Blood transcriptome of Rasa Aragonesa rams with different sexual behavior phenotype reveals CRYL1 and SORCS2 as genes associated with this trait.

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 (FDR < 0.10) in the A vs. NA rams comparison. The genes, acrosin inhibitor 1 (ENSOARG00020023278) and SORCS2, were upregulated (log2FC > 1) in active rams, whereas the CRYL1 and immunoglobulin lambda-1 light chain isoform X47 (ENSOARG00020025518) genes were downregulated (log2FC < -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.

Exploring differentially expressed genes in hypothalamic, pars tuberalis and pineal gland transcriptomes in different sexual behavior phenotypes in rams using RNA-Seq.

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.

Characterization of the pars tuberalis and hypothalamus transcriptome in female sheep under different reproductive stages.

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.

Genome-Wide Association Study Demonstrates the Role Played by the CD226 Gene in Rasa Aragonesa Sheep Reproductive Seasonality.

A genome-wide association study (GWAS) was used to identify genomic regions influencing seasonality reproduction traits in Rasa Aragonesa sheep. Three traits associated with either ovarian function based on blood progesterone levels (total days of anoestrus and progesterone cycling months) or behavioral signs of oestrous (oestrous cycling months) were studied. The GWAS included 205 ewes genotyped using the 50k and 680k Illumina Ovine Beadchips. Only one SNP associated with the progesterone cycling months overcame the genome-wide significance level (rs404991855). Nine SNPs exhibited significant associations at the chromosome level, being the SNPs rs404991855 and rs418191944, that are located in the CD226 molecule (CD226) gene, associated with the three traits. This gene is related to reproductive diseases. Two other SNPs were located close to the neuropeptide Y (NPY) gene, which is involved in circadian rhythms. To validate the GWAS, partial characterization of both genes by Sanger sequencing, and genotyping of two synonymous and two nonsynonymous SNPs in the NPY and CD226 genes, respectively, were performed. SNP association analysis showed that only SNP rs404360094 in the exon 3 of the CD226 gene, which produces an amino acid substitution from asparagine (uncharged polar) to aspartic acid (acidic), was associated with the three seasonality traits. Our results suggest that the CD226 gene may be involved in the reproductive seasonality in Rasa Aragonesa.

The LEPR Gene Is Associated with Reproductive Seasonality Traits in Rasa Aragonesa Sheep.

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.

A new allele in the BMP15 gene (FecXRA) that affects prolificacy co-segregates with FecXR and FecXGR in Rasa aragonesa sheep.

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.

Ovine oocytes display a similar germinal vesicle configuration and global DNA methylation at prepubertal and adult ages.

Epigenetic mechanisms are thought to be involved in the reduced developmental capacity of early prepubertal ewe oocytes compared to their adult counterparts. In this study, we have analyzed the global DNA methylation pattern and in vitro meiotic and developmental competence of oocytes at the germinal vesicle (GV) stage obtained from adult and 3-month-old donors. All oocytes were aspirated from antral follicles with a diameter ≥3 mm, and DNA methylation on 5-methylcytosine was detected by immunofluorescence using an anti-methyl cytosine antibody. The main global chromatin configuration pattern shown by both prepubertal and adult ovine oocytes corresponded to condensed chromatin localized close to the nuclear envelope (the SNE pattern). Immunofluorescence showed that a global bright nuclear staining of 5-methylcytosine (5-mC) occurred in all germinal vesicle stage oocytes and matched the propidium iodide staining pattern. The total fluorescence intensity values of lamb GVs were not lower than those observed in adult GVs. The meiotic competence and cleavage rates were similar in adult and prepubertal oocytes, however, the developmental competence of embryos to reach blastocysts was higher for adult oocytes than lamb oocytes (p<0.0001). In conclusion, our results indicate that adult-size oocytes derived from 3 to 4 month old prepubertal ewes show similar GV morphology and DNA methylation staining patterns to those obtained from adult animals, despite exhibiting a lower developmental competence.

Genome-wide association studies for reproductive seasonality traits in Rasa Aragonesa sheep breed.

