Functional Fertility Genomics in Sheep

The conservation, characterization, and sustainable utilization of farm animal genetic resources have far-reaching socioeconomic effects. Finnsheep, which is native to Finland, is globally known for its exceptional prolificacy and has been exported to several countries to improve the productivity of local breeds. Prolificacy, mainly determined by the ovulation rate and litter size phenotypes, has high economic importance in sheep farming systems. The ovulation rate is associated with the number of ovulatory follicles produced during the follicular growth phase of the estrous cycle, and successful implantation of the conceptus ensures maximum litters. The primary aim of this study was to evaluate the exceptional prolificacy of Finnsheep. Another important and overarching aim was to explore the genetic aspects of two important reproductive events leading to ovulation and successful pregnancy in sheep. To gain an insight into how these two processes are regulated genetically and non-genetically, the whole transcriptomes of selected reproductive tissues relevant to folliculogenesis (first phase, ovary) and early pregnancy prior to implantation (second phase, corpus luteum (CL) and endometrium) were analyzed. In the main experiment, the ovaries of 31 Finnsheep (n = 11) and Texel (n = 11) ewes as well as their F1 crosses (n = 9) collected during surgery were biopsied; other tissues, including the CL and endometrium, were collected at the slaughterhouse. Half of the animals were maintained on a flushing diet during the entire experiment to study the influence of nutrition during early pregnancy. In addition, the transcriptomes of the ovarian and endometrial tissues of Finnsheep ewes were compared to those of European feral mouflons. For transcriptomic analysis, cDNA libraries targeting mRNAs and microRNAs were sequenced using the Illumina HiSeq 2000 system with 100-base pair (bp) paired-end and 50-bp single-end techniques, respectively. Gene expression results from the first phase of the experiment revealed that the flushing diet strongly affected the Texel and, to some extent, the F1-crosses of sheep; however, Finnsheep were not affected by the diet. Likewise, the gene expression profiles of the F1 crosses were more similar to those of Finnsheep than of Texel, possibly because of the parent-of-origin effects, such as genomic imprinting. While no major candidate genes reportedly associated with prolificacy were differentially or highly expressed, FecGF polymorphism (V371M) in GDF9 was present in half of the experimental Finnsheep and F1 crosses but completely absent in the Texel sheep. The second phase of the experiment provided an insight on the cross-talk between the CL and endometrium, as revealed by both the shared and tissue-specific genes. Finnsheep had a higher embryo mortality rate than Texel. The top expressed genes were associated with progesterone formation and events (elongation and attachment), leading to blastocyst implantation. Several endogenous retroviruses (ERVs) were expressed in the endometrium samples. Immune-related genes and pathways appeared to be differentially expressed and/or regulated in the CL of pure-breds. During the preimplantation stage, the immune system of the Finnsheep ewes appeared superior to that of the Texel ewes. Owing to the influence of interferon tau in massive gene expression changes starting around day 13, the gene expression profiles of the endometrium samples in response to elongated embryos were remarkably different from those in response to spherical embryos. Moreover, several interferon-stimulated genes were upregulated in the endometrium samples having elongated embryos. In addition to differentially expressed genes between the reproductive tissues of domestic (Finnsheep) and feral (European mouflon) sheep, we identified novel genes and miRNAs and their regulatory mechanisms associated with reproductive traits. A few novel structural findings were obtained as part of this work. Maternally imprinted miRNA cluster on chromosome 18 (C18MC), which is conserved