J. M. Feugang

Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility.

BackgroundMale infertility is a major problem for mammalian reproduction. However, molecular details including the underlying mechanisms of male fertility are still not known. A thorough understanding of these mechanisms is essential for obtaining consistently high reproductive efficiency and to ensure lower cost and time-loss by breeder.ResultsUsing high and low fertility bull spermatozoa, here we employed differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) and identified 125 putative biomarkers of fertility. We next used quantitative Systems Biology modeling and canonical protein interaction pathways and networks to show that high fertility spermatozoa differ from low fertility spermatozoa in four main ways. Compared to sperm from low fertility bulls, sperm from high fertility bulls have higher expression of proteins involved in: energy metabolism, cell communication, spermatogenesis, and cell motility. Our data also suggests a hypothesis that low fertility sperm DNA integrity may be compromised because cell cycle: G2/M DNA damage checkpoint regulation was most significant signaling pathway identified in low fertility spermatozoa.ConclusionThis is the first comprehensive description of the bovine spermatozoa proteome. Comparative proteomic analysis of high fertility and low fertility bulls, in the context of protein interaction networks identified putative molecular markers associated with high fertility phenotype.

Transcriptome analysis of bull spermatozoa: implications for male fertility

Spermatozoa deliver more than the paternal genome into the oocyte; they also carry remnant messenger RNA from spermatogenesis. The RNA profiles of spermatozoa from high-fertility and a low-fertility Holstein bulls were analysed using Affymetrix bovine genechips. A total of 415 transcripts out of approximately 24,000 were differentially detected in spermatozoa collected from both bulls (fold change > or =2.0; P<0.01). These transcripts were associated with different cellular functions and biological processes. Spermatozoa from high-fertility bulls contained higher concentrations of transcripts for membrane and extracellular space protein locations, while spermatozoa from the low-fertility bulls were deficient of transcripts for transcriptional and translational factors. Quantitative real-time PCR was used on three low-fertility and four high-fertility bulls to validate the microarray data. Two highly represented transcripts in the microarray analysis (protamine 1 and casein beta 2) were validated, as well as a third transcript (thrombospondin receptor CD36 molecule) that showed a lower concentration in low-fertility bulls. This study presents the global analysis of spermatozoa originating from bulls with opposite fertility. These results provide some specific transcripts in spermatozoa that could be associated with bull fertility.

Green Tea Compound in Chemoprevention of Cervical Cancer

Objectives: Human papillomavirus (HPV) infection is closely associated with the development of more than 95% of cervical cancer. Clinical trials using several chemopreventive agents are underway, but results are inconclusive. Most agents used in trials inhibited the growth of cancer cells in vitro, and about half of patients had some degree of clinical responses; however, the therapeutic effect was confounded by high rates of spontaneous regression and relapse. The selection of nontoxic agents especially food, beverage, and natural products that suppress oncogenic HPV, inhibit malignant transformation, and can additionally be used long term may be important for cervical cancer prevention. Methods: We evaluated green tea compound (epigallocatechin gallate and polyphenols E) effects on immortalized cervical epithelial and cervical cancer cells. HPV-immortalized cervical epithelial cells, TCL1, and HPV-positive cervical cancer cells, Me180 and HeLa, were used in the study. The effects of green tea compounds on cell growth, apoptosis, cell cycle, and gene expression were examined and characterized. Results: Both epigallocatechin gallate and polyphenols E inhibited immortalized cervical epithelial and cancer cell growth. Apoptosis induction and cell cycle changes were observed in a dose-dependent manner. Western blot analysis of apoptosis-related proteins, p53 and p21, showed dose-dependent increase, whereas p27 was not affected. HPV-E7 protein expression was decreased by green tea compounds. Conclusions: This study provides information on the potential mechanisms of action of green tea compounds in suppression of HPV-related cervical cells, and it will enable us to assess the feasibility of using these agents.

Application of quantum dot nanoparticles for potential non-invasive bio-imaging of mammalian spermatozoa.

BackgroundVarious obstacles are encountered by mammalian spermatozoa during their journey through the female genital tract, and only few or none will reach the site of fertilization. Currently, there are limited technical approaches for non-invasive investigation of spermatozoa migration after insemination. As the knowledge surrounding sperm behavior throughout the female genital tract still remains elusive, the recent development of self-illuminating quantum dot nanoparticles may present a potential means for real-time in vitro and in vivo monitoring of spermatozoa.ResultsHere, we show the ability of boar spermatozoa to harmlessly interact and incorporate bioluminescent resonance energy transfer-conjugated quantum dot (BRET-QD) nanoparticles. The confocal microscope revealed in situ fluorescence of BRET-QD in the entire spermatozoon, while the ultra-structural analysis using the transmission electron microscope indicated BRET-QD localization on the sperm plasma membrane and intracellular compartment. In controlled-in vitro assays, bioluminescent imaging demonstrated that spermatozoa incubated with BRET-QD and luciferase substrate (coelenterazine) emit light (photons/sec) above the background, which confirmed the in situ fluorescence imaging. Most importantly, sperm motility, viability, and fertilizing potential were not affected by the BRET-QD incorporation when used at an appropriated ratio.ConclusionsOur results demonstrate that pig spermatozoa can incorporate BRET-QD nanoparticles without affecting their motility and capacity to interact with the oocyte when used at an appropriated balance. We anticipate that our study will enable in-depth exploration of the male components of in vivo migration, fertilization, and embryonic development at the molecular level using this novel approach.

