Eleni Dovolou

Effects of guaiazulene on in vitro bovine embryo production and on mRNA transcripts related to embryo quality.

Reactive oxygen species (ROS) are between the major contributors for the reduced rate of in vitro bovine embryo production. It is believed that they can cause abnormal meiosis of oocytes, developmental arrest or cell death of embryos. Reports on the effectiveness of various antioxidants on embryo yield are rather conflicting mainly due to the nature and the concentration of the substances used. Here we report the effects of guaiazulene--an exogenous antioxidant, without known properties that could interfere with the biological process of IVF--on embryo development and on the quality of the produced blastocysts. Bovine cumulus oocyte complexes (COCs) were aspirated from abattoir ovaries and COCs were matured in TCM199 with FCS and EGF at 39 °C under an atmosphere of 5% CO(2) in air, with maximum humidity. After 24 h oocytes were inseminated with frozen/thawed semen and co-incubated for further 24 h. Zygotes were cultured in groups of 25 in 25 μl of SOF with 5% FCS at 39 °C under an atmosphere of 5% CO(2) , 5% O(2) in air with maximum humidity. In the first experiment the maturation medium was modified with addition of 0.1 mM of G (n = 497), or 0.01 mM of guaiazulene (n = 468), 0.05% DMSO--the guaiazulene diluent (Control(+), n = 467), and 459 oocytes were used as Control(-). In the second experiment, the culture medium was modified with the addition of 0.1 mM of guaiazulene (n = 344), 0.01 mM of guaiazulene (n = 345), 0.05% DMSO (Control(+), n = 347) and 355 were the Control(-). Blastocyst yield was recorded on days 6, 7, 8 and 9. Day 7 blastocysts from each experiment and group were snap frozen and stored for mRNA extraction. Quantification of transcripts for mRNA of genes related to metabolism (AKR1B1, PTGS2, GADPH, SLC2A5, G6PD); oxidation (GPX1); and implantation (PLAC8) was carried out by real time quantitative RT-PCR. Data for embryo development and on transcript abundance were analysed by χ(2) and anova respectively. In the first experiment no differences were found between groups in terms of cleavage rate (Control(-): 74.20%; Control(+): 74.58%; 0.01 mM: 71.61%; 0.1 mM: 71.63%) or day 9 blastocyst yield (Control(-): 28.26%; Control(+): 25.80%; 0.01 mM: 25.86%; 0.1 mM: 25.25%). In the second experiment, cleavage rate tended to be higher in 0.01 mM group than in Control(-) (77.87% vs 71.41% respectively, p = 0.07). No other differences were detected in cleavage rate (Control(+): 71.32%; 0.1mM: 72.75%) or in the overall blastocyst yield on day 9 (Control(-): 25.50%; Control(+): 26.71%; 0.01 mm: 29.58%; 0.1 mM: 25.75%). In both experiments the relative abundance of genes studied varied between groups but these differences were not statistically significant. Our results imply that oxidation has minimal effect on the in vitro embryo production. Guaiazulene, a compound possessing no biological properties other than those of a strong antioxidant, while it increased cleavage rate, it failed to improve either the blastocyst formation rate, or the quality of the produced embryos under 5% O(2) .

Daily supplementation with ghrelin improves in vitro bovine blastocysts formation rate and alters gene expression related to embryo quality

Ghrelin is a gastric peptide having regulatory role in the reproductive system functionality, acting mainly at central level. Because the expression of ghrelin system (ghrelin and its receptor) has been detected in the bovine ovary, the objectives of the present study were to investigate whether ghrelin can affect the developmental potential of in vitro-produced embryos, and to test their quality in terms of relative abundance of various genes related to metabolism, apoptosis and oxidation. In the first experiment, in vitro-produced zygotes were cultured in the absence (control [C]) and in the presence of three concentrations of acylated ghrelin (200 pg/mL [Ghr200], 800 pg/mL [Ghr800]; and 2000 pg/mL [Ghr2000]); blastocyst formation rates were examined on Days 7, 8, and 9. In the second experiment, only the 800 pg/mL dose of ghrelin was used. Zygotes were produced as in experiment 1 and 24 hours post insemination they were divided into 4 groups; in two groups (C; without ghrelin; Ghr800 with ghrelin), embryos were cultured without medium replacement; in the remaining two groups (Control N and GhrN), the culture medium was daily renewed. A pool of Day-7 blastocysts were snap frozen for relative mRNA abundance of various genes related to metabolism, oxidation, implantation, and apoptosis. In experiment 3, embryos were produced as in experiment 2, but in the absence of serum (semi-defined culture medium). In experiment 1, no differences were detected between C, Ghr200, and Ghr2000, although fewer blastocysts were produced in group Ghr800 compared with C. In experiment 2, the lowest blastocysts yield was found in Ghr800, whereas daily renewal of ghrelin (Ghr800N) resulted to increased blastocysts formation rate, which on Day 7 was the highest among groups (P < 0.05). In experiment 3, ghrelin significantly suppressed blastocysts yield. Significant differences were detected in various relative mRNA abundance, giving an overall final notion that embryos produced in the presence of ghrelin were of better quality than controls. Our results imply a specific role of ghrelin in early embryonic development; however, the specific mode of its action needs further investigation.

