Reproductive Performance of Mares Fed Dietary Zearalenone.

It is known that zearalenone (ZON) interacts directly with estrogen receptors, and its in vivo effects on reproduction have been well-documented in several species. In contrast, reports of ZON's impact on horse reproduction are conflicting and inconclusive, some studies confounded by the presence of mycotoxins such as deoxynivalenol in the feed. This study assesses the effect of chronic consumption of zearalenone on reproduction in cycling mares fed >95% pure ZON (0, 2, or 8 mg/da; n = 7 mares/treatment) for three estrous cycles, followed by artificial insemination, through 16 days of pregnancy. Animals were on ZON treatment for between 70 and 121 days (average 84) depending on individual cycle patterns. ZON-induced changes in serum concentration of estradiol (E2) and progesterone (P4), and total estrogenicity were measured using RIAs and the E-screen assay, respectively. Effects on reproductive physiology and pregnancy were monitored by ultrasound and clinical parameters. No significant changes were found in reproductive hormone levels of E2, or P4 for mares on ZON treatments compared to controls, although there was a significant (P < 0.01) increase in P4 levels across Cycle number in High ZON (8 mg/da) treated mares. There was also an increasing trend in the interovulatory interval in the High ZON treatment group. The overall estrogenicity was similar across treatments and over time, not differing from controls or between ZON treatment groups. Adverse uterine and ovarian effects were also not observed, but pregnancy rates were mixed with only 4 of 7 mares on Low ZON becoming pregnant, and only 3 maintaining pregnancy and fetal heartbeat by Day 30, compared to 5 of 6 control mares and all 7 mares on High ZON. Because reproductive efficiency and hormone concentrations are highly variable across individuals, this study did not demonstrate that ZON at 2 or 8 mg/da was detrimental to mares' reproduction. Yet, inferring that ZON treatments were completely without effect is also not appropriate, as the absence of measurable significant differences could be attributed to the limited sample size. Most importantly, there were no extreme signs of toxicology, in contrast to previous reports when ZON was fed at these "doses."

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

BACKGROUND: L-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. RESULTS: L-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. CONCLUSIONS: In 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 Fisher's 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 vivo monitoring of fetoplacental Vegfr2 gene activity in a murine pregnancy model using a Vegfr2-luc reporter gene and bioluminescent imaging.

BACKGROUND: Vascular 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. METHODS: Imaging 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. RESULTS: Fetoplacental 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). CONCLUSIONS: In 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.