RESUMEN
Reproductive fitness of rams is seasonal, showing the highest libido during short days coinciding with the ovarian cyclicity resumption in the ewe. However, the remarkable variation in sexual behavior between rams impair farm efficiency and profitability. Intending to identify in vivo sexual behavior biomarkers that may aid farmers to select active rams, transcriptome profiling of blood was carried out by analyzing samples from 6 sexually active (A) and 6 nonactive (NA) Rasa Aragonesa rams using RNA-Seq technique. A total of 14,078 genes were expressed in blood but only four genes were differentially expressed (FDRâ <â 0.10) in the A vs. NA rams comparison. The genes, acrosin inhibitor 1 (ENSOARG00020023278) and SORCS2, were upregulated (log2FCâ >â 1) in active rams, whereas the CRYL1 and immunoglobulin lambda-1 light chain isoform X47 (ENSOARG00020025518) genes were downregulated (log2FCâ <â -1) in this same group. Gene set Enrichment Analysis (GSEA) identified 428 signaling pathways, predominantly related to biological processes. The lysosome pathway (GO:0005764) was the most enriched, and may affect fertility and sexual behavior, given the crucial role played by lysosomes in steroidogenesis, being the SORCS2 gene related to this signaling pathway. Furthermore, the enriched positive regulation of ERK1 and ERK2 cascade (GO:0070374) pathway is associated with reproductive phenotypes such as fertility via modulation of hypothalamic regulation and GnRH-mediated production of pituitary gonadotropins. Furthermore, external side of plasma membrane (GO:0009897), fibrillar center (GO:0001650), focal adhesion (GO:0005925), and lamellipodium (GO:0030027) pathways were also enriched, suggesting that some molecules of these pathways might also be involved in rams' sexual behavior. These results provide new clues for understanding the molecular regulation of sexual behavior in rams. Further investigations will be needed to confirm the functions of SORCS2 and CRYL1 in relation to sexual behavior.
Analyzing ram sexual behavior via blood transcriptome profiling can help to identify in vivo sexual behavior biomarkers as an innovative alternative to invasive and time-consuming methods in farms. Using RNA-sequencing technique, we compared 12 Rasa Aragonesa rams with different sexual behavior (6 sexually active and 6 nonactive) to identify differentially expressed genes (DEGs) in peripheral blood putatively responsible of libido differences between rams. Comparative analysis revealed four candidate genes and several signaling pathways related to sexual behavior such as lysosome, and positive regulation of the extracellular signal-regulated kinase 1/2 (ERK1 and ERK2) cascade. This data will be helpful for further investigations to understand the differences of sheep sexual behavior.
Asunto(s)
Conducta Sexual Animal , Transcriptoma , Animales , Femenino , Masculino , Fenotipo , Reproducción/genética , Conducta Sexual Animal/fisiología , Ovinos/genética , Oveja Doméstica , Cristalinas/genética , Receptores de Superficie Celular/genéticaRESUMEN
Reproductive seasonality is a limiting factor in sheep production. Sexual behavior is a key element in reproductive efficiency, and this function is regulated by the hypothalamus-pituitary-gonadal (HPG) axis. To understand the mechanisms of sexual behavior, transcriptomic sequencing technology was used to identify differentially expressed genes (DEGs) in the hypothalamus (HT), pars tuberalis (PT) and pineal gland (PG) in Rasa Aragonesa rams with different sexual behavior. Bioinformatics analysis of the 16,401 identified genes by RNA-Seq revealed 103 and 12 DEGs in the HT and the PG, respectively, at a false discovery rate (FDR) of 5% with an absolute value of expression ≥ 1 (log2FC). However, no DEGs were found in the PT. Functional annotation and pathway enrichment analysis showed that DEGs of HT were enriched mainly in neuroactive ligand-receptor interactions and signaling pathways, including notable candidate genes such as MTNR1A, CHRNA2, FSHB, LHB, GNRHR, AVP, PRL, PDYN, CGA, GABRD, and TSHB, which play a crucial role in sexual behavior. The GnRH and cAMP signaling pathways were also highlighted. In addition, gene set enrichment analysis (GSEA) identified potential pathways, dominated mainly by biological process category, that could be responsible for the differences in sexual behavior observed in rams. The intracellular protein transport and pattern specification process were enriched within the PT and the transcription factor binding and protein ubiquitination pathways for the PG. Thus, these pathways together may play an important role in the regulation of the sexual behavior in Rasa Aragonesa rams through the hypothalamic-pituitary-gonadal axis. The validation of 5 DEGs using reverse transcription quantitative polymerase chain reaction (RT-qPCR) showed expression patterns like the found with RNA-Seq. Overall, these results contribute to understanding the genomic basis of sexual behavior in rams. Our study demonstrates that multiple networks and pathways orchestrate sexual behavior in sheep.
