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.