Combined administration of gonadotropin-releasing hormone, progesterone, and meloxicam is an effective treatment for the repeat-breeder cow

In practice, the etiologic treatment of the repeat-breeder cow is nearly infeasible. In this study, we tested the hypothesis that a combined treatment would benefit the conception rate of repeat-breeder cows. The components of this regimen target ovulation defects, late progesterone (P4) rise, and premature luteolysis. In a 5-year period, 402 repeat-breeder cows were divided in five groups, and treatment regimens consisted of the following: gonadotropin-releasing hormone (GnRH; Group 1, n=115, 0.012mg buserelin im 4 to 6h before artificial insemination); P4 (Group 2, n=51, 100mg P4 intravaginally, on Days 5 to 7); meloxicam (Group 3, n=31, 0.5mgkg(-1), 24h(-1) melomicronxicam sc, on Days 16 to 18); GnRH+P4+meloxicam (Group 4, n=98); and no treatment (Group 5, control, n=107). Artificial insemination was conducted only after overt estrus; thereafter, the duration of the estrous cycle was assessed in all cows that were detected to return to heat. The conception and pregnancy rate was compared among groups. The proportion of cows that returned to estrus after artificial insemination did not differ among groups; the duration of estrous cycle was the shortest in Group 1 and the longest in Group 4. In Group 4, pregnancy rate was higher (P<0.05) than that of Groups 1 and 5 (35.71% vs. 20.00% and 17.76% for Groups 4, 1, and 5, respectively), but though numerically higher, it did not differ statistically from that of Groups 2 (27.45%) and 3 (22.58%). Our results imply that a multifaceted protocol has to be applied for the successful treatment of the repeat-breeder cow.

EFFECTS OF MELATONIN TREATMENT ON FOLLICULAR DEVELOPMENT AND OOCYTE QUALITY IN CHIOS EWES - SHORT COMMUNICATION

Follicular development and oocyte quality were assessed by laparoscopic observation and in vitro fertilisation, respectively, in melatonin-treated (Group M) and control (Group C) anoestrous Chios ewes (n = 10 in each group). Fourteen days after melatonin insertion, all ewes had laparoscopic evaluation of the follicular population followed by oocyte pick-up (OPU); on day 22 intravaginal progestagen sponges were inserted for 14 days. Two days after sponge removal the follicular population was re-evaluated and a second follicular aspiration was performed. Collected oocytes from the second OPU underwent in vitro maturation, fertilisation and culture. The number of large follicles was higher in Group M than in the control ewes during the first OPU and tended to be so (P = 0.06) at the second. Morphologically, oocytes collected from controls were of better quality than those from Group M; however, more oocytes collected from melatonintreated animals fertilised and developed in vitro . These results indicate that melatonin is a potent regulator of follicular development and oocyte competence during the anoestrous period of the ewe.

Effects of Addition of Tissue-Type Plasminogen Activator in In Vitro Fertilization Medium on Bovine Embryo Development and Quality

Plasminogen activators/Plasmin system plays pivotal role in regulating reproductive functions of mammals. Here, we examined the effects of modification of in vitro fertilization medium (IVF medium) with the addition of tissue-type plasminogen activator (t-PA), on bovine embryo development and quality, assessed by quantification of expression of various genes related to metabolism, oxidation, implantation and apoptosis. In addition, plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were measured in the spent media. After conventional IVM, 2016 cumulus-oocyte complexes (COCs) were divided into four groups with modified composition of the IVF medium containing t-PA and/or its inhibitor epsilon-aminocaproic acid (control, t-PA, t-PA+ε-ACA, ε-ACA). Presumptive zygotes were cultured for 8 days in synthetic oviductal fluid (SOF) medium; gene expression studies were carried out on morulae and blastocysts. t-PA alone significantly suppressed cleavage and blastocyst formation rates, but this effect was neutralized by the addition of ε-ACA. PAA in the treated group was significantly reduced by ε-ACA, but without total elimination. Significant differences were detected in the expression of genes related to apoptosis and/or cell cycle arrest (BAX, BCL2L1, KAT2B) between embryos produced in t-PA-modified media and controls, giving an overall notion that the inferior developmental competence of treated embryos may be attributed to apoptotic phenomena induced by t-PA. In conclusion, it appears that excessive t-PA content in the IVF media, suppresses blastocyst formation rate, possibly due to induction of apoptotic phenomena.

