"促育生精方" 对环磷酰胺致大鼠不育模型CatSper1 基因和CatSper2基 因的影响

目的：研究" 促育生精方" 对精子特异性钙通道蛋白CatSper1、CatSper2 表达的影响。方法：采用Real-time PCR 法检测 CatSper1 mRNA、CatSper2 mRNA 在各组大鼠（模型组、低剂量组、中剂量组、高剂量组、空白对照组）精子中的表达;用Western blot 检测各组CatSper1 蛋白,CatSper2 蛋白的表达。结果：成功建立大鼠不育模型。CatSper1 mRNA、CatSper2 mRNA表达量为： 中、高剂量组显著高于模型组（P＜0.05）。CatSper1、CatSper2蛋白表达量：中、高剂量组显著高于模型组（P＜0.05）。结论：促育生精 方能有效提高少弱精症模型大鼠精子特异性钙通道CatSper1、CatSper2 基因及其蛋白的表达。     

CatSperbeta, a novel transmembrane protein in the CatSper channel complex

Four CatSper ion channel subunit genes (CatSpers 1-4) are required for sperm cell hyperactivation and male fertility. The four proteins assemble (presumably as a tetramer) to form a sperm-specific, alkalinization-activated Ca(2+)-selective channel. We set out to identify proteins associating with CatSper that might help explain its unique role in spermatozoa. Using a transgenic approach, a CatSper1 complex was purified from mouse testis that contained heat shock protein 70-2, a testis-specific chaperone, and CatSperbeta, a novel protein with two putative transmembrane-spanning domains. Like the CatSper ion channel subunits, CatSperbeta was restricted to testis and localized to the principal piece of the sperm tail. CatSperbeta protein is absent in CatSper1(-/-) sperm, suggesting that it is required for trafficking or formation of a stable channel complex. CatSperbeta is the first identified auxiliary protein to the CatSper channel.     

Identification of human and mouse CatSper3 and CatSper4 genes: Characterisation of a common interaction domain and evidence for expression in testis

Background  

CatSper1 and CatSper2 are two recently identified channel-like proteins, which show sperm specific expression patterns. Through targeted mutagenesis in the mouse, CatSper1 has been shown to be required for fertility, sperm motility and for cAMP induced Ca²⁺ current in sperm. Both channels resemble a single pore forming repeat from a four repeat voltage dependent Ca²⁺ /Na⁺ channel. However, neither CatSper1 or CatSper2 have been shown to function as cation channels when transfected into cells, singly or in conjunction. As the pore forming units of voltage gated cation channels form a tetramer it has been suggested that the known CatSper proteins require additional subunits and/or interaction partners to function.     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

The CatSper channel: a polymodal chemosensor in human sperm

The sperm-specific CatSper channel controls the intracellular Ca(2+) concentration ([Ca(2+)](i)) and, thereby, the swimming behaviour of sperm. In humans, CatSper is directly activated by progesterone and prostaglandins-female factors that stimulate Ca(2+) influx. Other factors including neurotransmitters, chemokines, and odorants also affect sperm function by changing [Ca(2+)](i). Several ligands, notably odorants, have been proposed to control Ca(2+) entry and motility via G protein-coupled receptors (GPCRs) and cAMP-signalling pathways. Here, we show that odorants directly activate CatSper without involving GPCRs and cAMP. Moreover, membrane-permeable analogues of cyclic nucleotides that have been frequently used to study cAMP-mediated Ca(2+) signalling also activate CatSper directly via an extracellular site. Thus, CatSper or associated protein(s) harbour promiscuous binding sites that can host various ligands. These results contest current concepts of Ca(2+) signalling by GPCR and cAMP in mammalian sperm: ligands thought to activate metabotropic pathways, in fact, act via a common ionotropic mechanism. We propose that the CatSper channel complex serves as a polymodal sensor for multiple chemical cues that assist sperm during their voyage across the female genital tract.     

二甲双胍联合枸橼酸氯米芬对多囊卵巢综合征不孕患者性激素和胰岛素水平的影响

目的:探讨二甲双胍、枸橼酸氯米芬联合治疗对多囊卵巢综合征(PCOS)不孕患者性激素和胰岛素水平的影响。方法:选取我院于2017年1月到2018年7月期间收治的101例PCOS不孕患者,采用数字表法将患者随机分为对照组(n=50)和研究组(n=51),对照组给予枸橼酸氯米芬治疗,研究组在对照组基础上联合二甲双胍治疗,采用门诊复查等方式随访6个月,记录两组患者排卵率及妊娠率,比较两组患者治疗前、治疗后的性激素、胰岛素以及血管活性因子水平,记录不良反应发生情况。结果:两组患者治疗后黄体生成素(LH)、睾酮(T)、胰岛素(INS)、LH/卵泡刺激素(FSH)均较治疗前降低,且研究组低于对照组(P0.05)。研究组排卵率、妊娠率均高于对照组(P0.05)。两组患者治疗后血管紧张素(AT-Ⅱ)、血管内皮生长因子(VEGF)均较治疗前降低,且研究组低于对照组(P0.05)。两组不良反应发生率对比无统计学差异(P0.05)。结论:二甲双胍联合枸橼酸氯米芬治疗PCOS不孕,安全有效,可有效调节患者胰岛素、性激素水平,提高排卵率、妊娠率,改善血管活性因子水平。     

CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

Ca²⁺-dependent mechanisms are critical for successful completion of fertilization. Here, we demonstrate that CRISP1, a sperm protein involved in mammalian fertilization, is also present in the female gamete and capable of modulating key sperm Ca²⁺ channels. Specifically, we show that CRISP1 is expressed by the cumulus cells that surround the egg and that fertilization of cumulus–oocyte complexes from CRISP1 knockout females is impaired because of a failure of sperm to penetrate the cumulus. We provide evidence that CRISP1 stimulates sperm orientation by modulating sperm hyperactivation, a vigorous motility required for penetration of the egg vestments. Moreover, patch clamping of sperm revealed that CRISP1 has the ability to regulate CatSper, the principal sperm Ca²⁺ channel involved in hyperactivation and essential for fertility. Given the critical role of Ca²⁺ for sperm motility, we propose a novel CRISP1-mediated fine-tuning mechanism to regulate sperm hyperactivation and orientation for successful penetration of the cumulus during fertilization.     

Pharmacological Targeting of Native CatSper Channels Reveals a Required Role in Maintenance of Sperm Hyperactivation

The four sperm-specific CatSper ion channel proteins are required for hyperactivated motility and male fertility, and for Ca²⁺ entry evoked by alkaline depolarization. In the absence of external Ca²⁺, Na⁺ carries current through CatSper channels in voltage-clamped sperm. Here we show that CatSper channel activity can be monitored optically with the [Na⁺]_i-reporting probe SBFI in populations of intact sperm. Removal of external Ca²⁺ increases SBFI signals in wild-type but not CatSper2-null sperm. The rate of the indicated rise of [Na⁺]_i is greater for sperm alkalinized with NH₄Cl than for sperm acidified with propionic acid, reflecting the alkaline-promoted signature property of CatSper currents. In contrast, the [Na⁺]_i rise is slowed by candidate CatSper blocker HC-056456 (IC₅₀ ∼3 µM). HC-056456 similarly slows the rise of [Ca²⁺]_i that is evoked by alkaline depolarization and reported by fura-2. HC-056456 also selectively and reversibly decreased CatSper currents recorded from patch-clamped sperm. HC-056456 does not prevent activation of motility by HCO₃ ⁻ but does prevent the development of hyperactivated motility by capacitating incubations, thus producing a phenocopy of the CatSper-null sperm. When applied to hyperactivated sperm, HC-056456 causes a rapid, reversible loss of flagellar waveform asymmetry, similar to the loss that occurs when Ca²⁺ entry through the CatSper channel is terminated by removal of external Ca²⁺. Thus, open CatSper channels and entry of external Ca²⁺ through them sustains hyperactivated motility. These results indicate that pharmacological targeting of the CatSper channel may impose a selective late-stage block to fertility, and that high-throughput screening with an optical reporter of CatSper channel activity may identify additional selective blockers with potential for male-directed contraception.     

Up-regulation of CatSper genes family by selenium

Background  

CatSper1-4 are a unique family of sperm cation channels, which are exclusively expressed in the testis and play an important role in sperm motility and male fertility. Despite their vital role in male fertility, almost nothing is known about the factors regulating their expression. Here, we investigated the effects of selenium (Se) on the expression of CatSper genes and sperm parameters in aging versus young male mice.     

ARF6 activated by the LHCG receptor through the cytohesin family of guanine nucleotide exchange factors mediates the receptor internalization and signaling

The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a G(s)-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization.     

The CatSper channel controls chemosensation in sea urchin sperm

Sperm guidance is controlled by chemical and physical cues. In many species, Ca²⁺ bursts in the flagellum govern navigation to the egg. In Arbacia punctulata, a model system of sperm chemotaxis, a cGMP signaling pathway controls these Ca²⁺ bursts. The underlying Ca²⁺ channel and its mechanisms of activation are unknown. Here, we identify CatSper Ca²⁺ channels in the flagellum of A. punctulata sperm. We show that CatSper mediates the chemoattractant-evoked Ca²⁺ influx and controls chemotactic steering; a concomitant alkalization serves as a highly cooperative mechanism that enables CatSper to transduce periodic voltage changes into Ca²⁺ bursts. Our results reveal intriguing phylogenetic commonalities but also variations between marine invertebrates and mammals regarding the function and control of CatSper. The variations probably reflect functional and mechanistic adaptations that evolved during the transition from external to internal fertilization.     

