Antioxidative Potential of Red Deer Embryos Depends on Reproductive Stage of Hind as a Oocyte Donor

The aim was to compare the blastocyst stages of red deer embryos in respect of in vitro fertilization (IVF) efficiency, morphology, apoptotic and proliferative abilities, and antioxidative potential according to the reproductive status of hinds. We used three experimental groups, including the ovaries collected post mortem on the 4th and 13th days of the estrous cycle and during pregnancy (n = 18). After oocyte maturation, frozen-thawed epididymal semen was used for IVF. Blastocyst quality, apoptotic potential by determining the mRNA expression of BAX, BCL-2, OCT4, SOX2, and placenta-specific 8 gene (PLAC8), and antioxidative potential of blastocysts were evaluated by determining the mRNA expression of CuSOD, MnSOD, and GPX as well as the enzymatic activity of superoxide dismutase and reduced glutathione. The highest development rate of expanded blastocyst, mRNA expression of BCL-2, OCT4, SOX2, and PLAC8 and mRNA expression and enzymatic activity of the antioxidative factors increased (p < 0.05) in blastocysts developed from the oocytes collected on the 4th day, compared to those developed from the oocytes collected on the 13th day of the cycle and during pregnancy. Our study indicates that the 4th day of the estrous cycle is the most effective period for oocyte collection for IVF and embryo development in hinds, considering quality parameters and antioxidative potential of the blastocysts.

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Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

10.21203/rs.3.rs-330029/v1 ◽

2021 ◽

Author(s):

Jozsef Bodis ◽

Endre Sulyok ◽

Akos Varnagy ◽

Viktória Prémusz ◽

Krisztina Godony ◽

...

Keyword(s):

Gene Expression ◽

In Vitro Fertilization ◽

Mrna Expression ◽

Granulosa Cells ◽

Follicular Fluid ◽

Vitro Fertilization ◽

Expression Levels ◽

The Impact ◽

Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

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Prospective randomized trial of the effect of two flushing media on oocyte collection and fertilization rates after in vitro fertilization

Fertility and Sterility ◽

10.1016/s0015-0282(97)00391-9 ◽

1997 ◽

Vol 68 (6) ◽

pp. 1132-1134 ◽

Cited By ~ 5

Author(s):

Marinko M Biljan ◽

Nicola Dean ◽

Robert Hemmings ◽

Francois Bissonnette ◽

Seang Lin Tan

Keyword(s):

In Vitro Fertilization ◽

Randomized Trial ◽

Prospective Randomized Trial ◽

Vitro Fertilization ◽

Fertilization Rates ◽

Oocyte Collection

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Does prostacyclin (PGI2) support porcine corpus luteum function? – data from in vitro study

Problems of Endocrinology ◽

10.14341/probl201662549 ◽

2016 ◽

Vol 62 (5) ◽

pp. 49

Author(s):

Magdalena Julia Szymańska ◽

Agnieszka Blitek

Keyword(s):

