Fertilization following intracytoplasmic sperm injection of in vivo and in vitro matured oocytes from an Australian marsupial, the tammar wallaby (Macropus eugenii)

Conventional in vitro fertilization (IVF) techniques have been unable to produce normal embryos in any Australian marsupial, largely owing to problems with the early stages of sperm–oocyte binding. This study has used intracytoplasmic sperm injection (ICSI) of in vivo and in vitro matured tammar wallaby oocytes to bypass these processes and achieve fertilization in vitro. The fertilization rate (i.e. development to the two-pronuclei stage) of in vivo and in vitro matured oocytes following ICSI and sham injection was assessed at 17–19 h after injection. Fertilization occurred in 48% (45/93) of in vivo matured oocytes that were injected with spermatozoa. Significantly fewer sham-injected oocytes (6/82, P<0.005) and uninjected control oocytes (5/84, P<0.005) formed two pronuclei. In a direct comparison, the numbers of in vivo and in vitro matured oocytes that formed two pronuclei after ICSI were 22/28 (78.6%) and 23/40 (57.6%), respectively, which are not significantly different. There was also no significant difference in the nuclear response of in vivo and in vitro matured oocytes to sham injection. The numbers of oocytes forming a single pronucleus after sham injection were 10/24 (41.7%) and 24/37 (64.9) for in vivo and in vitro matured oocytes, respectively. Immature germinal-vesicle-stage oocytes were unable to decondense sperm injected during ICSI or to form pronuclei. These results demonstrate that both in vitro and in vivo matured tammar wallaby oocytes can be fertilized by ICSI. The success of ICSI not only offers the opportunity for fundamental analysis of marsupial fertilization but could, in conjunction with development of appropriate culture conditions and embryo transfer technologies, contribute to increased production of offspring from rare or valuable marsupials.

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326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab326 ◽

2006 ◽

Vol 18 (2) ◽

pp. 270

Author(s):

C. Hanna ◽

C. Long ◽

M. Westhusin ◽

D. Kraemer

Keyword(s):

In Vitro Maturation ◽

Calf Serum ◽

Cumulus Cell ◽

Germinal Vesicle ◽

Cumulus Cells ◽

Culture Conditions ◽

Germinal Vesicle Breakdown ◽

Significant Difference ◽

Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

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Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽

10.1530/rep.0.1230455 ◽

2002 ◽

pp. 455-465 ◽

Cited By ~ 74

Author(s):

YH Choi ◽

CC Love ◽

LB Love ◽

DD Varner ◽

S Brinsko ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Polar Body ◽

Cumulus Cells ◽

Fertilization Rate ◽

Developmental Competence ◽

Cleavage Rate ◽

First Polar Body ◽

Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

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Fertilization and early embryonic development of in vitro matured metaphase I oocytes in patients with unexpected low oocyte maturity rate

Zygote ◽

10.1017/s0967199421000654 ◽

2021 ◽

pp. 1-5

Author(s):

Nafiye Yılmaz ◽

Şebnem Özyer ◽

Derya Taş ◽

Mehmet Caner Özer ◽

Ayten Türkkanı ◽

...

Keyword(s):

Embryonic Development ◽

Fertilization Rate ◽

Early Embryonic Development ◽

Oocyte Maturity ◽

Significant Difference ◽

Maturation Rate ◽

Developmental Capacity ◽

Metaphase I

Summary To determine the fertilization and embryonic potential of immature metaphase I (MI) oocytes in patients with low oocyte maturity rate in whom the percentage of mature oocytes obtained was less than 75% of the total retrieved ones. In vivo matured metaphase II (MII) oocytes (MII-ICSI, n = 244), and in vitro matured MI oocytes (MI-MII-ICSI, n = 202) underwent an intracytoplasmic sperm injection (ICSI) procedure. Maturation rate, fertilization rate and early embryonic development were compared in both groups. In total, 683 oocytes were collected from 117 ICSI cycles of 117 patients. Among them, 244 (35.7%) were mature MII and 259 (37.9%) were MI after the denudation process. Of those 259 MI oocytes, 202 (77.9%) progressed to MII oocytes after an incubation period of 18–24 h. The maturation rate was 77.9%. Fertilization rate was found to be significantly higher in the rescued in vitro matured MI oocyte group when compared with the in vivo matured MII oocyte group (41.6% vs 25.8%; P = 0.0006). However, no significant difference was observed in terms of cleavage rates on days 2 and 3 between the groups (P = 0.9126 and P = 0.5031, respectively). There may be unidentified in vivo factors on the oocyte maturation causing low developmental capacity in spite of high fertilization rates in the group of patients with low oocyte maturity rate. Furthermore, studies are needed to determine the appropriate culture characteristics as well as culture period and ICSI timing of these oocytes.

