生物钟在蛋鸡排卵-产蛋过程中的调控作用

王晓鹃，刘磊，焦洪超，赵景鹏，林海



生物钟在蛋鸡排卵-产蛋过程中的调控作用

王晓鹃，刘磊，焦洪超，赵景鹏，林海

（山东农业大学动物科技学院/山东省动物生物工程与疾病防治重点实验室，山东泰安 271018）

生物体内源性的昼夜节律使其能够预测周边环境周期性的变化，使机体的内在代谢和周边环境保持一致。在禽类卵泡的成熟、排卵和蛋的形成过程中，不同生理进程在时间上的吻合显示了机体自身以及机体与环境之间的协调统一。动物对营养物质的摄入、内分泌激素的生成、能量代谢等一系列的行为和生理过程都有生物钟参与调控。文章从光照和营养两种因素入手，综述了生物钟在神经内分泌、能量摄入和能量代谢中的调控作用，揭示了蛋鸡的排卵和产蛋机制。1.光信号通过调控生物钟影响下丘脑-垂体-性腺轴（HPG轴），从而调控机体的繁殖活动。在光信号刺激下，位于禽类视交叉上核（SCN）和松果体的中枢生物钟作用于下丘脑，使下丘脑定时性释放促性腺激素释放激素（GnRH）和促性腺激素抑制激素（GnIH），GnRH和GnIH继而作用于垂体调节释放促性腺激素-促黄体生成素（LH）和促卵泡激素（FSH），卵巢中存在的外周生物钟接受中枢的同步化信号来维持生物节律，促使禽类的卵泡成熟和定时排卵；2.除了受到HPG的神经内分泌调控之外，蛋鸡的排卵-产蛋过程还受到机体能量代谢的影响。中枢和外周的生物钟基因能够调控食欲调节系统，从而影响能量摄入；生物钟能够通过调控代谢过程中重要限速酶的表达、整合核受体和营养信号蛋白、调节代谢感受器和代谢物、影响肠道微生物等途径来调节能量代谢，影响卵黄前体物质的合成、转运和沉积；禽类松果体分泌的褪黑素可通过介导降钙素、甲状旁腺素（PTH）及雌激素分泌，节律性地调节体内钙代谢，影响蛋壳的形成。能量摄入的时间和行为、机体能量代谢和能量状态也可以通过腺苷酸活化蛋白激酶（AMPK）、过氧化物酶体增殖物激活受体α（PPARα）等一些与食欲调控和能量代谢相关的细胞因子反过来调控生物钟。营养-生物钟-能量代谢三者之间相互作用，使生物体适应环境的能力增强，能量利用达到最优。因此，通过调整进食时间和食物组分（如饲料能量水平和钙水平），能够改变能量代谢从而调节生物钟的功能。将环境（光照管理）和营养（饲喂时间、饲料配方）综合研究并加以运用，使机体生物钟成为连接外部环境信号和内部能量代谢的纽带，既能响应外界环境刺激，又能同时调控机体能量代谢进程，从而使各项生理功能得到更好地发挥，这将为蛋鸡的产蛋调控机制研究提供新的视角。

生物钟；蛋鸡；产蛋；光照；能量

昼夜节律是生命的基本特征之一，它几乎影响了生物体生命活动的方方面面，帮助其实现自身和外界环境的同步和适应。在自然状态下, 生物钟接受外界光/暗、食物、温度以及化学因子等环境信号，调整自身节律保持与外界环境的同步[1-5]，从而适应环境。蛋鸡的排卵-产蛋循环具有明显的节律性和环境适应性。家禽在解剖学和生理学上与哺乳动物有很大的不同，性成熟的蛋鸡卵巢内含有大量各种级别和各种状态的卵泡。卵泡的成熟、排卵和蛋的形成是多组织、多过程、多层次参与的生理事件，在此过程中不同生理进程在时间上的吻合显示了机体自身以及机体与环境之间的协调统一。已有的研究表明，内分泌激素的生成、禁食/采食、葡萄糖和脂质代谢、体温的维持等一系列的行为和生理过程都有生物钟参与调控[6-9]。生物钟可使生物体预见环境的改变，从而调整它们的行为和生理机能来适应每天的环境变化。在哺乳动物中，中枢生物钟位于下丘脑前段的视交叉上核（suprachiasmatic nucleus, SCN），外周生物钟（peripheral clock）几乎遍布全身各组织器官。中枢和外周生物钟组成了一个有等级梯度的生物钟系统，他们既相对独立又互相联系，共同维护机体各项生命活动的协调一致。哺乳动物的中枢生物钟只有一个即SCN，而禽类的中枢生物钟至少位于三处，分别是松果体、视网膜和SCN[10-11]。禽类和哺乳动物的生物节律分子机制高度保守[12]。笔者从昼夜生物钟系统切入，整合生命过程中的节律现象，能够全面地解析排卵-产蛋这一复杂又特殊的生理过程。因此，探索生物钟系统在蛋鸡生殖系统中的调控作用对于提高蛋鸡产量、揭示生物体对外在环境的适应机制都有着重要意义。