Endothelial Cells ◽

Mrna Expression ◽

Corpus Luteum ◽

Estrous Cycle ◽

In Vitro Study ◽

Luteal Cells ◽

Enzymatic Isolation ◽

Effective Doses ◽

And Function

Background. Prostacyclin (PGI2) of luteal origin is involved in the control of corpus luteum (CL) development and function in cattle. PGI2 may regulate the process of angiogenesis and may stimulate progesterone (P4) secretion by luteal cells via its specific receptors, PTGIR. In contrast to cattle, the role of PGI2 in the pig CL has not yet been described.Aim. The present study aimed to investigate the effect of PGI2 on 1) P4 secretion by luteal cells, and 2) the expression of angiogenesis-related genes in endothelial cells of the porcine CL.Methods. CL collected from gilts on day 5-7 of the estrous cycle were used for enzymatic isolation of luteal (Experiment 1) and endothelial (Experiment 2) cells. In Exp. 1, cultured luteal cells were incubated with increasing (0, 0.01, 0.1, 1, 5 µM) doses of PGI2 analogues: iloprost (ILO) and carbaprostacyclin (cPGI2) for 8 h. To determine the effective doses of PGI2 analogues, P4 concentration in culture medium was examined by RIA. Thereafter, luteal cells were treated with ILO and cPGI2 at the concentration of 1 and 5 µM in the presence or absence of PTGIR antagonist (CAY10441). After 8 h of incubation the medium was collected for P4 determination. In Exp. 2, isolated endothelial cells were treated for 24 h with ILO and cPGI2 at doses of 1 and 5 µM. Then, cells were collected for analysis of Ang-1 and -2 mRNA expression using qPCR.Results. Both, ILO and cPGI2 affected P4 secretion by luteal cells. Elevated levels of P4 were observed in medium after treatment of luteal cells with 1 µM of ILO and 0.1, 1 and 5 µM of cPGI2 compared with control values (p<0.05). The addition of CAY10441 inhibited the stimulatory effect of ILO on P4 secretion, while did not change P4 production by luteal cells incubated with cPGI2. Moreover, PGI2 analogues differentially affected (p<0.05) the expression of proangiogenic factors. ILO stimulated Ang-2, whereas cPGI2 positively affected Ang-1 mRNA expression in endothelial cells at concentrations of 1 µM and 5 µM, respectively.Conclusion. PGI2 affects P4 secretion during luteal phase of the estrous cycle and may regulate the process of angiogenesis in the porcine CL.

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Manganese Provides Antioxidant Protection for Sperm Cryopreservation that May Offer New Consideration for Clinical Fertility

Oxidative Medicine and Cellular Longevity ◽

10.4161/oxim.2.3.8804 ◽

2009 ◽

Vol 2 (3) ◽

pp. 152-159 ◽

Cited By ~ 24

Author(s):

Ranjna S. Cheema ◽

Amrit K. Bansal ◽

Gurmail Singh Bilaspuri

Keyword(s):

Protective Effect ◽

Semen Quality ◽

Maximum Level ◽

Egg Yolk ◽

Quality Parameters ◽

Vitro Fertilization ◽

Bull Semen ◽

Protein Leakage ◽

A Chain

Reactive oxygen species (ROS) are generated by sperm metabolism. While, ROS are required for maturation, capacitation and acrosome reaction, they also modify many peroxidable cellular compounds. There is production of ROS during cryopreservation and frozen spermatozoa are highly sensitive to lipid peroxidation (LPO). Antioxidants exert a protective effect on the plasma membrane of frozen bovine sperm preserving both metabolic activity and cellular viability. Manganese (Mn++) is proved to be a chain breaking antioxidant in biological system. Therefore, we examined the role of (Mn++) during cryopreservation of cattle bull semen. Semen was divided into four parts and cryopreserved in egg-yolk-citrate extender + glycerol (EYC-G), EYC-G + 100 µM of Mn++, EYC-G + 150 µM of Mn++and EYC-G + 200 µM of Mn++. After four hours of cooling and 24 hrs of freezing, the spermatozoa were examined for percentage motility, Hypo-osmotic swelling (HOS), LPO and protein leakage. Addition of manganese to the semen during cryopreservation showed a protective effect and accounted for an increase in semen quality parameters [percentage motility, HOS percent and decrease in malondialdehyde (MDA) production and protein leakage]. The effect of manganese on motility and HOS was non-significant (p < 0.05) in cooled spermatozoa but significant with 150 µM of Mn++in frozen-thawed spermatozoa. MDA production and protein leakage decreased to a significant and maximum level (p < 0.05) on addition of 200 µM of manganese. The addition of manganese to EYC-G dilutor will improve the quality/fertility of semen, which will result in improvement of in vitro fertilization and artificial insemination success rate.

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Beneficial Effects of Melatonin in the Ovarian Transport Medium on In Vitro Embryo Production of Iberian Red Deer (Cervus elaphus hispanicus)

Animals ◽

10.3390/ani10050763 ◽

2020 ◽

Vol 10 (5) ◽

pp. 763 ◽

Cited By ~ 2

Author(s):

Irene Sánchez-Ajofrín ◽

María Iniesta-Cuerda ◽

Patricia Peris-Frau ◽

Alicia Martín-Maestro ◽

Daniela-Alejandra Medina-Chávez ◽

...