Male sexual behavior is a key factor in reproduction, especially in seasonal breeders such as sheep. The identification of differentially expressed genes (DEGs) in brain regions involved in male reproduction and sexual behavior between rams with different sexual activity by RNA high-throughput sequencing can provide useful information to the sheep meat industry. This work aimed to determine the possible molecular mechanisms underlying the sexual behavior of Rasa Aragonesa rams by investigating transcriptional changes in the hypothalamus (HT), pars tuberalis (PT) and pineal gland (PG) between active (A) and nonactive (NA) rams. Comparative analysis revealed 103 and 12 DEGs between the A vs. NA comparison in the HT and the PG, respectively, but no DEGs were found in the PT. Gene ontology (GO) enrichment analysis of DEGs in HT samples revealed significant pathways, associated mainly with neuroactive ligand-receptor interactions, and the GnRH and cAMP signaling pathways. Furthermore, gene set enrichment analysis (GSEA) detected many overrepresented pathways related to sexual behavior via an interaction network within the hypothalamic-pituitary-gonadal axis. These data will be helpful for further investigations to look for mutations or functional single nucleotide polymorphisms (SNPs) that may be used for genetic assisted selection to improve sexual behavior in sheep.
Asunto(s)
Glándula Pineal , Transcriptoma , Ovinos/genética , Animales , Masculino , RNA-Seq/veterinaria , Hipotálamo/metabolismo , Oveja Doméstica , Fenotipo , Perfilación de la Expresión Génica/veterinariaRESUMEN
For understanding the molecular events underlying the follicular (F) and luteal (L) phases of estrous cycle, and anestrous (A) phase, the pars tuberalis (PT), and hypothalamus (HT) transcriptomes of 21 ewes were studied. In HT, 72 and 3 differential expression genes (DEGs) were found when comparing F vs. A and L vs. A, respectively. In PT, 6 and 4 DEGs were found in F vs. A and L vs. A comparisons, respectively. Enrichment analysis for DEGs between the F and A phases in the HT revealed significant clusters, mainly associated with actin-binding, and cytoskeleton, that are related to neural plasticity modulated by gonadal steroid hormones, as well as with oxytocin signaling. We found that DEGs in PT had higher differences in expression levels than those found in HT. In this sense, the ITLN was highly upregulated in the F and L vs. A phases, being MRPL57 and IRX4 highly downregulated in L vs. A comparison. The DDC gene in PT, related to LH regulation, was upregulated in the F phase. The gene set enrichment analysis (GSEA) revealed multiple pathways related to neurotransmission and neuronal plasticity. Our study reveals new candidate genes involved in the reproductive stages' transitions in seasonal sheep.
RESUMEN
The aim of this study was to characterize and identify causative polymorphisms in the leptin receptor (LEPR) gene responsible for the seasonal variation of reproductive traits in sheep. Three reproductive seasonality traits were studied: the total days of anoestrous (TDA), the progesterone cycling months (P4CM) and the oestrous cycling months (OCM). In total, 18 SNPs were detected in 33 ewes with extreme values for TDA and OCM. Six SNPs were non-synonymous substitutions and two of them were predicted in silico as deleterious: rs596133197 and rs403578195. These polymorphisms were then validated in 239 ewes. The SNP rs403578195, located in exon 8 and leading to a change of alanine to glycine (Ala284Gly) in the extracellular domain of the protein, was associated with the OCM trait, being the G allele associated with a decrease of 12 percent of the OCM trait. Haplotype analyses also suggested the involvement of other non-synonymous SNP located in exon 20 (rs405459906). This SNP also produces an amino acid change (Lys1069Glu) in the intracellular domain of the protein and segregates independently of rs403578195. These results confirm for the first time the role of the LEPR gene in sheep reproductive seasonality.
RESUMEN
A FecX-mutated allele called FecXR in the BMP15 gene has been described in Rasa aragonesa sheep. FecXR causes increased prolificacy when heterozygous and sterility when homozygous in ewes. However, highly prolific ewes without the FecXR allele have been found in this breed. Therefore, a genome-wide association study (GWAS) was performed in 158 ewes (tail H: N = 73, mean prolificacy ± standard deviation = 2.14 ± 0.26; tail L: N = 85, mean prolificacy = 1.06 ± 0.08) with the Ovine HD SNP BeadChip. In this analysis, the FecXGR allele was found to have genome-wide significance associated with prolificacy, first described in the Grivette sheep breed. We also identified a novel polymorphism in exon 2 of BMP15 in 9 high prolific ewes by Sanger sequencing. This new mutation, called FecXRA, is a SNP (Oar3.1_X: g. 50970948C > T; NM_001114767.1: c.1172C > T) that produces an amino acid substitution (ENSOART00000010201: p.T400I) that is predicted to be deleterious and to alter the predicted secondary structure of the mature protein. To confirm if this SNP had any the effect on prolificacy, we genotyped sires with known EBVs (Estimated Breeding Values), finding one hemizygous sire for the FecXRA allele with the highest EBV in the breeding program (effect on litter size at + 0.39 lamb per lambing). A very low frequency, ranging from 0.13 to 2%, was found for the FecXGR and FecXRA alleles in 3428 animals belonging to four different flocks. Finally, an association study was performed to validate and quantify the effects of the FecXGR and FecXRA alleles. Significant increased prolificacy of 0.52 ± 0.05, 0.42 ± 0.05 and 0.32 ± 0.01 were found when comparing FecXGR, FecXRA and FecXR heterozygous ewes to wild type homozygous ones. These effects are of the same order of magnitude as the effect of most of other known major genes for prolificacy. Only significant differences between FecXGR and FecXR were found among the three alleles associated with increased prolificacy. However, we cannot confirm the effect of the FecXRA allele at homozygous state because we did not find any homozygous ewes. These results confirm that these three alleles in the BMP15 gene that affect prolificacy co-segregate in Rasa aragonesa sheep.