上皮细胞离子通道对雌性生殖道内液体微环境的调节作用:对生殖与不孕的影响

雌性生殖道内适宜的液体微环境对一系列生殖事件起至关重要的作用。位于生殖道上皮细胞顶膜或基底膜的一系列离子通道和转运体,通过对水、电解质的跨膜转运,从而调节雌性生殖道内液体的分泌与吸收。本综述着重探讨了上皮细胞钠离子通道和囊性纤维化跨膜电导调节体对雌性生殖道内液体容量和成分的调节以及它们在不同生殖事件,比如精子获能及着床中的重要作用。同时对因离子通道失活或失调引起的雌性生殖道内液体微环境稳态失衡导致的一系列病理改变,如卵巢过度刺激综合征、输卵管积水以及不孕提出了新的见解。     

精子功能相关的蛋白质调控受精过程的研究进展

哺乳动物的受精过程涉及到精子一系列的功能活动,如精子在雌性生殖道的运行、精子的超活化与获能、顶体反应以及精卵融合等。在精子经历的这一系列过程中,精子功能相关的蛋白质发挥着不可或缺的作用,这些蛋白分子的正常与否与雄性个体的繁殖力高低密切相关,因此精子功能相关的蛋白质能够作为评定哺乳动物精液受精能力的生物标记。文章主要对哺乳动物精子功能相关的蛋白质进行了综述,以阐述相关蛋白分子对精子运动活力、精子获能、顶体反应、透明带穿入和精卵融合等方面的重要作用以及这些蛋白分子在家畜遗传改良上的潜在应用。     

哺乳动物精子获能过程中信号通路研究进展

哺乳动物精子在雌性生殖道内及体外获能培养过程中伴随着胆固醇外流、质膜重组、离子通道调节及获能相关蛋白磷酸化状态改变等相关生理调节过程,其中信号通路及相应信号分子对精子获能及功能修饰起到重要调节作用,成为精子细胞超激活运动及完成受精作用的关键环节。根据近年来的研究报道,对哺乳动物精子获能过程中已知的信号通路、信号分子及调节因子、离子通道、存在的问题及未来研究主要方向进行综述,为精子体外培养及辅助生殖等提供理论参考。     

胞外ATP在男性生殖道中的作用

胞外ATP除了能广泛作为神经递质外,还被认为是一种旁分泌或自分泌因子。ATP从男性生殖道中的精子或上皮细胞中释放,在调节各种生殖生理功能中起多种作用。本文综述了ATP调节附睾上皮细胞阴离子分泌的信号通路,阐述了ATP对依赖上皮细胞的输精管平滑肌收缩的调节机制,讨论了ATP在男性生殖道中的功能和作用。     

哺乳动物受精与透明质酸

哺乳动物精子在睾丸内产生之后,经历了附睾内成熟,雌性生殖道中获能以及超激活运动,最终获得了受精能力。在透明带或其他因素诱导下,发生顶体反应。１精子的成熟与获能精子的成熟与获能,均与精子头部膜的变化有关,精子从附睾头向附睾尾的运动过程中,逐步获得受精能力（Ｙａｎａｇｉｍａｃｈｉ,１９９４）。附睾内精子成熟的变化主要发生在以下几个方面：１．质膜上胆固醇含量增多。２．由于吸附了附睾分泌的大量蛋白质...     