Keyword(s):

Red Deer ◽

Oocyte Quality ◽

Scale Production ◽

Post Mortem ◽

Embryo Production ◽

Transport Medium ◽

Iberian Red Deer ◽

Blastocyst Quality ◽

In Vitro Embryo Production

A major limiting factor for the development of in vitro embryo production (IVP) in wild species, such as Iberian red deer, compared to livestock animals is the poor availability and limited access to biological material. Thus, the use of post-mortem ovaries from slaughtered animals represent a source of oocytes for the large scale production of embryos needed for research and to improve the efficiency of IVP. However, these oocytes are not as developmentally competent as their in vivo counterparts. Moreover, oocytes are usually obtained from ovaries that have been transported for long distances, which may also affect their quality. In order to overcome the issues associated with prolonged storage times of post-mortem material, in this study we examined the effect of melatonin supplementation to the ovary transport medium on oocyte quality, embryo yield, and blastocyst quality in Iberian red deer. When necessary, sheep was used as an experimental model due to the large number of samples required for analysis of oocyte quality parameters. Oocytes were in vitro matured and assessed for early apoptosis; DNA fragmentation; reactive oxygen species (ROS); reduced glutathione (GSH) content, mitochondrial membrane potential, and distribution; and relative abundance of mRNA transcript levels. After in vitro fertilization, embryo rates and blastocyst quality were also investigated. The results revealed that melatonin treatment significantly increased intracellular level of GSH in sheep oocytes. Moreover, the percentage of cleavage and blastocyst yield in red deer was greater compared to the Control group and there was lower abundance of oxidative stress- and apoptosis-related SHC1, TP53, and AKR1B1 mRNA transcripts in blastocysts for the Melatonin group. In conclusion, the supplementation of melatonin to the ovary storage medium had a positive effect on the developmental competence and quality of resulting blastocysts in Iberian red deer.

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Effect of nucleocytoplasmic ratio on the in vitro porcine embryo development after in vitro fertilization or parthenogenetic activation

Zygote ◽

10.1017/s0967199421000381 ◽

2021 ◽

pp. 1-7

Author(s):

Nguyen Thi Men ◽

Thanh Quang Dang-Nguyen ◽

Tamas Somfai ◽

Hiep Thi Nguyen ◽

Junko Noguchi ◽

...

Keyword(s):

Formation Rate ◽

Cell Number ◽

Control Group ◽

Total Cell Number ◽

Vitro Fertilization ◽

Blastocyst Quality ◽

Porcine Embryo ◽

Oocyte Cytoplasm

Summary This study was conducted to examine whether the nuclear to cytoplasmic (N/C) ratio had any influence on the timing of embryo compaction and blastocoel formation, as well as formation rate and quality of blastocyst. First, we produced embryos with increased N/C ratio by removal of approximately one-third of the cytoplasm and with decreased N/C ratio by doubling the oocyte cytoplasm with an enucleated oocyte. The initiation of compaction and cavitation in reduced cytoplasm group was significantly earlier (P < 0.05) compared with the control and doubled cytoplasm groups. The rate of blastocysts in the reduced cytoplasm and doubled cytoplasm groups was significantly lower (P < 0.05) compared with the control group. Blastocyst quality in terms of total cell number in the reduced cytoplasm group was significantly lower (P < 0.05) compared with the doubled cytoplasm group, but not different from the control group. Next, we produced embryos with various N/C ratios by oocyte fusion combined with cytochalasin D treatment. The onset of compaction and cavitation in the 2N/2C group (decreased N/C ratio) was significantly delayed (P < 0.05) or had the tendency to be delayed (P = 0.064), respectively, compared with the control group (2N/1C). A significantly higher rate of blastocyst was observed in the 4N/2C group compared with the 1N/1C group (P < 0.05) but not different from the remaining groups. These results demonstrated that an increase in N/C ratio caused an earlier occurrence of morula compaction and blastocyst formation in both in vitro fertilization (IVF) and parthenogenetically activated pig embryos.

