Recent advances in early pregnancy loss diagnosis in dairy cows: New approaches.

Early pregnancy loss is a primary cause of low reproductive rates in dairy cows, posing severe economic losses to dairy farming. The accurate diagnosis of dairy cows with early pregnancy loss allows for oestrus synchronization, shortening day open, and increasing the overall conception rate of the herd. Several techniques are available for detecting early pregnancy loss in dairy cows, including rectal ultrasound, circulating blood progesterone, and pregnancy-associated glycoproteins (PAGs). Yet, there is a need to improve on existing techniques and develop novel strategies to identify cows with early pregnancy loss accurately. This manuscript reviews the applications of rectal ultrasound, circulating blood progesterone concentration, and PAGs in the diagnosis of pregnancy loss in dairy cows. The manuscript also discusses the recent progress of new technologies, including colour Doppler ultrasound (CDUS), interferon tau-induced genes (ISGs), and exosomal miRNA in diagnosing pregnancy loss in dairy cows. This study will provide an option for producers to re-breed cows with pregnancy loss, thereby reducing the calving interval and economic costs. Meanwhile, this manuscript might also act as a reference for exploring more economical and precise diagnostic technologies for early pregnancy loss in dairy cows.

ABA-CsABI5-CsCalS11 module upregulates Callose deposition of citrus infected with Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) primarily caused by Candidatus Liberibacter asiaticus (CLas) has been threatening citrus production globally. Under HLB conditions, an excessive accumulation of the polysaccharide callose in citrus phloem occurs, leading to phloem blockage and starch accumulation in leaves. The callose production is controlled by callose synthases (CalS), which have multiple members within plants. However, the knowledge of callose production in the citrus upon infection with CLas is limited. In this study, we firstly identified 11 CalSs in the Citrus sinensis genome through bioinformatics and found the expression pattern of CsCalS11 exhibited a positive correlation with callose deposition in CLas-infected leaves (correlation coefficient of 0.77, P ≤ 0.05). Knockdown of CsCalS11 resulted in a reduction of callose deposition and starch accumulation in CLas-infected citrus. Interestingly, we observed significantly higher concentrations of abscisic acid (ABA) in HLB-infected citrus leaves compared to uninfected ones. Furthermore, the expressions of CsABI5, CsPYR, and CsSnRK2 in the ABA pathway substantially increased in citrus leaves upon CLas infection. Additionally, the expression of CsCalS11 was significantly upregulated in citrus leaves following the application of exogenous ABA. We confirmed that CsABI5, a pivotal component of the ABA signaling pathway, regulates CsCalS11 expression by binding to its promoter using yeast one-hybrid assay, dual luciferase assay, and transient expression in citrus leaves. In conclusion, our findings strongly suggest that the CsABI5-CsCalS11 module plays a crucial role in regulating callose deposition through the ABA signaling pathway during CLas infection. The results also revealed new function of the ABA signaling pathway in plants under biotic stress.

Rfamide-related peptide-3(RFRP-3) receptor gene is expressed in mouse ovarian granulosa cells: Potential role of RFRP-3 in steroidogenesis and apoptosis.

RFRP-3 is a functional ortholog of avian GnIH and regulates reproductive activities in the gonads of animals. However, the role of RFRP-3 in the function of ovarian granulosa cells in mice remains unclear. First, we detected the expression of the RFRP-3 receptor (GPR147) in the ovarian granulosa cells of mice. Second, the effect of RFRP-3 treatment on estradiol and progesterone secretions from granulosa cells was tested by ELISA. Meanwhile, the expression of genes and proteins regulating steroid hormone synthesis was respectively examined by qPCR and western blot. Furthermore, the effect of RFRP-3 treatment on the apoptosis of granulosa cells was analyzed. The results revealed that the GPR147 protein (a RFRP-3 receptor) was expressed in the ovarian granulosa cells of mice. Low and medium doses RFRP-3 treatment significantly reduced progesterone secretion in the granulosa cells (P < 0.05), while RFRP-3 suppressed p450scc, 3ß-HSD, StAR, and FSHR expression in a non-dose-dependent manner. Moreover, RFRP-3 treatment might induce the apoptosis of granulosa cells. Additionally, low doses RFRP-3 significantly reduced p-ERK1/2 protein expression (P < 0.05) in the ovarian granulosa cells. We here, for the first time, confirmed that GPR147 was expressed in the ovarian granulosa cells of mice. Our findings suggested that and RFRP-3 regulates the granulosa cell function through the ERK signaling pathway, which will lay the foundation for uncovering molecular mechanisms by which RFRP-3 regulates follicle development in future.

Study on the Pattern of Postpartum Uterine Involution in Dairy Cows.

