GATA4: Regulation of expression and functions in goat granulosa cells.

GATA4 plays a pivotal role in the reproductive processes of mammals. However, the research on GATA4 in goat ovary is limited. This study aimed to study the expression and function of GATA4 in goat ovary. Utilizing real-time PCR and western blot analysis, we studied the expression and regulatory mechanisms of GATA4 in goat ovary and granulosa cells (GCs). We found that GATA4 was expressed in all follicle types in the goat ovary, with significantly higher levels in GCs of larger follicles (>3 mm) compared to those in smaller follicles (<3 mm). Additionally, we demonstrated that human chorionic gonadotrophin (hCG) induced GATA4 mRNA expression via the activation of PKA, MEK, p38 MAPK, PKC, and PI3K pathways in vitro. Our study also showed that hCG suppressed the levels of miR-200b and miR-429, which in turn directly target GATA4, thereby modulating the basal and hCG-induced expression of GATA4. Functionally, we examined the effect of siRNA-mediated GATA4 knockdown on cell proliferation and hormone secretion in goat GCs. Our results revealed that knockdown of GATA4, miR-200b, and miR-429 suppressed cell proliferation. Moreover, knockdown of GATA4 decreased estradiol and progesterone production by inhibiting the promoter activities of CYP11A1, CYP19A1, HSD3B, and StAR. Collectively, our findings suggest a critical involvement of GATA4 in regulating goat GC survival and steroidogenesis.

Chi-miR-3880 mediates the regulatory role of interferon gamma in goat mammary gland.

A healthy mammary gland is a necessity for milk production of dairy goats. The role of chi-miR-3880 in goat lactation is illustrated in our previous study. Among the differentially expressed genes regulated by chi-miR-3880, one seventh were interferon stimulated genes, including MX1, MX2, IFIT3, IFI44L, and DDX58. As the inflammatory cytokine interferon gamma (IFNγ) has been identified as a potential marker of caseous lymphadenitis in lactating sheep, the interaction between IFNγ and immune-related microRNAs was explored in this study. Chi-miR-3880 was found to be one of the microRNAs downregulated by IFNγ in goat mammary epithelial cells (GMECs). The study illustrated that IFNγ/chi-miR-3880/DDX58 axis modulates GMEC proliferation and lipid formation through PI3K/AKT/mTOR pathway, and regulates apoptosis through Caspase-3 and Bcl-2/Bax pathways. The role of the axis in mammary involution was reflected by the expression of p53 and NF-κB. In conclusion, IFNγ/chi-miR-3880/DDX58 axis plays an important part in lactation.

miR-486 Responds to Apoptosis and Autophagy by Repressing SRSF3 Expression in Ovarian Granulosa Cells of Dairy Goats.

The accumulation of ovarian granulosa cell (GC) apoptosis underlies follicular atresia. By comparing the previous sequencing results, miR-486 was found to be differentially expressed at higher levels in the monotocous goat than in the polytocous goat. Unfortunately, the miRNA-mediated mechanisms by which the GC fate is regulated are unknown in Guanzhong dairy goats. Therefore, we investigated miR-486 expression in small and large follicles, as well as its impact on normal GC survival, apoptosis and autophagy in vitro. Here, we identified and characterized miR-486 interaction with Ser/Arg-rich splicing factor 3 (SRSF3) using luciferase reporter analysis, detecting its role in GC survival, apoptosis and autophagy regulation through qRT-PCR, Western blot, CCK-8, EdU, flow cytometry, mitochondrial membrane potential and monodansylcadaverine, etc. Our findings revealed prominent effects of miR-486 in the regulation of GC survival, apoptosis and autophagy by targeting SRSF3, which might explain the high differential expression of miR-486 in the ovaries of monotocous dairy goats. In summary, this study aimed to reveal the underlying molecular mechanism of miR-486 regulation on GC function and its effect on ovarian follicle atresia in dairy goats, as well as the functional interpretation of the downstream target gene SRSF3.