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P–233 The spatial arrangement of blastomeres and time of cavitation forming as predictors of blastocyst quality

Human Reproduction ◽

10.1093/humrep/deab130.232 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

N Sayme ◽

T Krebs ◽

M Kasoha ◽

D H A Maas ◽

E F Solomayer ◽

...

Keyword(s):

Pregnancy Rate ◽

Statistical Significance ◽

Spatial Arrangement ◽

Time Frame ◽

Classification Model ◽

Time Duration ◽

Vitro Fertilization ◽

Morphokinetic Parameters ◽

Blastocyst Quality

Abstract Study question Does the spatial arrangement of blastomeres and the start of blastulation affect blastocyst quality? Summary answer Better blastocyst quality is associated with the spatial arrangement of the embryo and the shorter time frame of blastulation (cavitation). What is known already The ability to select the human embryo with the highest implantation potential remains one of the greatest challenges in the management of In Vitro Fertilization patients. Several publications have proposed that additional morphological evaluations of blastomere arrangement and the dynamics of late-stage embryonic divisions might be a useful non-invasive way for embryo selection. In the last decade, the introduction of time-lapse technology enables continuous monitoring of embryo development, which leads to better outcomes than a selection based on the traditional morphology assessment. Study design, size, duration The spatial arrangement was defined as tetrahedrally if the cleavage planes were perpendicularly orientated, while embryos with rather parallelly orientated cleavage axes were considered as non-tetrahedral embryos. The injection time of ICSI was designated as “time zero” (t0), and EmbryoViewer software was used to calculate the time duration between injection and start of blastulation (cavitation). Obtained results were later correlated with the embryo’s capability to form a blastocyst as well as with blastocyst quality. Participants/materials, setting, methods A total of 195 oocytes from 40 patients undergoing the antagonist cycle for ICSI treatment were evaluated. All blastocysts were cultured in Embryoscope™ according to the manufacturer’s specifications (Vitrolife, Sweden). The Gardner and Schoolcraft scoring system was used to describe blastocyst quality. Statistical analyses were performed using IBM SPSS version 24. Data were reported as median and range. Differences between groups were tested using the Mann-Whitney U test. Statistical significance was defined as p < 0.05. Main results and the role of chance Obtained data showed that 83.6% (61/73) of embryos with tetrahedral arrangement formed blastocysts compared to 42.4% (50/116) of embryos with the non-tetrahedral arrangement (p < 0,001). Moreover, tetrahedral embryos more frequently formed good quality blastocyst compare to the non-tetrahedral [59% (36/61) vs 18 (9/50)% respectively; p < 0,001]. In addition, we found that good quality blastocyst had a significantly shorter time frame between injection and blastulation start, compared with blastocysts which did not reach good quality [95.00h (84–118) vs 102h (77–121) respectively; p = 0,006]. Limitations, reasons for caution The limitation of the present study was that due to the double-embryo transfer correlation between those morphokinetic parameters and pregnancy rate can not be calculated. Further research should link these morphokinetic parameters with pregnancy rate and live birth rate as well. Wider implications of the findings: The potential of our findings is considerable, especially for countries with strict Embryo Law Regulation. Obtained results might be highly useful for selecting embryos with high implantation potential. In addition, the present work illustrates the possibility of additional information that can potentially be incorporated into an embryo classification model. Trial registration number Not applicable

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21-Hydroxylase-Derived Steroids in Follicles of Nonobese Women Undergoing Ovarian Stimulation for In Vitro Fertilization (IVF) Positively Correlate With Lipid Content of Luteinized Granulosa Cells (LGCs) as a Source of Cholesterol for Steroid Synthesis

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2013-3204 ◽

2014 ◽

Vol 99 (4) ◽

pp. 1299-1306 ◽

Cited By ~ 12

Author(s):

Marli Amin ◽

Ariel Simerman ◽

Michele Cho ◽

Prapti Singh ◽

Christine Briton-Jones ◽

...

