Deciphering the miRNA transcriptome of granulosa cells from dominant and subordinate follicles at first follicular wave in goat.

In goats, most follicles in the ovaries will be atresia and only a few dominant follicles (DFs) may eventually mature and ovulate at a follicular wave. To investigate the potential microRNAs (miRNAs) that regulate the expression of genes associated with follicular dominance or atresia, small RNA sequencing was performed on granulosa cells of DF and subordinate follicle at the first follicular wave in goats. A total of 108 differentially expressed miRNAs were detected in the two types of follicle granulosa cells: 16 upregulated miRNAs and 92 downregulated miRNAs. Kyoto Encyclopedia of Genes and Genomes analysis of the target genes showed that TKTL1, LOC102187810, LOC102184409 and ALDOA are closely associated with follicle dominance and are involved in the pentose phosphate pathway. Furthermore, a coexpression network of miRNAs and follicular dominance-related genes was constructed. The qPCR results well correlated with the small RNA sequencing data. Our findings provide new insight for exploring the molecular mechanism of miRNAs in regulating follicular development in goats.

Granulosa cell transcriptomic study reveals the differential regulation of lncRNAs and mRNAs related to follicle development in goat.

Mammalian follicle development is a complex biological process regulated by several factors. More than 99% of the follicles in goat ovaries will be atresia and only a few will eventually mature and ovulate. To investigate the potential long non-coding RNAs (lncRNAs) that regulate the expression of genes associated with follicular dominance or atresia, RNA-seq was performed on dominant follicles (DFs) and subordinate follicles (SFs) of granulosa cells from goats at the first follicular wave. A total of 92 differentially expressed lncRNAs and 676 differentially expressed mRNAs were detected in both types of follicles. The qRT-PCR results were consistent with the transcriptome sequencing data. Kyoto Encyclopedia of Genes and Genomes analysis of the differentially expressed mRNAs revealed that LHR and LDLR are associated with follicle dominance and are involved in the ovarian steroidogenesis pathway. The co-located mRNAs CALM2 and PPP1CA were significantly enriched during oocyte meiosis and in the cAMP and oxytocin signalling pathways. The co-expressed mRNAs were significantly enriched in the oestrogen signalling pathway and in ovarian steroidogenesis and progesterone-mediated oocyte maturation. A co-expression network of lncRNAs, target genes and differentially expressed genes was constructed. Follicle development-related genes, such as LDLR, NOTCH1 and FGF12, were included. These findings expand the lncRNA catalogue and provide a basis for further studies on the mechanism of regulating follicular development in goats.

Study on the relationship between expression patterns of cocaine-and amphetamine regulated transcript and hormones secretion in porcine ovarian follicles.

BACKGROUND: Cocaine-and amphetamine regulated transcript (CART) is an endogenous neuropeptide, which is widespread in animals, plays a key role in regulation of follicular atresia in cattle and sheep. Among animal ovaries, CART mRNA was firstly found in the cattle ovaries. CART was localized in the antral follicles oocytes, granulosa and cumulus cells by immunohistochemistry and in situ hybridization. Further research found that secretion of E2 was inhibited in granulosa cells with a certain dose of CART, the effect depends on the stage of cell differentiation, suggesting that CART could play a crucial role in regulating follicle atresia. The objective of this study was to characterize the CART expression model and hormones secretion in vivo and vitro in pig follicle granulosa cells, preliminarily studied whether CART have an effect on granulosa cells proliferation and hormones secretion in multiparous animals such as pigs. METHODS: The expression levels of CART mRNA in granulosa cells of different follicles were analyzed using qRT-PCR technology. Immunohistochemistry technology was used to localize CART peptide. Granulosa cells were cultured in medium supplemented with different concentrations of CART and FSH for 168 h using Long-term culture system, and observed using a microscope. The concentration of Estradiol (E2) and progesterone (P) in follicular fluids of different test groups were detected by enzyme linked immunosorbent assay (ELISA). RESULTS: Results showed that expression level of CART mRNA was highest in medium follicles, and significantly higher than that in large and small follicles (P < 0.05). Immunohistochemical results showed that CART were expressed both in granulosa cells and theca cells of large follicles, while CART were detected only in theca cells of medium and small follicles. After the granulosa cells were cultured for 168 h, and found that concentrations of E2 increase with concentrations of follicle-stimulating hormone (FSH) increase when the CART concentration was 0 µM. And the concentration of FSH reached 25 ng/mL, the concentration of E2 is greatest. It shows that the production of E2 needs induction of FSH in granulosa cells of pig ovarian follicles. With the increasing of CART concentrations (0.01, 0.1, 1 µM), E2 concentration has a declining trend, when the FSH concentrations were 25 and 50 ng/mL in the medium, respectively. CONCLUSIONS: These results suggested that CART plays a role to inhibit granulosa cells proliferation and E2 production, which induced by FSH in porcine ovarian follicular granulosa cells in vitro, but the inhibition effect is not significant. So we hypothesis CART maybe not a main local negative regulatory factor during porcine follicular development, which is different from the single fetal animals.

