Research area SABRE WP6
Efficient and unrestricted reproduction is important for the viability of livestock production enterprises. In dairy cattle, for example, selection for improved milk yield has seen concurrent reductions in fertility, especially expressing itself as abnormal ovarian cyclicity, poor oestrus expression and failure to establish successful pregnancy after insemination (fertilization failure and embryonic loss). Getting cows pregnant again at an optimum time after calving is considered a major problem for the dairy industry. Direct genetic selection for fertility is hampered by a relatively low heritability of the trait and the lack of easy-to-measure parameters for fertility with higher heritabilities. Knowledge of the regulation of the cascade of processes underlying successful reproduction (resumption of ovarian cyclicity after calving, maintenance of regular cycles until pregnant, oestrus expression, folliculogenesis, ovulation, luteal function, embryonic developmental competence, embryo-maternal interactions, foetal development and survival) will improve the possibilities for breeders to increase the genetic merit of farm animals for fertility and reproductive success while further improving the genetic merit for productivity in general.
Successful pregnancy at an optimum stage of lactation depends on many factors. Critical factors are currently oestrus expression and embryonic survival. The transcriptomic research in this WP will therefore focus on the genetic regulation of female sexual behaviour (related to oestrus expression) and oocyte and embryo developmental competence (related to survival in the course of mbryogenesis). Further, this WP will include fine-mapping and further identification of a segregating QTL (BTA7) for conception rate. This work package will generate fundamental knowledge of the genomics of important aspects of reproduction and fertility in large domestic animals. This knowledge will enable the development of successful breeding strategies for improved reproduction efficiency. As such, it will contribute to more sustainable livestock production.
SABRE WP 6 Objectives
• Identification in dairy cattle of genes involved in female sexual behaviour
• Identification of genes involved in folliculogenesis
• Identification of genes involved in embryonic developmental competence
• Development of a protocol for in vitro production of well-defined preimplantation embryos for transcriptome analysis
• Fine mapping of a segregating QTL (BTA7) affecting conception rate in dairy cows
• Contribution to the development of a phenotype ontogeny database concerning reproduction traits of interest for livestock production (in collaboration with WP3)
Description of the work
Task 6.1 Identification of genes involved in female sexual behaviour (ASG-WUR)
Task 6.1.1 Collection of brain area samples (M0 to M0+6)
Samples of different areas of the brains of 24 first lactation dairy cows with either a high (n=12) or a low (n=12) genetic merit for milk yield will be collected. The selection of brain areas to be sampled is based on our current knowledge of brain areas involved in female sexual behaviour in rodents. These areas are the hypothalamus, the medial preoptic area, the stria terminalis, the ventral tegmental area and the pituitary. Within each genetic merit group, 6 cows will be sacrificed on the first day of their second post partum oestrus and 6 cows on day 10 after their second post partum oestrus.
Task 6.1.2 Transcriptome analysis (M0+7 to M0+12)
The collected material will be used to study the expression of genes at the transcriptomic level (microarray analyses, using either an oligo microarray or the Affymetrix microarray). Comparisons will be made between high and low fertile cows and between cows in oestrus and cows in the luteal phase of the ovarian cycle. These comparisons will allow the identification of genes expressed during oestrus but not during the luteal phase of the cycle and will indicate whether these genes are differentially expressed in relation to genetic merit for fertility.
Task 6.1.3 Pathway analysis and literature review (M0+7 to M0+18)
The neuro-endocrine and biochemical pathways in which genes and differentially expressed genes that have been identified to be of importance during oestrus and for oestrus expression will be described and evaluated. An elaborate literature study of the neuro-endocrine regulation of female sexual behaviour (mainly studies in rodents) will be performed to strengthen the pathway analysis.