哺乳动物精子超激活运动研究进展

哺乳动物精子经过雌性生殖道与卵子发生融合完成受精过程,需要正常的精子运动能力作为保障。超激活运动可以帮助精子穿透卵子透明带,与卵子发生结合形成受精卵。精子超激活运动的激发和维持是通过自身生理和雌性生殖道内生化环境共同调节完成的。"超激活运动"自发现起到现在已成为生殖学研究的重要部分,现对精子超激活运动与受精的关系、调节因子、鞭毛结构、技术手段及未来研究方向进行综述,为男性不育及避孕等相关临床研究提供理论依据。     

精子的运动特性

精子的运动特性与生育力有密切关系。本文从超微结构水平和分子生物学基础出发,阐述精子运动机制的微管滑动学说;介绍了精子在液体介质中的运动形式、检测精子活动力的主要技术、影响精子活动力的因素、从异常精液中选择正常精子的技术,以及精子活动力在雄性和雌性生殖道中的演变过程。     

调控精子成熟和功能的生殖道胞外囊泡研究进展

哺乳动物精子从发生到具备受精能力到达受精部位是一个漫长的过程。在此过程的不同环境中存在可调控精子成熟和功能的胞外囊泡,这些胞外囊泡包括来自雄性生殖道内的附睾小体和前列腺小体,以及来自雌性生殖道内的阴道小体、输卵管小体及子宫小体。现介绍胞外囊泡的概况、生殖道内的胞外囊泡特征、精子摄取胞外囊泡的可能机制以及生殖道胞外囊胞研究的未来展望,以期更好地理解在受精过程中精子功能如何受胞外囊泡调控,为男性不育诊治提供线索。     

哺乳动物睾丸细胞间连接的动态调控

精子的发生过程是一个受多种因素(包括由细胞间连接的动态变化所形成的微环境等)精确调控的过程。细胞因子及睾酮以自分泌、旁分泌的形式对该微环境中的细胞连接水平如:生殖细胞穿越血睾屏障(the blood-testis barrier,BTB)的开闭机制等进行调控,从而对精子的发生起到重要调节作用。本文讨论了各种因素对细胞间连接的自分泌、旁分泌调控方式的影响,并简要介绍了近腔细胞外质特化(apical ectoplasmic specialization,近腔ES)-BTB-半桥粒/基底膜功能调控轴模型在生精细胞穿越BTB及精子释放等生精过程中的作用,为人们进一步认识精子发生过程中细胞间联系的功能及其调控提供了新的视角。     

Dicer调节生殖功能的研究进展

Dicer是微小非编码RNA生成的关键内切酶,介导微小RNA(micro RNA,miRNA)和小干扰RNA(small interfering RNA,siRNA)的产生,通过RNA干扰(RNA interference,RNAi)途径实现转录或转录后水平基因调控,在调节细胞增殖、分化、凋亡等方面起重要作用。近年来Dicer基因在生殖领域的研究越来越受关注,最近的研究表明Dicer与男性生精细胞发育、精子形成及成熟、精子活力和形态生成、卵泡发育、排卵及黄体形成、性激素合成、输卵管功能、子宫内膜容受性等方面都有密切关系。繁衍后代需要精子和卵子的共同参与,Dicer可能通过影响精子和卵子的数量或者质量进而导致胚胎发育异常,因此理解Dicer在雄性与雌性生殖的重要调节作用对于理解生殖调节异常相关的疾病如无精子症、复发性流产等的发病机制具有重要的作用。本文对Dicer在雄性生殖道与雌性生殖中的关键作用进行了综述,旨在进一步从分子层面深入理解Dicer与生殖相关疾病的关系。     

The physiology of bicarbonate transporters in mammalian reproduction

HCO(3)(-) plays critically important roles during virtually the entire process of reproduction in mammals, including spermatogenesis, sperm capacitation, fertilization, and development of early stage embryos. Therefore, the acid-base balance in the male and female reproductive tracts must be finely modulated. The fluid milieu in the epididymis is acidic, containing very low concentration of HCO(3)(-). In this acidic low HCO(3)(-) environment, mature sperm are rendered quiescent in the epididymis. In contrast, the luminal fluid in the female uterus and oviduct is alkaline, with very high concentration of HCO(3)(-) that is essential for sperm to fulfill fertilization. HCO(3)(-) transporter of solute carrier 4 (SLC4) and SLC26 families represent the major carriers for HCO(3)(-) transport across the plasma membrane. These transporters play critical roles in intracellular pH regulation and transepithelial HCO(3)(-) transport. The physiological roles of these transporters in mammalian reproduction are of fundamental interest to investigators. Here we review recent progress in understanding the expression of HCO(3)(-) transporters in reproductive tract tissues as well as the physiological roles of these transporters in mammalian reproduction.     