Expression Profiling and Identification of Novel SNPs in CatSper2 Gene and Their Influence on Sperm Motility Parameters in Bovines

122 randomly selected Vrindavani cattle were studied to detect polymorphism in four fragments of the CatSper2 gene that were comprised of exon 2, 4, 5, and 6 with flanking regions. Using PCR-SSCP and sequencing analysis, three SNPs (T157C, C273A, and A274C) in the first fragment, one SNP (C30G) in the second fragment, and two SNPs (T86G and T292C) in the fourth fragment were identified. The third fragment did not reveal any polymorphism. The SNPs were used for construction of haplotypes and three haplotypes were found. The least square analysis of variance revealed a significant (P?G or C>T SNPs may not play a role in sperm motility. However, when the comparison was made between haplotype I and II, it can be inferred that C>T SNP may have a role in sperm motility, as haplotype II has better motility parameters. Expression profiling of Catper2 gene revealed nonsignificant down regulation of CatSper2 gene in poor motility sperm compared to good motility sperm.     

In vitro fertilization analysis of squirrel monkey oocytes produced by various follicular induction regimens and the incidence of triploidy

An in vitro fertilization system utilizing squirrel monkeys was used to compare follicle-stimulating hormone, clomiphene citrate and prostaglandin E₁ as follicular induction regimens, analyze culture medium characteristics, and examine the physiological phenomenon of polyspermy. Induction of follicular growth was poor with clomiphene citrate when compared to the control group and increased the incidence of atretic follicles at all levels tested. When prostaglandin E₁ was administered, larger numbers of mature oocytes were recovered at laparoscopy. There was no difference in fertilization rate between the treatment and control groups. Homologous serum was an adequate protein source in TC-199 fertilization medium for squirrel monkey oocytes. Although the rate of triploidy was increased with in vitro fertilization, there was no relationship between sperm concentration and the incidence of polyspermy. These findings demonstrate that the squirrel monkey is a valuable primate system for studies of in vitro fertilization and preimplantation development. © 1993 Wiley-Liss, Inc.     

The effect of luteinizing hormone, human chorionic gonadotropin and adrenocorticotropic hormone on puberty in gilts reared in confinement

Three trials were conducted to determine the effect of human chorionic gonadotropin (HCG), luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) on the incidence of estrus in gilts which were reared in confinement, relocated and exposed to a boar. In trial 1, 33 gilts were given saline or 250 IU HCG at an average age of 191 days and then relocated and observed for estrus twice daily for 10 days. Treatment with HCG did not increase the proportion of gilts that exhibited estrus. In trial 2, 42 gilts were relocated at an average age of 200 days. The gilts were assigned to three treatment groups and injected with saline, 68 mug LH or 1 mg LH. After 10 days of estrous detection, a laparoscopic examination of the ovaries was conducted on all gilts failing to exhibit estrus. In groups 1 to 3, the proportions of gilts exhibiting estrus or ovulating during the 10 days after treatment were 13 of 21, 6 of 10, and 5 of 11, respectively. In trial 3, 12 gilts were relocated to pasture lots, given saline or 80 IU ACTH twice daily for 2 days and checked for estrus for 14 days. The proportions of gilts that exhibited estrus after the administration of saline or ACTH were 4 of 6 and 6 of 6, respectively. The results indicate that the incidence of estrus in gilts reared in confinement, relocated and exposed to a boar was not affected by pre-treatment with exogenous HCG, LH or ACTH.     

复方玄驹胶囊联合枸橼酸氯米芬和维生素E治疗男性肾阳虚型不育症的临床研究

摘要 目的：探讨复方玄驹胶囊联合枸橼酸氯米芬和维生素E对男性肾阳虚型不育症的治疗效果。方法：选择2017年1月-2019年12月于我院就诊并辨证为肾阳虚型男性不育症患者120例,采用随机数字表法分为研究组和对照组。对照组58例患者给予枸椽酸氯米芬和维生素E治疗,研究组62例患者在此基础上加用复方玄驹胶囊,比较两组临床疗效、治疗前后精子形态正常率、精液量、精子密度、前向运动率、精子液化时间、睾酮(testosterone,T)、黄体生成激素(luteotropic hormone,LH)、卵泡刺激素(follicle stimulatin hormone,FSH)及雌二醇(17β-estrodiol,E2)水平的变化。结果：治疗后,研究组患者的总有效率为85.48 %,显著高于对照组(72.41 %,P＜0.05)。与治疗前相比,两组患者精子形态正常率、精液量、精子密度和前向运动率均显著升高,而精子液化时间明显缩短(P＜0.05),研究组患者精子形态正常率、精液量、精子密度和前向运动率均显著高于对照组,而精子液化时间较对照组显著缩短(P＜0.05)。与治疗前相比,两组患者血清T、LH、FSH水平显著增加,E2水平无明显变化;与对照组相比,研究组血清LH水平显著增加(P＜0.05),T和FSH水平无显著差异。结论：复方玄驹胶囊联合枸橼酸氯米芬和维生素E对男性肾阳虚型不育症患者有较好的疗效,可显著提高精子质量,改善患者生殖激素水平。     