Effects of treatment with Astragalus Membranaceus on function of rat leydig cells.

BACKGROUND: Astragalus membranaceus (AM) is a Chinese traditional herb which has been reported to have broad positive effects on many diseases, including hepatitis, heart disease, diabetes and skin disease. AM can promote cell proliferation, increase the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx), and inhibit apoptosis by regulating the transcription of proto-oncogenes controlling cell death. While AM is included in some commercially available "testosterone boosting supplements", studies directly testing ability of AM to modulate testosterone production are lacking. In the present study, we examined the effects of AM on Leydig cell function in vitro. METHODS: Rat Leydig cells were purified and treated with AM at different concentrations (0 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL, 100 µg/mL and 150 µg/mL) and cell counting-8 (CCK-8) assay, Enzyme-linked immunosorbent assay, quantitative real time PCR and analysis of activities of SOD and GPx were done respectively. RESULTS: Treatment with 100 µg/mL (P<0.05) and 150 µg/mL AM (P<0.01) significantly increased Leydig cell numbers. Treatment with AM (20 µg/mL, 50 µg/mL and 100 µg/mL) significantly increased testosterone production (P<0.01). In addition, increased Leydig cell SOD and GPx activities were observed in response to 20 µg/mL and 50 µg/mL AM treatment (P<0.01). Furthermore, expression of Bax mRNA was significantly decreased (P<0.01), and the ratio of Bcl-2/Bax mRNA was significantly increased in response to 20 µg/mL AM in the culture medium (P<0.05). CONCLUSIONS: Results supported a beneficial effect of AM on multiple aspects of rat Leydig cell function in vitro including testosterone production.

MicroRNA-27a-3p Inhibits Melanogenesis in Mouse Skin Melanocytes by Targeting Wnt3a.

MicroRNAs (miRNAs) play an essential role in the regulation of almost all the biological processes, including melanogenesis. MiR-27a-3p is nearly six times higher in white alpaca skin compared to brown skin, which indicates that miR-27a-3p may be a candidate regulator for melanogenesis. Wnt3a plays an important role in promoting melanoblasts to differentiate into melanocytes and melanogenesis. To confirm the function of miR-27a-3p to melanogenesis in mammals, miR-27a-3p mimic, inhibitor and their negative control were transfected into mouse melanocytes. As a result, miR-27a-3p inhibits melanogenesis by repressing Wnt3a at post-transcriptional level. A significant decrease in Wnt3a luciferase activity was observed in 293T cells co-transfected with the matched luciferase reporter vector and pre-miR-27a. Furthermore, the presence of exogenous miR-27a-3p significantly decreased Wnt3a protein expression rather than mRNA and reduced ß-catenin mRNA levels in melanocytes. The over-expression of miR-27a-3p significantly increased the melanin content of melanocytes. However, miR-27a-3p inhibitor performs an opposite effect on melanogenesis. Wnt3a is one target of miR-27a-3p. MiR-27a-3p could inhibit Wnt3a protein amount by post-transcriptional regulation and melanogenesis in mouse melanocytes. Previous studies reported that Wnt3a promoted melanogenensis in mouse melanocytes. Thus, miR-27-3p inhibits melanogenesis by repressing Wnt3a protein expression.

