339. THE RELATIONSHIP BETWEEN DNA FRAGMENTATION IN MOUSE OOCYTES AND GENITAL TRACT MICROORGANISMS

2010 ◽  
Vol 22 (9) ◽  
pp. 139
Author(s):  
J. E. Harris ◽  
E. S. Pelzer ◽  
J. A. Allan ◽  
E. Whiteside ◽  
L. De Boer ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Virulence Factors ◽  
Follicular Fluid ◽  
Genital Tract ◽  
Oocyte Quality ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Mouse Oocytes ◽  
Tract Infections

Colonising bacteria detected within the follicular fluid of women undergoing assisted reproductive technology cycles has been associated with decreased embryo transfer rates and decreased pregnancy rates. The bacteria isolated from the follicular fluid of these women, at the time of trans-vaginal oocyte retrieval include Gram-positive anaerobic rods, Gram-negative anaerobic rods, streptococci, staphylococci, and lactobacilli. Some of these bacteria are opportunistic pathogens in the female genital tract. The expression of virulence factors including hyaluronidase and DNase, could affect the structural integrity of the oocyte and induce DNA fragmentation and apoptosis. Hyaluronin is a major carbohydrate component of the cumulus oocyte complex that could be a target for bacterial hyaluronidase. Other virulence factors associated with bacteria detected in cases of genital tract infections include the hydrolytic enzymes sialidase, β-galactosidase and β-N-acetylhexosaminidase (Howe 1999). Production of reactive oxygen species as a result of the presence of bacteria and bacterial heat shock proteins have been suggested as mechanisms responsible for increased DNA fragmentation and apoptotic progression of male gametes in colonised semen. Previous studies have not confirmed a direct relationship between sperm morphology and the level of sperm DNA fragmentation. DNA fragmentation in oocytes in relation to the presence of bacteria remains to be investigated. Oocyte quality is only determined by the assessment of cumulus cell morphology prior to in-vitro fertilisation. In-vitro testing of oocyte DNA integrity under various conditions may further the understanding of oocyte quality. In this study, mouse oocytes were used to investigate the structural and DNA integrity of oocytes after in vitro exposure to Lactobacillus spp., Streptococcus spp., and Staphylococcus spp. and determine whether the morphological appearance of the oocyte was predictive of the level of DNA fragmentation and whether DNA fragmentation can be attributed to certain bacterial species found colonising follicular fluid. (1) Howe L (1999). Mucinase and sialidase activity of the vaginal microflora: implications for the pathogenesis of preterm labour. International Journal of STD and AIDS 10(7), 442–446.

174 Follicular fluid extracellular vesicles obtained from Holstein cows kept under thermoneutral or heat stress conditions modify gene expression of in vitro-matured oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 211
Author(s):  
F. M. Dalanezi ◽  
R. A. Ferrazza ◽  
J. C. Ochoa ◽  
H. D. Mogllón ◽  
F. C. Destro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Follicular Fluid ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Holstein Cows ◽  
Dna Integrity ◽  
Meiotic Progression ◽  
Expression Of Genes

Heat stress (HS) has a massive impact on bovine reproduction. In cows, some of these deleterious effects involve follicular development and oocyte quality. Extracellular vesicles (EV) secreted by granulosa cells play a critical role in the intrafollicular environment by directly influencing cumulus cells and oocyte functions. The objective of this study was to investigate the effect of follicular fluid EV obtained from Holstein cows kept under thermoneutral (TN) or HS conditions, on in vitro bovine oocyte maturation. Non-lactating Holstein cows were synchronized with the Ovsynch protocol and also received an intravaginal progesterone device. From ovulation day (Day 1), cows were randomly assigned to TN (26°C, 73% humidity; n=12) or HS (36°C, 70% humidity; n=12) environments. On Day 9, 2 follicles (F1 and F2) were individually aspirated and all remained follicles were ablated. Then, on Day 14, newly formed F1 and F2 were also aspirated. Follicular fluid from all follicles from each treatment was pooled and EV were obtained according to Silveira et al. (2017 PLoS One 12, 1-25) and diluted in PBS (100μL of PBS per mL of follicular fluid centrifuged). Pools of 20 cumulus-oocyte complexes (COC) grade 1 or 2 (Stojkovic et al. Biol Reprod 200164, 904-992], predominantly from Bos indicus, were submitted to the following treatment groups: Control (n=4): matured in 90μL of TCM-199 with Eagles’ salts, glutamine, NaHCO3, pyruvate, amikacin, and FSH (base medium); TN (n=4): matured in 81μL of base medium+9μL of TN EV suspension; and HS (n=4): matured in 81μL+9μL of HS EV suspension. All treatments were carried out at 38.5°C for 24h in a humid atmosphere with 5% CO2. After 24h of maturation, COC were evaluated for meiotic progression (Hoechst 33342 stain), DNA integrity (TUNEL stain), and expression of genes related to oocyte quality (TaqMan assay, Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Results were analysed using ANOVA followed by Tukey post hoc test (P<0.05). When the experimental groups were compared with the control group, there was no treatment effect on meiotic progression, DNA integrity, or gene expression of cumulus cells. In the oocytes of the TN group, the genes HSF1, IGFBP2, BMP15, GDF9, CDCA8, HAS2, RPL15, STAT3,and PFKP were less expressed. We concluded that oocytes matured in the presence of EV from follicular fluid of cows kept under TN conditions had lesser expression of genes related to oocyte quality. This study was supported by FAPESP (Grant #2012/18297-7) and CAPES Foundation of Brazil.