exclusively among placental mammals, was reported for the first time in sheep. With 46 expressed miRNAs, sheep C18MC may be the largest miRNA cluster among all mammalian species. The miRNAs (>500) quantified in this thesis are valuable, given that only 153 sheep miRNAs have been identified till date. A novel ERV transcript with high similarity to the genomic region within the FecL locus and known to affect prolificacy was identified, indicating that ERV plays an important role in reproduction and may even contribute to litter size differences. Overall, the work presented in this thesis explored two critically important aspects of reproduction, i.e., ovulation and preimplantation, in sheep using state-of-the-art genomics and bioinformatics tools. The resources and findings from this thesis are highly relevant to both breeders and researchers studying sheep. The comprehensive list of genes and miRNAs expressed in the three key reproductive tissues is a useful resource for understanding transcriptional patterns during the follicular growth phase and preimplantation stage leading to pregnancy in sheep. The diet- and F1 cross-related experimental results will be valuable for implementing sheep breeding strategies aimed at achieving an optimum reproductive capacity. Furthermore, these results will provide a foundation for future research, and the data may have additional applications following the advancement of analysis tools and technologies.Lammas on ollut ihmisen kotieläimenä 9 000 – 11 000 vuotta. Se on yksi varhaisimmista kotieläimistä. Lampaan ja muiden kotieläinten geenivarojen tutkimisella, säilyttämisellä ja kestävällä hyödyntämisellä on pitkäaikaisia taloudellisia ja yhteiskunnallisia merkityksiä. Suomen alkuperäinen lammasrotu, suomenlammas, ja rodun geenit ovat maailmanlaajuisesti tärkeä eläingeenivara. Rotu on tunnettu poikkeuksellisesta hedelmällisyydestä ja sitä on viety useisiin maihin monien lammasrotujen hedelmällisyyden parantamiseksi. Naarashedelmällisyydellä, joka ilmenee ovulaatioasteena eli kiimakierrossa irtoavien munasolujen lukumääränä ja vuonuekokona eli karitsoiden lukumääränä, on huomattava taloudellinen merkitys lammastaloudelle. Suomenlampaan hedelmällisyyteen vaikuttavat geenit ovat kuitenkin huonosti tunnettuja. Tämän tutkimuksen tavoitteena oli tarkastella, mitkä geenit edistävät lampaan ja erityisesti suomenlampaan poikkeuksellista hedelmällisyyttä ja miten nämä geenit toimivat. Lisäksi tutkittiin, miten ruokinta vaikuttaa geenien toimintaan ja siten naarashedelmällisyyteen. Tutkimuksen pääasiallinen eläinaineisto koostui ruokintakokeessa olleista suomenlammas- ja texelrotuisista uuhista sekä näiden kahden rodun ensimmäisen polven risteytysuuhista (50% suomenlammas ja 50% texel). Uuhia oli ruokintakokeessa yhteensä 31 ja puolet kunkin roturyhmän (suomenlammas, texel ja F1-polvi) uuhista sai lisäenergiaa rehustuksessa toisen puolen ollessa vertailuryhmänä, joka oli ilman lisäenergiaa. Lisäenergian tarjoamisen on todettu nostavan uuhen ovulaatioastetta. Geenien toimintaa tarkasteltiin kahdessa eri lisääntymiskauden vaiheessa. Ensimmäinen vaihe oli follikkeleiden kasvuvaihe, jossa munasarjojen munarakkuloissa (follikkeleissa) kypsyy munasoluja. Tässä vaiheessa tutkimus kohdistui geenien toimintaan uuhien munasarjoissa. Toinen näytteiden keruuvaihe oli preimplantaatiovaihe eli vaihe ennen hedelmöittyneiden munasolujen kiinnittymistä kohdun limakalvoon eli endometriumiin. Tällöin tarkasteltiin geenien ilmentymistä munasarjojen keltarauhasissa (corpus luteum, CL) ja endometriumissa. Kudosnäytteistä eristettiin RNA. RNA-näytteet sekvensoitiin eli määritettiin geenien ilmentyneet osat uuden sukupolven sekvensointilaitteella. Näiden RNA-sekvenssien lisäksi tarkasteltiin niin sanottuja lyhyitä mikro-RNA-molekyylejä, jotka toimivat geenien säätelyssä vaikuttaen geenien toiminnan hiljentämiseen, toisin sanoen siihen, mistä perimän DNA:sta koodatuista RNA-sekvensseistä tuotetaan proteiineja. Bioinformatiikan menetelmiä hyödyntäen voitiin määrittää, mitkä geenit ovat ilmentyneet eri kudosnäytteissä ja mikä kunkin geenin