1 生物钟通过HPG轴影响排卵

动物的生殖系统发育和功能维持受到下丘脑-垂体-性腺（HPG）轴的调控。HPG轴启动后，首先，下丘脑合成分泌促性腺激素释放激素（GnRH）；其次，GnRH与受体结合，刺激垂体释放促性腺激素，包括促黄体生成素（LH）和促卵泡激素（FSH）；最后，促性腺激素激活性腺的发育和性类固醇激素的分泌，如雌二醇和睾酮。下丘脑、垂体、性腺在中枢神经系统的调控下形成一个封闭的自动反馈系统，三者相互协调、相互制约使动物的生殖分泌系统保持相对稳定。

排卵最主要的诱发因素是来自垂体的LH峰。LH受体（LHR）的表达分布显示，等级前卵泡和刚刚进入等级序列的F6和F5卵泡上LHR均较少，而在F1中最高。LH峰的释放可以追溯到上游GnRH峰的生成，GnRH峰的定时性释放在多种动物中都有过报道[13]。早期研究发现，切除SCN后性腺轴上的激素失去了正常状态时的昼夜节奏性，并且扰乱了排卵的正常发生，这证明SCN参与了排卵调控。SCN处衍生出两种神经元直接连接到GnRH神经元[14]，其中一种神经元-前腹侧室周核神经元（AVPV），是雌激素反馈信号与昼夜节律信号的汇合点，即AVPV不仅受SCN昼夜节律性调控还可接受性激素的反馈性调控，使下游的GnRH峰表现为在特定时期（由生物钟调控）由雌激素触发（由性激素反馈性调控）的现象[15]。除SCN外，由禽类另一处中枢，即生物钟-松果体合成分泌的褪黑素会直接作用于促性腺激素抑制激素（GnIH）神经元并调控GnIH的生成，GnIH既可以作用于GnRH神经元又可直接作用于脑垂体，从而抑制FSH和LH峰的生成。

中枢生物钟可以直接感受外部环境信号的刺激[16]，并通过下游的神经内分泌系统向相应的靶组织传递输出信号，所以说，外周生物钟接受中枢的同步化信号来维持生物节律。卵巢中存在外周生物钟己经在多个物种上被报道过[17]，但是卵巢是一个多组分的复杂组织，禽类尤其如此。研究发现，蛋禽排卵前卵泡F1-F3的颗粒细胞受到生物节律的直接调控，并在F1中节律震荡最为强烈[18]。LH、FSH都可以影响小鼠卵巢颗粒细胞中生物钟基因的表达[19-20]，而在禽类上的研究发现，只有LH具有同样的作用[21]。

光照是影响动物繁殖的主要的环境调控因子[22]，光信号通过颅骨和视网膜，通过一系列神经信号转导引起下丘脑血管活性肠肽（VIP）和催乳素（PRL）分泌上升，最终通过影响下丘脑GnRH和垂体FSH和LH来调控繁殖活动[23]。禽类的繁殖活动对光照是很敏感的，Hahn等[24]研究证明，将成年家雀由16L﹕8D的光照环境转移到13D﹕11D后，下丘脑视前区和正中隆起的GnRH神经元与神经纤维增多，表明鸟类下丘脑GnRH的表达受光照时间的影响。长光照使鸟类脑内的GnRH表达以及外周血中LH和FSH的含量显著下降[25]。光周期也能引起家禽PRL的分泌和浓度的改变[26]，随着光照时间的增长，处于繁殖期的家禽PRL分泌不断上升[27]。上述研究表明，光信号通过调控生物钟影响HPG轴，从而调控机体的繁殖活动。

