scholarly journals Difference in Developmental Kinetics of Y-Specific Monoclonal Antibody Sorted Male and Female In Vitro Produced Bovine Embryos

2019 ◽  
Vol 21 (1) ◽  
pp. 244 ◽  
Author(s):  
Tabinda Sidrat ◽  
Rami Kong ◽  
Abdul Khan ◽  
Muhammad Idrees ◽  
Lianguang Xu ◽  
...  
Keyword(s):  
Oxygen Species ◽  
Bovine Embryos ◽  
Male And Female ◽  
Endogenous Factors ◽  
Epigenetic Events ◽  
Mitochondrial Distribution ◽  
Growth Differences ◽  
Kinetics Of ◽  
Implantation Period

Sex-related growth differences between male and female embryos remain an attractive subject for reproductive biologists. This study aimed to investigate the endogenous factors that play a crucial role in the pace of early development between male and female bovine embryos. Using sex pre-selected semen by Y-specific monoclonal antibodies for the production of bovine embryos, we characterized the critical endogenous factors that are responsible for creating the development differences, especially during the pre-implantation period between male and female embryos. Our results showed that at day seven, (57.8%) Y-sperm sorted in vitro cultured embryos reached the expanded blastocyst (BL) stage, whereas the X-sperm sorted group were only 25%. Y-BLs showed higher mRNA abundance of pluripotency and developmental competency regulators, such as Oct4 and IGF1-R. Interestingly, Y-sperm sorted BLs had a homogeneous mitochondrial distribution pattern, higher mitochondrial membrane potential (∆Ѱm), efficient OXPHOS (oxidative phosphorylation) system and well-encountered production of ROS (reactive oxygen species) level. Moreover, Y-blastocysts (BLs) showed less utilization of glucose metabolism relative to the X-BLs group. Importantly, both sexes showed differences in the timing of epigenetic events. All these factors directly or indirectly orchestrate the whole embryonic progression and may help in the faster and better quality yield of BL in the Y-sperm sorted group compared to the X counterpart group.

Developmental kinetics and gene expression in male and female bovine embryos produced in vitro with sex-sorted spermatozoa

2010 ◽  
Vol 22 (2) ◽  
pp. 426 ◽  
Author(s):  
Pablo Bermejo-Álvarez ◽  
Patrick Lonergan ◽  
Detlef Rath ◽  
Alfonso Gutiérrez-Adan ◽  
Dimitrios Rizos
Keyword(s):  
Gene Expression ◽  
Mrna Abundance ◽  
Bovine Embryos ◽  
Same Sex ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Female Embryo ◽  
Male And Female ◽  
Kinetics Of

Using bovine embryos generated in vitro from IVF with X-sorted, Y-sorted and unsorted spermatozoa, we compared the kinetics of male and female embryo development and gene expression between male and female blastocysts. Bovine in vitro-matured oocytes (n = 8858) were fertilised with spermatozoa from each of three different bulls (X-sorted, Y-sorted or unsorted spermatozoa depending on the experiment). The cleavage rate was assessed 24, 27, 30, 33, 36, 40, 44 and 48 h post insemination (h.p.i.) and blastocyst development was recorded on Days 6–9. The relative mRNA abundance of nine genes (GSTM3, DNTM3A, PGRMC1, TP53, BAX, COX2, IGF2R, AKR1B1 and PLAC8) was analysed in male and female Day 7 blastocysts produced with sorted and unsorted spermatozoa from one bull. Cumulative cleavage rate and blastocyst yield were significantly higher in the unsorted group compared with the X- or Y-sorted group from the same bull (P ≤ 0.05). Although differences existed between bulls in terms of cleavage rate, no differences were observed in cleavage rate between X- and Y-sorted spermatozoa within a bull. The blastocyst yield was significantly higher only for Bull 3 when the Y-sorted spermatozoa were used (27.1+2.8 v. 19.1+1.4 for Y- and X-sorted spermatozoa, respectively; P < 0.05). There were no differences in the mRNA abundance of the nine genes analysed between embryos of the same sex produced with sorted or unsorted spermatozoa. However, significant differences in polyA mRNA abundance were observed between male and female blastocysts for three genes (GSTM3, DNMT3A and PGRMC1; P ≤ 0.05). In conclusion, the use of sorted rather than unsorted spermatozoa in IVF significantly delays the onset of first cleavage. Differences were noted between bulls, but not between X- and Y-sorted spermatozoa, and although no differences were found in terms of the mRNA abundance of the nine genes tested between sorted and unsorted spermatozoa, sex-related differences were found in the case of three genes.

