Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.     

The proteomic characterization of ram sperm during cryopreservation analyzed by the two-dimensional electrophoresis coupled with mass spectrometry

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.     

Effect of Uremia on Semen Quality and Reproductive Function in Humans

In this study, we sought to evaluate the effect of uremia on semen quality and reproductive function in humans. For this purpose, 53 end-stage uremic patients were randomly selected. The semen samples were produced by masturbation. Fertility index (FI) was calculated according to the following formula: sperm density (×10⁶/ml) × sperm motility (%) × normal sperm morphology rate (% per 10,000). The semen samples of uremic patients were compared with those of fertile and infertile males. The results show that three patients failed to produce semen. There were no sperm found in four semen samples. The sperm motility, survival rate, sperm density, and normal sperm morphology rate of the remaining 46 patients were found to be significantly lower than those of controls. The uremic patients had the FI of 0.68(2.08) which was obviously lower than that of fertile 7.7(13.51) and infertile 4.13(5.77) males. It was, therefore, concluded that uremia caused a significant decline in sperm quality and reproductive function which resulted in consequential infertility in humans.     

Lactotransferrin in Asian Elephant (Elephas maximus) Seminal Plasma Correlates with Semen Quality

Asian elephants (Elephas maximus) have highly variable ejaculate quality within individuals, greatly reducing the efficacy of artificial insemination and making it difficult to devise a sperm cryopreservation protocol for this endangered species. Because seminal plasma influences sperm function and physiology, including sperm motility, the objectives of this study were to characterize the chemistry and protein profiles of Asian elephant seminal plasma and to determine the relationships between seminal plasma components and semen quality. Ejaculates exhibiting good sperm motility (≥65%) expressed higher percentages of spermatozoa with normal morphology (80.3±13.0 vs. 44.9±30.8%) and positive Spermac staining (51.9±14.5 vs. 7.5±14.4%), in addition to higher total volume (135.1±89.6 vs. 88.8±73.1 ml) and lower sperm concentration (473.0±511.2 vs. 1313.8±764.7×10⁶ cells ml⁻¹) compared to ejaculates exhibiting poor sperm motility (≤10%; P<0.05). Comparison of seminal plasma from ejaculates with good versus poor sperm motility revealed significant differences in concentrations of creatine phosphokinase, alanine aminotransferase, phosphorus, sodium, chloride, magnesium, and glucose. These observations suggest seminal plasma influences semen quality in elephants. One- and two-dimensional (2D) gel electrophoresis revealed largely similar compositional profiles of seminal plasma proteins between good and poor motility ejaculates. However, a protein of ∼80 kDa was abundant in 85% of ejaculates with good motility, and was absent in 90% of poor motility ejaculates (P<0.05). We used mass spectrometry to identify this protein as lactotransferrin, and immunoblot analysis to confirm this identification. Together, these findings lay a functional foundation for understanding the contributions of seminal plasma in the regulation of Asian elephant sperm motility, and for improving semen collection and storage in this endangered species.     

Quantitative Proteomic Analysis of Seminal Plasma,Sperm Membrane Proteins,and Seminal Extracellular Vesicles Suggests Vesicular Mechanisms Aid in the Removal and Addition of Proteins to the Ram Sperm Membrane

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction‐specific proteins that show demonstrable links to sperm fertility. Many of these membrane‐bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.     

Sperm phosphoproteome profiling by ultra performance liquid chromatography followed by data independent analysis (LC-MS(E)) reveals altered proteomic signatures in asthenozoospermia

Sperm motility is an important prerequisite for successful fertilization and is regulated by cyclic AMP activated protein kinase A which phosphorylates flagella proteins like axonemal dynein and initiates motility. Increase in calcium influx reverses this process by dephosphorylation that is mediated by calcineurin. Analyzing the phosphoenriched fractions of spermatozoa lysates from eight normozoospermic-, and asthenozoospermic-samples, respectively, by Nano UPLC-MS(E), the present study investigates the phosphoproteins involved in sperm motility in an attempt to identify the key pathways regulating sperm motility and likely to be altered in spermatozoa of asthenozoospermic individuals. 66 phosphoproteins were differentially regulated in asthenozoospermia. The deregulated proteins comprised predominantly the HSPs, cytoskeletal proteins, proteins associated with the fibrous sheath, and those associated with energy metabolism. EM analysis of these spermatozoa demonstrated significant defects in mitochondria, and fibrous sheath and these defects could be correlated with the altered proteome. Pathway analysis revealed that carbohydrate and energy metabolism, cAMP mediated PKA signaling, PI3K/AKT signaling and pathway regulating actin based motility by Rho were significantly altered indicating that motility in spermatozoa is regulated through the concerted effort of these pathways. The data identified signature molecules that have the potential as biomarkers for diagnosing etiology of asthenozoospermia.     