FecB mutation and litter size are associated with a 90-base pair deletion in BMPR1B in East Friesian and Hu crossbred sheep.

Litter size is a critical economic trait in livestock, but only a few studies have focused on associated indel mutations in BMPR1B, a key regulator of ovulation and litter size in sheep. We evaluated the effects of BMPR1B mutations on the reproductive performance of sheep. We used Hu, East Friesian, and East Friesian/Hu crossbred sheep as experimental subjects and identified a novel 90 bp deletion in BMPR1B, which coincides with the c.746A > G (FecB mutation) genotype. The correlation between the two loci and litter size was then evaluated. We identified three genotypes for the Del-90bp locus, namely, II, ID, and DD, and three genotypes for the c.746A > G locus, namely ++, B+, and BB. Both Del-90bp and c.746A > G significantly affected the litter size of Hu and East Friesian/Hu crossbred sheep. Linkage disequilibrium analysis revealed a strong linkage disequilibrium between these loci in Hu sheep and the F1 population (r2 > 0.33), which suggests that detecting this 90 bp deletion might be a simple method to identify the likely carriers of c.746A > G. However, the function of this 90-bp deletion still needs further exploration. We provide genetic data that can be used as a reference for the breeding of improved prolific traits in sheep.

Effect of MiR-100-5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo.

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR-100-5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR-100-5p was significantly inhibited in the receptive phase (RE) rather than in the pre-receptive phase (PE). Overexpression of miR-100-5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR-100-5p, HOXA1, was confirmed by 3'-UTR assays. Meanwhile, the product of HOXA1 mRNA RT-PCR increased in the RE more than that in the PE. The HOXA1-siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR-100-5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ-9110 which acted as a sponge for miR-100-5p reversed the relevant biological effects of miR-100-5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ-9110/miR-100-5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR-100-5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR-100-5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR-100-5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ-9110/miR-100-5p/HOXA1 axis in vivo.

PITX2 regulates steroidogenesis in granulosa cells of dairy goat by the WNT/ß-catenin pathway.

Paired-like homeodomain transcription factor 2 (PITX2), a major driver of multiple tissue development, is a transcription factor that regulates gene expression in organisms. However, it is unknown if PITX2 regulates goat granulosa cell (GC) steroidogenesis. Therefore, we investigated the role and mechanism of PITX2 in GC steroidogenesis. In our study, PITX2 significantly facilitated the secretion level of estrogen and progesterone through increasing CYP11A1, CYP19A1, and STAR mRNA and protein expressions in GCs. Furthermore, PITX2 participated in the WNT pathway, enhancing the production of E2 and P4 in GCs. PITX2 in GCs increased the DVL-1 and CTNNB1 expression, involved in the WNT/ß-catenin signaling pathway related to steroidogenesis. Moreover, GC steroidogenesis-related gene translation was decreased by CTNNB1-siRNA but enhanced when transfected with PITX2. PITX2 regulates secretion of E2 and P4 from GCs via the WNT/ß-catenin pathway and alters GC proliferation and steroidogenesis. These findings will help understand the role of PITX2 in goat ovarian follicular development and oocyte maturation.

Pou2F3 silencing enhanced the proliferation of mammary epithelial cells in dairy goat via PI3K/AKT/mTOR signaling pathway.

Pou2F3 (POU class 2 homeobox 3) is found to be ubiquitously expressed in multiple epidermal layer cells to mediating proliferation. Although some POU factors exert a crucial regulation in mammary epithelial cells (MECs), the biological function of Pou2F3 is unclear. In this study, we aimed to investigate the endogenous potential effects of Pou2F3 on the proliferation and the roles of PI3K/AKT/mTOR signaling pathway in MECs. We used small interfering RNA to silence Pou2F3 expression. The interfering efficiency of Pou2F3 was confirmed by using RT-qPCR and Western blot. The cell viability and proliferation were indicated by Cell Counting Kit-8 and EdU assays. Flow cytometry was performed to evaluate the cell apoptosis in MECs. These results demonstrated that Pou2F3 potently suppressed the proliferation and induced the apoptosis of MECs. Consistently, the primary protein expressions of PI3K/AKT/mTOR signaling pathway were examined by Western blot. Pou2F3 silencing significantly increased the phosphorylation of PI3K, AKT and mTOR expressions. Moreover, Pou2F3 silencing reduced the ratio of BCL-2/BAX protein expression. Our findings show that Pou2F3 silencing can induce the proliferation of MECs and decrease the cell apoptosis, which suggest that Pou2F3 may serve as a potential upstream regulator of PI3K/AKT/mTOR signaling pathway in MECs.

