Long noncoding RNA (lncRNA) plays a crucial role in the hypothalamic-pituitary-testis (HPT) axis associated with sheep reproduction. The pituitary plays a connecting role in the HPT axis. However, little is known of their expression pattern and potential roles in the pituitary gland. To explore the potential lncRNAs that regulate the male sheep pituitary development and sexual maturation, we constructed immature and mature sheep pituitary cDNA libraries (three-month-old, TM, and nine-month-old, NM, respectively, n = 3) for lncRNA and mRNA high-throughput sequencing. Firstly, the expression of lncRNA and mRNA were comparatively analyzed. 2417 known lncRNAs and 1256 new lncRNAs were identified. Then, 193 differentially expressed (DE) lncRNAs and 1407 DE mRNAs were found in the pituitary between the two groups. Moreover, mRNA-lncRNA interaction network was constructed according to the target gene prediction of lncRNA and functional enrichment analysis. Five candidate lncRNAs and their targeted genes HSD17B12, DCBLD2, PDPK1, GPX3 and DLL1 that enriched in growth and reproduction related pathways were further filtered. Lastly, the interaction of candidate lncRNA TCONS_00066406 and its targeted gene HSD17B12 were validated in in vitro of sheep pituitary cells. Our study provided a systematic presentation of lncRNAs and mRNAs in male sheep pituitary, which revealed the potential role of lncRNA in male reproduction. Full article

Baculoviral inhibitor of apoptosis repeat-containing 5 (Birc5), also known as survivin, is a member of the inhibitor of apoptosis (IAP) family of proteins and regulates the size of tissues through cell division control. The uterus is the most dynamically sized organ among tissues during the estrous cycle. Although Birc5 is expressed in some terminally differentiated cells, the regulation of its expression in the uterus remains unknown. We investigated the regulation of Birc5 expression in the mouse uterus. RT-PCR analysis showed that Birc5 was expressed in various tissues, including the uterus; the expression level of Birc5 was significantly higher at the diestrus stage. Immunohistochemistry and Western blotting analysis revealed that Birc5 was more active in luminal and glandular epithelium than in endometrial stroma. In ovariectomized mice, Birc5 expression in the uterus was gradually increased by estrogen treatment; however, progesterone injection decreased its expression. Estrogen-induced Birc5 expression was blocked by treatment with estrogen receptor antagonist, ICI 182, 780 and progesterone-reduced Birc5 expression was inhibited by the progesterone receptor antagonist RU486. These results suggest that Birc5 expression is dynamically regulated by a combination of estrogen and progesterone via their receptor-mediated signaling. Full article

Disorders of sex development (DSD) and reproduction are not uncommon among horses, though knowledge about their molecular causes is sparse. Here we characterized a ~200 kb homozygous deletion in chromosome 29 at 29.7–29.9 Mb. The region contains AKR1C genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens. Mutations in AKR1C genes are associated with human DSDs. Deletion boundaries, sequence properties and gene content were studied by PCR and whole genome sequencing of select deletion homozygotes and control animals. Deletion analysis by PCR in 940 horses, including 622 with DSDs and reproductive problems and 318 phenotypically normal controls, detected 67 deletion homozygotes of which 79% were developmentally or reproductively abnormal. Altogether, 8–9% of all abnormal horses were homozygous for the deletion, with the highest incidence (9.4%) among cryptorchids. The deletion was found in ~4% of our phenotypically normal cohort, ~1% of global warmblood horses and ponies, and ~7% of draught breeds of general horse population as retrieved from published data. Based on the abnormal phenotype of the carriers, the functionally relevant gene content, and the low incidence in general population, we consider the deletion in chromosome 29 as a risk factor for equine DSDs and reproductive disorders. Full article

