfully established. In the brain level, kisspeptin expression was attenuated than control. In the ovary level, the stressed mice displayed irregularly shrunk oocytes with broken zona pellucida throughout the follicle stages, pyknotic granulosa cells, decreased number of developing follicles and increased number of atretic follicles than the control. In case of kisspeptin expression in the whole ovary tissue, the expression level was decreased in the stressed mice. In detail, the less intensity of kisspeptin expression in the antral follicles phase was observed in the stressed mice than control mice, indicating that local function of kisspeptin during ovary cycle is highly associated with development of ovarian follicles. We expect that the present study has important implications for the fields of reproductive biology.

Previous studies have shown that kisspeptin (Kp-10) is expressed in mammalian ovaries; however, the expression and role of Kp-10 in bovine ovarian granulosa cells are still unclear. In this study, we assessed the expression of Kp-10 and its effects on the proliferation and apoptosis of bovine granulosa cells. Immunohistochemical analysis showed that Kp-10 was expressed in the cytoplasm of bovine ovarian granulosa cells. Moreover, MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2- H-tetrazolium bromide) assays showed that 100 nM Kp-10 significantly inhibited the viability of granulosa cells (P<0.01). Flow cytometry analysis showed that Kp-10 could significantly increase accumulation of cells in the G1 phase, decrease accumulation of cells in the S phase, and promote apoptosis in bovine granulosa cells (P<0.05). Additionally, Kp-10 decreased the mRNA levels of Bcl-2, an anti-apoptotic gene; increased the mRNA levels of caspase-3, a pro-apoptotic gene; and increased the mRNA levels of Fas and Fasl, two membrane surface molecule genes (P<0.05). Thus, our findings demonstrated for the first time that Kp-10 inhibited proliferation and promoted apoptosis in bovine ovarian granulosa cells. These findings provide insights into our understanding of the role of Kp-10 in mediating the proliferation of bovine granulosa cells.

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.

Kisspeptin (Kiss) and its cognate receptor, kisspeptin receptor (KissR; G protein coupled receptor 54, GPR54), have recently been recognized as potent regulators of reproduction in teleosts. Additionally, leptin plays an important role in energy homeostasis and reproductive function in teleosts. The purpose of this study was to examine differences in the concentration of the hormones of the Kiss/KissR system and leptin and the expression of their underlying genes, all of which are involved in the sexual maturation of female goldfish, Carassius auratus, following treatment with Kiss. The expression levels of KissR increased after the Kiss injection. Furthermore, the peptide hormone leptin also increased after the injection (in vivo and in vitro). Additionally, the expression of GnRH and GTHs (GTHα, FSHβ, and LHβ) increased in the brain and pituitary (in vitro and in vitro). These results support the hypothesis that Kiss plays important roles in the direct regulation of the hypothalamus-pituitary-gonad axis and leptin in goldfish. Therefore, we suggest that Kiss system gene expression is correlated with energy balance and reproduction.

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.