2 生物钟整合能量/物质代谢影响产蛋

除了受到HPG的神经内分泌调控之外，蛋鸡的排卵-产蛋过程还受到机体能量代谢的影响。卵泡吸收的卵黄来源于肝脏合成的卵黄前体物质-极低密度脂蛋白（VLDL）和卵黄蛋白原（VTG）。肝脏合成卵黄前体物质后, 经血液转运至卵巢。卵泡中的初级卵母细胞不断聚集卵黄，使卵泡体积增大，经成熟分化后排出。蛋形成时要分泌大量的卵清蛋白并动员大量的钙去形成蛋壳，营养或钙的缺乏可能会延长该过程。研究也发现，在能量缺乏时（禁食状态或采食基础日粮），应激激素抑制卵泡发育和产蛋性能；而在能量充足时（饲喂状态或采食高脂日粮），这种作用会减弱[28]。这表明蛋鸡的卵泡发育和产蛋性能与机体的能量状态有关。最近的研究发现，产蛋鸭卵巢的生物钟基因表达水平与产蛋量密切相关[29]，产蛋鸡漏斗部（捕获蛋黄的部位）和子宫部（形成蛋壳的部位）的生物钟基因Bmal1、Clock、Per2和Per3在排卵过程中发挥了重要作用[30]。

2.1 生物钟调控能量摄入

随着一天中能量需求的波动，动物的采食行为也呈现节律性。研究发现，几乎80%的食物消耗于小鼠活跃的夜间。禽类胃肠道长度相对较短，食糜通过消化道速度较快，因此禽类有频繁采食的习性，其累积采食量较高。自然光照下，鸡采食高峰发生在清晨和黄昏[31]。通过调整光照改变昼夜节律，能够调节鸡的采食量[32]，这表明生物体的钟基因调控着食欲和采食行为。研究证明，中枢和外周的生物钟基因Bmal1能够调控食欲调节系统[33-34]。穹隆周区和下丘脑背内侧核的食欲素（orexin）神经元具有昼夜节律性活动[35-36]，穹隆周区orexin神经元受视交叉上核谷氨酸能和γ-氨基丁酸能神经元的支配，而视交叉上核同时也是生物钟的中枢调节部位；进一步研究证明，orexin神经元还参与了睡眠/觉醒周期、食欲、自主神经活动以及昼夜节律性的调节[37-38]。鸡与哺乳动物的orexin同源性很高[39]。在哺乳动物中，瘦素在机体食欲调控和能量代谢中发挥着重要作用，敲除SCN会破坏瘦素表达的节律性[40]。小鼠敲除生物钟基因Bmal1后导致瘦素的分泌和基因表达发生改变[41]。敲除生物钟基因Clock后，与食欲调控有关的神经肽orexin和胃饥饿素ghrelin 的 mRNA 表达水平均下降[42-43]。

代谢物和进食行为也可以反过来调控生物钟[44]，其中进食时间可能比食物组分更重要[45-46]。在小鼠不活跃的光照时期给予它们食物，此时能量消耗低、呼吸交换率高，将导致生物时钟的不同步以及代谢紊乱[47-48]。摄食时间的改变使外周生物钟基因与中枢生物钟基因表达的相位发生解偶联[45]。对丧失了基本生物节律的小鼠在特定的时间给予食物，可以恢复其肝脏中某些基因表达的节律性[49]。鸡上的研究也发现，限饲能够改变鸡的生物节律和活动[50]。采食时间和采食行为对生物钟的影响可能是通过一些与食欲调控和能量代谢相关的细胞因子实现的。研究发现，食物消耗可能通过腺苷酸活化蛋白激酶（AMPK）改变生物钟基因的表达，AMPK作为细胞能量感受器，在缺失时将导致肝脏中生物钟基因Cry1的稳定性和时钟节律性消失[51]。此外，食物还可能通过瘦素影响生物钟。小鼠肝脏和脂肪组织中缺失瘦素，其正常活动和时钟基因表达节律减弱[52]，瘦素受体缺失的小鼠在脂肪组织中也表现出生物钟基因功能损伤[53]，补充瘦素能够恢复生物钟的功能并改善代谢指标[52]。

