Jonathan M Greene

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.

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.

In vivo monitoring of fetoplacental Vegfr2 gene activity in a murine pregnancy model using a Vegfr2-luc reporter gene and bioluminescent imaging

BackgroundVascular endothelial growth factor receptor-2 (VEGFR2) plays a pivotal role in angiogenesis by eliciting vascular endothelial cell growth when bound to VEGF, a powerful pro-angiogenic ligand. While Vegf and Vegfr2 are expressed throughout gestation, the latter third of gestation in mice is characterized by a marked increase in fetoplacental angiogenesis. Thus, the objective of this study was to determine the feasibility of monitoring fetoplacental Vegfr2 gene activity non-invasively using a Vegfr2-luc reporter transgenic mouse and bioluminescent imaging.MethodsImaging parameters were optimized using two wild-type (WT) females, bearing Vegfr2-luc fetuses. Then, seven WT females, bred to Vegfr2-luc males, were imaged from gestational day (GD) 12 to 18 to determine the usefulness of the Vegfr2-luc mouse as a model for studying fetoplacental Vegfr2 activity during pregnancy. Semi-quantitative RT-PCR of Vegfr2 was also performed on whole fetoplacental units during this time. Additionally, resultant neonates were imaged at postnatal day (PND) 7, 14 and 21 to monitor Vegfr2 activity during post-natal development.ResultsFetoplacental Vegfr2 gene activity was detected as light emissions beginning on GD 12 of gestation and increased throughout the imaging period (P < 0.05), and this paralleled the Vegfr2 mRNA data obtained from RT-PCR analysis. A decline in fetoplacental light emissions was associated with a poor pregnancy outcome in one pregnancy, indicating that this approach has potential use for studies monitoring pregnancy well being. Additionally, neonatal Vegfr2 activity was detected at PND 7, 14 and 21 but declined with time (P < 0.0001).ConclusionsIn utero fetoplacental Vegfr2 gene activity was monitored longitudinally in a quantitative manner using a luciferase reporter gene and bioluminescent imaging during the latter third of gestation. This study demonstrates the feasibility of using the Vegfr2-luc mouse to monitor late gestation fetoplacental angiogenic activity under normal and experimental conditions. Additionally, neonatal Vegfr2 gene activity was monitored for three weeks postpartum, allowing continuous monitoring of Vegfr2 activity during the latter third of gestation and postnatal development within the same animals.

l-Arginine in the Uterus and Placenta and During Gestation in Mammals

Often considered to be one of the most versatile amino acids, l-arginine is classified as a basic, cationic amino acid with three amine groups comprising a guanidino group in the side chain. l-arginine was first isolated from lupin seedlings by Schulze and Steiger (Z Physiol Chem 11:43–65, 1886), and shortly thereafter, Hedin (Z Physiol Chem 21:297–305, 1895) discovered that l-arginine is a component of animal proteins (as reviewed by Wu and Morris, Biochem J 336(Pt 1):1–17, 1998). Following the discovery of l-arginine, many efforts to determine its essentiality or dispensability were undertaken with a definitive answer still being debated today. The results from Scull and Rose (J Biol Chem 89(1):109–123, 1930) suggested that l-arginine was a dispensable or nonessential amino acid. This finding was repeated in humans by Rose and colleagues (J Biol Chem 206(1):421–430, 1954) who reported that removal of l-arginine from the diet did not result in a negative nitrogen balance in adult males.