Surrogate Indexes of Insulin Resistance in Dairy Goats: Transitional Variation in Subclinical Hyperketonemia.

BACKGROUND: Dairy goats are highly susceptible to subclinical hyperketonemia (SCHK) during the transition period. This study aimed to compare the variation in metabolic parameters and surrogate indexes of insulin resistance (sIR) between goats with SCHK and clinically healthy (HEAL) goats during the transition period. METHODS: Twenty Guanzhong dairy goats were assorted to HEAL (n = 10) and SCHK (n = 10) groups according to the blood ß-hydroxybutyrate (BHBA) concentrations. The blood samples were taken from the jugular vein of each goat at -3, -2, -1, 0 (partum), +1, +2, and +3 weeks relative to kidding to analyses GLU and INS. The sIR was calculated from blood metabolic parameters. RESULTS: Compared with the HEAL goats, the insulin concentrations were significantly higher in SCHK goats during the first three weeks postpartum. The QUICKI, revised QUICKI (RQUICKI), and RQUICKIBHBA were significantly lower in goats with SCHK at 1 week postpartum, while the homeostasis model assessment-IR (HOMA-IR) was significantly higher. CONCLUSION: Goats with SCHK made more efforts through elevated insulin levels at early lactation than HEAL goats, thereby maintaining the normal glucose concentrations.

EGF-Induced miR-223 Modulates Goat Mammary Epithelial Cell Apoptosis and Inflammation via ISG15.

The health of mammary gland is essential for lactation. Epidermal growth factor (EGF) is reported to play an important role in lactation initiation and miR-223 is a conserved microRNA in anti-inflammation. In this study, EGF was found to induce a higher expression of miR-223 in goat mammary epithelial cell (gMEC). The downstream genes of miR-223 were screened by RNA sequencing, including Interferon-stimulated gene product 15 (ISG15), a pivotal immune responder, which was detected to be downregulated by EGF and miR-223. Due to the correlation between inflammation and apoptosis, the gMEC apoptosis modulated by EGF, miR-223, and ISG15 was investigated, and the protein expressions of Bcl-2/Bax, Caspase 3 and p53 were examined to evaluate the apoptosis of gMEC. The protein expressions of p-STAT3/STAT3, PR, FOXC1, and HOXA10, which had been shown to be related to inflammation, were detected to assess the inflammation of gMEC. This study provided a regulation axis, EGF/miR-223/ISG15, and illustrated its regulation to gMEC apoptosis and inflammation.

Oxidative status in dairy goats: periparturient variation and changes in subclinical hyperketonemia and hypocalcemia.