Sheep breeds from Mediterranean area show reproductive seasonal patterns of oestrous behaviour and ovulatory activity, mainly regulated by variation in the photoperiod. Maximal reproductive activity is associated with short days from August to March. The aim of this study therefore was, to identify new SNPs and genes associated to reproductive seasonality in sheep by using the Illumina OvineSNP50 Beadchip. A total of 239 adult Rasa Aragonesa breed ewes from one flock were controlled from January to August. Three reproductive seasonality traits were considered: the total days of anoestrus (TDA), based on weekly individual plasma progesterone levels and defined as the sum of days in anoestrus, considering anoestrus those periods with three or more consecutive P4 concentrations lower than 0.5 ng/ml; the progesterone cycling months (P4CM), defined for each ewe as the rate of cycling months between January and August based on progesterone determinations and the oestrus cycling months (OCM), defined for each ewe as the rate of months cycling between January and August based on oestrus records. Genotyping of 123 ewes was performed with the OvineSNP50 Infinium Beadchip. After the quality control (QC) performed on the raw genotypes, a total of 47,206 SNPs distributed over the 27 ovine chromosomes and 110 ewes were included in subsequent analyses. Principal component analysis revealed a substructure within the total dataset and identified 4 principal clusters in the experimental flock. None of the SNPs overcame the genome-wide significance level (P = 1.06 × 10-6). However, the SNPs OAR4_66002395 (9.41E-6), and OAR8_25877010 (1.86E-5) reached the genome-wide suggestive significance level (set to 2.32 × 10-5) for TDA and P4CM traits, respectively, while OAR23_14608581 was significant for both TDA (2.02E-5) and P4CM (1.05E-5) traits. Five SNPs evidenced association at chromosome-wise level: SNPs OAR4_66002395, OAR23_14608581 and s20800 (DTA), and OAR8_25877010, OAR23_14608581 and s48474 (P4CM). Several genes related to circadian and circannual rhythms were found close to these SNPs: NPSR1 (SNP OAR4_66002395), HS3ST5 (SNP OAR8_25877010), RPTOR (SNP s48474), and NPTX1 (SNP s48474) and could be considered as candidate gene related to TDA and P4CM traits.

BMP15 regulates the inhibin/activin system independently of ovulation rate control in sheep.

Polymorphisms in the gene encoding bone morphogenetic protein 15 (BMP15) have been associated with multiple ovulations in sheep. As BMP15 regulates inhibin expression in rodents, we assumed that the ovarian inhibin/activin system could mediate part of the effect of BMP15 mutations in the regulation of ovulation rate in sheep. To answer this question, we have studied the effects of two natural loss-of-function mutations of BMP15 on the expression of components of this system. The FecXR and the FecXGr mutations, when present respectively in Rasa Aragonesa ewes at the heterozygous state and in Grivette ewes at the homozygous state, were associated with a twofold increase in ovulation rate. There were only small differences between mutant and wild-type ewes for mRNA expression of INHA, INHBA, ACVR1B, ACVR2A, FST or TGFBR3 in granulosa cells and inhibin A or activin A concentrations in follicular fluid. Moreover, the effects of mutations differed between breeds. In cultures of granulosa cells from wild-type ewes, BMP15, acting alone or in synergy with GDF9, stimulated INHA, INHBA and FST expression, but inhibited the expression of TGFBR3 Activin A did not affect INHBA expression, but inhibited the expression of ACVR2A also. The complexity of the inhibin/activin system, including positive and antagonistic elements, and the differential regulation of these elements by BMP15 and activin can explain that the effects of BMP15 mutations differ when present in different genetic backgrounds. In conclusion, the ovarian inhibin/activin system is unlikely to participate in the increase of ovulation rate associated with BMP15 mutations in sheep.

The Bone Morphogenetic Protein 15 Up-Regulates the Anti-Müllerian Hormone Receptor Expression in Granulosa Cells.

