García-Ortega J, Pinto F M, Fernández-Sánchez M, Prados N, Cejudo-Román A, Almeida T A, Hernández M, Romero M, Tena-Sempere M, Candenas L
Instituto Valenciano de Infertilidad, Seville, Spain.
Instituto de Investigaciones Químicas, CSIC, Seville, Spain.
Hum Reprod. 2014 Dec;29(12):2736-46. doi: 10.1093/humrep/deu247. Epub 2014 Oct 14.
Are neurokinin B (NKB), NK3 receptor (NK3R), kisspeptin (KISS1) and kisspeptin receptor (KISS1R) expressed in human ovarian granulosa cells?
The NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and functionally active in ovarian granulosa cells.
The NKB/NK3R and KISS1/KISS1R systems are essential for reproduction. In addition to their well-recognized role in hypothalamic neurons, these peptide systems may contribute to the control of fertility by acting directly on the gonads, but such a direct gonadal role remains largely unknown.
STUDY DESIGN, SIZE, DURATION: This study analyzed matched mural granulosa cells (MGCs) and cumulus cells (CCs) collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval.
PARTICIPANTS/MATERIALS, SETTING, METHODS: The samples were provided by 56 oocyte donor women undergoing ovarian stimulation treatment. Follicular fluid samples containing MGCs and cumulus-oocyte complexes were collected after transvaginal ultrasound-guided oocyte retrieval. RT-PCR, quantitative real-time PCR, immunocytochemistry and western blot were used to investigate the pattern of expression of the NKB/NK3R and KISS/KISS1R systems in MGCs and CCs. Intracellular free Ca(2+) levels, [Ca(2+)]i, in MGCs after exposure to NKB or KISS1, in the presence or not of tachykinin receptor antagonists, were also measured.
NKB/NK3R and KISS1/KISS1R systems were expressed, at the mRNA and protein levels, in MGCs and CCs, with significantly higher expression in CCs. Kisspeptin increased the [Ca(2+)]i in the cytosol of human MGCs while exposure to NKB failed to induce any change in [Ca(2+)]i. However, the [Ca(2+)]i response to kisspeptin was reduced in the presence of NKB. The inhibitory effect of NKB was only partially mimicked by the NK3R agonist, senktide and marginally suppressed by the NK3R-selective antagonist SB 222200. Yet, a cocktail of antagonists selective for the NK1, NK2 and NK3 receptors blocked the effect of NKB.
LIMITATIONS, REASONS FOR CAUTION: The granulosa and cumulus cells were obtained from oocyte donors undergoing ovarian stimulation, which in comparison with natural cycles, may have affected gene and protein expression in granulosa cells.
Our data demonstrate that, in addition to their indispensable effects at the central nervous system, the NKB/NK3R and kisspeptin/KISS1R systems are co-expressed and are functionally active in non-neuronal reproductive cells of the female gonads, the ovarian granulosa cells.
STUDY FUNDING/ COMPETING INTERESTS: This work was supported by grants from Ministerio de Economía y Competitividad (CTQ2011-25564 and BFI2011-25021) and Junta de Andalucía (P08-CVI-04185), Spain. J.G.-O., F.M.P., M.F.-S., N.P., A.C.-R., T.A.A., M.H., M.R., M.T.-S. and L.C. have nothing to declare.
神经激肽B（NKB）、NK3受体（NK3R）、亲吻素（KISS1）及亲吻素受体（KISS1R）是否在人卵巢颗粒细胞中表达？
NKB/NK3R及亲吻素/KISS1R系统在卵巢颗粒细胞中共表达且具有功能活性。
NKB/NK3R及KISS1/KISS1R系统对生殖至关重要。除了在下丘脑神经元中广为人知的作用外，这些肽系统可能通过直接作用于性腺来参与生育控制，但这种性腺直接作用在很大程度上仍不清楚。
研究设计、规模、持续时间：本研究分析了在取卵时从卵母细胞供体的排卵前卵泡中收集的配对壁层颗粒细胞（MGCs）和卵丘细胞（CCs）。
参与者/材料、环境、方法：样本由56名接受卵巢刺激治疗的卵母细胞供体女性提供。经阴道超声引导取卵后，收集含有MGCs和卵丘-卵母细胞复合体的卵泡液样本。采用逆转录-聚合酶链反应（RT-PCR）、定量实时聚合酶链反应（qRT-PCR）、免疫细胞化学和蛋白质印迹法研究NKB/NK3R及KISS/KISS1R系统在MGCs和CCs中的表达模式。还测量了在存在或不存在速激肽受体拮抗剂的情况下，NKB或KISS1作用于MGCs后细胞内游离钙离子水平（[Ca(2+)]i）。
NKB/NK3R及KISS1/KISS1R系统在mRNA和蛋白质水平上均在MGCs和CCs中表达，在CCs中的表达显著更高。亲吻素可增加人MGCs胞质中的[Ca(2+)]i水平，而NKB作用后[Ca(2+)]i未发生任何变化。然而，在存在NKB的情况下，亲吻素引起的[Ca(2+)]i反应降低。NK3R激动剂senktide仅部分模拟了NKB的抑制作用，而NK3R选择性拮抗剂SB 222200对其抑制作用微弱。然而，NK1、NK2和NK3受体选择性拮抗剂的混合物可阻断NKB的作用。
局限性、谨慎的原因：颗粒细胞和卵丘细胞取自接受卵巢刺激的卵母细胞供体，与自然周期相比，这可能影响颗粒细胞中的基因和蛋白质表达。
我们的数据表明，除了在中枢神经系统中不可或缺的作用外，NKB/NK3R及亲吻素/KISS1R系统在女性性腺的非神经元生殖细胞即卵巢颗粒细胞中共表达且具有功能活性。
研究资金/利益冲突：本研究得到西班牙经济与竞争力部（CTQ2011 - 25564和BFI2011 - 25021）以及安达卢西亚自治区政府（P08 - CVI - 04185）的资助。J.G.-O.、F.M.P.、M.F.-S.、N.P.、A.C.-R.、T.A.A.、M.H.、M.R.、M.T.-S.和L.C.无利益冲突声明。