Postpartum uterine involution is necessary for the normal reproduction of dairy cows. The study aimed to investigate the pattern of postpartum uterine involution and the impact of parity on uterine involution in Chinese Holstein dairy cows. The diameter of the uterine cervix, pregnant uterine horn, and non-pregnant uterine horn were monitored using a B-mode veterinary ultrasound scanner at 5, 10, 15, 20, 25, 30, 35, and 40 days, respectively, after parturition in both multiparous and primiparous dairy cows. Meanwhile, the concentrations of hydroxyproline, E2, and IGF-1 were detected using ELISA at 5, 10, 15, 20, 30, and 40 d after parturition in both multiparous and primiparous dairy cows. Furthermore, the duration of uterine involution was compared in the multiparous and primiparous dairy cows. The results demonstrated that the diameter of the uterine cervix and the pregnant uterine horn did not decrease any further at 25 days postpartum for both the multiparous cows and the primiparous cows. Hydroxyproline levels gradually decreased with uterine involution; however, there was no significant variation in IGF-1 concentrations during uterine involution in the dairy cows. Although E2 concentrations of the peripheral plasma displayed an upward trend from day 5 to day 15 in the two groups of postpartum cows, there was no significant difference between the two groups during uterine involution. These results suggest that postpartum uterine involution was around 25 days postpartum in both the primiparous dairy cows and the multiparous Chinese Holstein dairy cows. Parity did not affect uterine involution in the postpartum Chinese Holstein dairy cows. The hydroxyproline levels of the peripheral blood may be an indicator of uterine involution in postpartum cows. Nonetheless, IGF-1 and E2 levels of the periphery blood are not associated with uterine involution in Chinese Holstein dairy cows.

Exosomes: The role in mammalian reproductive regulation and pregnancy-related diseases.

Exosomes are a kind of extracellular vesicles that are produced and secreted by different mammalian cells. They serve as cargo proteins and can transfer different kinds of biomolecules, including proteins, lipids, and nucleic acids, which consequently act on target cells to exert different biological effects. Recent years have witnessed a significant increase in the number of studies on exosomes due to the potential effects of exosomes in the diagnosis and treatment of cancers, neurodegenerative diseases, and immune disorders. Previous studies have demonstrated that exosomal contents, especially miRNAs, are implicated in numerous physiological processes such as reproduction, and are crucial regulators of mammalian reproduction and pregnancy-related diseases. Here, we describe the origin, composition, and intercellular communication of exosomes, and discuss their functions in follicular development, early embryonic development, embryonic implantation, male reproduction and development of pregnancy-related diseases in humans and animals. We believe this study will provide a foundation for revealing the mechanism of exosomes in regulating mammalian reproduction, and providing new approaches and ideas for the diagnosis and treatment of pregnancy-related diseases.

Analysis of reproduction-related transcriptomes on pineal-hypothalamic-pituitary-ovarian tissues during estrus and anestrus in Tan sheep.

Seasonal estrus is an important factor limiting the fertility of some animals such as sheep. Promoting estrus in the anestrus season is one of the major ways in improving the fecundity of seasonally breeding animals. The pineal-hypothalamus-pituitary-ovary (PHPO) axis plays a decisive role in regulating animal reproduction. However, the molecular mechanisms by which the PHPO axis regulates seasonal reproduction in animals are not well understood, especially in Tan sheep. To this end, we collected pineal, hypothalamus, pituitary and ovary tissues from Tan sheep during estrus and anestrus for RNA-Sequencing, and performed bioinformatics analysis on the entire regulatory axis of the pineal-hypothalamic-pituitary-ovary (PHPO). The results showed that 940, 1,638, 750, and 971 DEGs (differentially expressed genes, DEGs) were identified in pineal, hypothalamus, pituitary and ovary, respectively. GO analysis showed that DEGs from PHPO axis-related tissues were mainly enriched in "biological processes" such as transmembrane transport, peptide and amide biosynthesis and DNA synthesis. Meanwhile, KEGG enrichment analysis showed that the bile acid secretion pathway and the neuroactive ligand-receptor interaction pathway were significantly enriched. Additionally, four potential candidate genes related to seasonal reproduction (VEGFA, CDC20, ASPM, and PLCG2) were identified by gene expression profiling and protein-protein interaction (PPI) analysis. These findings will contribute to be better understanding of seasonal reproduction regulation in Tan sheep and will serve as a useful reference for molecular breeding of high fertility Tan sheep.

Integrative Analysis of miRNA-mRNA in Ovarian Granulosa Cells Treated with Kisspeptin in Tan Sheep.

Kisspeptin is a peptide hormone encoded by the kiss-1 gene that regulates animal reproduction. Our studies revealed that kisspeptin can regulate steroid hormone production and promote cell proliferation in ovarian granulosa cells of Tan sheep, but the mechanism has not yet been fully understood. We speculated that kisspeptin might promote steroid hormone production and cell proliferation by mediating the expression of specific miRNA and mRNA in granulosa cells. Accordingly, after granulosa cells were treated with kisspeptin, the RNA of cells was extracted to construct a cDNA library, and miRNA-mRNA sequencing was performed. Results showed that 1303 expressed genes and 605 expressed miRNAs were identified. Furthermore, eight differentially expressed miRNAs were found, and their target genes were significantly enriched in progesterone synthesis/metabolism, hormone biosynthesis, ovulation cycle, and steroid metabolism regulation. Meanwhile, mRNA was significantly enriched in steroid biosynthesis, IL-17 signaling pathway, and GnRH signaling pathway. Integrative analysis of miRNA-mRNA revealed that the significantly different oar-let-7b targets eight genes, of which EGR1 (early growth response-1) might play a significant role in regulating the function of granulosa cells, and miR-10a regulates lipid metabolism and steroid hormone synthesis by targeting HNRNPD. Additionally, PPI analysis revealed genes that are not miRNA targets but crucial to other biological processes in granulosa cells, implying that kisspeptin may also indirectly regulate granulosa cell function by these pathways. The findings of this work may help understand the molecular mechanism of kisspeptin regulating steroid hormone secretion, cell proliferation, and other physiological functions in ovarian granulosa cells of Tan sheep.