Comparative Study on the Sperm Proteomes of Horses and Donkeys.

The reproductive performance of horse sperm and donkey sperm has been reported to differ. Sperm proteins play a crucial role in sperm viability and fertility. Although differences between species are known, no prior study has investigated disparities in the sperm proteome between horses and donkeys. Therefore, this study characterized and compared the sperm proteomes of horses and donkeys using 4D-DIA mass spectrometry technology. We identified 3436 proteins in horse sperm and 3404 proteins in donkey sperm. Of these, 3363 proteins were expressed in both horse and donkey sperm, with 73 proteins being specifically expressed in horse sperm, and 41 in donkey sperm. According to data analysis, donkeys exhibited a greater percentage of motility and progressive movement in straight-line sperm than horses, as well as lower percentages of static and slow sperm than horses. Joint analysis of the results from the horse and donkey sperm proteomes and their CEROS II-read parameters demonstrated a possible association between sperm proteins and their sperm viability patterns. These findings suggest that there are discrepancies in the expression levels and protein compositions of horse and donkey sperm and that certain specific proteins may be responsible for the differences in performance between these two species.

A Comparative Analysis of the Gene Expression Profiles in the Mammary Glands of Lactating and Nonlactating Mares at the Second Month of Gestation.

To investigate molecular regulation involved in lactation during pregnancy, this study focused on the transcriptomic profiles of mammary tissue from lactating and non-lactating Mongolian mares at the second month of gestation. A total of 4197 differentially expressed genes were identified by comparing mammary tissues from pregnant mares at two different states, including 1974 differentially expressed genes such as the milk protein-related genes a-s1-casein (CSN1S1), k-casein (CSN3), lactalbumin (LALBA), and lactoferrin (LTF), which were highly expressed in the lactating mares group, and overall, these differentially expressed genes were mainly associated with biological processes such as endoplasmic reticulum protein processing, the Toll-like receptor signaling pathway, steroid biosynthesis, cytokine-cytokine receptor interactions, and amino sugar and nucleotide glycolysis. These findings serve as a foundation for investigating the molecular underpinnings of lactation in pregnant equids.

Optimizing the preparation of paraffin sections from stallion testes.

The preparation of paraffin sections is an important experimental technique in animal histological research, and key factors that determine the quality of a section include the dehydration time, waxing time, and drying temperature of the paraffin section. Paraffin sections obtained from testis tissue of adult horses exhibited higher quality with clear tissue structure and complete cell morphology after they underwent gradient dehydration for 6 hours, were immersed in wax for 60 minutes, and were dried in a 75-degree oven for 15 minutes. The detailed, optimized procedures that are developed in the current study may simplify histological experiments and research on equine testes.

Different expression patterns of DNA methyltransferases during horse testis development.

DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was decreased and became similar to that at 0.5y. Immunofluorescence of DNMT1, DNMT3a and DNMT3b on testis samples confirmed the differential expression and localization of these three DNA methylation transferases during horse development. Further molecular biological studies are needed to understand the implications of the expression patterns of these DNMTs in horse testes.

Condition optimization for electroporation transfection in horse skeletal muscle satellite cells.

Satellite cells are an important cellular model for studying muscle growth and development and mammalian locomotion-related molecular mechanisms. In this study, we investigated the effects of voltage, pulse duration, and DNA dosage on horse skeletal muscle satellite cells' electroporation transfection efficiency using the eukaryotic expression plasmid Td Tomato-C1 (5.5 kb) encoding the red fluorescent protein gene mainly based on fluorescence-positive cell rate and cell survival rate. By comparison of different voltages, pulse durations, and DNA doses, horse skeletal muscle satellite cells have nearly 80% transfection efficiency under the condition of voltage 120 V, DNA dosage 7 µg/ml, and pulse duration 30 ms. This optimized electroporation condition would facilitate the application of horse skeletal muscle satellite cells in genetic studies of muscle function and related diseases.

Comprehensive analysis of the whole-transcriptome landscape of the ovarian cortex from Mongolian horses that reproduce seasonally.

The reproductive cycle of equines tends to be seasonal and is influenced by factors such as light and temperature. The process and methods of regulating the mare oestrous cycle in the anestrus period are still immature. The effects of noncoding RNAs and mRNAs on the oestrous cycle have aroused much interest, but corresponding analyses of seasonal mare ovaries have not been reported. Here, we report a whole transcriptome analysis of the Mongolian horse ovarian cortex collected in anestrus and diestrus periods. In total, 1081 mRNAs, 205 lncRNAs, 54 circRNAs, and 13 miRNAs were upregulated in winter anestrus ovarian cortex (WAO), and 1261 mRNAs, 90 lncRNAs, 29 circRNAs, and 40 miRNAs were upregulated in summer diestrus ovarian cortex (SDO). The GO and KEGG enrichment analysis of differentially expressed mRNAs and target genes of differentially expressed lncRNAs, circRNAs, and miRNAs revealed some key functions and pathways that may be related to follicle and oocyte development. We found that estrogen-related pathways were enriched in different RNAs. Our data were used to generate miRNA, circRNA, lncRNA, and mRNA databases from the Mongolian horse ovary and differential expression profiles between WAO and SDO; these results provide clues for exploring methods of estrus regulation in mares during the anestrus period.