Comprehensive analysis of LncRNAs modified by m6A methylation in sheep skin.

Objective: N6-methyladenosine (m6A) is the most prevalent methylation of mRNA and plays crucial roles in various physiological processes, including pigmentation. Yet, the regulatory mechanisms, including long noncoding RNAs (lncRNAs) m6A methylation contributing to pigmentation in sheep skin remains unclear. The purpose of this study was to identify potential lncRNAs and the m6A methylation of lncRNAs associated with pigmentation. Methods: RNA-seq and MeRIP-seq were performed to study the expression of lncRNAs and the m6A methylation of lncRNAs in black and white sheep skin. Furthermore, quantitative real-time polymerase chain reaction (qRT‒PCR) was used to verify the consistency with the RNA-seq and MeRIP-seq data. Results: 168 differentially expressed lncRNAs were detected between the two sheep skin colors. The differentially expressed lncRNAs enriched in the pathway of ECM-receptor interaction, Rap1 signaling pathway, and Non-homologous end-joining may play essential roles in pigmentation. We identified 577 m6A peaks and 617 m6A peaks in black and white sheep skin, respectively, among which 20 m6A peaks showed significant differences. The enriched motif in sheep skin was "GGACU", which aligned with the consensus motif "RRACH" (R=A or G, H=A, C or U). Differently methylated lncRNAs enriched in PI3K-Akt signaling pathway and Wnt signaling pathway might participate in skin pigmentation. ENSOARG00020015168 was the unique lncRNA with high expression and methylation (Hyper-Up) in black sheep shin. A lncRNA-mRNA network was constructed, with pigmentation-related genes, such as PSEN2, CCND3, COL2A1, and ERCC3. Conclusion: The m6A modifications of lncRNAs in black and white colored sheep skin were analyzed comprehensively, providing new candidates for the regulation of pigmentation.

Porcine granulosa cell transcriptomic analyses reveal the differential regulation of lncRNAs and mRNAs in response to all-trans retinoic acid in vitro.

Objective: The active metabolite of vitamin A, all-trans retinoic acid (ATRA), is involved in the proliferation and differentiation of granulosa cells, and promotes the follicular development, oocyte maturation, and ovulation in mammals. This study aims to investigate the ATRA induced potential long noncoding RNAs (lncRNAs) that regulate the expression of genes associated with granulosa cell proliferation and follicular development. Methods: The lncRNA and mRNA profiles of porcine granulosa cells from ATRA treatment and control group in vitro were constructed through RNA sequencing. Meanwhile, the sequencing data were verified using quantitative polymerase chain reaction (qPCR). Results: A total of 86 differentially expressed lncRNAs and 128 differentially expressed genes (DEGs) were detected in granulosa cells after ATRA treatment. The qRT-PCR results were consistent with the RNA-seq data. Functional annotation analysis revealed that the DEGs were remarkably enriched in ovary function and reproduction which contained FoxO, Hippo, Oocyte meiosis, mTOR signaling pathway, as well as several pathways associated with hormone regulation like Oxytocin signaling pathway and Steroid hormone biosynthesis. Moreover, an interaction network of lncRNAs and their cis-target DEGs was constructed, and 7 differentially expressed lncRNAs and 6 cis-target DEGs were enriched in ovarian steroidogenesis and reproduction. Conclusion: These findings expand the lncRNA catalogue and provide a basis for further studies on the mechanism of ATRA-mediated lncRNA regulation of follicular development in pigs.

Identification of FASN Gene Polymorphisms, Expression and Their Relationship with Body Size Traits in Guizhou White Goat (Capra hircus) with Different Genders.