Task 6.2 Identification of genes involved in folliculogenesis (INRA)
Task 6.2.1 Designs for inter-species experiments Preliminary experiments (M0+1 to M0+3)Preliminary experiments will be done to estimate how many follicles must be pooled to obtain an amount of RNA compatible with hybridisation on membranes (5µg of total RNA). Ovarian follicles from adult sexually synchronous female (at 24h after Regumate retrieval, and at 96h) will be collected in both species: pigs and cattle. The follicles will be individually dissected. After extraction the amount of RNA of each follicle will be evaluated. Sampling of biological material for transcriptome analysis: (M0+3 to M0+8). Transcriptome analysis will concern granulosa cells from small healthy, small atretic, large healthy and large atretic follicles of cattle and pig.According to the preliminary experiment, ovarian follicles from two adult sexually synchronous female (at 24h after Regumate retrieval, and at 96h) will be collected in both species: pigs and cattle. The follicles will be individually dissected, follicular fluid will be recovered and stored for further analysis (see below), a smear of granulosa cells will be done for Feulgen staining. The rest of granulosa cells will be individually recovered in 96-well plates and frozen at -80°C for future RNA extraction. Examination of the smears of granulosa cells will allow classifying follicles as healthy (mitoses) or atretic (pycnoses) follicles. Granulosa cells from small and maybe large follicles will then be pooled a posteriori in one of the four classes of studied follicles. A total of five independent RNA samples per class of follicles will be prepared for hybridisation. Tanscriptome analysis (M0+5 to M0+18). The membranes will be scanned using a highresolution BAS5000 scanner (Fuji, Génopôle de Toulouse) and the data will be managed using BASE software. Species-specific or non-specific regulated genes and gene networks will probably be identified. The regulation data will be further analysed using real-time quantitative PCR analysis (50 genes). Proteome analysis (M0+5 to M0+18). In parallel, the transcriptomic study will be completed by proteome analysis. Follicular fluids from each above-mentioned class of follicles will be analysed by bidimensional electrophoresis (2D-PAGE; five samples per class). First, 5µl follicular fluid will be loaded onto immobilised pH gradient strips (pH 4-7 and 6-11) from Amersham, and submitted to electrofocalisation (BioRad) and then SDS-PAGE (15x15 cm; Hoeffer). After colloidal blue staining (Invitrogen), all class-specific spots will be analysed by mass spectrometry (INRA Nouzilly). Our preliminary data demonstrates that mass spectrometry will concern around 100 spots per species. Alternatively, follicular fluids will be submitted to albumin and IgG depletion before 2D-PAGE (ProteoPrep Blue Albumin Depletion kit, Sigma). Actually follicular fluid is, at least in part, transudate of serum and thus contains high level of albumin and immunoglobulins. The depletion in both of these proteins will allow to load higher volumes, and
thus to visualize minor proteins.
Task 6.3 Designs for intra species experiments (INRA)
Task 6.3.1 Design for Booroola Granulosa cell culture (M0+9 to M0+12)
Granulosa cells from 12 wild type and 12 homozygous Booroola ewes will be cultured in the presence or absence of BMP-4 as previously described (Fabre et al., 2003). After 96h of culture, media will be recovered for progesterone assay. Transcriptome of ++/BB BMP-4 treated cells (M0+9 to M0+18); Cells (see above) will be scraped and RNA will be extracted and hybridised on bovine cDNA membranes. A total of 5 cultures will be performed, with four conditions (wild type granulosa
cells with and without BMP-4, and Booroola granulosa cells with and without BMP-4). Subtractive Suppressive Hybridisation approach (Diatchenko et al., 1996) will be used to generate a cDNA probe enriched in differentially expressed genes between the two conditions studied here: control and BMP-4-treated cells. These probes (control/BMP-4-treated and reverse experiment) will be used to hybridise the same bovine cDNA membranes.
Task 6.3.2 Design for Lacaune Granulosa cell culture (M0+9 to M0+12)
As for Booroola granulosa cells, granulosa cells from 12 wild type and 12 Lacaune autosomal homozygous ewes will be cultured in the presence or absence of BMP-4. After 96h of culture, media will be recovered for progesterone assay. Study of expression of target genes of BMP (M0+13 to M0+18). Cells (see above) will be scraped to study the expression of known target genes of BMP, i.e. the Star protein as well as P450scc and 3beta-HSD steroidogenic enzymes. If clear differences between genotypes are observed, later on we will plan comparative transcriptomic studies in response to BMP-4 as proposed above for Booroola ewes.