L-arginine enhances cell proliferation and reduces apoptosis in human endometrial RL95-2 cells

BackgroundL-arginine is considered to be one of the most versatile amino acids due to the fact that it serves as a precursor for many important molecules in cellular physiology. When supplemented in the diet, L-arginine can increase the number of implantation sites in mice and rats, suggesting an effect at the level of the endometrium. To this end, this study determined the effect that L-arginine has on apoptosis and cell proliferation in human endometrial RL95-2 cells.ResultsL-arginine at physiological (200 micromol/L) and supra-physiological (800 micromol/L) concentrations increased cell proliferation at days 2 and 4 post-treatment with a dose-dependent effect being observed on day 2. Additionally, inhibition of nitric oxide (NO) synthase and arginase, which are responsible for the conversion of L-arginine to NO and polyamines, respectively, reduced the proliferative effect of L-arginine. L-arginine also decreased the proportion of cells with TUNEL positive nuclei and increased the ratio of cells with healthy mitochondria compared to cells with a disrupted mitochondrial membrane potential, indicating that L-arginine prevents mitochondrial mediated apoptosis in endometrial RL95-2 cells. Furthermore, exposure to L-arginine did not affect total BAD protein expression; however, L-arginine increased the abundance of phosphorylated BAD protein.ConclusionsIn summary, L-arginine added to the culture media at physiological (200 micromol/L) and supraphysiological concentrations (800 micromol/L) enhanced endometrial RL95-2 cell proliferation through mechanisms mediated by NO and polyamine biosynthesis. In addition, L-arginine reduced endometrial RL95-2 mitochondrial mediated apoptosis through increased phosphorylation of BAD protein.

Dietary l-Arginine Supplementation during Gestation in Mice Enhances Reproductive Performance and Vegfr2 Transcription Activity in the Fetoplacental Unit

Regarded as one of the most versatile amino acids, arginine serves as a precursor for many molecules and has been reported to improve the reproductive performance of rats and pigs. To this end, we sought to determine if dietary L-arginine alters fetoplacental vascular endothelial growth factor receptor-2 (Vegfr2) transcription activity. Eighteen wild-type FVB/N female mice were bred to homozygous FVB/N-Tg(Vegfr2-luc)-Xen male mice. Bred female mice received 1 of 2 experimental diets: one supplemented with 2.00% (wt:wt) L-arginine (+Arg) or 1 supplemented with 4.10% (wt:wt) alanine (+Ala) to serve as an isonitrogenous control for +Arg. In addition, 6 mice were fed a nonsupplemented control (Con) diet to normalize bioluminescent imaging data. All data were analyzed using ANOVA followed by Fishers least significant difference. Total feed intake did not differ between groups; however, mice in the +Arg group consumed more arginine (P < 0.05). Arginine supplementation increased weight gain during the latter one-third of gestation (d 12- 18), total litter size, number of pups born alive, number of placental attachment sites, litter birth weight, and litter weight of pups born alive but decreased the individual birth weights (P < 0.05). During d 12-18, arginine supplementation increased (P < 0.05) the mean total Vegfr2 transcription activity and Vegfr2 transcription activity corrected for fetoplacental mass. Moreover, mice in the +Arg group had an earlier rise in Vegfr2 transcription activity. In conclusion, our results demonstrate that the beneficial effect of dietary L-arginine supplementation on mammalian reproduction is associated with enhanced Vegfr2 transcription activity in fetoplacental tissues.

In vitro effects of relaxin on gene expression in porcine cumulus-oocyte complexes and developing embryos