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The capability of reprogramming the male chromatin after fertilization is dependent on the quality of oocyte maturation

Reproduction ◽

10.1530/rep.1.00550 ◽

2005 ◽

Vol 130 (1) ◽

pp. 29-39 ◽

Cited By ~ 51

Author(s):

Luisa Gioia ◽

Barbara Barboni ◽

Maura Turriani ◽

Giulia Capacchietti ◽

Maria Gabriella Pistilli ◽

...

Keyword(s):

Dna Methylation ◽

Histone Acetylation ◽

Hdac Inhibitors ◽

Germinal Vesicle ◽

Fertilization Rate ◽

Sperm Nucleus ◽

Sperm Chromatin ◽

Germinal Vesicle Breakdown

The present experiments compared the ability of pig oocytes matured eitherin vivoorin vitroto structurally reorganize the penetrated sperm chromatin into male pronucleus (PN) and to carry out, in parallel, the epigenetic processes of global chromatin methylation and acetylation, 12–14 h afterin vitrofertilization (IVF). In addition, PN distribution of histone deacetylase (HDAC), a major enzyme interfacing DNA methylation and histone acetylation, was investigated. The ability of the oocyte to operate an efficient block to polyspermy was markedly affected by maturation. The monospermic fertilization rate was significantly higher forin vivothan forin vitromatured (IVM) oocytes(P< 0.01) which, furthermore, showed a reduced ability to transform the chromatin of penetrated sperm into male PN(P< 0.01). Indirect immunofluorescence analysis of global DNA methylation, histone acetylation and HDAC distribution (HDAC-1, -2 and -3), carried out in monospermic zygotes that reached the late PN stage, showed that IVM oocytes also had a reduced epigenetic competence. In fact, while in about 80% ofin vivomatured and IVF oocytes the male PN underwent a process of active demethylation and showed a condition of histone H4 hyperacetylation, only 40% of IVM/IVF zygotes displayed a similar PN remodelling asymmetry. Oocytes that carried out the first part of maturationin vivo(up to germinal vesicle breakdown; GVBD) and then completed the processin vitro, displayed the same PN asymmetry as oocytes matured entirelyin vivo. A crucial role of HDAC in the establishment of PN acetylation asymmetry seems to be confirmed by the use of HDAC inhibitors as well as by the abnormal distribution of the enzyme between the two PN in IVM zygotes. Collectively, these data demonstrated that some pig IVM oocytes fail to acquire full remodelling competence which is independent from their ooplasmic ability to morphologically reorganize the sperm nucleus into PN.

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Production of viable bovine embryos by intracytoplasmic sperm injection of oocytes harvested from slaughtered old cows

Cluj Veterinary Journal ◽

10.52331/cvj.v26i1.8 ◽

2021 ◽

Vol 26 (1) ◽

pp. 7-14

Author(s):

Mihai Cenariu ◽

Mihai Borzan ◽

Sorin Dan ◽

Remus Chiorean ◽

Emoke Pall

Keyword(s):

In Vitro Fertilization ◽

Intracytoplasmic Sperm Injection ◽

Fertilization Rate ◽

Fertilization Success ◽

Vitro Fertilization ◽

Bull Semen ◽

Intracytoplasmic Injection ◽

Lower Fertility

Abstract: (1) Background: Intracytoplasmic sperm injection (ICSI) is currently used to increase fertilization success by avoiding several oocyte or sperm deficiencies that would normally prevent conception after in vivo fertilization or classical in vitro fertilization. This paper aimed at improving the in vitro fertilization protocol of bovine oocytes, harvested from old cows after slaughtering, using intracytoplasmic sperm injection; (2) Methods: Oocytes were harvested by puncture of follicles from ovaries obtained from slaughtered old cows, followed by aspiration. Out of the 127 cumulus-oocyte complexes that were harvested, 84 (66.14%) were declared suitable for cultivation, after morphological evaluation. Following oocyte maturation for 22 hours, 77 cumulus-oocyte complexes were morphologically intact and could undergo the steps required for intracytoplasmic injection of spermatozoa. Frozen-thawed bull semen was used for ICSI and the 77 fertilized oocytes were kept for 24 hours in an atmosphere enriched with 5% CO2.; (3) Results: Fertilized oocytes transformed into 46 zygotes (fertilization rate of 59.74%), while after 168 h of cultivation 38 transferable compact morulae or early blastocysts were obtained; (4) Conclusions: Intracytoplasmic sperm injection can represent a viable alternative to classical IVF, when oocytes or sperm with lower fertility are used.