ilmentymistaso on ollut eri roturyhmissä. Tutkimus tuotti runsaasti uutta tietoa lampaan naarashedelmällisyyteen liittyvästä geenien toiminnasta ja alkuperäisen suomenlampaan perinnöllisistä erityisominaisuuksista. Follikkeleiden kasvuvaiheen tarkastelu osoitti, että lisäenergian antaminen rehustuksessa vaikutti voimakkaimmin texel-lampaiden geenitoimintoihin ja ovulaatioasteen, lievästi F1-risteytysuuhin, mutta ei juurikaan suomenlammasuuhiin. Lisäksi F1-uuhien geenien toiminta muistutti enemmän suomenlampaan kuin texelin geenien ilmentymistä. Tämä viittaa niin sanottuun ei-mendelistiseen, epigeneettiseen periytymiseen (imprinting), jossa suomenlampaasta peräisin olevien geenien toiminta on ollut aktiivisempaa risteytys-F1-uuhien munasarjoissa kuin texelin geenien. Lisäksi todettiin tunnetusta hedelmällisyysgeenistä, GDF9:stä (Growth Differentiation Factor 9), pistemutaatio suomenlampaalla, minkä oletetaan kohottavan ovulaatioastetta. Preimplantaatiovaiheessa suomenlampaasta todettiin yli kaksi kertaa enemmän keltarauhasia munasarjassa (keskimäärin 4.1/munasarja) kuin texelillä (1.7) todistaen alkuperäisrodun korkeaa ovulaatioastetta. Sitä vastoin suomenlammas osoittautui hedelmöittyneiden munasolujen suhteen ”tuhlailevammaksi” kuin texeluuhet, sillä suomalampaalla ilmeni kohdussa enemmän alkiokuolemia kuin texelillä. Tutkimus antoi todistetta siitä, että suomenlampaalla on geneettisesti tehokas immuunijärjestelmä. Suomenlampaan keltarauhasessa MHC-proteiinien ja sytokiinien tuottamiseen vaikuttavien geenien ilmenemistaso oli korkea edistäen organismin puolustuskykyä haitallisia bakteereja ja viruksia vastaan. Tutkimus antoi alustavaa viitettä siitä, että suomenlampaalla voisi olla kyky alkioiden diapaussiin eli alkioiden myöhästyneeseen kiinnittymiseen eli implantaatioon kohdun limakalvoon. Alkioiden diapaussin säätelyssä yhdeksi tärkeäksi geeniksi on aikaisemmissa tutkimuksissa todettu TXNL1 (Thioredoxin-like protein 1), jonka geenitoiminta oli huomattavan aktiivista suomenlammas- ja F1-uuhilla. Geeniperusteinen todiste diapaussin mahdollisuudesta suomenlampaalla voi johtua rodun korkeasta ovulaatioasteesta ja pitkästä lisääntymiskaudesta. Kiinnostava havainto oli niin ikään endogeenisistä retroviruksista (ERV:ista) lähtöisin olevat RNA-sekvenssit kohdun endometriumnäytteissä ja niiden rooli hedelmällisyydessä. Endogeeniset retrovirukset ovat viruksesta lähtöisin olevaa perimän osia, jotka ovat liittyneet isäntälajin perimään evoluution kuluessa. Tutkimuksessa tunnistettiin uusi ERV-transkriptio, joka oli hyvin samankaltainen X-kromosomissa sijaitsevan FecL-lokuksen genomialueen kanssa ja jonka tiedettiin vaikuttavan hedelmällisyysominaisuuksiin. Tulos osoittaa, että kyseisellä ERV:llä on tärkeä rooli lisääntymisessä ja se voi selittää vuonuekoon eroja eri lammasroduilla. Lisäksi endogeenisillä retroviruksilla on osoitettu olevan merkitystä kohdun suojaamisessa virusinfektioilta, istukan kehityksessä ja alkioiden varhaiskehityksessä kohdussa. Väitöskirja tuotti uutta tietoa myös geenitoimintojen säätelystä lampaalla. Lyhyiden mikro-RNA-molekyylien tarkastelu osoitti, että maternaalisesti leimautunut mikro-RNA-klusteri (”rypäs”) sijaitsee lampaan kromosomissa 18, josta löydettiin 46:n mikro-RNA:n muodostama klusteri. Tämä mikro-RNA-klusteri on konservoitunut yksinomaan istukallisilla nisäkkäillä, ja se raportoitiin nyt ensimmäisen kerran lampaalla. Tässä tutkimuksessa tunnistettiin yli 500 erilaista mikro-RNA:ta, mikä on huomattavan paljon enemmän, mitä aiemmin on tunnistettu lampaalla (vain 153 lampaiden mikro-RNA:ta oli tunnistettu ennen tätä tutkimusta). Tämän väitöskirjan tuottama kattava luettelo geeneistä ja mikro-RNA:ista, jotka ilmentyvät kolmessa keskeisessä lisääntymiskudoksessa kahdessa eri lisääntymisvaiheessa, on hyödyllinen tutkimusresurssi geenien funktioiden ymmärtämiseen follikkeleiden kasvuvaiheessa ja alkionkehityksen preimplantaatiovaiheessa, mitkä lopulta johtavat uuhien tiinehtymiseen. Nämä tulokset tarjoavat perustan tuleville tutkimuksille ja niitä voidaan hyödyntää genomitutkimuksen analyysimenetelmien kehityksessä