219 SORTING OF EQUINE SPERM USING A MICROFLUIDIC DEVICE AS A METHOD OF SPERM SELECTION FOR IN VITRO FERTILIZATION AND INTRACYTOPLASMIC SPERM INJECTION

2016 ◽  
Vol 28 (2) ◽  
pp. 241 ◽  
Author(s):  
R. Gonzalez ◽  
E. Carnevale
Keyword(s):  
Dna Fragmentation ◽  
Microfluidic Device ◽  
Membrane Integrity ◽  
Sperm Parameters ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Normal Morphology ◽  
Vitro Fertilization ◽  
Sperm Sorting

Microfluidic technology can be used for sperm separation. Microfluidic devices generate a fluid flow to sort sperm from a media reservoir into a collection chamber. In the human and mouse, the use of microfluidic devices resulted in the selection of sperm with improved sperm motility, normal morphology, and DNA integrity for in vitro fertilization (IVF), intrauterine insemination (IUI), and intracytoplasmic sperm injection (ICSI). With the use of microfluidic sperm separation, centrifugation can be eliminated, diminishing the risk of reactive oxygen species exposure and DNA damage. We hypothesised that equine sperm can be separated using a microfluidic sorting device (Fertile PlusTM Sperm Sorting Chip; DxNow, Worcester, MA, USA) to improve the quality of sperm for ICSI. The aim of our research was to evaluate sperm parameters, including motility, morphology, membrane integrity, and DNA integrity, in frozen-thawed samples of equine semen before and after sorting using the Fertile Plus Sperm Sorting Chip. Two experiments were performed. In Experiment 1, the microfluidic device was used to separate frozen-thawed semen samples (n = 10) from research stallions (n = 3) with good quality frozen semen; all semen was frozen by one method in our laboratory. In Experiment 2, clinical samples of frozen-thawed semen (n = 11) from 7 stallions were evaluated. The semen was of variable quality and frozen at different facilities. Sperm analyses included (1) motility, (2) morphology (Hancock stain, Animal Reproduction Systems, Chino, CA, USA), (3) live-dead sperm (Hancock stain), (4) membrane integrity (HOS, hypo-osmotic swelling test), and (5) DNA fragmentation (SCD, sperm chromatin dispersion). Two sample t-tests were used to compare sperm parameters. In Experiment 1, use of the Fertile Plus Sperm Sorting Chip improved sperm parameters between the original and sorted samples, respectively: sperm motility (37.2 ± 13.0% and 62.2 ± 15.6%; P = 0.002), normal morphology (60.1 ± 12.2% and 75.5 ± 9.7%; P = 0.006), percentage live sperm (55.8 ± 16.0% and 73.6 ± 12.9%; P = 0.03), HOS (33.7 ± 7.2% and 48 ± 9.7%; P = 0.001) and sperm DNA fragmentation (12.3 ± 4.4% and 5.6 ± 4.4%; P = 0.004). When the Fertile Plus Sperm Sorting Chip was used in Experiment 2 to separate frozen-thawed semen from various sources, improvements were noted between the original and sorted samples, respectively, with increased motility (22.0 ± 13.0% and 57.0 ± 11.6%; P = 0.0009), normal morphology (58.4 ± 9.6% and 74.0 ± 10.3%; P = 0.005), a higher percentage of live sperm (55.5 ± 11.2% and 68.3 ± 14.2%; P = 0.04), and decreased sperm DNA fragmentation (22.3 ± 14.7% and 8.2 ± 8.3%; P = 0.004); no effect was observed on HOS (21.2 ± 6.0% and 24.9 ± 11.5%; P = 0.19). Our results demonstrate that use of the Fertile Plus Sperm Sorting Chip resulted in a subpopulation of sperm with improved quality parameters. Separation of sperm using a microfluidic device has the potential to select sperm with desirable characteristics for equine assisted reproductive techniques.

Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome

Reproduction ◽  
2013 ◽  
Vol 146 (5) ◽  
pp. 433-441 ◽  
Author(s):  
Renata Simões ◽  
Weber Beringui Feitosa ◽  
Adriano Felipe Perez Siqueira ◽  
Marcilio Nichi ◽  
Fabíola Freitas Paula-Lopes ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Cleavage Rate ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Significant Difference

Sperm chromatin fragmentation may be caused by a number of factors, the most significant of which is reactive oxygen species. However, little is known about the effect of sperm oxidative stress (OS) on DNA integrity, fertilization, and embryonic development in cattle. Therefore, the goal of this study was to evaluate the influence of sperm OS susceptibility on the DNA fragmentation rate and in vitro embryo production (IVP) in a population of bulls. Groups of cryopreserved sperm samples were divided into four groups, based on their susceptibility to OS (G1, low OS; G2, average OS; G3, high OS; and G4, highest OS). Our results demonstrated that the sperm DNA integrity was compromised in response to increased OS susceptibility. Furthermore, semen samples with lower susceptibility to OS were also less susceptible to DNA damage (G1, 4.06%; G2, 6.09%; G3, 6.19%; and G4, 6.20%). In addition, embryo IVP provided evidence that the embryo cleavage rate decreased as the OS increased (G1, 70.18%; G2, 62.24%; G3, 55.85%; and G4, 50.93%), but no significant difference in the blastocyst rate or the number of blastomeres was observed among the groups. The groups with greater sensitivity to OS were also associated with a greater percentage of apoptotic cells (G1, 2.6%; G2, 2.76%; G3, 5.59%; and G4, 4.49%). In conclusion, we demonstrated that an increased susceptibility to OS compromises sperm DNA integrity and consequently reduces embryo quality.

Effect of in vitro exposure to lead chloride on semen quality and sperm DNA fragmentation

Zygote ◽  
2014 ◽  
Vol 23 (3) ◽  
pp. 384-393 ◽  
Author(s):  
M. Gomes ◽  
A. Gonçalves ◽  
E. Rocha ◽  
R. Sá ◽  
A. Alves ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Room Temperature ◽  
Semen Quality ◽  
Lead Chloride ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Progressive Motility ◽  
Sperm Dna ◽  
In Vitro Exposure

SummaryExposure to lead may cause changes in the male reproductive system. We evaluated the effect of lead chloride (PbCl2) in vitro on semen quality from 31 individuals. Samples were incubated at room temperature for two exposure times (4 h and 8 h) and with two concentrations of PbCl2 (15 μg/ml or 30 μg/ml). Results showed that PbCl2 significantly inhibited rapid progressive motility and caused an increase in the percentage of tail anomalies in both times and concentrations assessed, as well as a decrease in vitality in the group exposed to 30 μg/ml PbCl2. A significant increase in immotile sperm was also observed between the group control and the groups submitted to lead. Total motility and DNA fragmentation also showed a significant decrease and increase, respectively, after 4 h of incubation in the group exposed to 30 μg/ml and in both groups after 8 h of incubation. In conclusion, PbCl2 affected sperm parameters and DNA integrity, which are essential for male fertility.

Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection

Zygote ◽  
2007 ◽  
Vol 15 (1) ◽  
pp. 15-24 ◽  
Author(s):  
M. Nakai ◽  
N. Kashiwazaki ◽  
A. Takizawa ◽  
N. Maedomari ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Freeze Drying ◽  
Chelating Agents ◽  
Sperm Head ◽  
Control Group ◽  
Sperm Dna Fragmentation ◽  
Freeze Dried ◽  
Boar Sperm

SUMMARYSuccessful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

P–080 Higher sperm DNA fragmentation reduces the proportion of good quality embryos at day 5 on IVF and ICSI cycles from unselected males

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
I Hervá. Herrero ◽  
A Pacheco ◽  
R Rivera-Egea ◽  
M Gi. Julia ◽  
A Navarro-Gomezlechon ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Dna Integrity ◽  
Embryo Morphology ◽  
Sperm Dna Fragmentation ◽  
Cleavage Stage ◽  
Sperm Dna ◽  
Embryo Fragmentation ◽  
The Impact

Abstract Study question Does sperm DNA fragmentation (SDF) reduce the ratio of good-quality embryos in day 3 (D3) and day 5 (D5) of embryonic development? Summary answer High sperm DNA fragmentation (SDF &gt;15%) is associated with poor embryo quality at blastocyst-stage per cycle in unselected patients undergoing IVF and ICSI. What is known already It has been shown that the proportion of spermatozoa with DNA fragmentation is higher in infertile men than in semen from fertile men. However, the controversy regarding the impact that sperm genome damage can have on IVF or ICSI treatments is evident in the published literature. The effects of SDF would become evident after activation of the embryonic genome at 8-cell stage, compromising not only the quality of the embryos obtained, but also the reproductive outcomes, as reduced implantation rates, higher miscarriages rates and thus, a decreased chance of pregnancy. Study design, size, duration This multicentric observational retrospective study included 1339 couples who underwent 2759 IVF-ICSI cycles using autologous oocytes from January 2000 to March 2019. All men have an SDF test in their ejaculated spermatozoa by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling). The subjects were divided into two groups according to their sperm DNA integrity: low (≤15%) (n = 2287 cycles) or high (&gt;15%) (n = 472) SDF. Participants/materials, setting, methods Embryo quality was assessed complying morphological standards at cleavage-stage on D3 and at blastocyst-stage on D5 (inner cell mass (ICM) and trophectoderm (TE) grade (A, B, C or D)) in according to ASEBIR’s embryo selection criteria, being embryos of good quality those categorized as A+B. The outcomes were calculated in relation to the total number of zygotes obtained. The results were compared by Student t test; p value &lt;0.05 was considered significant. Main results and the role of chance The SDF average of the low group was 5.8% (95% CI 5.6–5.9) whereas in high group was 23.7% (95% CI 23.0–24.4). The female age was equal, 37.1 years (95%CI 37.0–37.2) and 37.1 years (95% CI 36.8–37.4) respectively. A total of 9796 embryos were evaluated. The optimal cleavage-stage embryo rate per cycle was 25.0% (95% CI 21.7–28.3) (8.0 average cells number, 1.5 embryo fragmentation average, symmetry 1, mononucleated cells) versus 26.7% (95%CI 19.1–34.2) (7.9 average cells number, 1.8 embryo fragmentation average, symmetry 1, mononucleated cells) when comparing between groups (p &lt; 0.001). Blastocyst-stage arrival rate (number of embryos at D5) per cycle was 55.8% (95% CI 54.3–57.2) in ≤ 15% SDF group (embryo quality score was ICM A:12.1%, B:69.5%, C:8.8%, D:4.5%; TE A: 7.5%, B:42.2%, C:42.2%, D:8.1%) and 55.9% (95% CI 52.8–59.1) in the &gt;15% SDF group (ICM A:12.0%, B:68.7%, C:10.6%, D: 5.2%; TE A:9.1%, B:44.8%, C:37.8%, D:8.3%) (p &lt; 0.001). The good quality blastocyst rate per cycle was significantly higher in the group with SDF ≤15%, 27.7% (95%CI 26.5–29.0) versus SDF &gt;15% (27.4% (95%CI 24.6–30.2)). Of the total number of blastocysts, the proportion of A+B blastocyst was 60.5% (95% CI 58.3–62.7) and 64.2% (95% CI 59.2–69.2) (p &lt; 0.001), respectively. Limitations, reasons for caution The retrospective and multicenter nature of this study leads to uncontrolled biases derived from the clinical practice. Although the results were not adjusted for female’s age, it was not statistically different between groups. Embryo morphology evaluation was performed by senior embryologists, it still remains a subjective evaluation, though. Wider implications of the findings: In this study, a higher amount of data was compiled so that a large number of embryos were analyzed. The DNA integrity of the sperm may be an important consideration when poor quality embryos were obtained in the previous cycle when deciding on the next clinical strategy to apply. Trial registration number NA

