Differentially expressed transcripts study during pregnancy and postpartum anestrus of yak (Bos grunniens).

Background: Yak is the main livestock species in the plateau area, and its reproductive performance is low, usually two years or three years. A very few of yaks recover within a certain period of time after delivery and smoothly enter the next estrous cycle, while most of them enter the postpartum anestrus and show no estrus performance. However, the key biological factors and influencing mechanisms that cause postpartum anestrus in yaks are not clear. Objective: To study the expression of differential transcripts in ovaries of yak during pregnancy and postpartum anestrus. Methods: Each three yaks in pregnancy and anestrus under natural grazing conditions in Haiyan County, Qinghai Province were selected and slaughtered, and their ovaries were collected and sent to Biomarker Technologies. Oxford Nanopore Technologies single-molecule real-time electrical signal sequencing technology was used to perform full-length transcriptome sequencing. Astalavista software was used to identify the types of alternative splicing events in yak estrus and pregnancy, and TAPIS pipeline was used to identify alternative polyadenylation. Results: The results showed that there were 1751 differentially expressed transcripts (DETs) between pregnancy and anestrus in yak, of which 808 were upregulated and 943 were downregulated. GO analysis showed that the biological processes of DETs were mainly reproductive, reproductive and rhythmic processes. KEGG analysis showed that the DET cell junction-related adhesion junction protein (ß-catenin) and amino terminal kinase (JNK) were involved in FAs (local adhesion). Phosphatidylinositol-3-kinase (PI3K) is involved in the PI3K/AKT/mTOR signaling pathway. Circadian rhythm output cycle failure (Clock) and brain and muscle tissue aromatic hydrocarbon receptor nuclear transporter-like protein 1 (Bmal1) are involved in circadian rhythm signaling pathway. Conclusion: This study found that ß-catenin, JNK, PI3K, Clock and Bmal1 were closely related to postpartum anestrus in yak.

MEL regulates miR-21 and let-7b through the STAT3 cascade in the follicular granulosa cells of Tibetan sheep.

Under physiological and pathological conditions, melatonin (MEL) can regulate microRNA (miRNA) expression. However, the mechanisms underlying the regulatory effects of MEL on miRNAs in ovaries are not understood. Firstly, by using fluorescence in situ hybridisation, we found that in ovaries and follicular granulosa cells (FGCs), MT1 co-located with miR-21 and let-7b. Additionally, immunofluorescence revealed that MT1, STAT3, c-MYC and LIN28 proteins co-located. The mRNA and protein levels of STAT3, c-MYC and LIN28 increased under treatment with 10-7 M MEL. MEL induced an increase in miR-21 and a decrease in let-7b. The LIN28/let-7b and STAT3/miR-21 axes are related to cell differentiation, apoptosis and proliferation. We explored whether the STAT3/c-MYC/LIN28 pathway was involved in miRNA regulation by MEL to explore the putative mechanism of the above relationship. AG490, an inhibitor of the STAT3 pathway, was added before MEL treatment. AG490 inhibited the MEL-induced increases in STAT3, c-MYC, LIN28 and MT1 and changes in miRNA. Through live-cell detection, we discovered that MEL enhanced the proliferation of FGCs. However, the ki67 protein levels decreased when AG490 was added in advance. Furthermore, the dual-luciferase reporter assay verified that STAT3, LIN28 and MT1 were target genes of let-7b. Furthermore, STAT3 and SMAD7 were target genes of miR-21. In addition, the protein levels of the STAT3, c-MYC, LIN28 and MEL receptors decreased when let-7b was overexpressed in FGCs. Overall, MEL might regulate miRNA expression through the STAT3 pathway. In addition, a negative feedback loop between the STAT3 and miR-21 formed; MEL and let-7b antagonized each other in FGCs. These findings may provide a theoretical basis for improving the reproductive performance of Tibetan sheep through MEL and miRNAs.

Effects of melatonin on development and hormone secretion of sheep theca cells in vitro.

Theca cells (TCs) play a unique role in the structure and function of the ovary. They are not only the structural basis of the follicle but also the androgen-secreting cells in female mammals, which can affect the normal development and atresia of the follicle. The results showed that melatonin receptor (MTR) MT1 and MT2 were expressed on sheep TCs. In the present study, the effects of different concentrations of MT at 0, 10-10, 10-8, 10-6 and 10-4 M/L on sheep TCs with regards to the antioxidant levels, proliferation, apoptosis and steroid hormone secretion were investigated. The results showed that in sheep TCs, all concentrations of MT significantly decreased reactive oxygen species (ROS) concentration and BAX expression; increased Cat, Sod1, and BCL-2 expression. The proliferation viability of TCs was significantly inhibited in all groups except for 10-10 M/L MT, and the expression of cyclin D1 and CDK4 was significantly reduced. MT significantly increased StAR expression and progesterone secretion in TCs, but there was no significant effect on androgen secretion and CYP11A1, CYP17A1 and 3ß-HSD expression in all groups. MT-induced progesterone secretion was completely inhibited by Luzindole (a nonspecific MT1 and MT2 inhibitor) and partially inhibited by 4p-PDOT (specific MT2 inhibitor). MT-induced progesterone secretion can be inhibited by LY294002 (PI3K/AKT pathway inhibitor). This study indicated that MT inhibits apoptosis and proliferation of in vitro cultured sheep TCs, which has implications for slowing ovarian atresia and aging. MT activates the PI3K/Akt pathway to mediate the synthesis and secretion of progesterone by TCs. This study provides a basis for further exploration of the role of TCs on follicle development and ovarian steroid hormone secretion.

miR-21, miR-125b, and let-7b contribute to the involution of atretic follicles and corpus lutea in Tibetan sheep ovaries.

