Transcriptome analysis reveals immune function-related mRNA expression in donkey mammary glands during four developmental stages.

The low susceptibility to mastitis of female donkey (jenny) mammary glands and the strong immune properties of donkey milk are acknowledged, but little is known about the genes involved in mammary gland immunity in jennies. Herein, we used RNA-sequencing and bioinformatics analyses to explore jenny mammary gland transcriptomes and detect potential functional differentially expressed (DE) mRNAs related to immunity during four specific developmental stages: foetal (F), pubertal (P), adult parous nonlactation (N) and lactation (L). A total of 2497, 583 and 1820 DE mRNAs were identified in jenny mammary glands at F vs. P, P vs. N, and N vs. L, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) analyses revealed numerous GO terms related to immune function, especially between F and P. Seven significantly enriched profiles were identified, among which 497 and 1261 DE mRNAs were upregulated in profiles 19 and 17. Eleven mRNAs were enriched in over 10 KEGG pathways. ß-2-microglobulin (B2M), immunoglobulin heavy constant mu (IGHM), toll like receptor 2 (TLR2), toll like receptor 4 (TLR4) and myeloid differentiation factor 88 (MYD88) were mainly involved in phosphoinositide 3-kinase (PI3K)-Akt signalling, phagosome and nuclear factor kappa-B (NF-kappa B) signalling pathways. The findings provide insight into the molecular features underpinning the low prevalence of intramammary infections (i.e., mastitis) in donkeys.

An Integrated Analysis of Lactation-Related miRNA and mRNA Expression Profiles in Donkey Mammary Glands.

Donkey milk is consumed by humans for its nutritional and therapeutic properties. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been implicated in the regulation of milk component synthesis and mammary gland development. However, the regulatory profile of the miRNAs and mRNAs involved in lactation in donkeys is unclear. We performed mRNA-seq and miRNA-seq and constructed coexpression regulatory networks for the mammary glands during the lactating and nonlactating period of jennies. We identified 3144 differentially expressed (DE) mRNAs (987 upregulated mRNAs and 2157 downregulated mRNAs) and 293 DE miRNAs (231 upregulated miRNAs and 62 downregulated miRNAs) in the lactating group compared to the nonlactating group. The DE miRNA target mRNA were significantly associated with pathways related to RNA polymerase, glycosphingolipid biosynthesis, mRNA surveillance, ribosome biogenesis in eukaryotes, glycerophospholipid metabolism, Ras signaling, and the fly hippo signaling pathway. The mRNA-miRNA coregulation analysis showed that novel-m0032-3p, miR-195, miR-26-5p, miR-23-3p, miR-674-3p, and miR-874-3p are key miRNAs that target mRNAs involved in immunity and milk lipid, protein, and vitamin metabolism in the jenny mammary gland. Our results improve the current knowledge of the molecular mechanisms regulating bioactive milk component metabolism in the mammary glands and could be used to improve milk production in donkeys.

Quantitative analysis of the serum proteome during early pregnancy in mares.

Equine pregnancy is currently diagnosed by rectal palpation, ultrasonographic examination, or by measuring changes in hormones in the blood. In the present study, we identified proteins that are differentially expressed in the sera of early pregnant and non-pregnant mares in order to develop a novel method for diagnosing equine pregnancy. Serum samples were obtained from 18 adult mares, pregnancy at day 32 after ovulation (n = 9) and in diestrus (n = 9). Proteomic analysis of the samples was conducted using liquid chromatography-electrospray ionization-tandem mass spectrometry. We identified 467 proteins from a total of 3514 peptides. Thirty-two proteins (15 upregulated and 17 downregulated) were significantly differentially expressed between the two groups. The Gene Ontology enrichment analysis revealed that they are related to extracellular matrix assembly, blood coagulation, and hemostasis, and the prominent molecular functions were integrin binding, cell adhesion molecule binding, and glycine C-acetyltransferase activity. The pathway analysis of Kyoto Encyclopaedia of Genes and Genomes showed that the top three pathways identified were glycine, serine, and threonine metabolism; cysteine and methionine metabolism; and ether lipid metabolism. The selected five serum proteins were newly potential candidates for pregnancy diagnosis in mares.

Comparative Analysis of Serum Mineral and Biochemical Parameter Profiles Between Late Pregnant and Early Lactating Jennies.

