Oviductal response to gametes and early embryos in mammals

The oviduct is a complex and organized thin tubular structure connecting the ovary with the uterus. It is the site of final sperm capacitation, oocyte fertilization and, in most species, the first 3–4days of early embryo development. The oviductal epithelium is made up of ciliary and secretory cells responsible for the secretion of proteins and other factors which contribute to the formation of the oviductal fluid. Despite significant research, most of the pathways and oviductal factors implicated in the crosstalk between gametes/early embryo and the oviduct remain unknown. Therefore, studying the oviductal environment is crucial to improve our understanding of the regulatory mechanisms controlling fertilization and embryo development. In vitro systems are a valuable tool to study in vivo pathways and mechanisms, particularly those in the oviducts which in livestock species are challenging to access. In studies of gamete and embryo interaction with the reproductive tract, oviductal epithelial cells, oviductal fluid and microvesicles co-cultured with gametes/embryos represent the most appropriate in vitro models to mimic the physiological conditions in vivo.

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Loss of Bmal1 decreases oocyte fertilization, early embryo development and implantation potential in female mice

Zygote ◽

10.1017/s0967199416000083 ◽

2016 ◽

Vol 24 (5) ◽

pp. 760-767 ◽

Cited By ~ 8

Author(s):

Jian Xu ◽

Yan Li ◽

Yizi Wang ◽

Yanwen Xu ◽

Canquan Zhou

Keyword(s):

Embryo Development ◽

Biological Clock ◽

Fertilization Rate ◽

Early Embryo ◽

Wild Type ◽

Early Embryo Development ◽

Female Mice ◽

Oocyte Fertilization

SummaryBiological clock genes expressed in reproductive tissues play important roles in maintaining the normal functions of reproductive system. However, disruption of female circadian rhythm on oocyte fertilization, preimplantation embryo development and blastocyst implantation potential is still unclear. In this study, ovulation, in vivo and in vitro oocyte fertilization, embryo development, implantation and intracellular reactive oxygen species (ROS) levels in ovary and oviduct were studied in female Bmal1+/+ and Bmal1−/− mice. The number of naturally ovulated oocyte in Bmal1−/− mice decreased (5.2 ± 0.8 vs 7.8 ± 0.8, P < 0.001), with an increasing abnormal oocyte ratio (20.4 ± 3.5 vs 11.7 ± 2.0%, P = 0.001) after superovulation. Significantly lower fertilization rate and obtained blastocyst number were observed in Bmal1−/− female mice either mated with wild-type in vivo or fertilized by sperm from wild-type male mice in vitro (all P < 0.05). Interestingly, in vitro fertilization rate of oocytes derived from Bmal1−/− increased significantly compared with in vivo study (P < 0.01). After transferring blastocysts derived from Bmal1+/+ and Bmal1−/− female mice to pseudopregnant mice, the implantation sites of the latter decreased 5 days later (8.0 ± 0.8 vs 5.3 ± 1.0, P = 0.005). The intracellular ROS levels in the ovary on proestrus day and in the oviduct on metestrus day increased significantly in Bmal1−/− mice compared with that of Bmal1+/+ mice. Deletion of the core biological clock gene Bmal1 significantly decreases oocyte fertilization rate, early embryo development and implantation potential in female mice, and these may be possibly caused by excess ROS levels generated in ovary and oviduct.

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Single cell RNA-seq reveals genes vital to in vitro fertilized embryos and parthenotes in pigs

Scientific Reports ◽

10.1038/s41598-021-93904-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhi-Qiang Du ◽

Hao Liang ◽

Xiao-Man Liu ◽

Yun-Hua Liu ◽

Chonglong Wang ◽

...

Keyword(s):