Effects of ghrelin on activation of Akt1 and ERK1/2 pathways during in vitro maturation of bovine oocytes.

The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus-oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml-1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

The In Vitro Impact of the Herbicide Roundup on Human Sperm Motility and Sperm Mitochondria

Toxicants, such as herbicides, have been hypothesized to affect sperm parameters. The most common method of exposure to herbicides is through spraying or diet. The aim of the present study was to investigate the effect of direct exposure of sperm to 1 mg/L of the herbicide Roundup on sperm motility and mitochondrial integrity. Sperm samples from 66 healthy men who were seeking semen analysis were investigated after written informed consent was taken. Semen analysis was performed according to the World Health Organization guidelines (WHO, 2010). Mitochondrial integrity was assessed through mitochondrial staining using a mitochondria-specific dye, which is exclusively incorporated into functionally active mitochondria. A quantity of 1 mg/L of Roundup was found to exert a deleterious effect on sperm’s progressive motility, after 1 h of incubation (mean difference between treated and control samples = 11.2%) in comparison with the effect after three hours of incubation (mean difference = 6.33%, p < 0.05), while the relative incorporation of the mitochondrial dye in mitochondria of the mid-piece region of Roundup-treated spermatozoa was significantly reduced compared to relative controls at the first hour of incubation, indicating mitochondrial dysfunction by Roundup. Our results indicate that the direct exposure of semen samples to the active constituent of the herbicide Roundup at the relatively low concentration of 1 mg/L has adverse effects on sperm motility, and this may be related to the observed reduction in mitochondrial staining.

UROKINASE-TYPE PLASMINOGEN ACTIVATOR DOES NOT AFFECT IN VITRO BOVINE EMBRYO DEVELOPMENT AND QUALITY

The effects of modification of the in vitro embryo culture media (IVC) with the addition of urokinase-type plasminogen activator (u-PA) on the yield and/or quality of bovine embryos were examined in two experiments. In Experiment 1, denuded embryos were cultured in semi-defined synthetic oviductal fluid (SOF) for seven days, while in Experiment 2 embryos were co-cultured with cumulus cell monolayer in a serum-containing SOF medium. Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were determined in all spent IVC media. At the activity used (5 IU/ml), u-PA had no effect either on in vitro embryo production rates or on embryo quality as revealed by gene expression analysis of 10 important mRNA transcripts related to apoptosis, oxidation, implantation and metabolism. PAA and PAI analysis indicated the need for wellbalanced plasminogen activators and inhibitors as a culture environment for embryo development. However, more research is needed to unveil the mechanism by which u-PA is involved in in vitro embryo production systems.

Association between three glycosidases activity [α-mannosidase (α-ΜΑΝ), β-Ν-acetyloglucosaminidase (NAGASE) and β-galactosidase (β-GAL)] and in vitro fertilization of bovine oocytes collected from different-sized follicles

We studied the role of three glycosidases (α- mannosidase - α-ΜΑΝ, β-N-acetyloglucosaminidase - NAGASE and β-galactosidase - β-GAL) in follicular fluid (FF) and in fertilization medium (FM) of bovine oocytes. Oocytes were allocated into 3 groups according to the follicular size (controls - CF: 2-8 mm, small follicle group - SF: 2-5 mm, large follicle group - LF: >5-8 mm). Bovine embryos were produced in vitro either in groups (experiment 1, n = 2099 oocytes) or individually (experiment 2, n = 79 oocytes). In both experiments, the activity of all glycosidases in the FF of large follicles was significantly lower than in the FF of small follicles group. In the FM of LF-group oocytes, α- MAN and NAGASE were significantly higher compared to SF- and CF-group oocytes (experiment 1) and β-GAL was significantly higher in SF- compared to CF-group oocytes (experiment 2). Cleavage rate was similar among all groups in both experiments; however significantly higher blastocyst formation was noted in CF-group compared to LF- (days 7, 8, 9) and SF- (days 8, 9) groups (experiment 1). In follicular fluid of small follicle group, β-GAL was associated positively with degenerating oocytes’ number and negatively with blastocyst rate at days 7, 8 (P = 0.065) and 9 (experiment 1). In fertilization medium of control group, α-MAN related negatively to cleavage rate (P < 0.05) and β-GAL to blastocyst rate at day 8 (P = 0.089) or day 9 (P = 0.072) (experiment 1). During fertilization, in experiment 1 all oocytes consumed β-GAL and only control or small follicle oocytes consumed α-MAN; in experiment 1, only large follicle oocytes released NAGASE, whereas all oocytes released all three glycosidases in experiment 2. In conclusion, glycosidases affect the developmental competence of oocytes collected from different sized follicles during in vitro fertilization, performed either in groups or individually; their role in follicular fluid is different from that in fertilization medium.