Abstract Simple Summary Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction. However, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. In this study, we performed a correlation analysis of transcriptomics and proteomics, and the results suggested several differentially expressed genes (DEGs)/differentially expressed proteins (DEPs), including galectin 3 (LGALS3), aspartoacylase (ASPA) and transthyretin (TTR), could be candidate genes influencing ovine litter size. Further analysis suggested that AS events, single nucleotide polymorphisms (SNPs) and microRNA (miRNA)-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. This study provides a new insight into ovine and even other mammalian reproduction. Abstract Previous studies revealed that alternative splicing (AS) events and gene variants played key roles in reproduction; however, their location and distribution in hypothalamic fecundity-related genes in sheep without the FecB mutation remain largely unknown. Therefore, in this study, we described the hypothalamic AS events and variants in differentially expressed genes (DEGs) in Small Tail Han sheep without the FecB mutation at polytocous sheep in the follicular phase vs. monotocous sheep in the follicular phase (PF vs. MF) and polytocous sheep in the luteal phase vs. monotocous sheep in the luteal phase (PL vs. ML) via an RNA-seq study for the first time. We found 39 DEGs with AS events (AS DEGs) in PF vs. MF, while 42 AS DEGs were identified in PL vs. ML. No DEGs with single nucleotide polymorphisms (SNPs) were observed in PF vs. MF, but five were identified in PL vs. ML. We also performed a correlation analysis of transcriptomics and proteomics, and the results suggested several key DEGs/differentially expressed proteins (DEPs), such as galectin 3 (LGALS3) in PF vs. MF and aspartoacylase (ASPA) and transthyretin (TTR) in PL vs. ML, could be candidate genes influencing ovine litter size. In addition, further analyses suggested that AS events, SNPs and miRNA-binding sites existed in key DEGs/DEPs, such as ASPA and TTR. All in all, this study provides a new insight into ovine and even other mammalian reproduction. Keywords: alternative splicing, transcriptomics, proteomics, SNPs, hypothalamus, sheep 1. Introduction In recent decades, many interesting phenomena have been revealed. The prominent one is the fact that the complexity of diverse organisms is not correlated with the number of genes coding proteins based on genome sequencing, which has gained much attention. Alternative splicing (AS) was then found to be responsible for this difference. AS is a mechanism by which primary transcripts stemming from protein-coding genes could be spliced into distinct isoforms and lead to the generation of diverse variants of proteins [[38]1]. Therefore, comparatively fewer protein-coding genes can generate many proteins with different functions, due to AS events, and maintain the cellular complexity of mammals. The outcomes of complexity caused by AS events could be increasing proteome diversity; introducing the terminal codons responsible for downregulation of mRNA expression; varying the untranslated regions (UTRs) which may influence the binding of non-coding RNAs and RNA stability [[39]2,[40]3,[41]4]. Several methods can be used to detect AS events, such as the analysis of expressed sequence tags or cDNA [[42]1], microarrays technology [[43]5] and recently developed in-depth genome-wide sequencing [[44]6]. AS events play major roles in diverse mammalian physiological activities. Detecting these AS events may, therefore, facilitate a better understanding of the functional specialization of cell types and tissues [[45]7]. Researchers have suggested that AS events were associated with diseases, including a novel biomarker for predicting triple-negative breast cancer [[46]8] and AS-associated genetic variants that increase the risks of bladder cancer in human [[47]9]. Reproduction is a key process for the generation of offspring in mammals. Ongoing research has found close relationships between AS events and reproductive events. Li et al. [[48]10] revealed that AS events found in the testes of Mongolian horses may be a key factor affecting spermatogenesis. A novel splice variant was discovered in Hydroxysteroid 17-Beta Dehydrogenase 3 genes in the testes of pigs and was highly expressed in Leydig cells, suggesting its key role in male reproductive traits [[49]11]. Efforts were also directed to explore the roles of AS events in female reproduction. Estrogen receptor alpha isoform delta 7 (ERΔ7) generated by AS events in the female myometrium functioned as a modulator of myometrial quiescence, which may facilitate fetal development [[50]12]. Importantly, Miao et al. [[51]13] uncovered many AS events associated with fecundity in different sheep breeds via ovarian transcriptomic analysis, suggesting the key roles of AS events in ovine reproduction. Single nucleotide polymorphisms (SNPs) and indels in genes can significantly influence gene and protein functions. FecB, one of the most famous genes influencing ovine fecundity, was a point mutation (from A to G) that occurred at base 746 of the bone morphogenetic protein receptor, type 1B gene [[52]14]. Most importantly, FecB has a dosage effect, which indicated that ewes with one copy of the FecB mutation could increase litter size by 1, while two copies of this mutations could significantly increase litter size by 1.5 [[53]15]. Further studies suggested that SNPs in fecundity genes, such as the growth differentiation factor 9 [[54]16], bone morphogenetic protein 15 (BMP15) [[55]17], BMP2, BMP7 [[56]18], the NLR family pyrin domain-containing 5 (NLRP5) and NLRP9 [[57]19], were highly associated with litter size in sheep. Regarding indels, RNA-seq can be an effective method to detect indels as demonstrated by different bioinformatics strategies in many fields such as clinical decision-making [[58]20], and many studies also found their key roles in litter size. The 11-bp insertion variant of intron 22 in DNA methyltransferase 3β was highly associated with litter size in goats [[59]21]. Three indels were detected in down syndrome cell adhesion molecule like 1, and further association analysis suggested that these indels were highly associated with litter size in goats [[60]22]. All in all, RNA-seq can be used as a reliable and effective method to detect AS events, SNPs and indels. With the development of sequencing technology, RNA-sequencing has widely been used to identify key genes and non-coding RNAs affecting complex traits such as reproduction. Zou et al. [[61]23] identified 289 differentially expressed genes (DEGs) from uniparous and multiple goats, and further analysis indicated that CD36, TNFAIP6, CYP11A1, SERPINA5 and PTGFR may be the candidate genes associated with fecundity. The pituitary is a key reproductive organ controlling hormone activities; previous pituitary transcriptomics studies identified SMAD2, NMB and EFNB3 as the key genes affecting ovine prolificacy in different fecundity sheep [[62]24]. Our previous study also indicated that GNRH1, PRL, GH, TRH and TTR may regulate the gonadotropin-releasing hormone (GnRH) activities in the hypothalamus [[63]25]. Proteins are the real embodiment of gene functions, and therefore, identifying candidate genes from the translational level can be more biologically informative than from the transcriptional level. Tang et al. [[64]26] uncovered several fecundity proteins, such as StAR and HSD3B, in sheep with different fecundities by ovarian proteomics. Our previous hypothalamic proteomics studies also suggested that (growth hormone) GH, insulin-like growth factor 1 receptor (IGF1R) and Transthyretin (TTR) played key roles in GnRH release [[65]27]. Importantly, transcription and translation are under dynamic changes and are different processes controlled by many factors. Therefore, combining transcriptomics and proteomics data can be an effective way to identify key genes associated with fecundity. Sheep are one of the animals whose reproduction was mainly controlled by hormones activities, and one of the key roles of the hypothalamus is initiating reproduction by releasing GnRH. Therefore, in this study, we combined the hypothalamic transcriptomic and proteomic data to identify key genes at transcriptional and translational levels and to try to explore the AS events and variants associated with reproduction occurred in hypothalami of sheep with different fecundities via hypothalamic RNA-seq. Our study may provide new insights and references