238 CAFFEINE PREVENTS A LOSS OF CALCIUM OSCILLATORY RESPONSE ASSOCIATED WITH POSTOVULATORY AGING OF MOUSE OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 217
Author(s):  
T. Wakai ◽  
N. Zhang ◽  
R. A. Fissore
Keyword(s):  
Subsequent Development ◽  
Oocyte Quality ◽  
Developmental Competence ◽  
Oscillatory Activity ◽  
Strontium Chloride ◽  
Oocyte Activation ◽  
Mouse Oocytes ◽  
Oocyte Aging ◽  
Postovulatory Aging

Numerous studies have demonstrated that postovulatory aging of oocytes prior to fertilization has detrimental effects on oocyte quality and developmental competence. Oocyte aging is accompanied by abnormal oocyte activation and subsequent development, suggesting a disruption of Ca2+ oscillations after fertilization. The inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in mammals is responsible for the majority of Ca2+ release during fertilization (Miyazaki S et al. 1993 Dev. Biol.). Previously, we reported that phosphorylation of IP3R1 at an MPM-2 epitope may play an important role in facilitating the induction of Ca2+ oscillations at the MII stage (Lee B et al. 2006 Development), indicating that IP3R1 phosphorylation may be a good indicator of the health of the oocyte. However, few studies have investigated the alteration of the Ca2+ signaling and IP3R1 function associated with oocyte aging. On the other hand, a previous report showed that caffeine increased MPF activity and suppressed fragmentation after parthenogenetic activation of aged oocytes (Kikuchi K et al. 2000 Biol. Reprod.). Therefore, the purpose of the present study was to examine whether and how Ca2+ oscillatory activity changes during oocyte aging and to test if caffeine prevents the negative effects of oocyte aging. MII mouse oocytes were collected 14 h after hCG injection and cultured in vitro for 8, 24 or 48 h with or without caffeine (5 or 10 mm). Oocyte quality was assessed by the occurrence of spontaneous fragmentation, monitoring of Ca2+ oscillations after exposure to 10 mm strontium chloride, Western blot analysis of IP3R1 phosphorylation and immunostaining of IP3R1. In oocytes in vitro aged for 8 h, the duration of the first Ca2+ rise was significantly decreased compared with fresh MII oocytes, although this reduction was not observed in MII oocytes treated with 5 mm caffeine. The phosphorylation of IP3R1 at the MPM-2 epitope was slightly decreased during oocyte aging in both caffeine and noncaffeine treatment. Importantly, whereas IP3R1 in MII oocytes treated for 8 h with 5 mm caffeine displayed the typical cortical cluster organization, IP3R1 in aged oocytes without caffeine became dispersed in the cytoplasm. In addition, caffeine significantly suppressed the spontaneous fragmentation that is normally observed by 48 h of in vitro culture. These results suggest that the Ca2+ oscillatory activity is compromised during oocyte aging and caffeine prevents the loss of integrity of Ca2+ signaling possibly by keeping the cortical distribution of IP3R1.

2 SPECIFIC FATTY ACID FOLLOW-UP REVEALS RUMEN-PROTECTED FAT SUPPLEMENTATION EFFECTS ON BOVINE OOCYTE QUALITY AND EMBRYO DEVELOPMENT

2014 ◽  
Vol 26 (1) ◽  
pp. 115 ◽  
Author(s):  
A. F. González-Serrano ◽  
C. R. Ferreira ◽  
V. Pirro ◽  
J. Heinzmann ◽  
K.-G. Hadeler ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Follicular Fluid ◽  
Oocyte Quality ◽  
Lipid Profiles ◽  
Dependent Manner ◽  
Fat Supplementation