Thymic degeneration and regeneration are regulated by estrogen and androgen. Recent studies have found that long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are involved in organ development. In this study, RNA sequencing (RNA-seq) results showed that ovariectomy significantly affected 333 lncRNAs, 51 miRNAs, and 144 mRNAs levels (p < 0.05 and |log2fold change| > 1), and orchiectomy significantly affected 165 lncRNAs, 165 miRNAs, and 208 mRNA levels in the thymus. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differentially expressed genes (DEGs) were closely related to cell development and immunity. Next, we constructed two lncRNA–miRNA–mRNA networks using Cytoscape based on the targeting relationship between differentially expressed miRNAs (DEMs) and DEGs and differentially expressed lncRNAs (DELs) analyzed by TargetScan and miRanda. Besides, we screened DEGs that were significantly enriched in GO and in ceRNA networks to verify their expression in thymocytes and thymic epithelial cells (TECs). In addition, we analyzed the promoter sequences of DEGs, and identified 25 causal transcription factors. Finally, we constructed transcription factor-miRNA-joint target gene networks. In conclusion, this study reveals the effects of estrogen and androgen on the expression of miRNAs, lncRNAs, and mRNAs in mice thymus, providing new insights into the regulation of thymic development by gonadal hormones and non-coding RNAs. Full article

In the domestic horse; failure of normal masculinization and virilization due to deficiency of androgenic action leads to a specific disorder of sexual development known as equine androgen insensitivity syndrome (AIS). Affected individuals appear to demonstrate an incoherency between their genetic sex and sexual phenotype; i.e., XY-sex chromosome constitution and female phenotypic appearance. AIS is well documented in humans. Here we report the finding of two novel genetic variants for the AR-gene identified in a Tennessee Walking Horse and a Thoroughbred horse mare; each in individual clinical cases of horse AIS syndrome. Full article

Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 genes in bovine GCs which were isolated from 3–8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3–8 mm), medium (9–12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. AMH, AMHR-II, BMP2, and FSHR genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the AMH, AMHR-II, and BMP2 expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance AMH and BMP2 abundance (p < 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines. Full article

Sexual dimorphism is a relevant factor in animal science, since it can affect the gene expression of economically important traits. Eventually, the interest in the prenatal phase in a transcriptome study may not comprise the period of development in which male and female conceptuses are phenotypically divergent. Therefore, it would be interesting if sex differentiation could be performed using transcriptome data, with no need for extra techniques. In this study, the sex of pig conceptuses (embryos at 25 days-old and fetuses at 35 days-old) was determined by reads counts per million (CPM) of Y chromosome-linked genes that were discrepant among samples. Thus, ten genes were used: DDX3Y, KDM5D, ZFY, EIF2S3Y, EIF1AY, LOC110255320, LOC110257894, LOC396706, LOC100625207, and LOC110255257. Conceptuses that presented reads CPM sum for these genes (ΣCPM_chrY) greater than 400 were classified as males and those with ΣCPM_chrY below 2 were classified as females. It was demonstrated that the sex identification can be performed at early stages of pig development from RNA-sequencing analysis of genes mapped on Y chromosome. Additionally, these results reinforce that sex determination is a mechanism conserved across mammals, highlighting the importance of using pigs as an animal model to study sex determination during human prenatal development. Full article

Testis cords are the embryonic precursors of the seminiferous tubules. Development of testis cords is a key event during embryonic testicular morphogenesis and is regulated by multiple signaling molecules produced by Sertoli cells. However, the exact nature and the cascade of molecular events underlying testis cord development remain to be uncovered. In the current study, we explored the role of DNA damage binding protein 1 (DDB1) in Sertoli cells during mouse testis cord development. The genetic ablation of Ddb1 specifically in Sertoli cells resulted in the compromised Sertoli cell proliferation and disruption of testis cord remodeling in neonatal mice. This testicular dysgenesis persisted through adulthood, resulting in smaller testis and low sperm production. Mechanistically, we observed that the DDB1 degradation can stabilize SET domain-containing lysine methyltransferase 8 (SET8), which subsequently decreases the phosphorylation of SMAD2, an essential intracellular component of transforming growth factor beta (TGFβ) signaling. Taken together, our results suggest an essential role of Ddb1 in Sertoli cell proliferation and normal remodeling of testis cords via TGFβ pathway. To our knowledge, this is the first upstream regulators of TGFβ pathway in Sertoli cells, and therefore it furthers our understanding of testis cord development. Full article