2.2 生物钟调控能量代谢

生物钟可以调控机体多种代谢途径，它能有效调节整个代谢过程及相关信号以及组织的代谢功能。研究发现，能量代谢活跃的外周组织如肝脏、骨骼肌、脂肪组织中约有5%—10%的基因都呈节律表达，并且具有明显的组织特异性[47, 54]。与能量代谢相关的激素，如胰岛素、脂联素、肾上腺糖皮质激素、瘦素等[55]，能量代谢相关酶的表达和活性[56]，以及与糖脂代谢相关的核受体大多也呈节律表达[57]。禽类的血浆葡萄糖、甘油三酯和肌酐也呈现明显的昼夜节律性[58]。生物钟基因在上述代谢过程中发挥着重要的调控作用，其中在与卵泡发育密切相关的脂质稳态调控中，Clock 和Bmal1扮演着重要角色。Zvonic等[47]研究表明，20%以上的小鼠脂肪转录组表达受昼夜节律调控。机体通过调节Clock 和 Bmal1，能够驱动脂肪代谢关键酶ATGL和HSL的节律性表达[59]，使循环中游离脂肪酸水平保持节律性[59-61]；Bmal1的mRNA 水平在脂肪分化的过程中高度表达[62]，Bmal1通过激活视黄酸相关孤儿核受体α（RORα）调节骨骼肌的脂肪生成和贮存[63]，在Bmal1全身性敲除的小鼠中，瘦素、脂联素、抵抗素等脂肪细胞因子的分泌和基因的表达均发生改变[41]；Clock突变的小鼠比正常小鼠更胖, 并伴有高血脂、脂肪肝等症状，这主要归因于脂肪的沉积和脂肪细胞肥大[60]。鸡上的研究也发现，生物钟影响脂肪合成[64]，与脂肪合成密切相关的转录因子胆固醇调节元件结合蛋白（SREBP）及其下游的靶基因，也受到光照和生物钟的调控[65]。

生物钟对代谢过程的调控通过以下方式实现：①调节代谢途径中重要限速酶的表达。如胆固醇生物合成的限速酶HMG-CoA还原酶（HMGCR）的激活呈现节律性[66-67]。②整合核受体和营养信号蛋白。如过氧化物酶体增殖物激活受体α（PPARα）是脂肪代谢主要调节因子，生物钟基因Clock 和 Bmal1能够结合到PPARα启动子的 E-box上，直接调节PPARα的表达[68]；生物钟基因Per2与核受体REV-ERBα相互作用从而调控肝脏糖代谢[69]；生物钟基因Per3通过结合到PPARγ的靶位点来抑制其表达，从而阻碍脂肪生成[70]。③调节代谢感受器和代谢物。Minami等[71]研究表明，数百种代谢物的含量水平在小鼠胞质中表现出昼夜振荡，包括磷脂、氨基酸和尿素循环的中间产物；AMPK是细胞能量状态的感受器，在小鼠的肝脏、下丘脑等组织中，AMPK的活性也是有节律的[72]。

另一方面，能量代谢也可以反过来调控生物钟。如过氧化物酶体增殖物激活受体γ共激活因子α（PGC-1α）受控于生物钟，反过来它又可调控生物钟，是连接生物钟和能量代谢的重要调控因子[73]。核受体PPARα能够结合到Bmal1启动子的PPARα反应元件（PPRE）上，调控Bmal1的表达[68]。能量感受器AMPK可以通过磷酸化 Cry1[51]和 Ck1ε[74]来调节生物时钟。一些原本是生物钟的输出信号，也可作为后续时钟循环的输入信号，如cAMP和NAD+[75-77]。