Involvement of a Na+/HCO-3 cotransporter in mouse sperm capacitation

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of "sperm capacitation." We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO(3)(-)/cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO(3)(-) has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO(3)(-) but not Cl(-) induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO(3)(-)-dependent hyperpolarization was not observed when Na(+) was replaced by the non-permeant cation choline(+). Replacement of Na(+) by choline(+) also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO(3)(-) current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na(+) suggest that a Na(+)/HCO(3)(-) cotransporter is present in mouse sperm and is coupled to events regulating capacitation.     

Glucose is a pH-Dependent Motor for Sperm Beat Frequency during Early Activation

To reach the egg in the ampulla, sperm have to travel along the female genital tract, thereby being dependent on external energy sources and substances to maintain and raise the flagellar beat. The vaginal fluid is rich in lactate, whereas in the uterine fluid glucose is the predominant substrate. This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters. It is well documented that glycolysis yields ATP and that HCO(3)- is a potent factor in the increase of beat frequency. We here show for the first time a pathway that connects both parts. We demonstrate a doubling of beat frequency in the mere presence of glucose. This effect can reversibly be blocked by 2-deoxy-D-glucose, dichloroacetate and aminooxyacetate, strongly suggesting that it requires both glycolysis and mitochondrial oxidation of glycolytic end products. We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged. Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH(i)-dependent way to produce HCO(3)-(,) thus enhancing sperm beat frequency and contributing to fertility.     

Role of carbonic anhydrase IV in the bicarbonate-mediated activation of murine and human sperm

HCO(3) (-) is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO(3) (-) containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO(3) (-) stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO(3) (-) enters spermatozoa either directly by anion transport or indirectly via diffusion of CO(2) with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO(2) with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV(-/-) mice we found a 32.13% reduction in total CA activity in the latter. The CA IV(-/-) sperm also have a reduced response to CO(2). While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO(2) application, beat frequency of CA IV(-/-) sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO(3) (-), which is necessary for stimulation of sAC and hence early activation of spermatozoa.     

Acquisition,initiation and maintenance of sperm motility in the shark,Triakis scyllia

Shark sperm were immotile in the testis, but motile in the vesicula seminalis, suggesting that motility potential is acquired in the male reproductive tract. Although sperm acquired motility, they were immobile in the undiluted semen, however, motility occurred upon dilution of the semen in electrolyte solutions, whose concentrations are the same as seawater or uterus fluid, suggesting that exposure to these fluids at ejaculation causes the initiation of sperm motility. Duration of sperm motility was longer in glucose-rich uterus fluid than glucose-free media, suggesting the important role of hexose for maintaining sperm motility in the female reproductive tract.     

Bicarbonate actions on flagellar and Ca2+ -channel responses: initial events in sperm activation

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO(3)(-) and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25 degrees C. Local perfusion with HCO(3)(-) produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO(3)(-) begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8+/-0.2 seconds at 15 mM HCO(3)(-) and 17.5+/-0.4 seconds at 1 mM HCO(3)(-). Removal of external HCO(3)(-) allows a slow return to basal beat frequency over approximately 10 minutes. Increases in beat symmetry accompany the accelerating action of HCO(3)(-). As in our past work, HCO(3)(-) also facilitates opening of voltagegated Ca(2+) channels, increasing the depolarization-evoked rate of rise of intracellular Ca(2+) concentration by more than fivefold. This action also is detectable at 1 mM HCO(3)(-) and occurs with an apparent halftime of approximately 60 seconds at 15 mM HCO(3)(-). The dual actions of HCO(3)(-) respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO(3)(-) on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 micro M cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca(2+) concentration from 17+/-4 to 41+/-6 nM second(-1). However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO(3)(-), perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO(3)(-) produces during sperm activation.     