BackgroundRelaxin hormone peptide is found in porcine follicular and utero-tubal fluids, but its possible actions during early embryo development are still undetermined. Here, we investigated the effects of porcine relaxin during oocyte maturation and embryo development, and gene expression in the pig.MethodsImmature cumulus-oocyte complexes (COCs) were obtained from ovarian follicles of sows. In experiment 1, COCs were matured in the presence of 0, 20, or 40 ng relaxin/ml, or 10% (v/v) porcine follicular fluid. In experiment 2, COCs were in vitro matured, fertilized and resulting embryos were cultured in the presence of 0, 20, or 40 ng relaxin/ml. In experiment 3, COCs were matured in the presence of 40 ng relaxin/ml, fertilized and zygotes were cultured as indicated in experiment 2. We evaluated the proportions of matured oocytes in experiment 1, cleaved and blastocysts on Day 2 and Day 7 post insemination in all experiments. The total cell number of blastocysts was also evaluated. In parallel, transcription levels of both relaxin and its receptors (RXFP1 and RXFP2), as well as a pro- (Bax) and anti- (Bcl2-like 1) apoptotic-related genes were determined. All data were analyzed by ANOVA and significant differences were fixed for P < 0.05.ResultsIn experiment 1, relaxin significantly increased the proportions of matured oocytes and cleaved embryos, as well as the expression level of RXFP2 mRNA compared to RXFP1 (P < 0.05). There was no effect on endogenous expression of relaxin and Bcl2-like1/Bax ratios. In all experiments, relaxin did not affect the proportions of blastocysts, but did significantly increase their total cell numbers (P < 0.05). Furthermore, no effect of relaxin was observed on Bcl2-like1/Bax expression ratios, which were similar between groups.ConclusionsExogenous relaxin influences its own receptors expression, improves oocyte nuclear maturation. Its beneficial effect on total cell number of blastocysts appears to be through a Bcl2-like1/Bax-independent mechanism.

Bioluminescent magnetic nanoparticles as potential imaging agents for mammalian spermatozoa

BackgroundNanoparticles have emerged as key materials for developing applications in nanomedicine, nanobiotechnology, bioimaging and theranostics. Existing bioimaging technologies include bioluminescent resonance energy transfer-conjugated quantum dots (BRET-QDs). Despite the current use of BRET-QDs for bioimaging, there are strong concerns about QD nanocomposites containing cadmium which exhibits potential cellular toxicity.ResultsIn this study, bioluminescent composites comprised of magnetic nanoparticles and firefly luciferase (Photinus pyralis) are examined as potential light-emitting agents for imaging, detection, and tracking mammalian spermatozoa. Characterization was carried out using infrared spectroscopy, TEM and cryo-TEM imaging, and ζ-potential measurements to demonstrate the successful preparation of these nanocomposites. Binding interactions between the synthesized nanoparticles and spermatozoon were characterized using confocal and atomic/magnetic force microscopy. Bioluminescence imaging and UV–visible-NIR microscopy results showed light emission from sperm samples incubated with the firefly luciferase-modified nanoparticles. Therefore, these newly synthesized luciferase-modified magnetic nanoparticles show promise as substitutes for QD labeling, and can potentially also be used for in vivo manipulation and tracking, as well as MRI techniques.ConclusionsThese preliminary data indicate that luciferase-magnetic nanoparticle composites can potentially be used for spermatozoa detection and imaging. Their magnetic properties add additional functionality to allow for manipulation, sorting, or tracking of cells using magnetic techniques.

Lectin-Functionalized Magnetic Iron Oxide Nanoparticles for Reproductive Improvement

Background: Semen ejaculates contain heterogeneous sperm populations that can jeopardize male fertility. Recent development of nanotechnology in physiological systems may have applications in reproductive biology. Here, we used magnetic nanoparticles as a novel strategy for sperm purification to improve semen fertility. Methods: Boar semen was obtained in artificial insemination doses from a local boar stud. Doses were mixed with or without magnetic nanoparticles designed to target and deplete moribund and poor performing spermatozoa under an electromagnetic field. Sperm motility characteristics were assessed prior to insemination of open gilts with control (n=3 gilts) and nanopurified (n=4 gilts) semen. Pregnancies were verified 30 days post-insemination. Litter sizes and post-natal development of piglets were respectively evaluated at parturition and weekly until weaning. Results: Nanopurification significantly improved sperm motility. Two gilts in the control group were confirmed nonpregnant, but the remainder maintained pregnancies through to parturition (33% vs. 100%, control and nanopurified groups, respectively). At parturition, the number of piglets born to the control gilt was not significantly different from the average of the nanopurified group (17 ± 0.0 vs. 15 ± 2, respectively; P>0.05); however, in the latter group 78% of piglets remained alive compared to 76% of the control. Birth weight of control piglets was lower (1.18 ± 0.22 kg) than those in the nanopurified group (1.41 ± 0.14 kg). Both groups of piglets showed linear and parallel growth rates with respective weight gains of 4.4x and 4.1x from birth to weaning. Interestingly, piglets produced in the nanopurified group comprised of 55% males compared to 38% in the control group. Conclusions: Magnetic nanoparticles used in this preliminary study exhibited no toxic effects on sperm fertilization capacity and piglet viability. Beneficial effects may be seen in semen fertility, with possible use for gender selection. Further investigations on a large scale are needed to confirm the current findings, with potential application in clinical practice.

Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa

BackgroundRelaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars.MethodsSpermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA.ResultsBoth receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa.ConclusionsImmunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.