Task 6.3.3 Design for twinning cattle
Phenotype and segregation analysis for animal choice (M0+1 to M0+6), Granulosa cell culture (M0+1 to M0+>18). We will conduct experiments of granulosa cell culture as described above. Granulosa cells from mono- and polyovulating cows will be cultured during 96h or 144h in the presence or absence of BMP-4, and media will be recovered for progesterone assay to test the hypothesis of an alteration of BMP signalling in polyovulating animals. A total of ten cultures with granulosa cells from both groups will be performed. This experiment will be done separately on each animal, when it will become available from the experimental flock. T
Task 6.4 Identification of genes involved in embryonic developmental competence (MTT, Bonn University)
Task 6.4.1 Detailed documentation of environmental and genetic factors affecting developmental capacity and postimplantation survival of preimplantation bovine embryos (M0+1 to M0+18)
Task 6.4.2 Construction of bovine preimplantation specific cDNA array (M0+1 to M0+12)
A total of ~ 300 bovine preimplantation specific transcripts will be prepared to be used as targets to construct the preimplantation specific array. These transcripts include those generated by Bonn using various methods (stage specific cDNA library, differential display RT-PCR and suppression subtractive hybridization) and others from the available public gene bank. These transcripts are known to be involved in various developmentally important pathways during embryogenesis. A minimum of 200 ng/µl of each clone will be purified and prepared to be spotted on glass slides by core-service provided by commercial companies.
Task 6.4.3 Endometrium biopsy collection from pre-transfer cycles of recipients (M0+3 to M0+18)
A total of at least 100 dairy cows (both in Germany and Finland) will be used as recipients. To obtain insight into the transcriptional activity of recipient’s endometrium, biopsies will be taken on day 0 (day of ovulation), day 7 and day 14 of the last or last two consecutive cycles prior to transfer. To avoid any individual variations the collected biopsies will be later pooled for analysis based on the pregnancy results obtained after transfer of biopsied blastocysts. Embryo survival or mortality at different stages of gestation will be used as phenotypical characteristics for embryo survivability. Accordingly, recipient’s endometrium biopsies will be pooled in three groups namely; those resulted in successful pregnancy and calf delivery (pool 1), possibly those which contained viable foetus at day 30 but not at day 60 after transfer (pool 2, optional!), and those resulted in no pregnancy at all (pool 3). A total of 3-4 independent pools will be made in each group to be used as independent probes during global gene expression analysis using bovine Affymetrix GenChip (with ~ 22,000 transcripts).
Task 6.4.4 In vivo production of bovine embryos from donor animals (M0+3 to M0+18)
Twenty donor dairy animals will be used to produce day 7 blastocysts for biopsy collection and subsequent transfer to recipients. For this, the donor animals will be superovulated with i.m. injection of 2000 IU equine chorionic gonadotrophin (eCG) at day 9 of oestrus cycle. After luteolysis induction by PGF-alpha at day 13, 3000 IU hCG will be administered intravenously at day 15. All superovulated animals will be artificially inseminated using selected bulls spermatozoa. A conventional uterine flushing technique will be applied to obtain transferable day 7 blastocysts. The number of donor animals to be utilized at a time coincided with the number of recipients available at any specific time. Based on the past experiences of the working group the number of transferable blastocysts obtained per donor animal is 4-6.
Task 6.4.5 Embryo biopsy and transfer (M0+3 to M0+18)
Immediately after flushing,morphologically good quality blastocysts will be used for biopsy collection, which is performed by taking 30-40% (both ICM and TE cells) of the intact blastocyst using a Beaver micro blade fixed to micromanipulator under an inverse microscope. The biopsied blastocysts i.e. 60-70% part will be cultured for 1-2 h in vitro and only well re-expanded blastocysts will be transferred to recipient animals. The corresponding biopsies will be frozen for further transcriptional analysis. Once embryos are transferred, pregnancy monitoring will be done at days 30 and 60 post-transfer. As it is the case for endometrial biopsies, embryo biopsies will also be pooled based on the pregnancy diagnostic out come.