Background: The objectives of this study were to identify loci, positional candidate genes, gene-sets, and pathways associated with spontaneous abortion (SA) in cattle and compare these results with previous human SA studies to determine if cattle are a good SA model for humans. Pregnancy was determined at gestation day 35 for Holstein heifers and cows. Genotypes from 43,984 SNPs of 499 pregnant heifers and 498 pregnant cows that calved at full term (FT) were compared to 62 heifers and 28 cows experiencing SA. A genome-wide association analysis, gene-set enrichment analysis–single nucleotide polymorphism, and ingenuity pathway analysis were used to identify regions, pathways, and master regulators associated with SA in heifers, cows, and a combined population. Results: Twenty-three loci and 21 positional candidate genes were associated (p < 1 × 10⁻⁵) with SA and one of these (KIR3DS1) has been associated with SA in humans. Eight gene-sets (NES > 3.0) were enriched in SA and one was previously reported as enriched in human SA. Four master regulators (p < 0.01) were associated with SA within two populations. Conclusions: One locus associated with SA was validated and 39 positional candidate and leading-edge genes and 2 gene-sets were enriched in SA in cattle and in humans. Full article

Detection of selection footprints provides insight into the evolution process and the underlying mechanisms controlling the phenotypic diversity of traits that have been exposed to selection. Selection focused on certain characters, mapping certain genomic regions often shows a loss of genetic diversity with an increased level of homozygosity. Therefore, the runs of homozygosity (ROHs), homozygosity by descent (HBD), and effective population size (N_e) are effective tools for exploring the genetic diversity, understanding the demographic history, foretelling the signature of directional selection, and improving the breeding strategies to use and conserve genetic resources. We characterized the ROH, HBD, N_e, and signature of selection of six Chinese goat populations using single nucleotide polymorphism (SNP) 50K Illumina beadchips. Our results show an inverse relationship between the length and frequency of ROH. A long ROH length, higher level of inbreeding, long HBD segment, and smaller N_e in Guangfeng (GF) goats suggested intensive selection pressure and recent inbreeding in this breed. We identified six reproduction-related genes within the genomic regions with a high ROH frequency, of which two genes overlapped with a putative selection signature. The estimated pair-wise genetic differentiation (F_ST) among the populations is 9.60% and the inter- and intra-population molecular variations are 9.68% and 89.6%, respectively, indicating low to moderate genetic differentiation. Our selection signatures analysis revealed 54 loci harboring 86 putative candidate genes, with a strong signature of selection. Further analysis showed that several candidate genes, including MARF1, SYCP2, TMEM200C, SF1, ADCY1, and BMP5, are involved in goat fecundity. We identified 11 candidate genes by using cross-population extended haplotype homozygosity (XP-EHH) estimates, of which MARF1 and SF1 are under strong positive selection, as they are differentiated in high and low reproduction groups according to the three approaches used. Gene ontology enrichment analysis revealed that different biological pathways could be involved in the variation of fecundity in female goats. This study provides a new insight into the ROHs patterns for maintenance of within breed diversity and suggests a role of positive selection for genetic variation influencing fecundity in Chinese goat. Full article

High fertility and early puberty in Bos indicus heifers are desirable and genetically correlated traits in beef production. The hypothalamus–pituitary–ovarian (HPO) axis synthesizes steroid hormones, which contribute to the shift from the pre-pubertal state into the post-pubertal state and influence subsequent fertility. Understanding variations in abundance of proteins that govern steroid synthesis and ovarian signaling pathways remains crucial to understanding puberty and fertility. We used whole ovaries of six pre-pubertal and six post-pubertal Brahman heifers to conduct differential abundance analyses of protein profiles between the two physiological states. Extracted proteins were digested into peptides followed by identification and quantification with massspectrometry (MS) by sequential window acquisition of all instances of theoretical fragment ion mass spectrometry (SWATH-MS). MS and statistical analysis identified 566 significantly differentially abundant (DA) proteins (adjusted p < 0.05), which were then analyzed for gene ontology and pathway enrichment. Our data indicated an up-regulation of steroidogenic proteins contributing to progesterone synthesis at luteal phase post-puberty. Proteins related to progesterone signaling, TGF-β, retinoic acid, extracellular matrix, cytoskeleton, and pleiotrophin signaling were DA in this study. The DA proteins probably relate to the formation and function of the corpus luteum, which is only present after ovulation, post-puberty. Some DA proteins might also be related to granulosa cells signaling, which regulates oocyte maturation or arrest in ovaries prior to ovulation. Ten DA proteins were coded by genes previously associated with reproductive traits according to the animal quantitative trait loci (QTL) database. In conclusion, the DA proteins and their pathways were related to ovarian activity in Bos indicus cattle. The genes that code for these proteins may explain some known QTLs and could be targeted in future genetic studies. Full article