BACKGROUND: A better comprehension of the redox status during the periparturient period may facilitate the development of management and nutritional solutions to prevent subclinical hyperketonemia (SCHK) and subclinical hypocalcemia (SCHC) in dairy goats. We aimed to evaluate the variation in the redox status of dairy goats with SCHK and SCHC during their periparturient periods. Guanzhong dairy goats (n = 30) were assigned to SCHK (n = 10), SCHC (n = 10), and healthy (HEAL, n = 10) groups based on their blood ß-hydroxybutyrate (BHBA) and calcium (Ca) concentrations. Blood were withdrawn from goats every week from 3 weeks before the expected parturition date to 3 weeks post-kidding. On the same day, the body condition scores (BCS) were evaluated, and the milk yield was recorded for each goat. The metabolic profile parameters and the indicators of oxidative status were determined by using the standard biochemical techniques. RESULTS: In comparison with the HEAL goats, SCHK and SCHC goats presented with a more dramatic decline of BCS post-kidding and a significant decrease in the milk yield at 2- and 3-weeks postpartum, ignoring the obvious increase at 1-week postpartum. The levels of non-esterified fatty acids (NEFA) peaked at parturition, exhibiting significantly higher levels from 1-week prepartum to the parturition day in the SCHK and SCHC groups. The malondialdehyde (MDA) concentration was increased in the SCHK goats from 1-week antepartum until 3-weeks postpartum, with its concentration being significantly higher in the SCHC goats at parturition. The hydrogen peroxide (H2O2) concentration was significantly lower in the SCHK and SCHC goats from 2-weeks antepartum to 1-week post-kidding. The total antioxidant capacity (T-AOC) and the superoxide dismutase (SOD) level were decreased at 1-week antepartum in the SCHK and SCHC goats, respectively. The glutathione peroxidase (GSH-Px) level was increased in the SCHK and SCHC goats during the early lactation period. CONCLUSIONS: The SCHK and SCHC goats exerted more efforts to maintain their redox homeostasis and to ensure the production performance than the HEAL goats during their periparturient period, probably owing to more intense fat mobilization and lipid peroxidation in the former.

Whole Transcriptome Analysis: Implication to Estrous Cycle Regulation.

Estrous cycle is one of the placental mammal characteristics after sexual maturity, including estrus stage (ES) and diestrus stage (DS). Estrous cycle is important in female physiology and its disorder may lead to diseases, such as polycystic ovary syndrome, ovarian carcinoma, anxiety, and epilepsy. In the latest years, effects of non-coding RNAs and messenger RNA (mRNA) on estrous cycle have started to arouse much concern, however, a whole transcriptome analysis among non-coding RNAs and mRNA has not been reported. Here, we report a whole transcriptome analysis of goat ovary in estrus and diestrus periods. Estrus synchronization was conducted to induce the estrus phase and on day 32, the goats shifted into the diestrus stage. The ovary RNA of estrus and diestrus stages was respectively collected to perform RNA-sequencing. Then, the circular RNA (circRNA), microRNA (miRNA), long non-coding RNA (lncRNA), and mRNA databases of goat ovary were acquired, and the differential expressions between estrus and diestrus stages were screened to construct circRNA-miRNA-mRNA/lncRNA and lncRNA-miRNA/mRNA networks, thus providing potential pathways that are involved in the regulation of estrous cycle. Differentially expressed mRNAs, such as MMP9, TIMP1, 3BHSD, and PTGIS, and differentially expressed miRNAs that play key roles in the regulation of estrous cycle, such as miR-21-3p, miR-202-3p, and miR-223-3p, were extracted from the network. Our data provided the miRNA, circRNA, lncRNA, and mRNA databases of goat ovary and each differentially expressed profile between ES and DS. Networks among differentially expressed miRNAs, circRNAs, lncRNAs, and mRNAs were constructed to provide valuable resources for the study of estrous cycle and related diseases.

Circ-140/chi-miR-8516/STC1-MMP1 Regulates αs1-/ß-Casein Secretion and Lipid Formation in Goat Mammary Epithelial Cells.

MicroRNAs play an essential role in mammary gland development, and involution is a factor that limits lactation. Chi-miR-8516 is one of the validated microRNAs that regulates the expression of STC1 and MMP1, which surge during the involution of the mammary gland. This study aims to explore the direct or indirect regulation of STC1 and MMP1 by chi-miR-8516 and the regulation of chi-miR-8516 by circ-140. In goat mammary epithelial cells, we found that chi-miR-8516 takes circ-140 as a sponge and regulates MMP1 expression by targeting STC1 and promoting the phosphorylation of MAPK. The examination of αs1-/ß-casein and lipid showed the modulation of the circ-140/chi-miR-8516/STC1-MMP1 axis in casein secretion and lipid formation, which was regulated by the phosphorylation of mTOR and STAT5. This study illustrates an axis that regulates the synthesis of milk components, and explores the pathways in which the axis participates.