Keyword(s):

In Vitro Fertilization ◽

Mrna Expression ◽

Granulosa Cells ◽

Lipid Content ◽

Mineralocorticoid Receptor ◽

Steroid Synthesis ◽

Vitro Fertilization ◽

Receptor Mrna

Context: Mineralocorticoid synthesis by the nonhuman primate periovulatory follicle enhances luteinization. Whether a similar event occurs in women undergoing in vitro fertilization (IVF) is unknown. Objective: The objective of the study was to determine whether human luteinized granulosa cells (LGCs) produce mineralocorticoids derived from 21-hydroxylase activity and also express mRNA for 21-hydroxylase and the mineralocorticoid receptor. Design: This was a prospective cohort study. Setting: The study was conducted at an academic center. Patients: LGC lipid content and follicle fluid (FF) hormone analysis was performed on 27 nonobese IVF women. LGCs from six additional nonobese IVF women were used for gene expression studies. Intervention: At oocyte retrieval, FF was aspirated from the first follicle (≥16 mm in size) of each ovary and pooled LGCs were collected. Main Outcome Measures: FF steroid analysis was performed by liquid chromatography-tandem mass spectrometry. LGCs were stained with lipid fluorescent dye BODIPY FL C16 to estimate lipid content by confocal microscopy as a cholesterol source for steroidogenesis in vivo. Quantitative real-time PCR was performed using LGCs to detect 21-hydroxylase and mineralocorticoid receptor mRNA expression. Pearson correlation coefficients determined associations between FF steroid levels and LGC lipid content. Results: FF levels of the 21-hydroxylase-derived steroids, 11-deoxycorticosterone [DOC, 39.97, median (13.94–63.02) ng/mL] and 11-deoxycortisol [11DOC, 2.07 (0.69–5.01) ng/mL], along with the 21-hydroxylase precursor 17-hydroxyprogesterone [1268.21 (493.26–3558.39) ng/mL], positively correlated with LGC lipid content (84 ± 43 fluorescent units/sample) (P ≤ .05, all steroids). 21-Hydroxylase and mineralocorticoid receptor mRNA expression was detected in LGCs. Conclusions: Human LGCs likely synthesize 21-hydroxylase-derived mineralocorticoids from cholesterol-containing lipid in vivo to promote postovulatory luteinization via mineralocorticoid receptor-mediated events.

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Leukotrienes modulate secretion of progesterone and prostaglandins during the estrous cycle and early pregnancy in cattle: an in vivo study

Reproduction ◽

10.1530/rep-10-0202 ◽

2010 ◽

Vol 140 (5) ◽

pp. 767-776 ◽

Cited By ~ 13

Author(s):

Anna J Korzekwa ◽

Mamadou M Bah ◽

Andrzej Kurzynowski ◽

Karolina Lukasik ◽

Agnieszka Groblewska ◽

...

Keyword(s):

Mrna Expression ◽

Estrous Cycle ◽

Early Pregnancy ◽

Luteal Phase ◽

Reproductive Tract ◽

Cell Function ◽

Ovarian Cell

Recently, we showed that leukotrienes (LTs) regulate ovarian cell functionin vitro. The aim of this study was to examine the role of LTs in corpus luteum (CL) function during both the estrous cycle and early pregnancyin vivo. mRNA expression of LT receptors (BLTfor LTB4andCYSLTfor LTC4), and 5-lipoxygenase (5-LO) in CL tissue and their localization in the ovary were studied during the estrous cycle and early pregnancy. Moreover, concentrations of LTs (LTB4and C4) in the CL tissue and blood were measured.5-LOandBLTmRNA expression increased on days 16–18 of the cycle, whereasCYSLTmRNA expression increased on days 16–18 of the pregnancy. The level of LTB4was evaluated during pregnancy compared with the level of LTC4, which increased during CL regression. LT antagonists influenced the duration of the estrous cycle: the LTC4antagonist (azelastine) prolonged the luteal phase, whereas the LTB4antagonist (dapsone) caused earlier luteolysisin vivo. Dapsone decreased progesterone (P4) secretion and azelastine increased P4secretion during the estrous cycle. In summary, LT action in the bovine reproductive tract is dependent on LT type: LTB4is luteotropic during the estrous cycle and supports early pregnancy, whereas LTC4is luteolytic, regarded as undesirable in early pregnancy. LTs are produced/secreted in the CL tissue, influence prostaglandin function, and serve as important factors during the estrous cycle and early pregnancy in cattle.

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