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.     

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.     

Genome-wide profiling of gene expression in the epididymis of alpha-chlorohydrin-induced infertile rats using an oligonucleotide microarray

Background  

As one of the chlorinated antifertility compounds, alpha-chlorohydrin (ACH) can inhibit glyceraldehyde-3-phosphate dehydrogenase (G3PDH) activity in epididymal sperm and affect sperm energy metabolism, maturation and fertilization, eventually leading to male infertility. Further studies demonstrated that the inhibitory effect of ACH on G3PDH is not only confined to epididymal sperm but also to the epididymis. Moreover, little investigation on gene expression changes in the epididymis after ACH treatment has been conducted. Therefore, gene expression studies may indicate new epididymal targets related to sperm maturation and fertility through the analysis of ACH-treated infertile animals.     

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.     

Evolutionary Genomics Reveals Lineage-Specific Gene Loss and Rapid Evolution of a Sperm-Specific Ion Channel Complex: CatSpers and CatSperβ

The mammalian CatSper ion channel family consists of four sperm-specific voltage-gated Ca²⁺ channels that are crucial for sperm hyperactivation and male fertility. All four CatSper subunits are believed to assemble into a heteromultimeric channel complex, together with an auxiliary subunit, CatSperβ. Here, we report a comprehensive comparative genomics study and evolutionary analysis of CatSpers and CatSperβ, with important correlation to physiological significance of molecular evolution of the CatSper channel complex. The development of the CatSper channel complex with four CatSpers and CatSperβ originated as early as primitive metazoans such as the Cnidarian Nematostella vectensis. Comparative genomics revealed extensive lineage-specific gene loss of all four CatSpers and CatSperβ through metazoan evolution, especially in vertebrates. The CatSper channel complex underwent rapid evolution and functional divergence, while distinct evolutionary constraints appear to have acted on different domains and specific sites of the four CatSper genes. These results reveal unique evolutionary characteristics of sperm-specific Ca²⁺ channels and their adaptation to sperm biology through metazoan evolution.     

Sperm DNA fragmentation and oxidation are independent of malondialdheyde

Background  

There is clinical evidence to show that sperm DNA damage could be a marker of sperm quality and extensive data exist on the relationship between DNA damage and male fertility status. Detecting such damage in sperm could provide new elements besides semen parameters in diagnosing male infertility. We aimed to assess sperm DNA fragmentation and oxidation and to study the association between these two markers, routine semen parameters and malondialdehyde formation.     

Cytochrome P450arom, androgen and estrogen receptors in pig sperm

Background  

Androgens and estrogens are crucial for mammalian sperm differentiation but their role in biology of mature male gamete is not still defined. The expression of proteins involved in the biosynthesis and action of these steroid hormones has been demonstrated in human spermatozoa, but very few data have been reported in mature sperm from non human species. The purpose of the current study was to investigate the expression of aromatase (P450arom), estrogen (ERalpha/ERbeta) and androgen (AR) receptors in ejaculated spermatozoa of pig.     

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.     

Male sexual ornament size is positively associated with reproductive morphology and enhanced fertility in the stalk-eyed fly <Emphasis Type="Italic">Teleopsis dalmanni</Emphasis>

Background  

Exaggerated male ornaments and displays often evolve in species where males only provide females with ejaculates during reproduction. Although "good genes" arguments are typically invoked to explain this phenomenon, a simpler alternative is possible if variation in male reproductive quality (e.g. sperm number, ejaculate content, mating rate) is an important determinant of female reproductive success. The "phenotype-linked fertility hypothesis" states that female preference for male ornaments or displays has been selected to ensure higher levels of fertility and has driven the evolution of exaggerated male traits. Females of the stalk-eyed fly Teleopsis dalmanni must mate frequently to maintain high levels of fertility and prefer to mate with males exhibiting large eyespan, a condition-dependent sexual ornament. If eyespan indicates male reproductive quality, females could directly increase their reproductive success by mating with males with large eyespan. Here we investigate whether male eyespan indicates accessory gland and testis length, and then ask whether mating with large eyespan males affects female fertility.     

Evolutionary genomics reveals lineage-specific gene loss and rapid evolution of a sperm-specific ion channel complex: CatSpers and CatSperbeta

The mammalian CatSper ion channel family consists of four sperm-specific voltage-gated Ca2+ channels that are crucial for sperm hyperactivation and male fertility. All four CatSper subunits are believed to assemble into a heteromultimeric channel complex, together with an auxiliary subunit, CatSperbeta. Here, we report a comprehensive comparative genomics study and evolutionary analysis of CatSpers and CatSperbeta, with important correlation to physiological significance of molecular evolution of the CatSper channel complex. The development of the CatSper channel complex with four CatSpers and CatSperbeta originated as early as primitive metazoans such as the Cnidarian Nematostella vectensis. Comparative genomics revealed extensive lineage-specific gene loss of all four CatSpers and CatSperbeta through metazoan evolution, especially in vertebrates. The CatSper channel complex underwent rapid evolution and functional divergence, while distinct evolutionary constraints appear to have acted on different domains and specific sites of the four CatSper genes. These results reveal unique evolutionary characteristics of sperm-specific Ca2+ channels and their adaptation to sperm biology through metazoan evolution.     

"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。     

Molecular and functional characterization of voltage-gated sodium channels in human sperm

Background  

We have investigated the expression of voltage-gated sodium channels in human spermatozoa and characterized their role in sperm motility.     

Female-driven mechanisms,ejaculate size and quality contribute to the lower fertility of <Emphasis Type="Italic">sex-ratio</Emphasis> distorter males in <Emphasis Type="Italic">Drosophila simulans</Emphasis>

Background  

Sex-ratio meiotic drive refers to the preferential transmission of the X chromosome by XY males. The loss of Y-bearing sperm is caused by an X-linked distorter and results in female-biased progeny. The fertility of sex-ratio (SR) males expressing the distorter is usually strongly reduced compared to wild-type males, especially when they are in competition. The aim of this study was to identify the post-copulatory mechanisms that lower the fertility of SR males in Drosophila simulans. Parameters contributing to male fertility were measured in single and double mating conditions.     

Identification, cloning and functional characterization of novel beta-defensins in the rat (Rattus norvegicus)

Background  

beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation.     

<Emphasis Type="Italic">In vitro</Emphasis> antioxidant activity of the prostatic secretory granules in rabbit semen after exposure to organic peroxides

Background  

The prostate gland of rabbits produces numerous granules, which are specifically implicated in the inhibition of sperm capacitation during the first hours after mating. These granules are rich in vitamin E, but their role in the antioxidant protection of rabbit sperm has not been studied.     

Effect of estrogens on boar sperm capacitation <Emphasis Type="Italic">in vitro</Emphasis>

Background  

Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1), 17beta-estradiol (E2) and estriol (E3) as well as the synthetical one, 17alpha-ethynylestradiol (EE2) on boar sperm capacitation in vitro.