Effects of TG interaction factor 1 on synthesis of estradiol and progesterone in granulosa cells of goats through SMAD2/3-SP1 signaling pathway.

The TG interaction factor 1 (TGIF1) is of the TALE homologue domain protein family and is considered as a transcriptional repressor of SMAD protein that interacts with DNA through a specific consensus-binding site for TG and recruits mSin3A and histone deacetylases to the SMAD complex. In this study, there is the first detailed description of TGIF1 on steroidogenesis in goat granulosa cells. When there is a relatively greater expression of the TGIF1 gene, there is a lesser abundance of CYP11A1, CYP19A1, and StAR mRNA transcript and protein and 3ß-HSD mRNA transcript in granulosa cells of goats. Furthermore, there were lesser concentrations of 17ß-estradiol (E2) and progesterone (P4) in culture medium when there was greater TGIF1 gene expression and there were greater concentrations of these hormones in the culture medium when there was lesser TGIF1 gene expression. There may be functions of TGIF1, therefore, in suppression of SMAD-induced E2 and P4 production and in decreasing the phosphorylation of SMAD2/3 in granulosa cells of goats and relative abundance of the SMAD2/3 protein transcription factor, SP1. With suppression of TGIF1 gene expression, there was a reversal of SP1-induced suppression of steroidogenesis-related genes. Results of the present study provide insights about the potential mechanism underlying the regulation of granulosa cell steroidogenesis of goats by TGIF1.

OGR1 negatively regulates ß-casein and triglyceride synthesis and cell proliferation via the PI3K/AKT/mTOR signaling pathway in goat mammary epithelial cells.

Goat milk in some cases is less allergenic than cow milk, therefore, more people drink goat milk in the world, so it is necessary for us to improve the yield and quality of goat milk. Previous studies have shown that some genes are closely related to lactation. Ovarian cancer G protein-coupled 1 (OGR1) is a G protein-coupled receptor discovered recently. OGR1 is widely found in various tissues of organisms and is involved in cell skeleton reorganization, carcinogenesis, cell proliferation, and apoptosis by regulating multiple signaling pathways in cells. However, the modulating effect of OGR1 in lactation is still unknown. Therefore, the objective of this study is to investigate the function of OGR1 in goat mammary epithelial cells (GMECs). Flow cytometry, CCK8, EDU, enzyme-linked immunosorbent assay, and triglyceride test kit assays were performed and we found that OGR1 regulated Bcl-2/Bax ratio, Fas protein expression as well as the phosphorylation of AKT and mammalian target of rapamycin (mTOR). si-OGR1 could enhance the proliferation of GMECs by promoting G1/S phase progression and the synthesis of ß-casein and triglyceride. By contrast, OGR1 repressed GMECs proliferation and down-regulated the synthesis of ß-casein and triglyceride by blocking the PI3K/AKT/mTOR signaling pathway in GMECs.

Effects of miR-101-3p on goat granulosa cells in vitro and ovarian development in vivo via STC1.