Follicular granulosa cells (FGCs) are crucial for ovarian follicle functions, and miRNAs are differentially expressed at various stages of follicular developments. In this study, we confirmed that miR-21, miR-125b, and let-7b were located in FGCs/luteal cells by in situ hybridization experiments. Moreover, miR-21 and miR-125b expressions were upregulated in late corpus lutea (CL) and atretic follicles (AF); let-7b expression was increased in early AF. After transfected with inhibitor or mimic of miRNAs in FGCs, we found that FGCs apoptosis was decreased in the miR-21-mi group but increased in the miR-125b-mi group using flow cytometry. mRNA and protein expression levels were determined for apoptosis-related factors (e.g., Bcl-2 and Bax), the potential target genes of miRNAs (e.g., SMAD7, SP1, and STAT3), hormone receptors (e.g., FSHR and LHR), and genes related to hormone secretion (e.g., CYP19, CYP11, and 3ßHSD). The protein levels of SMAD7 were decreased in the miR-21-mi group but opposite to SP1 and FSHR. In the let-7b-mi group, Bcl-2, SMAD7, and FSHR were suppressed but not Bax, CYP11, and 3ßHSD. However, hormone secretion was not changed in the supernatant of transfected FGCs. This study provides information about ovarian miRNAs to improve the fertility in Tibetan sheep.

Transcriptome and proteome analysis of pregnancy and postpartum anoestrus ovaries in yak.

BACKGROUND: Domestic yaks are the most important livestock species on the Qinghai-Tibetan Plateau. Adult female yaks normally breed in the warm season (July to September) and enter anestrous in the cold season (November to April). Nevertheless, it is unclear how ovarian activity is regulated at the molecular level. OBJECTIVES: The peculiarities of yak reproduction were assessed to explore the molecular mechanism of postpartum anestrus ovaries in yaks after pregnancy and parturition. METHODS: Sixty female yaks with calves were observed under natural grazing in Haiyan County, Qinghai Province. Three yak ovaries in pregnancy and postpartum anestrus were collected. RNA sequencing and quantitative proteomics were employed to analyze the pregnancy and postpartum ovaries after hypothermia to identify the genes and proteins related to the postpartum ovarian cycle. RESULTS: The results revealed 841 differentially expressed genes during the postpartum hypoestrus cycle; 347 were up-regulated and 494 genes were down-regulated. Fifty-seven differential proteins were screened: 38 were up-regulated and 19 were down-regulated. The differential genes and proteins were related to the yak reproduction process, rhythm process, progesterone-mediated oocyte maturation, PI3K/AKT signaling pathway, and MAPK signaling pathway categories. CONCLUSIONS: Transcriptome and proteomic sequencing approaches were used to investigate postpartum anestrus and pregnancy ovaries in yaks. The results confirmed that BHLHE40, SF1IX1, FBPX1, HSPCA, LHCGR, BMP15, and ET-1R could affect postpartum hypoestrus and control the state of estrus.

A comparative transcriptome and proteomics study of post-partum ovarian cycle arrest in yaks (Bos grunniens).

Post-partum ovarian cycle arrest is the main factor affecting yak reproductive efficiency. There are few reports regarding the molecular regulatory mechanism of post-partum oestrus at transcriptome and proteome levels in yaks. Our previous studies focussed on the ovaries of yaks with post-partum ovarian cycle arrest and post-partum oestrus yaks. In this study, RNA sequencing transcriptomic study was combined with quantitative proteomic analyses to identify post-partum ovarian cycle-related genes and proteins. Consequently, 1,149 genes and 24 proteins were found to be up- or downregulated during post-partum oestrus. The analysis of differentially regulated genes identified three gene or protein pairs that were synchronously upregulated and no gene or protein pairs that were synchronously downregulated, suggesting that these upregulated genes may regulate the post-partum ovarian cycle. The functional classification of these differentially expressed genes and proteins indicated their connection with the oocyte meiosis, the oestrogen signalling pathway, the progesterone-mediated oocyte maturation and the gonadotrophin-releasing hormone (GnRH) signalling pathway. In this study, a total of six genes and two proteins involved in the oocyte meiosis, the oestrogen signalling pathway, the progesterone-mediated oocyte maturation and the GnRH signalling pathway were identified. The CSNK1A1, M91_09723, M91_11326, M91_21439, M91_19073, SHC2, Atf6b, M91_03062, HSPCA and calmodulin could regulate oestrus, respectively, in the post-partum so as to control the anoestrus status.