Information on how supplementation of high-yield dairy cows with rumen-protected fat affects fertility in cattle herds is scarce. Here, Holstein-Friesian heifers (n = 84) received a supplement consisting of either rumen-protected conjugated linoleic acid (CLA; cis-9,trans-11-CLA and trans-10,cis-12-CLA) or stearic acid 18 : 0 (SA) on top of an isocaloric grass silage diet. Two supplementation doses were used (100 and 200 g d–1). Blood and follicular fluid were collected at the start and end of the supplementation period for analysis of cholesterol, insulin-like growth factor (IGF), and nonesterified fatty acids (NEFA), and for fatty acid profiling. Although cholesterol, IGF, and NEFA levels did not differ among experimental groups, lipid profiles in blood and follicular fluid were affected in a dose-dependent manner by both supplements. After 45 days of supplementation, oocytes were collected by ovum pick-up (OPU). The mRNA relative abundance of target genes (IGF1r, GJA1, FASN, SREBP1, and SCAP) was analysed in single in vitro- (24 h IVM) and in vivo-matured (collected by OPU 20 h after GnRH injection) oocytes and in vitro-produced blastocysts (Day 8) by qPCR (n = 6/group). Lipid profiling of individual oocytes from the CLA-supplemented (n = 37) and the SA-supplemented (n = 50) was performed by desorption electrospray ionization mass spectrometry (DESI-MS). Oocytes from the CLA-supplemented (n = 413) and the SA-supplemented (n = 350) groups were used for assessing maturation and blastocysts development rates. In immature oocytes, CLA supplementation led to an increase of triacylglycerol 52 : 3 [TAG (52 : 3)] and TAG (52 : 2), squalene, palmitic acid 16 : 0, and oleic acid 18 : 1, and decreased abundance of TAG (56 : 3), TAG (50 : 2) and TAG (48 : 1). In vitro-matured oocytes showed different lipid profiles, with increased abundances of TAG (52 : 3), and TAG (52 : 2) as well as phosphatidylinositol 34 : 1 [Plo (34 : 1)], whereas phosphatidylglycerol (34 : 1) [PG (34 : 1)] and palmitic acid 16 : 0 were less abundant in in vitro-matured oocytes. SCAP was significantly down-regulated in in vitro-matured oocytes from supplemented heifers compared with their in vivo-matured counterparts. Maturation (CLA = 74% v. SA = 67%) and blastocyst rates (CLA = 22.4% v. SA = 12.7%) were different among experimental groups. One-way ANOVA and the Tukey-Kramer test were applied for a multiple comparison of means (P-value ≤ 0.05 was considered as statistically significant). In conclusion, we demonstrate here that fatty acid monitoring along different compartments (i.e. blood system, follicular fluid, and intra-oocyte) after rumen-protected fat supplementation of dairy heifer diet reveals nutritional footprints on oocyte quality and embryo development. These results demonstrate the close relationship between nutrition and cattle herd's fertility and, at the same time, support the role of the bovine model for understanding nutritional-dependent fertility impairments.

scholarly journals A Conserved PapB Family Member, TosR, Regulates Expression of the Uropathogenic Escherichia coli RTX Nonfimbrial Adhesin TosA while Conserved LuxR Family Members TosE and TosF Suppress Motility

2014 ◽  
Vol 82 (9) ◽  
pp. 3644-3656 ◽  
Author(s):  
Michael D. Engstrom ◽  
Christopher J. Alteri ◽  
Harry L. T. Mobley
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Virulence Factors ◽  
Upper Urinary Tract ◽  
Host Cells ◽  
Promoter Regions ◽  
Content Type ◽  
Tract Infections

ABSTRACTA heterogeneous subset of extraintestinal pathogenicEscherichia coli(ExPEC) strains, referred to as uropathogenicE. coli(UPEC), causes most uncomplicated urinary tract infections. However, no core set of virulence factors exists among UPEC strains. Instead, the focus of the analysis of urovirulence has shifted to studying broad classes of virulence factors and the interactions between them. For example, the RTX nonfimbrial adhesin TosA mediates adherence to host cells derived from the upper urinary tract. The associatedtosoperon is well expressedin vivobut poorly expressedin vitroand encodes TosCBD, a predicted type 1 secretion system. TosR and TosEF are PapB and LuxR family transcription factors, respectively; however, no role has been assigned to these potential regulators. Thus, the focus of this study was to determine how TosR and TosEF regulatetosAand affect the reciprocal expression of adhesins and flagella. Among a collection of sequenced UPEC strains, 32% (101/317) were found to encode TosA, and nearly all strains (91% [92/101]) simultaneously carried the putative regulatory genes. Deletion oftosRalleviatestosArepression. Thetospromoter was localized upstream oftosRusing transcriptional fusions of putative promoter regions withlacZ. TosR binds to this region, affecting a gel shift. A 100-bp fragment 220 to 319 bp upstream oftosRinhibits binding, suggesting localization of the TosR binding site. TosEF, on the other hand, downmodulate motility when overexpressed by preventing the expression offliC, encoding flagellin. Deletion oftosEFincreased motility. Thus, we present an additional example of the reciprocal control of adherence and motility.

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