BACKGROUND: MiRNAs act as pivotal post-transcriptional gene mediators in the regulation of diverse biological processes, including proliferation, development and apoptosis. Our previous study has showed that miR-101-3p is differentially expressed in dairy goat ovaries compared single with multiple litters. The objective of this research was to explore the potential function and molecular mechanism of miR-101-3p via its target STC1 in goat ovarian growth and development. RESULTS: cDNA libraries were constructed using goat granulosa cells transfected with miR-101-3p mimics and negative control by RNA-sequencing. In total, 142 differentially expressed unigenes (DEGs) were detected between two libraries, including 78 down-regulated and 64 up-regulated genes. GO and KEGG enrichment analysis showed the potential impacts of DEGs on ovarian development. STC1 was singled out from DEGs for further research owing to it regulates reproductive-related processes. In vitro, bioinformatics analysis and 3'-UTR assays confirmed that STC1 was a target of miR-101-3p. ELISA was performed to detect the estrogen (E2) and progesterone (P4) levels. CCK8, EdU and flow cytometry assays were performed to detect the proliferation and apoptosis of granulosa cells. Results showed that miR-101-3p regulated STAR, CYP19A1, CYP11A1 and 3ß-HSD steroid hormone synthesis-associated genes by STC1 depletion, thus promoted E2 and P4 secretions. MiR-101-3p also affected the key protein PI3K, PTEN, AKT and mTOR in PI3K-AKT pathway by STC1, thereby suppressing proliferation and promoting apoptosis of granulosa cells. In vivo, the distribution and expression levels of miR-101-3p in mouse ovaries were determined through fluorescence in situ hybridisation (FISH). Immunohistochemistry results showed that STC1 expression was suppressed in mouse ovaries in miR-101-3p-agonist and siRNA-STC1 groups. Small and stunted ovarian fragments, decreased numbers of follicles at diverse stages were observed using Hematoxylin-eosin (HE) staining, thereby showing unusual ovarian development after miR-101-3p overexpression or STC1 depletion. Inhibition of miR-101-3p manifested opposite results. CONCLUSIONS: Taken together, our results demonstrated a regulatory mechanism of miR-101-3p via STC1 in goat granulosa cells, and offered the first in vivo example of miR-101-3p and STC1 functions required for ovarian development.

MiR-184 Combined with STC2 Promotes Endometrial Epithelial Cell Apoptosis in Dairy Goats via RAS/RAF/MEK/ERK Pathway.

The endometrium undergoes a series of complex changes to form a receptive endometrium (RE) that allows the embryo to be implanted. The inability to establish endometrial receptivity of livestock causes embryo implantation failure and considerable losses to animal husbandry. MicroRNAs (miRNAs) are a class of noncoding RNAs. Studies have found that miRNAs can regulate many critical physiological processes, including the establishment of RE during embryo implantation. miR-184 is highly expressed in the endometrial receptive period of dairy goats. This study aimed to explore the effect of miR-184 on endometrial epithelial cell (EEC) apoptosis and RE establishment. Stanniocalcin2 (STC2) is a direct target of miR-184, and miR-184 decreases the expression of STC2 in dairy goat EECs. miR-184 can activate EECs apoptosis through the RAS/RAF/MEK/ERK pathway. Additionally, miR-184 increases the expression levels of RE marker genes, such as forkhead box M1 (FOXM1) and vascular endothelial growth factor (VEGF). These findings indicate that miR-184 promotes the apoptosis of endometrial epithelial cells in dairy goats by downregulating STC2 via the RAS/RAF/MEK/ERK pathway, and that it may also regulate the establishment of RE in dairy goats.

CircRNA8220 Sponges MiR-8516 to Regulate Cell Viability and Milk Synthesis via Ras/MEK/ERK and PI3K/AKT/mTOR Pathways in Goat Mammary Epithelial Cells.

Circular RNAs (circRNAs), which are considered a large class of endogenous noncoding RNAs, function as regulators in various biological procedures. In this study, the function and molecular mechanisms of circRNA8220 in goat mammary epithelial cells (GMECs) were explored. CircRNA8220 could spong miR-8516 and block the function of miR-8516 by binding to the target site of miR-8516 a negative feedback relationship existed between circRNA8220 and miR-8516. Stanniocalcin 2 (STC2) was a target gene of miR-8516. circRNA8220 could up-regulate the expression of STC2 by sponging miR-8516 in GMECs. circRNA8220/miR-8516/STC2 could promote proliferation and enhance the synthesis of ß-casein and triglycerides (TG) via Ras/MEK/ERK and PI3K/AKT/mTOR signaling pathways, respectively.

A Regulatory Circuit Orchestrated by Novel-miR-3880 Modulates Mammary Gland Development.