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L‐amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase‐like proteins, massive up‐regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self‐perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus; indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction‐oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.     

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。     

LCM纯化的人支气管上皮癌变各阶段组织的定量蛋白质组学研究

为分析支气管上皮癌变进程中的差异表达蛋白质,筛选肺鳞癌早期诊断标志物,以人支气管上皮癌变各阶段组织为研究对象,先采用激光捕获显微切割技术(LCM) 纯化人正常支气管上皮组织、鳞状化生、不典型增生、原位癌、浸润性肺鳞癌组织,再用同位素标记相对和绝对定量 (iTRAQ) 技术结合二维液相色谱串联质谱（2D LC-MS/MS）鉴定支气管上皮癌变进程中各阶段的差异表达蛋白质。结果共鉴定了1036个蛋白质,筛选出102个与人支气管上皮癌变相关的差异蛋白质,在这些差异蛋白质中,有的在支气管上皮癌变过程中进行性上调,有的在支气管上皮癌变过程中进行性下调,有的呈阶段特异性改变;功能分析表明,这些差异蛋白质涉及代谢、细胞凋亡、增殖、分化、信号传导、转录、翻译、细胞粘附、免疫反应与发育等。Western blotting 及免疫组织化学技术验证了其中 2个差异蛋白(S100A9和 CKB) 的表达,证实了定量蛋白质组学结果的可靠性。研究结果提示：这些差异表达蛋白质与支气管上皮癌变相关,并可成为肺鳞癌的早期诊断标志物,进一步研究差异蛋白的生物学功能,将有助于阐明支气管上皮的癌变机制,从而为肺鳞癌的早期诊断与发病机制研究提供新思路。     

Comparative proteomic analysis of early salt stress responsive proteins in roots and leaves of rice

Growth and productivity of rice (Oryza sativa L.) are severely affected by salinity. Understanding the mechanisms that protect rice and other important cereal crops from salt stress will help in the development of salt‐stress‐tolerant strains. In this study, rice seedlings of the same genetic species with various salt tolerances were studied. We first used 2DE to resolve the expressed proteome in rice roots and leaves and then used nanospray liquid chromatography/tandem mass spectrometry to identify the differentially expressed proteins in rice seedlings after salt treatment. The 2DE assays revealed that there were 104 differentially expressed protein spots in rice roots and 59 in leaves. Then, we identified 83 proteins in rice roots and 61 proteins in rice leaves by MS analysis. Functional classification analysis revealed that the differentially expressed proteins from roots could be classified into 18 functional categories while those from leaves could be classified into 11 functional categories. The proteins from rice seedlings that most significantly contributed to a protective effect against increased salinity were cysteine synthase, adenosine triphosphate synthase, quercetin 3‐O‐methyltransferase 1, and lipoxygenase 2. Further analysis demonstrated that the primary mechanisms underlying the ability of rice seedlings to tolerate salt stress were glycolysis, purine metabolism, and photosynthesis. Thus, we suggest that differentially expressed proteins may serve as marker group for the salt tolerance of rice.     

Organelle‐specific probe JC‐1 identifies membrane potential differences in the mitochondrial function of bovine sperm

The mitochondrial probe 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide (JC‐1) not only identifies mitochondria exhibiting low membrane potentials by the emission of green fluorescence (range, 510–520 nm) but also differentiates these from mitochondria exhibiting relatively high membrane potentials. This discrimination occurs because JC‐1 forms aggregates at high membrane potentials. These J‐aggregates emit a bright red‐orange fluorescence at 590 nm. In this study, JC‐1 was combined with the classical dead cell stain, propidium iodide (PI), to identify a spectrum of functional sperm along with degenerate sperm. Flow cytometric analysis of bull sperm showed that the aggregate:monomer ratio differed among bulls before cryopreservation (P < 0.001) but not afterwards (P > 0.05). The effects of stain equilibration time, sperm concentration, and live:dead ratios were examined. The addition of SYBR‐14 to the JC‐1 and PI combination enhanced the distinction between the red PI‐stained and red‐orange JC‐1–stained populations. This discrimination between J‐aggregates and the PI‐stained sperm was affected by sperm concentration. These studies show that JC‐1 can be useful in monitoring mitochondrial function in bovine sperm. Mol. Reprod. Dev. 53:222–229, 1999. © 1999 Wiley‐Liss, Inc.     