168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS

2012 ◽  
Vol 24 (1) ◽  
pp. 196
Author(s):  
A. R. Buzzo ◽  
A. R. Pupulim ◽  
J. Mazucheli ◽  
F. V. Meirelles ◽  
I. P. Emanuelli
Keyword(s):  
In Vitro Culture ◽  
Culture Medium ◽  
Culture Media ◽  
Proteinase K ◽  
Bovine Embryos ◽  
Male And Female ◽  
Stage Of Development ◽  
Males And Females ◽  
Kinetics Of

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO2) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O2, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.

scholarly journals Post-hatching development of bovine embryos in vitro: the effects of tunnel preparation and gender

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 123-134 ◽  
Author(s):  
Grazieli Marinheiro Machado ◽  
Ester Siqueira Caixeta ◽  
Carolina Madeira Lucci ◽  
Rodolfo Rumpf ◽  
Maurício Machaim Franco ◽  
...  
Keyword(s):  
Gene Expression ◽  
Morphological Characteristics ◽  
Tunnel Construction ◽  
Agarose Gel ◽  
Male And Female ◽  
Embryo Size ◽  
And Gender ◽  
Phosphate Buffered Saline ◽  
Kinetics Of

SummaryThe objective of this study was to compare morphological characteristics, kinetics of development, and gene expression of male and female IVP embryos that were cultured until day (D)15 (fertilization = D0), using either phosphate-buffered saline (PBS) or Milli-Q water (MQW) to dilute the agarose gel used for tunnel construction. On D11, embryos (n = 286) were placed in agarose gel tunnels diluted in PBS and MQW. Embryos were evaluated for morphology, and embryo size was recorded on D11, D12.5, D14 and D15. Then, embryos were sexed and used for gene expression analyses (G6PD, GLUT1, GLUT3, PGK1, PLAC8, KRT8, HSF1 and IFNT). The percentage of elongated embryos at D15 was higher (p < 0.05) in the PBS (54%) than in the MQW (42%) gel. However, embryos produced in MQW were bigger (p < 0.05) and had a lower expression of GLUT1 (p = 0.08) than those cultured in PBS. There was a higher proportion of male than female embryos at D15 in both treatments, MQW (65% vs. 35%; p < 0.05) and PBS (67% vs. 33%; p < 0.05); however, embryo size was not significantly different between genders. Moreover, D15 female embryos had greater expression of G6PD (p = 0.05) and KRT8 (p = 0.03) than male embryos. In conclusion, the diluent used for tunnel construction affected embryo development in the post-hatching development (PHD) system, and the use of MQW was the most indicative measure for the evaluation of embryo quality. Male and female embryos cultured from D11 to D15, either in an MQW or PBS agarose gel, demonstrated similar development but different gene expression.

129 EFFECT OF SERUM SUPPLEMENTATION ON AMP-ACTIVATED PROTEIN KINASE (AMPK) ACTIVITY AND LIPID METABOLISM OF IN VITRO-CULTURED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 156
Author(s):  
S. Prastowo ◽  
F. Rings ◽  
D. S. Wondim ◽  
E. Tholen ◽  
C. Looft ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mitochondrial Biogenesis ◽  
Reactive Oxygen ◽  
Rna Isolation ◽  
Mitochondrial Activity ◽  
Oxygen Species ◽  
Bovine Embryos ◽  
Fluorescent Intensity ◽  
Ampk Activity