To investigate the nucleotide variation sites (SNPs) and expression differences of the fatty acid synthase gene (FASN) in Guizhou white goats, the relationship between the variation and body size traits was investigated. In this study, DNA was extracted from the blood of 100 samples of white goats from different regions in Guizhou province, China, and the variation sites were screened using pooled sequencing by mixing DNA samples, and 242 blood samples with body size traits were used for association analysis. The allele frequency, genotype frequency, homozygosity, heterozygosity and effective gene number were calculated by using PopGene 32.0 software, the population polymorphism information content was calculated by using PIC software (Version 0.6), and the state of genetic balance of the genes was analyzed by using the chi-square test. The mRNA of FASN gene expression levels in male and female goats were investigated by using real-time fluorescence quantitative PCR (RT-qPCR). The general linear mixed model of MINTAB software (Version 16.0) was used to analyze the association between FASN gene nucleotide mutation sites and body size traits. The results showed that there was one nucleotide mutation site g.141 C/T in the target fragment of FASN gene amplification, and revealed two alleles, C and T, and three genotypes CC, CT and TT. The genotype frequencies for CC, CT and TT were 0.4308, 0.4205 and 0.1487, respectively. The allele frequencies for C and T were 0.6410 and 0.3590, respectively. The genetic homozygosity (Ho) was higher than the heterozygosity (He). The χ2 test showed that the mutation site was in the Hardy-Weinberg equilibrium state (p > 0.05). The RT-qPCR results showed that the FASN gene had different expression levels in the longissimus dorsi muscle of male and female goats, and its expression was significantly higher in male goats than in female goats. The association analysis results showed that the mutation of the FASN gene had different effects on body size traits of male and female goats, and the presence of the populations of the T allele and the TT genotype recorded higher body size traits (body weight, heart girth and wither height) in female populations. Therefore, the site of the FASN gene can be used as a candidate marker for the early selection of growth traits in Guizhou white goats.

Licorice Extract Supplementation Benefits Growth Performance, Blood Biochemistry and Hormones, Immune Antioxidant Status, Hindgut Fecal Microbial Community, and Metabolism in Beef Cattle.

This study aimed to evaluate the effects of licorice extract (LE) on growth performance, nutrient apparent digestibility, serum index (biochemistry, hormones, humoral immunity, and antioxidant function), hindgut fecal microbiota, and metabolism in beef cattle. In total, 12 male yellow cattle aged 12 months were divided into two groups (6 cattle per group): the basal diet (CK group) and the basal diet supplemented with 2 g/kg LE (CHM group). The entire experimental phase lasted for 120 days, including a 30-day pre-feeding period. Compared to the CK group, the average daily gain, crude fiber, calcium, and crude protein nutrient digestibility were greater on d 30 than d 60 (p < 0.05) and the feed meat ratio was lower for LE addition (p < 0.01). In terms of serum indexes, the insulin and nitric oxide contents were enhanced on d 30, the alkaline phosphatase level was improved on d 60, and the levels of albumin, immunoglobulin A, and catalase were increased on d 90 (p < 0.05). In contrast, the cholesterol content was lower on d 60 for LE addition compared with the CK group (p < 0.05). The higher enrichment of [Eubacterium]-oxidoreducens-group, p-2534-18b5-gut-group, and Ileibacterium were observed in the CHM group (p < 0.05), while the relative abundances of Gallibacterium and Breznakia in the CHM group were lower compared with the CK group (p < 0.05). In addition, the differential metabolites related to healthy growth in the CHM group were increased compared with the CK group. And there was a close correlation between hindgut microbiota and metabolic differentials. In general, LE has a promoting effect on the growth performance and health status of beef cattle over a period (30 to 60 days).

m6A mRNA Methylation Analysis Provides Novel Insights into Pigmentation in Sheep Skin.