Task 6.4.6 Development of a protocol for production of well-defined preimplantation embryos for use in transcriptome analysis
Preimplantation bovine embryos can be produced in vitro in large numbers, and they can be differing in developmental capacity and postimplantation survival based on either their different origin (e.g. from high yielding dairy cows at specified stress levels (milk yield level, distance from calving, feeding regimen etc.) or their different invitro period (being influenced by specified stress factors (reduced or increased temperature, addition of metabolic inhibitors etc.)). The research work will include documentation of the link between the different backgrounds of the preimplantation embryos and their subsequent developmental capacity and postimplantation survival. For this, established methods will be used with (a) morphological, functional and biochemical markers of the embryos in larger scales and (b) on their in-vivo survival after transfer to recipients for further development in-utero in small scale.
Task 6.5. Fine mapping of a segregating QTL (BTA7) affecting conception rate in dairy cows (ARO)
ARO completed a daughter design genome scan of the Israeli Holstein population for QTL affecting nine quantitative traits, including female fertility defined as the inverse of the number of inseminations to conception in percent. Eleven sire families including 5221 daughters were analyzed for 73 microsatellites. A highly significant QTL affecting female conception rate was identified on BTA 7 near position 15 cM, with an effect of 0.66 units of the cows’ genetic evaluations. This QTL is segregating in one sire family out of five analyzed for this chromosomal region. During the first 18 months of the project the QTL will be fine-mapped in the segregating sire family by means of interval mapping. Once the peak location and confidence interval have been clearly identified, six additional sire families will be analyzed for segregating markers near the peak location. Ten cows carrying the “low” sire haplotype, and ten cows carrying the “high” sire haplotype will be identified. There are very few remaining daughters of these sires in the population. Thus granddaughters carrying the two alternative sire haplotypes in the region of interest will be identified. If more than one sire is determined heterozygous for the QTL, then these cows will be split among the heterozygous sire families. Genetic material will be collected from 1000 granddaughters for each of the heterozygous sires by vaginal swabs. The granddaughter samples will also be used to determine the QTL allele frequencies, as described by Weller et al. (2002)50. If the favourable allele is relatively rare, then the economic value of this QTL will be much greater.
Task 6.6 Contribution to the development of a phenotype ontology database concerning reproduction traits of interest for livestock production (in collaboration with WP3)
Task 6.6.1 Development of reproductive trait vocabulary (M0+1 to M0+6)
To initiate the reproductive trait vocabulary, a high-level categorization of reproductive functions will be developed. Subsequently the vocabulary will be refined and the various phenotypes will be hierarchically structured.
Task 6.6.2 Phenotype data annotation (M0+ 7 to M0>18)
Phenotypes will be described relative to information on mutations, QTLs and the genetic background in which the phenotype was observed. In close collaboration with WP3 the phenotype data annotations will be included in a database system for phenotype ontologies.
(Preliminary) Task 6.7 Combine QTL mapping and gene expression results (DIAS)
Several partners in WP 6 work on functional genomics of genes related to fertility. The WP6 partner in Israel works on QTL detection and QTL data are also avaible to DIAS. Combining QTL mapping and gene expression results related to fertility is the aim of task 6.7. For example, by means of constructing
two complementary filters: QTL mapping filters on correct genomic location of genes, while geneexpression filters on the placing of genes in the correct regulatory pathway. This combined approach will reduce the number of candidate genes from perhaps several hundred to maybe a handful, while also filtering out false positives. Developed methods will be made available in a user-friendly format for application in other WPs and to the international user community. The task fits in with the work in WP1 that relates to integrating functional genomics and QTL results, and ensures the application of new tools on fertility data.