A major problem of embryos cultured in vitro with serum is cytoplasmic lipid accumulation resulting in lower cryotolerance compared with those derived from in vivo or in the absence of serum. AMPK is known as a master regulator of lipid, glucose, and protein metabolism in mammalian cells. Moreover, it has been reported as controller of acetyl-CoA carboxylase α (ACC), the gene responsible for lipid synthesis, and associated with mitochondrial biogenesis and activities in response to oxidative stress. In the present study we aimed to investigate the regulation of AMPK during serum supplementation in vitro. For this, bovine embryos were produced in vitro in SOF media supplemented with oestrous cow serum or fatty acid–free BSA as a system without serum. Triplicate pools (each 10 blastocysts) from each group were used for RNA isolation using Arcturus®PicoPure®RNA Isolation Kit (Life Technologies, USA). Reverse transcription was performed using a combination of Oligo(dT)23 and random primers. Quantification of AMPK catalytic α1 (AMPKA1), ACC, peroxisome proliferator-activated receptor gamma coactivator 1 α (PGC1A), and sterol regulatory element binding transcription factor 2 (SREBP2) transcripts were performed using ABI PRISM® 7000 SDS system (Applied Biosystems, Foster City, CA, USA) using GAPDH as internal control. Normalized log-transformed transcript amount data were statistically analysed using t-test. In addition, AMPK protein was detected by immunofluorescence, mitochondrial activity by MitoTracker® Red (Invitrogen, Carlsbad, CA, USA), and reactive oxygen species by H2DCFDA molecular probe (Life Technologies, USA), and fluorescent intensity signals were visualised under confocal laser scanning microscopy LSM 710 (Carl Zeiss, Germany). Results showed that the expression of AMPKA1, PGC1A, a mitochondrial biogenesis protein, and SREBP2, a regulator of lipid oxidation, were found to be lower (0.4-, 0.2-, and 0.7-fold, respectively; P < 0.05) in blastocysts derived from cultured with serum compared to without serum. By contrast, ACC was up-regulated in blastocysts cultured with serum by 1.8-fold (P < 0.05) compared to without serum. In comparison to blastocyst cultured without serum, a reduced fluorescent intensity was observed in AMPKA1 protein and mitochondrial activity in blastocyst cultured with serum. The presence of serum was also found to be involved in increasing reactive oxygen species accumulation in embryos cultured with serum. The reduced level of AMPK leads to increased ACC and subsequently enhanced conversion of fatty acids into lipid, which is associated with reduced mitochondrial biogenesis protein, elevated reactive oxygen species level, and reduced lipid oxidation by suppression of SREBP2. In conclusion, the presence of serum in in vitro culture environment affected the AMPK activity and thereby genes associated with lipid metabolism in early bovine embryos.

Effects of histone hyperacetylation on the preimplantation development of male and female bovine embryos

2010 ◽  
Vol 22 (6) ◽  
pp. 1041 ◽  
Author(s):  
Clara S. Oliveira ◽  
Naiara Z. Saraiva ◽  
Marcela M. de Souza ◽  
Tatiane A. D. Tetzner ◽  
Marina R. de Lima ◽  
...  
Keyword(s):  
Histone Deacetylases ◽  
Trichostatin A ◽  
Preimplantation Development ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Male And Female ◽  
Beneficial Effects ◽  
Histone Hyperacetylation ◽  
Vitro Production