此外，体内的能量状态也可以通过代谢信号反过来作用于生物钟。研究发现，营养水平直接影响SCN的时相。给予高脂饲料的小鼠，其生物节律发生改变，自发活动周期延长[78-79]，高胆固醇饮食不影响肝脏中生物钟基因（和）以及钟控基因（和）的节律表达, 但会使生物钟控制基因Pai-1的表达量上升[80]。高脂饮食能够显著抑制小鼠脂肪组织中时钟关键基因、及的表达[79]。饲喂低能饲料的鸡，其生物节律发生改变，活动减少[81]。PGC-1α和PPARα是连接生物钟和能量代谢的重要调控因子[68, 73]，因此推测，代谢物和进食行为对生物钟基因的影响可能是通过PGC- 1α和PPARα实现的。

近年来的研究发现，在生物钟和能量代谢的互作网络中，肠道微生物扮演了重要角色[82]。由大量微生物菌群组成的肠道微环境参与了机体的免疫调控及能量代谢等生理过程[83]。肠道内的微生物与宿主相互作用，共同维持机体动态的生物平衡。研究发现，肠道微生物也会受到生物钟的调控，这些肠道微生物的生物节律与其宿主具有同步性[84-89]。研究发现，在大鼠活跃的暗周期，肠道微生物主要负责消化营养物质、修复并延伸其DNA；在大鼠不活跃的亮周期，肠道微生物主要参与排毒、感知环境信号、长出鞭毛辅助移动等进程[90]。进一步研究发现，肠道微生物的这种节律性与生物钟基因Per1/2的调控有关[90]。肠道微生物的区系和多样性均具有生物节律[91]，并且会影响到机体代谢物、肝脏转录组和解毒功能的生物节律[92]，影响肝脏功能的节律性[93]。高脂饮食能干扰肠道微生物的这种节律，反过来，对肠道微生物的节律进行调控能改善因高脂饮食导致的肥胖[94]。因此，肠道微生物能同时响应并调控生物钟和能量代谢过程。

2.3 生物钟调控钙代谢

蛋壳的主要成分是碳酸钙，蛋鸡可从骨组织中动员8%—10%的钙用于形成蛋壳，所以钙在骨组织中的动员和在蛋壳腺中的沉积对蛋的形成非常重要。鸡蛋蛋壳的形成具有明显的生物节律，蛋壳形成的最活跃时期常处于光照周期的黑暗阶段。骨代谢的平衡也与生物钟基因的调控和支配有关，成骨细胞具有生物钟基因，其增殖活性表现为明显的昼低夜高的24h节律变化，这表明机体钙代谢是受到生物钟调控的。

松果体作为禽类的中枢生物钟之一，其分泌的褪黑素在主导生物节律、调控骨的代谢平衡和钙代谢方面具有重要作用。褪黑素可以直接作用于破骨细胞、成骨细胞及直接调节钙代谢平衡，或者通过增加非快动眼睡眠时相，增加生长激素的分泌，从而间接影响骨代谢。研究表明褪黑素可通过介导降钙素、甲状旁腺素（PTH）及雌激素分泌来调节体内钙代谢[95]。Conti等[96]研究发现骨髓细胞中含有高浓度的褪黑素，并对骨髓细胞增殖有积极作用。骨髓的褪黑素水平为夜间血浆褪黑素水平的2 倍[97]。蛋鸡上的研究也发现，褪黑素能调节钙的分配，从而影响骨强度和蛋壳重量[98]。除褪黑素外，PTH也是与钙代谢相关激素中研究最多的激素之一。研究显示，在生理情况下，PTH的分泌具有昼夜节律性，高峰出现在上午的0—6点[99]。PTH对钙代谢的影响主要表现为节律的紊乱，用磷酸盐或钙制剂进行时间疗法可以调整内源性PTH激素的昼夜节律，钙代谢紊乱也随之显著改善[100]。在产蛋期尤其是产蛋后期，产蛋鸡对钙的需要量增加，夜间补充光照和补充饲喂次数有利于鸡群在形成蛋壳期间摄取饲料中的钙，提高产蛋率、改善蛋壳质量[101]。

综上所述，营养-生物钟-能量代谢，三者之间相互作用，使生物体适应环境的能力增强，能量利用达到最优。因此，可以通过调整进食时间和食物组分（如饲料能量水平和钙水平），改变能量代谢从而调节生物钟的功能。