A single male domestic shorthair cat that did not complete puberty was reported. At four years of age, it still had primary dentition, testicular hypoplasia, and was relatively small for its age. We hypothesized that the phenotype might have been due to an inherited form of hypogonadotropic hypogonadism (HH). We sequenced the genome of the affected cat and compared the data to 38 genomes from control cats. A search for private variants in 40 candidate genes associated with human HH revealed a single protein-changing variant in the affected cat. It was located in the TAC3 gene encoding tachykinin 3, a precursor protein of the signaling molecule neurokinin B, which is known to play a role in sexual development. TAC3 variants have been reported in human patients with HH. The identified feline variant, TAC3:c.220G>A or p.(Val74Met), affects a moderately conserved region of the precursor protein, 11 residues away from the mature neurokinin B sequence. The affected cat was homozygous for the mutant allele. In a cohort of 171 randomly sampled cats, 169 were homozygous for the wildtype allele and 2 were heterozygous. These data tentatively suggest that the identified TAC3 variant might have caused the suppression of puberty in the affected cat. Full article

We have previously reported that glyoxalase domain-containing protein 4 (GLOD4) is expressed in sheep testes by proteome analysis, but its roles during testicular development remain unclear. The aim of this study was to understand the expression characteristics and biological functions of the GLOD4 gene in developmental Tibetan sheep testes. The cDNA sequence of the Tibetan sheep GLOD4 gene was cloned by the RT-PCR method, and the structural characteristics of the GLOD4 protein were analyzed using relevant bioinformatics software, including ProtParam, TMHMM, Signal P 4.1, SOPMA, and phyre2. The expression patterns and immunolocalization of GLOD4 were examined in developmental testes derived from three-month-old (3M), one-year-old (1Y), and three-year-old (3Y) Tibetan sheep using quantitative real-time PCR (qRT-PCR), Western blot, immunohistochemistry, and immunofluorescence staining. The sequence analysis showed that the coding sequence (CDS) region of the GLOD4 gene was 729 bp in length and encoded 242 amino acids. Bioinformatics analysis found that the nucleotide and amino acid sequence of Tibetan sheep GLOD4 exhibited the highest sequence similarity with goat and chiru, and the least with zig-zag eel, of the species compared. GLOD4 expressions at both the mRNA and protein levels were significantly higher in the testes of the 1Y and 3Y groups than those in the 3M group (p < 0.01). Immunohistochemistry and immunofluorescence results indicated that the GLOD4 protein was mainly localized in the cytoplasm of Leydig cells from Tibetan sheep testes throughout the development stages. These results taken together suggest that the GLOD4 gene may be implicated in the development of the Leydig cells of Tibetan sheep during different stages of maturity. Full article

Balanced chromosome rearrangements are one of the main etiological factors contributing to hypoprolificacy in the domestic pig. Amongst domestic animals, the pig is considered to have the highest prevalence of chromosome rearrangements. To date over 200 unique chromosome rearrangements have been identified. The factors predisposing pigs to chromosome rearrangements, however, remain poorly understood. Nevertheless, here we provide empirical evidence which sustains the notion that there is a non-random distribution of chromosomal rearrangement breakpoints in the pig genome. We sought to establish if there are structural chromosome factors near which rearrangement breakpoints preferentially occur. The distribution of rearrangement breakpoints was analyzed across three level, chromosomes, chromosome arms, and cytogenetic GTG-bands (G-banding using trypsin and giemsa). The frequency of illegitimate exchanges (e.g., reciprocal translocations) between individual chromosomes and chromosome arms appeared to be independent of chromosome length and centromere position. Meanwhile chromosome breakpoints were overrepresented on some specific G-bands, defining chromosome hotspots for ectopic exchanges. Cytogenetic band level factors, such as the length of bands, chromatin density, and presence of fragile sites, were associated with the presence of translocation breakpoints. The characteristics of these bands were largely similar to that of hotspots in the human genome. Therefore, those hotspots are proposed as a starting point for future molecular analyses into the genomic landscape of porcine chromosome rearrangements. Full article