The Drosophila melanogaster seminal fluid protein Acp62F is a protease inhibitor that is toxic upon ectopic expression.

Drosophila melanogaster seminal fluid proteins stimulate sperm storage and egg laying in the mated female but also cause a reduction in her life span. We report here that of eight Drosophila seminal fluid proteins (Acps) and one non-Acp tested, only Acp62F is toxic when ectopically expressed. Toxicity to preadult male or female Drosophila occurs upon one exposure, whereas multiple exposures are needed for toxicity to adult female flies. Of the Acp62F received by females during mating, approximately 10% enters the circulatory system while approximately 90% remains in the reproductive tract. We show that in the reproductive tract, Acp62F localizes to the lumen of the uterus and the female's sperm storage organs. Analysis of Acp62F's sequence, and biochemical assays, reveals that it encodes a trypsin inhibitor with sequence and structural similarities to extracellular serine protease inhibitors from the nematode Ascaris. In light of previous results demonstrating entry of Acp62F into the mated female's hemolymph, we propose that Acp62F is a candidate for a molecule to contribute to the Acp-dependent decrease in female life span. We propose that Acp62F's protease inhibitor activity exerts positive protective functions in the mated female's reproductive tract but that entry of a small amount of this protein into the female's hemolymph could contribute to the cost of mating.     

Seminal proteins but not sperm induce morphological changes in the Drosophila melanogaster female reproductive tract during sperm storage

In most insects, sperm transferred by the male to the female during mating are stored within the female reproductive tract for subsequent use in fertilization. In Drosophila melanogaster, male accessory gland proteins (Acps) within the seminal fluid are required for efficient accumulation of sperm in the female's sperm storage organs. To determine the events within the female reproductive tract that occur during sperm storage, and the role that Acps and sperm play in these events, we identified morphological changes that take place during sperm storage in females mated to wild-type, Acp-deficient or sperm-deficient males. A reproducible set of morphological changes occurs in a wild-type mating. These were categorized into 10 stereotypic stages. Sperm are not needed for progression through these stages in females, but receipt of Acps is essential for progression beyond the first few stages of morphological change. Furthermore, females that received small quantities of Acps reached slightly later stages than females that received no Acps. Our results suggest that timely morphological changes in the female reproductive tract, possibly muscular in nature, may be needed for successful sperm storage, and that Acps from the male are needed in order for these changes to occur.     

PKD2 cation channel is required for directional sperm movement and male fertility

Sperm of both mammals and invertebrates move toward specific sites in the female reproductive tract. However, molecular mechanisms for sperm to follow directional cues are unknown. Here, we report genetic analysis of Drosophila Pkd2 at 33E3 (Pkd2, CG6504), which encodes a Ca(2+)-activated, nonselective cation channel homologous to the human Pkd2 autosomal dominant polycystic kidney disease (ADPKD) gene. The PKD2 family of genes has been implicated in sensory responses through protein localization on primary cilia of epithelia and neurons. In renal tubules, cilium-associated PKD2 appears to mediate Ca(2+) influx in response to fluid flow, and the loss of fluid sensation probably contributes to cyst growth and ADPKD. Sperm tails or flagella are specialized cilia essential for movement. Drosophila Pkd2 is abundantly associated with the tail and the acrosome-containing head region of mature sperm. Targeted disruption of Pkd2 results in male sterility without affecting spermatogenesis. The mutant sperm are motile but fail to swim into the storage organs in the female. Rare mutant sperm that reach the storage organs are able to fertilize the egg and produce viable progeny. Our data demonstrate that the Drosophila PKD2 cation channel operates in sperm for directional movement inside the female reproductive tract.