Dynamics of apoptosis-related gene expression during follicular development in yak.

The potential reproduction power of domestic animals is limited by a complicated follicular atresia process. P53, caspase-9 (Casp9), Bax, Bcl-2 and Fas play a crucial role in the ovarian mitochondrion-dependent apoptosis and death receptor pathway. In accordance with this study, the expression levels of Casp9, Bax, Bcl-2 and Fas were analysed in ovaries and oviducts of yak by immunohistochemistry (IHC). P53 and the above in ovarian granulosa cells (GCs) from atretic (3-6 mm) to healthy follicles (6-8 mm) and in oviducts were examined from the luteal phase to the follicular phase during the oestrous circle by Western blot (WB) and real-time PCR (RT-PCR). Results demonstrated that typical classic apoptotic factors Casp9, Bax, Bcl-2 and Fas were expressed in the cytoplasm and zonal pellucida of oocytes, primordial follicles, primary follicles, ovarian surface epithelium, ovarian GCs, granular lutein cells, surface epithelia in oviduct uterotubal junction and oviduct ampulla during the luteal phase. RT-PCR and WB revealed that P53 and Fas significantly increased in GCs of atretic follicles. P53 and Casp9 increased in oviduct epithelium during the luteal phase, but Fas was unchanged. A contrary tendency was noted in Bcl-2 and Bax expression. Overall, P53 and Fas play an essential role in inducing GC apoptosis, and Bax, Bcl-2, Casp9 and P53 are involved in oviduct epithelial regeneration in yak.

Monascus sanguineus May Be a Natural Nothospecies.

The genus Monascus has important economic and ecological values. In 2016, we isolated a strain M. sanguineus. After studying the phylogenetic relationship of Monascus, we believe that M. sanguineus is an independent species and speculate that it is a natural nothospecies. Recently, the morphological characteristics and sequences of seven genes (ITS, LSU, ß-tubulin, calmodulin, RNA polymerase II subunit, ß-ketoacyl synthase, and mating-type locus 1-1) of 15 Monascus strains were analyzed, including sequencing of multiple clones of five protein genes in four M. sanguineus strains. Two types of haplotypes (A and B) were observed in the five protein genes of M. sanguineus. Haplotype A was closely related to M. ruber, and haplotype B may be derived from an unknown Monascus species. The results demonstrated that M. sanguineus including type strains may be a natural nothospecies. This study laid the foundation for further exploration of the M. sanguineus genome, and the study may be of significant importance for the Monascus fermentation industry.

Expression and localization of follicle-stimulating hormone receptor in the yak uterus during different stages of the oestrous cycle.

Morphological changes of the uterus and alterations in its secretory activity under the influence of steroid hormones been well documented. The oestrous cycle is also associated with significant changes in plasma follicle-stimulating hormone (FSH), whose effects are mediated through its receptor (FSHR). Reports showed that in many mammals, FSHR was expressed in gonadal and extragonadal tissues including cervix, female reproductive tract, and pituitary gland. Follicle-stimulating hormone (FSH) signals through endothelial FSHR directly stimulate angiogenesis and involved in the timing of birth in human, and the presence of FSHR in the placenta is essential for normal pregnancy in mice. But little is known about FSHR expression in the yak uterus. The main objective of the present study was to determine the expression and localization of FSHR in the yak uterus during different phases of the oestrous cycle. Results showed that FSHR protein was localized in the surface and glandular epithelial cells, stroma cells, myometrial smooth muscle cells and blood vessel endothelial cells. The expression of FSHR protein peaked at oestrus, significantly decreased at dioestrus (p < 0.05) and increased again at proestrus. FSHR mRNA was highly expressed at both proestrus and oestrus, and decreased at metestrus with the lowest values at dioestrus (p < 0.05). In conclusion, FSHR expression in the yak uterus changed with the stage of the oestrous cycle suggesting that FSHR plays an essential role in regulating the endometrial and myometrial functions during the oestrus cycle in the yak.

Immunogenicity of adenovirus and DNA vaccines co-expressing P39 and lumazine synthase proteins of Brucella abortus in BALB/c mice.

Brucella poses a great threat to animal and human health. Vaccination is the most promising strategy in the effort to control Brucella abortus (B. abortus) infection, but the currently used live vaccines interfere with diagnostic tests and could potentially result in disease outbreak. Therefore, new subunit vaccines and combined immunization strategies are currently under investigation. In this study, immunogenicity and protection ability of a recombinant adenovirus and plasmid DNA vaccine co-expressing P39 and lumazine synthase proteins of B. abortus were evaluated based on the construction of the two molecular vaccines. Four immunization strategies (single adenovirus, single DNA, adenovirus/DNA, DNA/adenovirus) were investigated. The results showed that the immunization strategy of DNA priming followed by adenovirus boosting induced robust humoral and cellular immune responses, and it significantly reduced the numbers of B. abortus in a mouse model. These results suggest that it could be a potential antigen candidate for development of a new subunit vaccine against B. abortus infection.