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Comparison of glucose metabolism in in vivo- and in vitro-matured tammar wallaby oocytes and its relationship to developmental potential following intracytoplasmic sperm injection

Reproduction Fertility and Development ◽

10.1071/rd03062 ◽

2004 ◽

Vol 16 (6) ◽

pp. 617 ◽

Cited By ~ 3

Author(s):

Genevieve M. Magarey ◽

Karen E. Mate

Keyword(s):

Glucose Metabolism ◽

Intracytoplasmic Sperm Injection ◽

Tammar Wallaby ◽

Blastocyst Stage ◽

In Vitro Fertilisation ◽

Cell Stage ◽

Developmental Potential ◽

Developmental Capacity

Although marsupial oocytes undergo nuclear maturation in vitro, there is, at present, no indication of their developmental potential, largely owing to the lack of in vitro fertilisation and related technologies for marsupials. Glucose metabolism has proven a useful indicator of oocyte cytoplasmic maturation and developmental potential in several eutherian species. Therefore, the aims of the present study were to compare: (1) the rates of glycolysis and glucose oxidation in immature, in vitro-matured and in vivo-matured tammar wallaby oocytes; and (2) the metabolic rate of individual oocytes with their ability to form pronuclei after intracytoplasmic sperm injection. The rates of glycolysis measured in immature (2.18 pmol oocyte–1 h–1), in vitro-matured (0.93 pmol oocyte–1 h–1) and in vivo-matured tammar wallaby oocytes (0.54 pmol oocyte–1 h–1) were within a similar range to values obtained in eutherian species. However, unlike the trend observed in eutherian oocytes, the glycolytic rate was significantly higher in immature oocytes compared with either in vivo- or in vitro-matured oocytes (P < 0.001) and significantly higher in in vitro-matured oocytes compared with in vivo-matured oocytes (P < 0.001). No relationship was identified between glucose metabolism and the developmental capacity of oocytes after intracytoplasmic sperm injection when assessed after 17–19 h. Oocytes that became fertilised (two pronuclei) or activated (one or more pronucleus) were not distinguished from others by their metabolic rates. Longer culture after intracytoplasmic sperm injection (e.g. blastocyst stage) may show oocyte glucose metabolism to be predictive of developmental potential; however, culture to the single-cell stage did not reveal any significant differences in normally developing embryos.

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183 CANINE EMBRYOS OBTAINED BY INTRACYTOPLASMIC SPERM INJECTION

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab183 ◽

2014 ◽

Vol 26 (1) ◽

pp. 206 ◽

Cited By ~ 1

Author(s):