N6-methyladenosine (m6A) is the most universal post-transcriptional modification of mRNA which may play important roles in verious species. However, the potential roles of m6A in the pigmentation of skin are not completely understood. To explore the role of m6A modification in pigmentation of sheep skin, we used MeRIP-seq and RNA-seq to profile the skin transcriptome in black and white coat color (n=3). Our results showed that an average of 7701 m6A peaks were obtained for all samples and the average length was 305.89 bp. The GGACUU sequence was the most enrichment motif and shared in black skin and white skin. The m6A peaks were mainly enriched in the CDS, 3'UTR and 5'UTR, especially in CDS region near the stop codon of the transcript. 235 significantly differential peaks were found in black skin vs. white skin. The KEGG signaling pathways of downregulated and upregulated m6A peaks were mainly enriched in AGE-RAGE signaling pathway in diabetic complications, Viral carcinogenesis, Transcriptional misregulation in cancer, ABC transporters, Basal transcription factors and Thyroid hormone synthesis (P value <0.05). For RNA-seq, 71 differently expressed genes (DEGs) were scanned in black skin vs. white skin. DEGs were significantly enriched in tyrosine metabolism, melanogenesis, neuroactive ligand-receptor interaction pathway (P value <0.05). Combined m6A-seq and RNA-seq analysis showed that the hyper-up genes and hypo-up genes were both enriched in ErbB signaling pathway (P value <0.05). In conclusion, it provide a basis for further research into the functions of m6A methylation modifications in pigmentation.

Study on the Mechanical Properties and Mechanism of a Nickel-Iron Slag Cement-Based Composite under the Action of Sodium Sulfate.

The accumulated amount of nickel-iron slag has increased with the rapid development of the nickel-iron industry. To determine a method for comprehensively utilizing nickel-iron slag, triaxial compression tests of nickel-iron slag cement-based composite materials under the action of sodium sulfate were conducted, and the effects of the sodium sulfate concentration on the stress-strain relation, shear strength, cohesion, and internal friction angle of the composite materials were analyzed. In addition, the influence mechanism of the nickel-iron slag content and sodium sulfate concentration on the composite was examined. The results revealed that the stress-strain curve of the nickel-iron slag cement-based composites reflected softening. With the increase in the sodium sulfate concentration, the brittleness increased, while the shear strength, cohesion, and internal friction angle decreased; the addition of nickel-iron slag slowed down the rate at which these parameters decrease. Scanning electron microscopy images revealed that nickel-iron slag can improve the internal structure of the cement composite soil, enhance its compactness, and improve its corrosion resistance. The optimum nickel-iron slag content of 14% can improve the cementitious composites' resistance to sodium sulfate erosion in terms of solid waste utilization and cementitious soil performance. The results obtained can provide technical parameters for preparing and designing cement-based composite materials as well as certain theoretical significance and engineering reference value.

Experimental Study on the Dissolution Characteristics and Microstructure of Carbonate Rocks under the Action of Thermal-Hydraulic-Chemical Coupling.

Microdamage in a rock induces a change in the rock's internal structure, affecting the stability and strength of the rock mass. To determine the influence of dissolution on the pore structure of rocks, the latest continuous flow microreaction technology was used, and a rock hydrodynamic pressure dissolution test device simulating multifactor coupling conditions was independently developed. The micromorphology characteristics of carbonate rock samples before and after dissolution were explored using computed tomography (CT) scanning. To conduct the dissolution test on 64 rock samples under 16 groups of working conditions, 4 rock samples under 4 groups were scanned by CT under working conditions, twice before and after corrosion. Subsequently, the changes in the dissolution effect and pore structure before and after dissolution were quantitatively compared and analyzed. The results show that the dissolution results were directly proportional to the flow rate, temperature, dissolution time, and hydrodynamic pressure. However, the dissolution results were inversely proportional to the pH value. The characterization of the pore structure changes before and after sample erosion is challenging. After erosion, the porosity, pore volume, and aperture of rock samples increased; however, the number of pores decreased. Under acidic conditions near the surface, carbonate rock microstructure changes can directly reflect structural failure characteristics. Consequently, heterogeneity, the presence of unstable minerals, and a large initial pore size result in the formation of large pores and a new pore system. This research provides the foundation and assistance for predicting the dissolution effect and evolution law of dissolved pores in carbonate rocks under multifactor coupling, offering a crucial guide for engineering design and construction in karst areas.

Immunization against inhibin DNA vaccine as an alternative therapeutic for improving follicle development and reproductive performance in beef cattle.