Embryo Development ◽

Gene Networks ◽

Regulatory Networks ◽

Rna Binding ◽

Expression Patterns ◽

Early Embryo ◽

Specific Factor ◽

Early Embryos ◽

Genome Activation

AbstractSuccessful early embryo development requires the correct reprogramming and configuration of gene networks by the timely and faithful execution of zygotic genome activation (ZGA). However, the regulatory principle of molecular elements and circuits fundamental to embryo development remains largely obscure. Here, we profiled the transcriptomes of single zygotes and blastomeres, obtained from in vitro fertilized (IVF) or parthenogenetically activated (PA) porcine early embryos (1- to 8-cell), focusing on the gene expression dynamics and regulatory networks associated with maternal-to-zygote transition (MZT) (mainly maternal RNA clearance and ZGA). We found that minor and major ZGAs occur at 1-cell and 4-cell stages for both IVF and PA embryos, respectively. Maternal RNAs gradually decay from 1- to 8-cell embryos. Top abundantly expressed genes (CDV3, PCNA, CDR1, YWHAE, DNMT1, IGF2BP3, ARMC1, BTG4, UHRF2 and gametocyte-specific factor 1-like) in both IVF and PA early embryos identified are of vital roles for embryo development. Differentially expressed genes within IVF groups are different from that within PA groups, indicating bi-parental and maternal-only embryos have specific sets of mRNAs distinctly decayed and activated. Pathways enriched from DEGs showed that RNA associated pathways (RNA binding, processing, transport and degradation) could be important. Moreover, mitochondrial RNAs are found to be actively transcribed, showing dynamic expression patterns, and for DNA/H3K4 methylation and transcription factors as well. Taken together, our findings provide an important resource to investigate further the epigenetic and genome regulation of MZT events in early embryos of pigs.

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Which Low-Abundance Proteins are Present in the Human Milieu of Gamete/Embryo Maternal Interaction?

International Journal of Molecular Sciences ◽

10.3390/ijms20215305 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5305 ◽

Cited By ~ 3

Author(s):

Canha-Gouveia ◽

Paradela ◽

Ramos-Fernández ◽

Prieto-Sánchez ◽

Sánchez-Ferrer ◽

...

Keyword(s):

Embryo Development ◽

Reproductive Tract ◽

Culture Media ◽

Adult Life ◽

Early Embryo ◽

Female Reproductive Tract ◽

High Throughput Analysis ◽

Maternal Interaction ◽

Oviductal Fluid ◽

Abundance Proteins

The improvement of the embryo culture media is of high relevance due to its influence on successful implantation rates, pregnancy, neonatal outcomes, and potential effects in adult life. The ideal conditions for embryo development are those naturally occurring in the female reproductive tract, i.e., the oviductal and uterine fluids. To shed light on the differences between chemical and natural media, we performed the first comparative study of the low abundance proteins in plasma, uterine, and oviductal fluid collected, simultaneously, from healthy and fertile women that underwent a salpingectomy. The rationale for this design derives from the fact that high-abundant proteins in these fluids are usually those coming from blood serum and frequently mask the detection of low abundant proteins with a potentially significant role in specific processes related to the embryo–maternal interaction. The proteomic analysis by 1D-nano LC ESI-MSMS detected several proteins in higher amounts in oviductal fluid when compared to uterine and plasma samples (RL3, GSTA1, EZRI, DPYSL3, GARS, HSP90A). Such oviductal fluid proteins could be a target to improve fertilization rates and early embryo development if used in the culture media. In conclusion, this study presents a high-throughput analysis of female reproductive tract fluids and contributes to the knowledge of oviductal and uterine secretome.

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293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab293 ◽

2007 ◽

Vol 19 (1) ◽

pp. 262 ◽

Cited By ~ 1

Author(s):

I. Dimitriadis ◽

E. A. Rekka ◽

E. Vainas ◽

G. S. Amiridis ◽

C. A. Rekkas

Keyword(s):

Lipid Peroxidation ◽

Embryo Development ◽

Antioxidant Properties ◽

Thiobarbituric Acid ◽

Early Embryo ◽

Bovine Embryo ◽

Embryo Production ◽

Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples' ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P < 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

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161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab161 ◽

2016 ◽

Vol 28 (2) ◽

pp. 210

Author(s):

P. Hugon ◽

J. Lamy ◽

E. Corbin ◽

P. Mermillod ◽

M. Saint-Dizier

Keyword(s):

Corpus Luteum ◽

Embryo Development ◽

Zona Pellucida ◽

Developmental Stages ◽

Early Embryo ◽

Developmental Competence ◽

Control Group ◽

Vitro Fertilization ◽

Oviductal Fluid

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P < 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

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112 EFFECTS OF GUAIAZULENE ON IN VITRO CULTURE OF BOVINE ZYGOTES, AND ON mRNA TRANSCRIPTS RELATED TO EMBRYO QUALITY

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab112 ◽

2009 ◽

Vol 21 (1) ◽

pp. 156

Author(s):

E. Dovolou ◽

M. Clemente ◽

G. S. Amiridis ◽

I. Messinis ◽

A. Kalitsaris ◽

...