Proteomic Differences Between the Ovulatory and Anovulatory Sides of the Mare's Follicular and Oviduct Fluid.

The follicular fluid and oviduct fluid play major roles in oocyte maturation, sperm activation, and fertilization. To better understand the physiological environments for equine oocyte maturation and fertilization, here we conducted the proteome analysis and comparison on follicular fluids and oviduct fluids from the ovulatory side and the anovulatory side. The results showed that there is no significant difference between two side oviduct fluids, but a total of 71 differential abundance proteins (DAPs) were identified between two side follicular fluids, of which 9 are up-regulated and 62 are down-regulated in ovulatory side follicle fluid versus anovulatory side follicle fluid. As we expected, the function classification and enrichment results indicate that up- and down-regulated proteins are largely related to oocyte meiosis, maturation and ovulation. Noticeably, among 9 up-regulated DAPs in ovulatory side follicle fluid, as the DAP with the greatest fold change, PLA2G1B may be a newly discovered component that influences the efficacy of horse IVM/IVF. The current findings add to our knowledge of the in vivo conditions and regulation of equine reproduction, as well as the regulatory mechanism underpinning alternative ovulation.

Lower expression of the equine maternally imprinted gene IGF2R is related to the slow proliferation of hinny embryonic fibroblast in vitro.

BACKGROUND: Proliferation of embryonic fibroblasts under the same cell culture conditions, hinny embryonic fibroblasts (HiEFs) was slower than horse embryonic fibroblast (HEFs), donkey embryonic fibroblasts (DEFs) and mule embryonic fibroblasts (MuEFs). The imprinted genes IGF2 and IGF2R are important for cell proliferation. Therefore, we investigated whether the slower proliferation of HiEFs is related to an aberrant gene expression of IGF2 or its receptors or genes influencing the expression of the IGF2 system. METHODS AND RESULTS: Real-time polymerase chain reaction, immunofluorescence and cell starving experiment in HEFs, DEFs, MuEFs and HiEFs revealed that the slower proliferation of HiEF in vitro was related to its lower expression of IGF2R (P < 0.001). Moreover, quantification of allele-specific expression and bisulfate assay confirmed that in both MuEFs and HiEFs, IGF2R had normal maternal imprinting, implying that the imprint aberrant was not involved in the lower IGF2R expression in HiEFs. CONCLUSIONS: The reduction of IGF2R expression in HiEFs is associated with its slower proliferation in vitro.

MSTN Regulatory Network in Mongolian Horse Muscle Satellite Cells Revealed with miRNA Interference Technologies.

Myostatin (MSTN), a member of the transforming growth factor-ß superfamily, inhibits the activation of muscle satellite cells. However, the role and regulatory network of MSTN in equine muscle cells are not well understood yet. We discovered that MSTN knockdown significantly reduces the proliferation rate of equine muscle satellite cells. In addition, after the RNA sequencing of equine satellite cells transfected with MSTN-interference plasmid and control plasmid, an analysis of the differentially expressed genes was carried out. It was revealed that MSTN regulatory networks mainly involve genes related to muscle function and cell-cycle regulation, and signaling pathways, such as Notch, MAPK, and WNT. Subsequent real-time PCR in equine satellite cells and immunohistochemistry on newborn and adult muscle also verified the MSTN regulatory network found in RNA sequencing analysis. The results of this study provide new insight into the regulatory mechanism of equine MSTN.

The transcriptome landscapes of allantochorion and vitelline-chorion in equine day 30 conceptus.

During equine early gestation, trophectoderm forms chorion tissue, which is composed of two parts that one is covering allantoin, called allantochorion (AC) and another is covering yolk sac, which here we call vitelline-chorion (VC). Given that little is known about the equine trophoblast-derived chorion differentiation at an early stage, we first compared the transcriptome of AC and VC of day 30 equine conceptus based on RNA-sequencing. As a result, we found that compared to VC, there are 484 DEGs, including 305 up- and 179 down-regulated genes in AC. GO and KEGG analysis indicated that up-regulated genes in AC are mainly cell proliferation and cell adhesion-related genes, participating in allantois expansion and allantochorionic-placenta formation; dominant genes in VC are extracellular exosome and other cell adhesion-related genes implicated in direct and indirect conceptus-maternal communication. Additionally, as for the progenitor chorion tissue of equine chorionic gonadotropin secreting endometrium cup-the chorionic girdle (CG), which locates at the junction of the dilating AC and regressing VC, we revealed its unique gene expression pattern and the gene regulation during its further differentiation in vitro. Collectively, this study sheds light on the molecular events regarding the trophoblast differentiation and function at an early stage of the equine preimplantation conceptus.