CONTEXT: Anti-Müllerian hormone (AMH) is produced by the granulosa cells (GCs) of growing follicles and inhibits follicular development. OBJECTIVE: This study aimed to investigate the regulation of the AMH-specific type 2 receptor (AMHR2) gene expression in GCs by bone morphogenetic protein (BMP)15, BMP4 and growth differentiation factor (GDF)9. DESIGN, SETTING, AND PATIENTS: Their effects on AMHR2 and AMH mRNAs were studied in luteinized human GCs and in ovine GCs (oGCs) from small antral follicles. The effects of BMPs on human AMHR2 and AMH promoter reporter activities were analyzed in transfected oGCs. The in vivo effect of BMP15 on GCs AMHR2 and AMH expression was investigated by using Lacaune and Rasa Aragonesa hyperprolific ewes carrying loss-of-function mutations in BMP15. MAIN OUTCOME MEASURES: mRNAs were quantified by real-time RT-PCR. Promoter reporter constructs activities were quantified by the measurement of their luciferase activity. RESULTS: BMP15 and BMP4 enhanced AMHR2 and AMH expression in human GCs and in oGCs, whereas GDF9 had no effect. In oGCs, GDF9 increased BMP15 effect on AMH expression. Consistent with these results, BMP15 and BMP4, but not GDF9, enhanced AMHR2 promoter activity in oGCs, whereas GDF9 increased BMP15 effect on AMH promoter activity. Moreover, oGCs from both BMP15 mutant ewes had reduced AMHR2 mRNA levels but unchanged AMH expression compared with wild-type ewes. CONCLUSIONS: Altogether, these results suggest that the mechanisms of action of BMP15 on AMHR2 and AMH expression are different, and that by stimulating AMHR2 and AMH expression in GCs BMP15 enhances AMH inhibitory actions in GCs.

Early pregnancy diagnosis in sheep using near-infrared spectroscopy on blood plasma.

The objective of this study was to evaluate the ability of near-infrared reflectance spectroscopy (NIRS) to discriminate between pregnant and nonpregnant ewes in early stages of pregnancy after artificial insemination (AI) from blood plasma. Samples were collected using jugular puncture at 18 and 25 days after AI from 188 Rasa Aragonesa and Ansotana ewes. Plasma samples were analyzed for pregnancy-associated glycoprotein (PAG) and progesterone (P4) using ELISA commercial kits. The spectra of plasma samples were recorded in the visible and near-infrared ranges. The performance of these tests were compared, using as criterion standard the pregnancy status determined using transabdominal ultrasonography at 45 days after AI. Pregnancy rate was 47.9% (90/188). At Day 18, sensitivity was similar in NIRS and P4 tests (98.9% vs. 100%; not significant) and greater than PAG (32.2%; both P < 0.001). Specificity was similar in NIRS and PAG tests (both 100%) and greater than that of P4 (84.7%; P < 0.001). At Day 25, sensitivity and specificity of NIRS and PAG were both 100%. It can be concluded that NIRS was an accurate method of diagnosis of pregnancy at Days 18 and 25 after AI in ewes.

Anti-Müllerian hormone plasma concentration in prepubertal ewe lambs as a predictor of their fertility at a young age.

BACKGROUND: In mammals, the ovarian follicular reserve is highly variable between individuals and impacts strongly on ovarian function and fertility. Nowadays, the best endocrine marker of this reserve in human, mouse and cattle is the anti-Müllerian hormone (AMH). The objectives of this work were to determine whether AMH could be detected in the plasma of prepubertal ewe lambs and to assess its relationship with their fertility at a young age. RESULTS: Plasma was taken from 76 Rasa Aragonesa ewe lambs at 3.6 months of age for AMH determination. Simultaneously, 600 IU equine chorionic gonadotropin (eCG) was administered and the number of ovulations recorded 6 days later. AMH was detected in 93% of the lambs, and the concentrations were about 3-4-fold higher in ovulating than in non-ovulating lambs (P < 0.004). Ewes aged around 10 months were mated, giving an overall fertility of 29%, and those failing to conceive were mated again 4 months later. Fertility at first mating was significantly correlated with plasma AMH concentration at 3.6 months (Spearman's ρ = 0.34; P < 0.01). To use plasma AMH concentration as a screening test, a value of 97 pg/mL was determined as the optimum cutoff value to predict fertility at first mating (sensitivity = 68.2%; specificity = 72.2%). Fertility at first mating was 34.8 percentage points higher in ewe lambs with an AMH ≥ 97 pg/mL than in those with lower AMH concentrations (50% vs. 15%; P < 0.001). CONCLUSIONS: Plasma AMH concentration might be a reliable marker of the ovarian status of prepubertal ewe lambs, reflecting their ability to respond to eCG stimulation. A single AMH measurement performed on ewe lambs early in age could be useful to select for replacement ewes with a higher predicted fertility at first mating.