Revealing the Inhibitory Effect of Ginseng on Mitochondrial Respiration through Synaptosomal Proteomics

Ginseng, the active ingredients of which are ginsenosides, is the most popular herbal medicine and has potential merit in the treatment of cerebral disorders. To better understand the function of Ginseng in the cerebral system, we examined changes in the protein expression profiles of synaptosomes extracted from the cerebral cortical and hippocampal tissues of rats administered a high or low dose of Ginseng for 2 weeks. More than 5000 proteins belonging to synaptosomes were simultaneously identified and quantitated by an approach combining tandem mass tags with 2D liquid chromatography‐mass spectrometry (LC‐MS). Regarding differentially expressed proteins, downregulated proteins were much more highly induced than upregulators in the cerebral cortical and hippocampal synaptosomes, regardless of the dose of Ginseng. Bioinformatic analysis indicated the majority of the altered proteins to be located in the mitochondria, directly or indirectly affecting mitochondrial oxidative respiration. Further functional experiments using the substrate‐uncoupler inhibitor titration approach confirmed that three representative ginsenosides were able to inhibit oxidative phosphorylation in mitochondria. Our results demonstrate that Ginseng can regulate the function of mitochondria and alter the energy metabolism of cells, which may be useful for the treatment of central nervous disorders.     

Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage

Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility.     

Proteomic analysis of proteins differentially expressed in uterine lymphocytes obtained from wild‐type and NOD mice

Non‐obese diabetic (NOD) mice exhibit impaired fertility and decreased litter size when compared to wild type (WT) mice. However, it is unclear why allogeneic pregnant NOD mice are prone to spontaneous embryo loss. Herein, two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry (MS) were used to detect differentially expressed proteins in the uterine lymphocytes isolated from these mice and WT BALB/c controls. We found 24 differentially expressed proteins. The differential expression of 10 of these proteins was further confirmed by Western blot analysis. Out of the 24 identified proteins, 20 were expressed in uterine lymphocytes of WT mice at a level at least 2 times higher than in NOD mice, whereas 4 were down‐regulated. Western blot analysis confirmed that 8 proteins were up‐regulated and 2 proteins were down‐regulated in WT mice compared with NOD mice, consistent with the results of 2‐DE and MS. Additionally, most of the highly expressed proteins in WT uterine lymphocytes were expressed at a significantly lower level in the corresponding splenic group (17/20). These results suggest that up‐regulated expression of these proteins may be specific to uterine lymphocytes. Reported functions of the highly expressed proteins affect key functions during pregnancy, including cell movement, cell cycle control, and metabolisms. Finally, we analyzed the constitutional ratio of CD3⁺ and CD49b⁺ cells in the isolated lymphocytes by flow cytometry. Our results suggest that the differentially expressed proteins may participate in the modulation of embryo implantation and early‐stage development of embryos, and subsequently influence pregnancy outcome. J. Cell. Biochem. 108: 447–457, 2009. © 2009 Wiley‐Liss, Inc.     

Cryopreservation‐induced alterations in protein composition of rainbow trout semen

The aim of this study was to detect cryopreservation‐induced alterations in the protein composition of rainbow trout semen using two independent methods 1DE SDS‐PAGE prefractionation combined with LC‐MS/MS and 2D difference gel electrophoresis followed by MALDI‐TOF/TOF identification. Here, we show the first comprehensive dataset of changes in rainbow trout semen proteome after cryopreservation, with a total of 73 identified proteins released from sperm to extracellular fluid, including mitochondrial, cytoskeletal, nuclear, and cytosolic proteins. Our study provides new information about proteins released from sperm, their relation to sperm structure and function, and changes of metabolism of sperm cells as a result of cryopreservation. The identified proteins represent potential markers of cryoinjures of sperm structures and markers of the disturbances of particular sperm metabolic pathways. Further studies will allow to decipher the precise function of the proteins altered during rainbow trout cryopreservation and are useful for the development of extensive diagnostic tests of sperm cryoinjures and for the successful improvement of sperm cryopreservation of this economically important species.     

Differential seminal plasma proteome signatures of acute lymphoblastic leukemia survivors

With advances in therapeutic methods, there is a high survival rate among leukemia patients, of an extent more than 80%. However, chemotherapeutic drugs used to treat these patients have adverse effects on their overall health profile including fertility. The primary aim of this study was to identify differentially expressed proteins in seminal plasma of acute lymphoblastic leukemia (ALL) survivors compared to age-matched healthy controls, which can provide molecular basis of idiopathic infertility in such survivors. Differential proteome profiling was performed by 2D–differential in-gel electrophoresis, protein spots were identified by mass spectrometry and selective differentially expressed proteins (DEPs) were validated by western blotting and ELISA method. Out of eight DEPs identified, five proteins (isocitrate dehydrogenase 1, semenogelin 1, lactoferrin, prolactin-inducible protein, and human serum albumin) were upregulated and three (pepsinogen, prostate specific antigen and prostatic acid phosphatase) were downregulated. Expression profiles of these proteins are suggestive of reduction in semen quality in ALL survivors and can further be explored to determine their fertility status.