3 结论与展望

目前我国蛋鸡养殖逐渐趋向规模化、集约化，蛋品市场也向品牌化方向发展。市场对鸡蛋质量的要求越来越高，尤其是在产蛋后期，蛋鸡机体老化和饲养管理落后等因素都会造成鸡蛋品质的下降。光照是影响家禽繁殖和生产的最重要的生态因子之一，光信号作用于中枢生物钟，通过神经内分泌机制影响HPG轴来调控繁殖活动。另外，食物组分以及进食时间可以显著调控机体的生物钟。因此，了解饲粮中各种营养素及进食时间与生物钟的相互关系，将环境（光照管理）和营养（饲喂时间、饲料配方）综合研究并加以运用，使机体生物钟成为连接外部信号和内部能量代谢的纽带，既能响应上游环境刺激，又能同时调控下游能量代谢进程，从而使各项生理功能得到更好地发挥，这将为蛋鸡的产蛋调控机制研究提供新的视角。

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（责任编辑 林鉴非）

Regulation of Biological Clock in Ovulation-Laying of Laying Hens

WANG XiaoJuan, LIU Lei, JIAO HongChao, ZHAO JingPeng, LIN Hai

(Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Department of Animal Science, Shandong Agricultural University, Tai’an 271018)

The endogenous circadian rhythm enables the organisms to predict the changes of environmental cycle, which maintains consistency between body metabolism and the external environment. During the maturation of follicular, ovulation, and the formation of egg in birds, the coincidence of the different physiological processes in time shows the unity of the body itself and the coordination between the body and the environment. Biological clock participates in a series of behavior and physiological processes such as nutrition intake, the production of endocrine hormones and energy metabolism. In the present review, the role of biological clock in neuroendocrine, energy intake and energy metabolism has been discussed, from the points of light factor and nutrition factor, to reveal the potential regulating mechanism underlying ovulation and egg laying of hens. (1) Light signal acts on hypothalamic- pituitary-gonadal axis (HPG) by regulating the biological clock to influence reproductive activities. Under the stimulation of light, the central clocks in suprachiasmatic nucleus (SCN) and pineal act on hypothalamus, and make it to release gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormones (GnIH) periodically. GnRH and GnIH then act on pituitary, and make it to release gonadotropin hormone, that is luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Periphery clocks in ovary receive the central synchronization signal to maintain the biological rhythm, thereby regulating the maturation of follicles and ovulation. (2) In addition to being regulated by the neuroendocrine system of HPG axis, the ovulation-egg production process of laying hens is also affected by the body's energy metabolism. The central and peripheral clock genes regulate the appetite regulation system and thus affect energy intake; Biological clock can regulate the expression of key enzymes in the process of metabolism, integrate the nuclear receptors and nutrition signaling proteins, regulate metabolism sensors and metabolites, affect gut microbes to regulate energy metabolism, and affect the synthesis, transport and deposition of yolk precursor;Melatonin secreted by bird's pineal can regulate calcium metabolism rhythmically by mediating the secretion of calcitonin, parathyroid hormone (PTH) and estrogen, and influence the formation of egg shell.The time and the behavior of energy intake, the body energy metabolism and energy status can also modulate biological clock, through some appetite regulation and energy metabolism related cytokines such as AMP-activated protein kinase (AMPK), and peroxisome proliferator-activated receptors α (PPARα). There are interactions between nutrient, biological clock and energy metabolism, which accommodate organisms with the surrounding and optimize the energy utilization. Therefore, by adjusting the time of eating and the composition of feed (such as the energy level of feed and calcium level), energy metabolism can be changed to regulate the function of the biological clock. In conclusion, it will provide a new perspective for researching regulation mechanism of egg laying, if we make an integrated study on environment factor (light management) and nutrition (feeding time and feed formula) in which biological clock linked external factors and internal energy metabolism, that is, biological clock can both response to environmental stimuli, and regulate the body's energy metabolism process, to optimize the various physiological functions.

biological clock; laying hen; egg laying; light; energy

2018-04-04；

2018-06-13

“十三五”国家重点研发计划（2016YFD0500510）、国家自然科学基金（31672441）、国家现代农业产业技术体系建设专项资金（CARS-41）、山东省“双一流”奖补资金、泰山学者项目（201511023）

王晓鹃，E-mail：wangxj@sdau.edu.cn。

林海，E-mail：hailin@sdau.edu.cn