Gene Expression Profiling of Corpus luteum Reveals Important Insights about Early Pregnancy in Domestic Sheep

The majority of pregnancy loss in ruminants occurs during the preimplantation stage, which is thus the most critical period determining reproductive success. Here, we performed a comparative transcriptome study by sequencing total mRNA from corpus luteum (CL) collected during the preimplantation stage of pregnancy in Finnsheep, Texel and F1 crosses. A total of 21,287 genes were expressed in our data. Highly expressed autosomal genes in the CL were associated with biological processes such as progesterone formation (STAR, CYP11A1, and HSD3B1) and embryo implantation (e.g., TIMP1, TIMP2 and TCTP). Among the list of differentially expressed genes, sialic acid-binding immunoglobulin (Ig)-like lectins (SIGLEC3, SIGLEC14, SIGLEC8), ribosomal proteins (RPL17, RPL34, RPS3A, MRPS33) and chemokines (CCL5, CCL24, CXCL13, CXCL9) were upregulated in Finnsheep, while four multidrug resistance-associated proteins (MRPs) were upregulated in Texel ewes. A total of 17 known genes and two uncharacterized noncoding RNAs (ncRNAs) were differentially expressed in breed-wise comparisons owing to the flushing diet effect. The significantly upregulated TXNL1 gene indicated potential for embryonic diapause in Finnsheep and F1. Moreover, we report, for the first time in any species, several genes that are active in the CL during early pregnancy (including TXNL1, SIGLEC14, SIGLEC8, MRP4, and CA5A).202

Functional profiling of the endometrium transcriptome during preimplantation development in Finnsheep, Texel and their F1 crosses