S. Chastant-Maillard ◽

K. Reynaud ◽

S. Thoumire ◽

M. Chebrout

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Fetal Calf Serum ◽

Selection Process ◽

Calf Serum ◽

Germinal Vesicle ◽

Sperm Head ◽

Cell Stage ◽

Vitro Fertilization

In vitro fertilization encounters 2 specific difficulties in the canine species, with no puppies born to date: low penetration rates (10–50%) and high polyspermia (around 50% of fertilized oocytes; Saint-Dizier et al. 2001 J. Reprod. Fert. Suppl. 57, 147–150). The objectives of the study were to test whether intracytoplasmic sperm injection (ICSI), which overcomes these 2 obstacles, could allow production of canine embryos, using in vivo- or in vitro-matured oocytes. The time of ovulation was determined on 8 Beagle bitches from our experimental kennel by blood progesterone assay and transabdominal ultrasound examination. After ovariohysterectomy 82 to 100 h after ovulation, 58 metaphase II (MII) oocytes were collected by tubal flushing. In parallel, 88 oocytes from 6 anoestrus bitches were matured in vitro (M199 + 20% fetal calf serum for 72 h in 5% CO2 at 38°C). Sperm was collected from 1 Beagle dog with excellent fertility record at natural mating. The sperm was diluted 1 : 100 in PBS/BSA without any selection process. Intracytoplasmic sperm injection was performed at 38°C in M199 HEPES + 20% BSA (4-μm injection pipette; 120-μm holding pipette). One motile spermatozoon of normal morphology was injected per oocyte. Injected oocytes were cultured in vitro for 48 h after injection (M199 + 20% fetal calf serum in 5% CO2 at 38°C) in 4-well open dishes. Oocytes were then fixed and DNA and tubulin were stained for observation by confocal microscopy (Chebrout et al. 2012 Microsc. Microanal. 18, 483–492). Among the 58 MII oocytes recovered in vivo, 7.4% lysed at injection and 20% degenerated during the 48 h after injection. Among the 40 injected oocytes still alive, 6 fragmented (15%) and 4 developed as embryos [10%; 2-pronuclei (n = 2), 2-cell and 6-cell). None of the other oocytes showed decondensed female chromatin. Among the 88 oocytes incubated for in vitro maturation, 13 (14.8%) reached MII. These were successfully injected; 48 h after injection, 3 were embryos at the 2-cell stage and 10 were at the MII stage with a condensed sperm head. Fifty-one non-mature oocytes were injected; 31 were at the germinal vesicle (GV) stage and the stage of others was not determined. Of the GV oocytes, 71% degenerated during culture after injection. The 9 surviving oocytes were still at the GV stage with condensed sperm head 48 h after injection. In conclusion, canine embryos can be obtained through ICSI. Nevertheless, this procedure induced low activation rates. Development at later stages, especially after transfer into a recipient female, is to be evaluated, in particular for in vitro-produced MII oocytes, of lower cytoplasmic competence (Viaris et al. 2008 Reprod. Fert. Dev. 20, 626–639).

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188 EFFECTS OF CULTURE MEDIUM AND PROTEIN SUPPLEMENTATION ON mRNA EXPRESSION OF IN VITRO-PRODUCED PREIMPLANTATION BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab188 ◽

2008 ◽

Vol 20 (1) ◽

pp. 173

Author(s):

M. N. Purpera ◽

C. B. Ballard ◽

A. M. Giraldo ◽

D. Hylan ◽

R. A. Godke ◽

...

Keyword(s):

Connexin 43 ◽

Mrna Level ◽

Culture Conditions ◽

Protein Supplementation ◽

Developmental Potential ◽

Glut 1 ◽

Culture Systems ◽

Significant Difference

Numerous studies have reported aberrant gene expression levels in embryos attributed to suboptimal culture conditions. This study investigated the effects of different culture systems and protein sources on the development of IVP embryos as measured by cleavage and blastocyst rates, cell number, and relative abundance levels of Oct-4, Connexin 43, Nanog, and glucose transporter-1 (Glut-1) when compared with in vivo embryos. Experiment (Exp) 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium (KSOMaa) supplemented with amino acids. Exp 2 compared the same two culture systems with and without the addition of calf serum (CS). Oocytes were matured for 22 h, fertilized in vitro, and then cultured in the appropriate treatment medium. RNA was extracted from pools of blastocysts, reverse transcribed to cDNA, and primer-specific amplified via Q-PCR. Exp 1 analyzed 10 pools per treatment, and either 10 (in vivo) or 11 pools were analyzed per treatment in Exp 2. One-way ANOVA followed by multiple pair-wise comparisons using Tukey's test was used to detect differences between treatments and in vivo embryos (P < 0.05). Results from both experiments indicated that, despite similar cleavage and blastocyst rates among treatments, significant differences were detected at the mRNA level and in cell numbers between treatments. In Exp 1, Oct-4 was found to have a mean abundance mRNA level significantly greater in KSOMaa-cultured blastocysts than in either SOFaa-cultured blastocysts or in vivo embryos. The same pattern of upregulation of Oct-4 in KSOMaa or KSOMaa with CS-cultured blastocysts was detected in Exp 2. In contrast to that reported by others, Connexin 43 was not expressed at detectable levels in the in vivo embryos analyzed in our studies. Connexin 43 was not detected in IVP blastocysts used in Exp 1. Conversely, Connexin 43 was detected in KSOMaa, SOFaa, and SOFaa with CS-cultured blastocysts in Exp 2. There was no significant difference in expression of the ICM-specific transcript Nanog in either experiment. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of Glut-1 when compared with other treatments and in vivo embryos. Overall, the transcript levels of the majority of the genes analyzed were altered by the in vitro culture condition. Differences continue to be observed between in vitro-cultured and in vivo embryos, and until these differences are minimized, aberrations in in vitro development will continue to arise. Further research will possibly modify the current culture conditions, allowing for the production of in vitro embryos of higher developmental potential similar to that observed in in vivo-derived embryos.