Milk casein and triglyceride content are important production traits in goats. Studies on mechanisms in milk casein secretion and mammary gland development is essential for milk goat breeding. miRNAs play an important role in goat lactation. While novel-miR-3880 is highly expressed at goat peak lactation stage, its molecular mechanism has not been studied. The purpose of the present study was to explore the relationship between novel-miR-3880 and lactation, as well as to construct a network among novel-miR-3880, ciRNA13761, and E74 like ETS transcription factor 2 (ELF2), thus further exploring their potential roles in milk components and mammary gland development. ELF2 was previously proven to be important in cell survival and proliferation, and 3'-UTR of ELF2 was predicted to have binding sites of novel-miR-3880. Our study found that the overexpression of novel-miR-3880 exerted anti-apoptotic and proliferative roles in GMEC, induced a boost in triglyceride synthesis, and caused a decrease in α s1-, α s2-, and ß-casein, but an increase in κ-casein secretion. Furthermore, treatment in mice indicated that novel-miR-3880 could promote mammary gland development and extend the lactation period, while novel-miR-3880 expression was found to be suppressed by ciRNA13761 as a miRNA sponge. The present study explores a mechanism of triglyceride synthesis and casein secretion, and reveals a crosstalk between ciRNA13761/novel-miR-3880/ELF2 axis and PI3K/AKT/mTOR/S6K1 pathway, to gain a better understanding of lactation traits in dairy goats.

CircRNA-006258 Sponge-Adsorbs miR-574-5p to Regulate Cell Growth and Milk Synthesis via EVI5L in Goat Mammary Epithelial Cells.

The development of the udder and the milk yield are closely related to the number and vitality of mammary epithelial cells. Many previous studies have proved that non-coding RNAs (ncRNAs) are widely involved in mammary gland development and the physiological activities of lactation. Our laboratory previous sequencing data revealed that miR-574-5p was differentially expressed during the colostrum and peak lactation stages, while the molecular mechanism of the regulatory effect of miR-574-5p on goat mammary epithelial cells (GMECs) is unclear. In this study, the targeting relationship was detected between miR-574-5p or ecotropic viral integration site 5-like (EVI5L) and circRNA-006258. The results declared that miR-574-5p induced the down-regulation of EVI5L expression at both the mRNA and protein levels, while circRNA-006258 relieved the inhibitory effect through adsorbing miR-574-5p. EVI5L blocked the G1 phase and promoted the S phase by activating the Rab23/ITGB1/TIAM1/Rac1-TGF-ß/Smad pathway in GMECs. By increasing the protein expression of Bcl2 and reducing the protein expression of Bax, EVI5L promoted cell growth and inhibited apoptosis. The activation of the PI3K/AKT-mTOR signaling pathway promoted the production of triacylglycerol (TAG) and ß-casein in GMECs. The circRNA-006258/miR-574-5p/EVI5L axis could regulate the cell growth and milk synthesis of GMECs by sponge-adsorbed miR-574-5p. These results would provide scientific evidence for precision animal breeding in the industry of dairy goats.

MiR-29 regulates the function of goat granulosa cell by targeting PTX3 via the PI3K/AKT/mTOR and Erk1/2 signaling pathways.

PTX3, a member of the pentraxin protein family, plays important roles in ovulation as a marker of cumulus cell-oocyte complex expansion. However, the expression and function of PTX3 in goat ovarian GCs remain unclear. We isolated GCs from small and large follicles and found that PTX3 expression was significantly decreased and miR-29 mRNA expression was significantly increased during the growth of antral follicles. MiR-29 decreased PTX3 expression by targeting its 3' untranslated. Furthermore, miR-29 promoted GC proliferation, suppressed steroidogenesis and apoptosis by targeting PTX3 via the activation of the PI3K/AKT/mTOR and Erk1/2 signaling pathways. Treatment with inhibitors also verified these results. Meanwhile, we found that PI3K/AKT/mTOR and Erk1/2 signaling pathways had different role in secretion of E2 and P4 by regulating differently various steroidogenic enzyme (CYP19A1, CYP11A1, StAR and HSD3B) expression. These outcomes indicate the potential role of PTX3 in goat follicular growth and atresia.