The objective of the present study was to investigate the potential role of immunization against INH on follicular development, serum reproductive hormone (FSH, E2, and P4) concentrations, and reproductive performance in beef cattle. A total of 196 non-lactating female beef cattle (4-5 years old) with identical calving records (3 records) were immunized with 0.5, 1.0, 1.5, or 2.0 mg [(T1, n = 58), (T2, n = 46), (T3, n = 42) and (T4, n = 36), respectively] of the pcISI plasmid. The control (C) group (n = 14) was immunized with 1.0 mL 0.9% saline. At 21d after primary immunization, all beef cattle were boosted with half of the primary immunization dose. On day 10 after primary immunization, the beef cattle immunized with INH DNA vaccine evidently induced anti-INH antibody except for the T1 group. The T3 group had the greatest P/N value peak among all the groups. The anti-INH antibody positive rates in T2, T3 and T4 groups were significantly higher than that in C and T1 groups. RIA results indicated that serum FSH concentration in T2 group increased markedly on day 45 after booster immunization; the E2 amount in T3 group was significantly increased on day 10 after primary immunization, and the levels of E2 also improved in T2 and T3 groups after booster immunization; the P4 concentration in T2 group was significantly improved on day 21 after primary immunization. Ultrasonography results revealed that the follicles with different diameter sizes were increased, meanwhile, the diameter and growth speed of ovulatory follicle were significantly increased. Furthermore, the rates of estrous, ovulation, conception, and twinning rate were also significantly enhanced. These findings clearly illustrated that INH DNA vaccine was capable of promoting the follicle development, thereby improving the behavioral of estrous and ovulation, eventually leading to an augment in the conception rates and twinning rate of beef cattle.

Development and Application of Carbonate Dissolution Test Equipment under Thermal-Hydraulic-Chemical Coupling Condition.

The latest continuous flow micro reaction technology was adopted to independently develop carbonate rock dissolution test equipment. Carbonate rock dissolution tests were conducted under different temperatures, flow rates, and dynamic water pressure conditions to study the dissolution process of carbonate rocks under the coupling of heat-water-chemistry. The dissolution effect and development law of carbonate rocks were explored by quantitatively studying carbonate rock dissolution rate and chemical composition of karst water. The results showed that the self-designed dissolution test equipment has obvious advantages. After dissolution, carbonate rock specimens were damaged to varying degrees. The dissolution rate was proportional to water velocity and hydrodynamic pressure, with the velocity effect being greater than the hydrodynamic pressure effect. The pH value, conductivity, and Ca2+ ion content of the reaction solution gradually increased after dissolution. The development and application of the equipment have proved that, at low dynamic water pressures (2 MPa), the water flow velocity effect on the dissolution velocity was 1.5 times that when the dynamic water pressure was high (6 MPa); at a low water flow velocity of 15 mL/min, the dynamic water pressure effect on the dissolution velocity was three times that when the water flow velocity was high (75 mL/min). The development process is gradually becoming strong and stable. Its research has important theoretical significance and engineering application value to provide technical means and guarantee for the early identification, karst development, and safety evaluation of karst geological disasters.

Milk fatty acids profiles and milk production from dairy cows fed different forage quality diets.

Thirty lactating Holstein cows were used to investigate the effects of different forages quality on milk fatty acids (FA) profiles and production. The cows were assigned to 3 dietary treatments (n = 10 per treatment) in a randomized block design with 3 repeated measures. They were fed the experimental diets for 90 d with 3 days of collection of samples for analysis at about 27 d intervals (samples were collected on days 28, 29, 30, 58, 59, 60, 88, 89 and 90). The treatments were (DM basis): 1) mixed forages diet (MF) consisting of 3.7% Chinese wild rye, 26.7% corn silage and 23.4% alfalfa hay; 2) corn stalk diet 1 (CS1) where corn stalk was used to formulate a similar chemical nutrient level to MF; 3) corn stalk diet 2 (CS2) which used corn stalk to formulate a similar forage level to MF for the diet. Dry matter intake and BW were similar between treatments, but daily milk yield, milk fat and protein yield decreased (P < 0.05) in CS1 and CS2 compared with MF, with CS2 being the lowest (P < 0.05). In total FA of milk, the compositions of C18:1c9, C18:3 and unsaturated FA increased (P < 0.05) in CS1 and CS2 compared with MF, and C18:0 and trans-C18:1 were trended to increase (P < 0.10), but C4:0-C16:0 were decreased (P < 0.05). Compared with cows fed CS2, cows receiving CS1 increased the compositions of C4:0 to C12:0 and C18:2 (P < 0.05). The results suggests feeding corn stalk could produce a greater proportion of unsaturated fatty acid (UFA) in milk fat without resulting in milk fat depression (MFD) in mid lactation cows, but simply increasing the ratio of concentrate in low forages diets is not an effective way to increase milk fat synthesis and milk production.