Trichostatin A (TSA) induces histone hyperacetylation by inhibiting histone deacetylases and consequently increasing gene expression. The hypothesis was that TSA supplementation during the in vitro culture (IVC) of bovine embryos would increase the blastocyst rate, particularly in low-quality and female embryos. Oocytes were fertilised separately with X and Y spermatozoa and, 70 h after IVF, the IVC medium was supplemented with 5 nM and 15 nM TSA for 48 or 144 h. Incubation of female embryos with 5 nM and 15 nM TSA resulted in similar increases in acetylated histone H3K9 levels. However, to see comparable effects on acetylated histone H3K9 levels in male embryos, the culture medium needed to be supplemented with 15 nM TSA (as opposed to 5 nM TSA for female embryos). Treatment of male and female embryos with 5 nM TSA for 48 h or female embryos with 5 nM for 144 h had no effect on blastocyst rates, although 15 nM TSA compromised embryonic development. The terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) assay revealed increased apoptosis in female embryos treated with 5 nM TSA for 144 h, as well as in male and female embryos treated with 15 nM TSA for 48 h, but this increase in apoptosis was not observed in low-quality embryos. The results of the present study suggest that TSA treatment promotes histone hyperacetylation, but has no beneficial effects on the in vitro production of male and female bovine embryos during preimplantation development.

The influence of sperm concentration, length of the gamete co-culture and the evolution of different sperm parameters on the in vitro fertilization of prepubertal goat oocytes

Zygote ◽  
2010 ◽  
Vol 18 (4) ◽  
pp. 345-355 ◽  
Author(s):  
M.J. Palomo ◽  
T. Mogas ◽  
D. Izquierdo ◽  
M.T. Paramio
Keyword(s):  
Acrosome Reaction ◽  
Penetration Rate ◽  
Sperm Concentration ◽  
Semen Parameters ◽  
Oxygen Species ◽  
Vitro Fertilization ◽  
Rate Of Penetration ◽  
Sperm Penetration ◽  
Kinetics Of

SummaryThe aims of the present study were: (1) to evaluate the influence of sperm concentration (ranging from 0.5 × 106 to 4 × 106 spermatozoa/ml) and length of the gamete co-incubation time (2, 4, 6, 8, 10, 12, 16, 20, 24 or 28 h) on in vitro fertilization (IVF), assessing the sperm penetration rate; (2) to investigate the kinetics of different semen parameters as motility, viability and acrosome status during the co-culture period; and (3) to analyse the effect of the presence of cumulus–oocytes complexes (COCs) on these parameters. To achieve these objectives, several experiments were carried out using in vitro matured oocytes from prepubertal goats. The main findings of this work are that: (1) in our conditions, the optimum sperm concentration is 4 × 106 sperm/ml, as this sperm:oocyte ratio (approximately 28,000) allowed us to obtain the highest penetration rate, without increasing polyspermy incidence; (2) the highest percentage of viable acrosome-reacted spermatozoa is observed between 8–12 h of gamete co-culture, while the penetration rate is maximum at 12 h of co-incubation; and (3) the presence of COCs seems to favour the acrosome reaction of free spermatozoa on IVF medium, but not significantly. In conclusion, we suggest that a gamete co-incubation for 12–14 h, with a concentration of 4 × 106 sperm/ml, would be sufficient to obtain the highest rate of penetration, reducing the exposure of oocytes to high levels of reactive oxygen species produced by spermatozoa, especially when a high sperm concentration is used to increase the caprine IVF outcome.

Developmental kinetics of in vitro produced bovine embryos: the effect of sex, glucose and exposure to time-lapse environment

Zygote ◽  
2001 ◽  
Vol 9 (2) ◽  
pp. 105-113 ◽  
Author(s):  
J. Peippo ◽  
M. Kurkilahti ◽  
P. Bredbacka
Keyword(s):  
Sex Determination ◽  
Video Recording ◽  
Time Lapse ◽  
Recording System ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential ◽  
Kinetics Of ◽  
Time Lapse Video