Keyword(s):

Embryo Development ◽

Embryo Quality ◽

Early Embryo ◽

Cleavage Rate ◽

Embryo Production ◽

Early Embryo Development ◽

Mrna Transcripts ◽

Oviductal Fluid ◽

Maximum Humidity

We have previously shown that follicular and oviductal fluid provide greater total protection against lipid peroxidation than the respective media used for the in vitro embryo production. Reactive oxygen species (ROS) production has been implicated as a major cause for the reduced in vitro bovine embryo production; it is believed that they participate in meiotic arrest of oocytes, embryonic block and cell death. The aim of this study was to determine whether guaiazulene (G), an exogenous antioxidant, added in the post fertilization culture medium would affect the early embryo development and the quality of the produced blastocysts in terms of mRNA expression of several important genes. In a previous study we had shown that media modified with 0.01 mm of G provided the same antioxidant protection as the respective in vivo environments (i.e. the follicular and the oviductal fluid). Bovine cumulus–oocyte complexes (COC) were aspirated from ovaries derived from slaughtered cows and matured in groups of 50 in 500 μL in TCM199 with 10% fetal calf serum and 10 ng mL–1 Epidermal Growth factor at 39°C in an atmosphere of 5% CO2 in air and maximum humidity. Twenty-four hours later matured oocytes were inseminated with frozen/thawed bull semen and co-incubated in the same conditions as maturation. Presumptive zygotes were divided into 4 groups and cultured in groups of 25 in 25 μL of SOF with 5% FCS (Control–, n = 355), supplemented with 0.01 mm of G (n = 344) or 0.1 mm of G (n = 345) or 0.05% DMSO – the G diluent–(Control+, n = 347) at 39°C in an atmosphere of 5% CO2, 5% O2 and maximum humidity. Blastocyst yield was recorded on Days 6, 7, 8 and 9; Day 7 blastocysts from each group were snap frozen and stored at –80°C for mRNA extraction. Quantification of transcripts for aldose reductase mRNA (AKRIBI), prostaglandin G/H synthase-2 (PGHS-2, COX-2), glyceraldehyde 3-phosphate dehydrogenase (GADPH), facilitated glucose/fructose transporter, member 5 (GLUT-5) genes related to metabolism, glutathione peroxidase 1 (GPX1), glucose-6-phosphate dehydrogenase (G6PD) antioxidant enzymes and placenta-specific 8 (PLAC8) related to implantation was carried out by real-time quantitative RT-PCR. Data for embryo development and on transcript abundance were analyzed by chi square and ANOVA, respectively. Cleavage rate tended to be higher in 0.01 mm group than in Control– (77.87% v. 71.41%, P = 0.07). Barring that, no other differences were detected in cleavage rate (Control+: 71.32%; 0.1 mm: 72.75%) or in the overall blastocyst yield on Day 9 (Control–: 25.50%; Control+: 26.71%; 0.1 mm: 25.75%; 0.01 mm: 29.58%). The relative abundance of genes studied varied among groups, but these differences were not significant. We infer that under the current culture conditions, G as an antioxidant has no serious direct effect on early embryo development or on embryo quality at least on the mRNA transcripts studied. Further studies using the same antioxidant in different atmospheric conditions are planed. ED and GSA were sponsored by COST (FAO702) and OECD fellowships, respectively.

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87 EVALUATION OF THE NUMERICAL CHROMOSOMAL ABNORMALITIES ON IN VITRO EARLY BOVINE EMBRYOS: EFFECT OF THE CELL CO-CULTURE WITH GRANULOSA CELLS

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab87 ◽

2014 ◽

Vol 26 (1) ◽

pp. 157

Author(s):

S. Demyda-Peyrás ◽

M. Hidalgo ◽

J. Dorado ◽

M. Moreno-Millan

Keyword(s):

Embryo Development ◽

Granulosa Cells ◽

Chromosomal Abnormalities ◽

Culture Media ◽

Gradient Centrifugation ◽

Early Embryo ◽

Bovine Embryos ◽

Humid Atmosphere ◽

Early Embryos

Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.

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Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo

Reproduction ◽

10.1530/rep-18-0561 ◽

2019 ◽

Vol 158 (1) ◽

pp. 85-94 ◽

Cited By ~ 7

Author(s):

Meriem Hamdi ◽

María J Sánchez-Calabuig ◽

Beatriz Rodríguez-Alonso ◽

Sandra Bagés Arnal ◽

Kalliopi Roussi ◽

...