Transcriptome Analysis of Bovine Ovarian Follicles at Predeviation and Onset of Deviation Stages of a Follicular Wave.

For two libraries (PDF1 and ODF1) using Illumina sequencing 44,082,301 and 43,708,132 clean reads were obtained, respectively. After being mapped to the bovine RefSeq database, 15,533 genes were identified to be expressed in both types of follicles (cut-off RPKM > 0.5), of which 719 were highly expressed in bovine follicles (cut-off RPKM > 100). Furthermore, 83 genes were identified as being differentially expressed in ODF1 versus PDF1, where 42 genes were upregulated and 41 genes were downregulated. KEGG pathway analysis revealed two upregulated genes in ODF1 versus PDF1, CYP11A1, and CYP19A1, which are important genes in the steroid hormone biosynthesis pathway. This study represents the first investigation of transcriptome of bovine follicles at predeviation and onset of deviation stages and provides a foundation for future investigation of the regulatory mechanisms involved in follicular development in cattle.

Study on the relationship between expression patterns of cocaine-and amphetamine regulated transcript and hormones secretion in porcine ovarian follicles

Abstract Background Cocaine-and amphetamine regulated transcript (CART) is an endogenous neuropeptide, which is widespread in animals, plays a key role in regulation of follicular atresia in cattle and sheep. Among animal ovaries, CART mRNA was firstly found in the cattle ovaries. CART was localized in the antral follicles oocytes, granulosa and cumulus cells by immunohistochemistry and in situ hybridization. Further research found that secretion of E2 was inhibited in granulosa cells with a certain dose of CART, the effect depends on the stage of cell differentiation, suggesting that CART could play a crucial role in regulating follicle atresia. The objective of this study was to characterize the CART expression model and hormones secretion in vivo and vitro in pig follicle granulosa cells, preliminarily studied whether CART have an effect on granulosa cells proliferation and hormones secretion in multiparous animals such as pigs. Methods The expression levels of CART mRNA in granulosa cells of different follicles were analyzed using qRT-PCR technology. Immunohistochemistry technology was used to localize CART peptide. Granulosa cells were cultured in medium supplemented with different concentrations of CART and FSH for 168 h using Long-term culture system, and observed using a microscope. The concentration of Estradiol (E2) and progesterone (P) in follicular fluids of different test groups were detected by enzyme linked immunosorbent assay (ELISA). Results Results showed that expression level of CART mRNA was highest in medium follicles, and significantly higher than that in large and small follicles (P < 0.05). Immunohistochemical results showed that CART were expressed both in granulosa cells and theca cells of large follicles, while CART were detected only in theca cells of medium and small follicles. After the granulosa cells were cultured for 168 h, and found that concentrations of E2 increase with concentrations of follicle-stimulating hormone (FSH) increase when the CART concentration was 0 μM. And the concentration of FSH reached 25 ng/mL, the concentration of E2 is greatest. It shows that the production of E2 needs induction of FSH in granulosa cells of pig ovarian follicles. With the increasing of CART concentrations (0.01, 0.1, 1 μM), E2 concentration has a declining trend, when the FSH concentrations were 25 and 50 ng/mL in the medium, respectively. Conclusions These results suggested that CART plays a role to inhibit granulosa cells proliferation and E2 production, which induced by FSH in porcine ovarian follicular granulosa cells in vitro, but the inhibition effect is not significant. So we hypothesis CART maybe not a main local negative regulatory factor during porcine follicular development, which is different from the single fetal animals.