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Developmental potential of in vitro or in vivo matured oocytes

Zygote ◽

10.1017/s0967199413000233 ◽

2013 ◽

Vol 23 (1) ◽

pp. 93-98 ◽

Cited By ~ 4

Author(s):

Diego D. Alcoba ◽

Anita M. Pimentel ◽

Ilma S. Brum ◽

Helena E. Corleta

Keyword(s):

Germinal Vesicle ◽

Fertilization Rate ◽

Cleavage Rate ◽

Suitable Alternative ◽

Developmental Potential ◽

Immature Oocytes ◽

Prophase I ◽

Intra Cytoplasmic Sperm Injection

SummaryThis study compared the embryological features of mature and immature oocytes (different stages) collected from stimulated cycles of in vitro fertilization (IVF). Immature oocytes were identified, classified as PI (prophase I – germinal vesicle, GV) or MI (metaphase I), were matured in vitro and fertilized using the intra-cytoplasmic sperm injection (ICSI) technique. Fertilization potential, cleavage, and subsequent transfer/cryopreservation of the embryos derived from these in vitro matured oocytes were compared with those of in vivo matured oocytes (collected at the MII stage). The characteristics of embryos derived from gametes recovered in the MI and MII stages were similar. The fertilization rate of immature oocytes recovered in PI was significantly lower than that of MII oocytes (P = 0.031), and the cleavage rate of the PI group was also lower than that of the MI (P = 0.004) and MII (P < 0.001) groups. In vitro maturation of MI oocytes is a suitable alternative when immature oocytes are recovered, as their characteristics and development are similar to those of in vivo matured oocytes. Optimization of outcomes for PI oocytes will require development of techniques that can distinguish which of these gametes will mature and fertilize.

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77 TRANSCRIPTOME PROFILING IN OOCYTES-EMBRYO AND GRANULOSA CELLS FROM BOVINE

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab77 ◽

2017 ◽

Vol 29 (1) ◽

pp. 146 ◽

Cited By ~ 1

Author(s):

M. A. Sirard ◽

É. Fournier ◽

I. Dufort ◽

I. Gilbert ◽

C. Robert

Keyword(s):

Web Application ◽

Graphical Representation ◽

Germinal Vesicle ◽

Transcriptome Profiling ◽

Cumulus Cells ◽

Blastocyst Stage ◽

Culture Conditions ◽

Dna Arrays

The EmbryoGENE Network (2008–2013) has assembled data from more than 764 DNA arrays covering the entire transcriptome of bovine oocytes, embryos, and somatic cells, such as granulosa and cumulus cells. The data were obtained using the same extraction-amplification-hybridization-analysis protocols enabling normalization and statistical analysis across time, tissues, and experiments. To provide the scientific community with this information in an organised and functional matter, we have created a web application (http:// http://emb-bioinfo.fsaa.ulaval.ca/granulosaIMAGE/) where the information on 40,000 genes products from 4 tissues are currently available: oocytes (mainly at the germinal vesicle stage), blastocysts in different culture conditions (namely in vitro, in vivo, different concentrations of lipids, free radicals and glucose, as well as blastocysts from cloning, vitrification, and different breeds). Also represented is the complete panel of late folliculogenesis from 3 mm upward including growing, plateau, and atretic phase oocytes, as well as their response to LH and several types of ovarian stimulation. In addition, a postpartum panel of dominant follicles (PGF2α synchronized at Day 30, 60, 90, 12) with high or low BHB (beta-hydroxybutyrate) or vitamin treated (B-6 and B-9) is represented. In each of the panels, a red background indicates differential expression for any given gene or isoform (40,000 on the EmbryoGENE array). To complete the information, a RNAseq experiment was performed on each stage from the germinal vesicle oocyte to the blastocyst (including early and late 8 cell) and presented as an absolute expression level, which is a measure of the total number of RNA molecules for the selected family of transcripts. The web application also provides relative expression values, which measure the proportion of the total transcripts represented by the selected family of transcripts. This information is provided as a graphical representation from the germinal vesicle to the blastocyst stage. This source of information may be cited either by referring to the website or by citing each individual analysis (>20 published papers from >20 principal investigators from Canada and abroad, all publicly available) used to assemble the profiles. We anticipate that this site will be useful to all scientists in bovine reproduction. The EmbryoGENE network was supported by NSERC Canada and Semex Canada for the bovine side.

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