Association of Catsper1 or -2 with Ca(v)3.3 leads to suppression of T-type calcium channel activity

Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Ca(v)3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Ca(v)3.3-evoked T-type calcium current without altering other biophysical properties of Ca(v)3.3. Immunostaining studies revealed co-localization of CatSper1 and Ca(v)3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Ca(v)3.3 channel, which might be important in the regulation of sperm function.     

Effect of gonadotropin-releasing hormone,stage of sexual maturation and 6-methoxybenzoxazolinone on plasma gonadotropins,ovarian development and uterine weight in prepuberal gilts

Two experiments were conducted with prepuberal gilts at 60, 120 and 160 days of age to a) determine the effect of 6-methoxybenzoxazolinone 6-MBOA) on reproductive plasma hormone concentrations and organ development, and b) determine how plasma follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations before and after injection of gonadotropin-releasing hormone (GnRH) or 6-MBOA varied in relation to ovarian development. In Exp. 1, 12 gilts were used in a 4×4 Latin square design. Four gilts/age group were injected once with: 1) vehicle, 2.5% propylene glycol in 50% ethanol, 2) 2 μg of GnRH/kg body weight (BW), 3) 0.2 mg of 6-MBOA/kg BW, and 4) 2 mg of 6-MBOA/kg BW on four successive days in random order. Blood was collected via indwelling vena cava catheters. Injection of GnRH into gilts increased plasma FSH and LH at each age compared with vehicle (P<0.05). Hormone profiles for FSH and LH differed among age groups (P<0.01), but area under curves did not differ significantly among age groups. Injection of 6-MBOA did not significantly affect plasma FSH and LH. Plasma FSH and LH before the GnRH injection or on days when GnRH was not injected were greater at 60 than at 120 and 160 days (FSH, 128 vs 54 and 42 ng/ml; LH, 0.38 vs 0.16 and 0.13 ng/ml for 60, 120 and 160 days, respectively (P<0.05). In Exp. 2, vehicle, 0.2 or 2 mg of 6-MBOA/kg BW were injected once daily for 7 days in 19 gilts. Injections of 6-MBOA had no detectable effects on gonadotropin secretion, ovarian development or uterine weight. Between 60 and 120 days of age, vesicular follicles developed, ovarian weight increased 20-fold, and uterine weight increased 10-fold (P<0.05); basal concentrations of plasma FSH and LH decreased three- and twofold, respectively.     

The G-Protein Coupled Estrogen Receptor (GPER/GPR30) is a Gonadotropin Receptor Dependent Positive Prognosticator in Ovarian Carcinoma Patients

Follicle stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) were demonstrated to impact upon survival of patients suffering from epithelial ovarian cancer (EOC). Though structure wise the G-protein coupled estrogen receptor (GPER/GPR30) is related to FSHR/LHCGR, its prognostic impact in EOC remains controversial. We recently found that FSHR negative patients represent a specific EOC subgroup that may behave differently in respect to both treatment response and prognosis. Hence, the current study aimed to analyze how GPER may interact with the FSHR/LHCGR system in EOC and whether the prognostic significance of GPER in EOC cases (n = 151) may be dependent on the FSHR/LHCGR immunophenotype of the tumor. Ovarian cancer cell lines were used to study how FSH and LH regulate GPER and whether GPER activation differentially affects in vitro cell proliferation in presence/absence of activated FSHR/LHCGR. In EOC tissue, GPER correlated with FSHR/LHCGR and was related to prolonged overall survival only in FSHR/LHCGR negative patients. Although GPER was found to be specifically induced by LH/FSH, GPER agonists (4-Hydroxy-Tamoxifen, G1) reduced EOC cell proliferation only in case of LH/FSH unstimulated pathways. To the same direction, only patients characterized as LHCGR/FSHR negative seem to gain from GPER in terms of survival. Our combined tissue and in vitro results support thus the hypothesis that GPER activation could be of therapeutic benefit in LHCGR/FSHR negative EOC patients. Further studies are needed to evaluate the impact of GPER activation on a clinical scheme.     

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.