Carefully coordinated interaction between the endometrium and embryo is critical for the establishment and maintenance of pregnancy in mammals. By exploring the gene expression dynamics of this tissue during preimplantation development, we may be able to get insight into the genetic mechanisms of reproduction during early pregnancy. Here, we have performed comparative transcriptome profiling of the endometrium in response to spherical (Day 7 to Day 12) and elongated (Day 13 to Day 17) embryos in Finnsheep, Texel and their F1 crosses using RNA sequencing (RNA-seq) approach. A total of 21125 genes were expressed in our dataset of which 554 were significantly (absolute log2 fold change > 2.5; adjusted p-value < 0.01) upregulated in the endometrium with elongated embryos. Highly abundant autosomal genes in the endometrium were associated with biological processes such as facilitation of maternal recognition of pregnancy, trophoblast elongation and implantation (LGALS15, CST3, CST6, and EEF1A1). Several endogenous retroviruses (ERVs) including a novel ERV gene located in a reduced FecL locus potentially associated with sheep prolificacy were expressed in our dataset. Comparative transcriptome profiling of the endometrium having spherical and elongated embryos revealed distinct gene expression patterns. Genes that were upregulated in response to elongated embryos indicated the importance of immune system at the maternal-embryo interface prior to implantation

Differences in Adipose Gene Expression Profiles between Male and Female Even Reindeer (Rangifer tarandus) in Sakha (Yakutia)

Reindeer are native to harsh northern Eurasian environments which are characterized by long and cold winters, short summers, and limited pasture vegetation. Adipose tissues play a significant role in these animals by modulating energy metabolism, immunity, and reproduction. Here, we have investigated the transcriptome profiles of metacarpal, perirenal, and prescapular adipose tissues in Even reindeer and searched for genes that were differentially expressed in male and female individuals. A total of 15,551 genes were expressed, where the transcriptome profile of metacarpal adipose tissue was found to be distinct from that of perirenal and prescapular adipose tissues. Interestingly, 10 genes, including PRDM9, which is known to have an important role in adaptation and speciation in reindeer, were always upregulated in all three tissues of male reindeer

Whole-Genome Sequencing of Three Native Cattle Breeds Originating From the Northernmost Cattle Farming Regions

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Ovarian mRNA and miRNA Transcriptome Profiling of Domestic Sheep Breeds

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Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries)

Background: The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. Results: Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. Conclusions: The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation.Peer reviewe

Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries)

Abstract Background The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. Results Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. Conclusions The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation

Comparative mRNA and miRNA expression in European mouflon (Ovis musimon) and sheep (Ovis aries) provides novel insights into the genetic mechanisms for female reproductive success

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Whole-genome sequencing provides novel insights into the evolutionary history and genetic adaptation of reindeer populations in northern Eurasia

Domestic reindeer (Rangifer tarandus) play a vital role in the culture and livelihoods of indigenous people across northern Eurasia. These animals are well adapted to harsh environmental conditions, such as extreme cold, limited feed availability and long migration distances. Therefore, understanding the genomics of reindeer is crucial for improving their management, conservation and utilisation. In this study, we have generated a new genome assembly for the Fennoscandian domestic reindeer with high contiguity, making it the most complete reference genome for reindeer to date. The new genome assembly was utilised to explore genetic diversity, population structure and selective sweeps in Eurasian Rangifer tarandus populations which was based on the largest population genomic dataset for reindeer, encompassing 58 individuals from diverse populations. Phylogenetic analyses revealed distinct genetic clusters, with the Finnish wild forest reindeer (Rangifer tarandus fennicus) standing out as a unique subspecies. Divergence time estimates suggested a separation of ~ 52 thousand years ago (Kya) between the northern European Rangifer tarandus fennicus and Rangifer tarandus tarandus. Our study identified four main genetic clusters: Fennoscandian, the eastern/northern Russian and Alaskan group, the Finnish forest reindeer, and the Svalbard reindeer. Furthermore, two independent reindeer domestication processes were inferred, suggesting separate origins for the domestic Fennoscandian and eastern/northern Russian reindeer. Notably, shared genes under selection, including retroviral genes, point towards molecular domestication processes that aided adaptation of this species to diverse environments