In this study, a simple time-lapse video recording system was used to compare developmental kinetics of female and male bovine embryos produced in vitro. Following embryo sex determination, the timing of each cleavage up to the 4-cell stage was compared between the sexes from the videotapes after culture in the presence and absence of glucose. In the second experiment, the consequences of exposure to a time-lapse video recording (TL) environment were studied by culturing embryos further until day 7 in an incubator, followed by collection and sex determination of morulae and blastocysts. In the absence of glucose, female embryos cleaved earlier than male ones. In the presence of glucose, however, male embryos cleaved earlier than female ones. There was no difference in the number of morulae/blastocysts in the absence of glucose, but in the presence of glucose more male than female embryos reached the morula and blastocyst stage. Exposure to the TL environment itself also had a sex-related effect, being more detrimental to male than female embryos. The difference in the number of functional X chromosomes between the sexes during early preimplantation development could explain these findings. In females, an increased capacity for oxygen radical detoxification through the pentose phosphate pathway could result in a reduced cleavage rate. Furthermore, glucose may influence the expression of enzymes located on the X chromosome. According to these results, a simple time-lapse video recording system is suitable for investigating embryo developmental kinetics and perhaps for the selection of embryos with the greatest developmental potential.

103 TRANSDUCTION PATHWAYS RELATED TO GLUCOSE METABOLISM IN MALE AND FEMALE BOVINE EMBRYOS PRODUCED IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 157
Author(s):  
M. Garcia-Herreros ◽  
I. M. Aparicio ◽  
D. Rath ◽  
T. Fair ◽  
P. Lonergan
Keyword(s):  
Glucose Metabolism ◽  
Protein Expression ◽  
Sodium Dodecyl ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Glucose Transporter 1 ◽  
Male And Female ◽  
Glut 1 ◽  
Kinetic Rates

Glucose metabolism plays an important role in energy balance control in mammalian cells and has been widely used as an indicator of embryo developmental competence. Previous studies have shown that developmental kinetic rates following IVF are lower in female than in male blastocysts, which may be related to differences in glucose consumption and metabolism. In addition, we have demonstrated that inhibition of phosphatidylinositol 3-kinase (PI3-K) with a structurally unrelated inhibitor, LY294002, suggests a negative role for PI3-K in the regulation of bovine embryo development (Aparicio et al. 2010 Reproduction 140, 83–92). The aim of this study was to determine whether PI3-K has a role in the regulation of glucose metabolism in Day 7 bovine blastocysts and to study the possible differential protein expression involved in glucose metabolism [hexokinase-I (HK-I), phosphofructokinase-1 (PFK-1), pyruvate kinase1/2 (PMK1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/C (LDHA/C), glucose transporter-1 (GLUT-1) and glycogen synthase kinase-3 (GSK-3A/B)] between in vitro produced male and female embryos derived from IVF with either X- or Y-sorted semen. Day 7 blastocysts derived from unsorted semen (n = 25 blastocysts per group) were incubated up to 12 h in SOF culture medium in the presence or absence of LY294002 (10 μM) and stored. Similarly, male and female Day 7 blastocysts derived from sorted semen were collected apart and stored at –80°C until proteomic analysis (Western blot analysis of proteins separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis). Inhibition of PI3K significantly decreased HK-I (P < 0.01), PFK-1 (P < 0.001), GAPDH (P < 0.05), GSK-3A/B (P < 0.001), and GLUT-1 (P < 0.01) protein levels. Interestingly, protein expression of HK-1 (P < 0.001), PFK-1 (P < 0.01), PMK1/2 (P < 0.05), GAPDH (P < 0.01), and GLUT-1 (P < 0.001) was significantly higher in male compared with female blastocysts. The significant increase in the phosphorylated forms (Ser21 and Ser9) of both isoforms (GSK-3A/B) in male compared with female embryos is indicative of a higher inactivation of GSK-3A/B in males (P < 0.001). The presence of LDHA/C activity was not detected in any blastocyst group, irrespective of the gender or treatment studied. In conclusion, our data suggest that PI3K plays a major role in the regulation of glucose metabolism in bovine embryos, because pretreatment with LY294002 significantly modified the protein expression of HK-I, PFK-1, GAPDH, GSK- 3A/B, and GLUT-1, and underline the possibility of modulating glucose metabolism via the PI3K cellular pathway. The differential glycolytic metabolism between male and female blastocysts might explain the higher developmental kinetic rates in males described by other authors, because the expression of proteins involved in glycolysis and glycogenogenesis was significantly higher in male than in female in vitro-produced bovine embryos.

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