Keyword(s):

Epithelial Cells ◽

Metabolic Response ◽

Early Embryo ◽

Bovine Embryo ◽

Cell Stage ◽

Transcriptomic Response ◽

Early Embryos ◽

Oviduct Epithelial Cells

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

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Adipokines Expression and Effects in Oocyte Maturation, Fertilization and Early Embryo Development: Lessons from Mammals and Birds

International Journal of Molecular Sciences ◽

10.3390/ijms21103581 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3581

Author(s):

Anthony Estienne ◽

Adeline Brossaud ◽

Maxime Reverchon ◽

Christelle Ramé ◽

Pascal Froment ◽

...

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Reproductive Tract ◽

Current Knowledge ◽

Oocyte Quality ◽

Early Embryo ◽

Early Embryo Development ◽

Vitro Fertilization ◽

Physiological Processes

Some evidence shows that body mass index in humans and extreme weights in animal models, including avian species, are associated with low in vitro fertilization, bad oocyte quality, and embryo development failures. Adipokines are hormones mainly produced and released by white adipose tissue. They play a key role in the regulation of energy metabolism. However, they are also involved in many other physiological processes including reproductive functions. Indeed, leptin and adiponectin, the most studied adipokines, but also novel adipokines including visfatin and chemerin, are expressed within the reproductive tract and modulate female fertility. Much of the literature has focused on the physiological and pathological roles of these adipokines in ovary, placenta, and uterine functions. The purpose of this review is to summarize the current knowledge regarding the involvement of leptin, adiponectin, visfatin, and chemerin in the oocyte maturation, fertilization, and embryo development in both mammals and birds.

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204 RELATIVE MESSENGER RNA ABUNDANCE OF HYALURONAN RECEPTORS AND SYNTHASES ON IN VITRO- AND IN VIVO-DERIVED DAY 7 AND DAY 13 BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab204 ◽

2009 ◽

Vol 21 (1) ◽

pp. 200

Author(s):

M. Clemente ◽

A. T. Palasz ◽

J. de La Fuente ◽

P. Lonergan ◽

A. Gutierrez-Adan ◽

...

Keyword(s):

Embryo Development ◽

Messenger Rna ◽

Developmental Stages ◽

Transcript Abundance ◽

Early Embryo ◽

Bovine Embryos ◽

Cd44 Receptor ◽

Mrna Extraction

Hyaluronan (HA), which progressively increases during embryogenesis, is a glycosaminoglycan that plays a major role in oocyte/embryo development (Fenderson et al. 1993 Differentiation 54, 85–95). One of the main functions of HA is to participate in the cell proliferation and migration that are controlled by HA receptors, RHAMM and C44, and by the presence of different HA synthases, Has1, Has2, and Has3. All have very distinctive features and functions at different stages of embryo development. The objective of this study was to determine the relative mRNA abundance of HA receptors and synthases in Day 7 and 13 bovine embryos derived in vitro or in vivo. In vitro embryos were produced by standard oocyte maturation and fertilization procedures. Presumptive zygotes were cultured in groups of 25 in 25-μL droplets of synthetic oviduct fluid supplemented with 5% FCS at 39°C, 5% CO2, and 5%O2 with maximum humidity. In vivo blastocysts were collected from superovulated heifers on Day 7 (estrus = Day 0) by uterine flushing and on Day 13 immediately after slaughter by flushing the dissected reproductive tracts. All embryos were frozen in LN2 and stored at –80°C for mRNA extraction. Quantification of transcripts for RHAMM and CD44 receptors and Has2 and Has3 synthases was performed on groups of ten Day 7 blastocysts (3 groups for in vivo or in vitro) and individual Day 13 embryos (7 embryos in vivo or in vitro) by real-time quantitative RT-PCR. Data on differences in transcript abundance were analyzed by ANOVA. The relative abundance of the Has2 and Has3 synthases was similar between in vivo and in vitro embryos, irrespective of their developmental stage. The quantity of CD44 was significantly higher in in vitro compared with in vivo embryos only on Day 7. However, the quantity of RHAMM receptor was higher on Day 13 in in vitro compared with in vivo embryos. When the comparison was done between developmental stages (Day 7 v. Day 13) for in vivo and in vitro embryos, we found that in vivo-produced Day 7 blastocysts expressed significantly more RHAMM receptor than embryos on Day 13. The reverse pattern of expression was shown for CD44 receptor. For in vitro embryos, the only difference observed was for Has3, which was up-regulated on Day 13 compared with Day 7 embryos. In conclusion, the present study demonstrates, for the first time, developmental changes in the abundance of RHAMM and CD44 receptor mRNA and Has2 and Has3 synthase mRNA in in vivo and in vitro bovine-derived embryos on Day 7 and 13. We believe that our results will provide new insight into the potential role of this intriguing multifunctional molecule in bovine early embryo development.

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