The purpose of this study was to investigate the profile change of serum trace and major elements, and biochemical and hematological parameters in jennies during late pregnancy and early lactation. Twenty-five healthy Chinese Liaoxi jennies were used in late pregnancy and early lactation. Results showed that the levels of Fe, total protein (TP), and aspartate aminotransferase (AST) were highly variable interindividual among the jennies. Early lactating jennies showed significantly lower serum levels of K, Se, AST, total cholesterol (TC), and triglyceride than late pregnant jennies (P < .05). Principal component analysis identified six and five principal components of serum mineral and biochemical parameters for late pregnant and early lactating jennies, respectively, which was supported by the cluster analysis findings. Strong clustering of serum Cu-Mn, iPhos-Se-TP, and Ca-Zn-alanine aminotransferase-TC was found in the late pregnant jennies, and strong clustering of serum Ca-Zn-Se-Mn-albumin, Na-Fe-AST-triglyceride, and K-Mg-Cu-TP was observed in the early lactating jennies. The study suggests a significant variation in the serum levels of mineral and biochemical parameters in late pregnant and early lactating jennies, which is valuable in estimating their physiological status and providing proper health care.

Label-Free Mass Spectrometry-Based Quantitative Proteomics Analysis of Serum Proteins During Early Pregnancy in Jennies (Equus asinus).

Early pregnancy in jennies is routinely determined by palpation per rectum or ultrasonography and also by detecting steroid hormone and chorionic gonadotropin levels in the blood, plasma, and serum. Herein we applied label-free mass spectrometry-based quantitative proteomics to identify serum proteins that were differentially expressed between early pregnant (day 45 after ovulation) and non-pregnant jennies. Bioinformatics analysis allowed illustration of pathways potentially involved in early pregnancy. We identified 295 proteins from a total of 2,569 peptides. Twenty-five proteins (22 upregulated and three downregulated) were significantly differentially expressed between the early pregnant and non-pregnant groups. The majority of the differentially expressed proteins were involved in defense response, early embryonic development, and hormone signaling pathways. Furthermore, functional protein analyses suggested that proteins were involved in binding, enzyme inhibitor activity, and enzyme regulator activity. Five serum proteins-granulin precursor/acrogranin, transgelin-2, fibronectin, fibrinogen-like 1, and thrombospondin 1-can be considered as novel, reliable candidates to detect pregnancy in jennies. To the best of our knowledge, this is the first study to use label-free mass spectrometry-based quantitative proteomics to analyze serum proteins during early pregnancy in jennies. Our results should facilitate the identification of valuable pregnancy diagnostic markers in early pregnant jennies.

Characterizing the Serum Proteome of Donkeys (Equus asinus).

Serum and plasma are commonly used in clinical practice considering the widely accepted fact that the "normal" protein expression pattern of a healthy animal changes under disease conditions. We herein used a label-free mass spectrometry-based quantitative proteomics approach to characterize the serum proteome of donkeys. A total of 277 unique proteins were identified from 2,388 unique peptides. Gene ontology analyses showed that the most frequent processes were related to metabolic activities and biological regulation, response to stimulus, and immune system processes. The main annotated areas of origin were the extracellular region, extracellular region part, and organelle, and their molecular functions included binding, catalytic activity, and molecular function regulator. Analyses using the Clusters of Orthologous Groups for Eukaryotic Complete Genomes database indicated that the identified proteins could be categorized into three main groups: signal transduction mechanisms, amino acid transport and metabolism, and defense mechanisms. Most of the unique proteins were associated with the complement and coagulation cascades, and they participated in several disease-related metabolic pathways. Our results should be crucial for further analyses of changes in different physiological and pathophysiological conditions in donkeys.

A Cross-Sectional Survey of Foaling-Related Parameters of Jennies (Equus asinus) Under Smallholder Farm Conditions in Northeast China.

It is important to assess the reproductive efficiency and improve the reproductive management to promote the donkey population development. The overall foaling-related parameters of jennies under smallholder farm conditions in China were hardly under investigation. A cross-sectional survey of randomly selected 694 smallholder farms was conducted in 40 villages in the north, east, and south areas of Western Liaoning Province and Eastern Inner Mongolia Autonomous Region between March and July, 2017. Foaling-related parameters were assessed such as the mean age at first foaling, foaling rate and foaling interval during 2014-2017. Only two-thirds of the adult jennies foaled during the 3-year survey period and delivered a foal every 1.9 years. The mean age at first foaling was 45.3 months. The lowest incidence of foaling rate was in coincidence with the peak of foaling. The overall mean values for the foaling rate and foaling interval were 75% and 500.5 days, respectively. Significant changes of foaling rates before the age of 15 years and foaling intervals in the first 8 parities were not observed. The results indicate that the jennies could maintain relatively fine foaling-related parameters under smallholder farm conditions in northeast of China. However, the foaling rates hardly remained high during the whole breeding season represented a major loss to the donkey breeding.

The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors.

Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.