Medium-sized Ovarian Follicles Research Articles

Transcriptome analysis of miRNAs and mRNAs of medium-sized ovarian follicles of Duroc and Meishan sows

Litter size is an economically important trait in the swine industry. A high ovulation rate is a prerequisite for a large litter size. It differs among pig breeds with different genetic backgrounds. During the middle-late follicular stage, the medium-sized follicles (3–6.9 mm) of sows with a high ovulation rate can maintain their potential for development into ovulatory follicles. In this study, Meishan and Duroc sows were utilized as model animals due to their extreme litter size phenotypes. M2 follicles (5–6.9 mm) were collected on Day 4 of the follicular phase for high-throughput sequencing of small RNAs and mRNAs. Differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) were analyzed. KEGG pathway and GO enrichment analyses were conducted to analyze the DEGs and the target genes of DEMs, resulting in the construction of a regulatory network of DEMs-DEGs. A total of 402 miRNAs were identified, with 8 upregulated and 3 downregulated miRNAs in the Meishan group. KEGG pathway analysis revealed significant enrichment of target genes of DEMs in pathway such as Apoptosis, GnRH signaling pathway, MAPK signaling pathway, and PI3K-Akt signaling pathway. GO enrichment analysis revealed 1930 significantly enriched GO terms. KEGG pathway analysis of the DEGs revealed 13 significantly enriched pathways, including those involved in complement and coagulation cascades, ECM-receptor interactions, and PI3K-Akt signaling pathway. A total of 642 GO terms were significantly enriched. Through Pearson correlation analysis, 67 DEM-DEG pairs were identified, and 62 DEGs were found to be regulated by 9 DEMs. This study provides valuable insights into the function of miRNAs and mRNAs in regulating the growth and development of follicles.

Comparison of superovulatory responses to a standardized hormonal superstimulation protocol among three indigenous breeds of sheep in Brazil

This study examined the differences in ovarian responses and in vivo embryo production in three naturalized Brazilian sheep breeds (Morada Nova, Santa Inês, and Somalis Brasileira). Thirty cycling, multiparous ewes (n = 10/breed) received intravaginal devices containing 0.33 g of progesterone (CIDR®) for 9 days and the twice-daily superovulatory treatment with 133 mg of porcine follicle-stimulating hormone (six decreasing doses) started on Day 7 (Day 0 PM = CIDR® insertion). On Days 11 (36 h after CIDR® removal) and 15 (12 h before non-surgical embryo recovery performed seven days after CIDR® removal), all ewes were examined using B-mode and Doppler ultrasonography. The number of medium-sized ovarian follicles (4.0–5.99 mm) on Day 11 was greater (P = 0.003) in Santa Inês and Somalis Brasileira compared with Morada Nova ewes. Small antral follicle numbers (2.0–3.99 mm in diameter) on Day 11 were directly related to ovulatory responses in all three breeds of superovulated ewes. The number of corpora lutea on Day 15 was greater (P < 0.0001) in Santa Inês, followed by Somalis Brasileira and Morada Nova ewes; however, the number of viable embryos and viability rate did not differ (P > 0.05) among the three genotypes of ewes studied. The total ovarian area (TA), color Doppler area (DA), and DA/TA × 100% increased from Day 11 to Day 15 in all breeds, and they were lowest (P < 0.001) in superovulated Morada Nova sheep. Significant positive correlations among ovarian antral follicle numbers in different size classes (Day 11) and ovulatory responses were recorded in all breeds, but associations between the ovarian blood perfusion and superovulatory outcomes were restricted to the Morada Nova and Santa Inês genotypes. In summary, ovarian follicle numbers and blood flow indices differed among the three naturalized Brazilian ewes, but no significant differences were noted in embryo yields and quality following superovulation. Small antral follicle count 36 h after CIDR® treatment was a reliable predictor of impending ovulation rates in all ewes under study.

Transcriptome analysis reveals that long noncoding RNAs contribute to developmental differences between medium-sized ovarian follicles of Meishan and Duroc sows

Ovulation rate is an extremely important factor affecting litter size in sows. It differs greatly among pig breeds with different genetic backgrounds. Long non-coding RNAs (lncRNAs) can regulate follicle development, granulosa cell growth, and hormone secretion, which in turn can affect sow litter size. In this study, we identified 3554 lncRNAs and 25,491 mRNAs in M2 follicles of Meishan and Duroc sows. The lncRNA sequence and open reading frame lengths were shorter than mRNAs, and lncRNAs had fewer exons, were less abundant, and more conserved than protein-coding RNAs. Furthermore, 201 lncRNAs were differentially expressed (DE) between breeds, and quantitative trait loci analysis of DE lncRNAs were performed. A total of 127 DE lncRNAs were identified in 119 reproduction trait-related loci. In addition, the potential target genes of lncRNAs in cis or trans configurations were predicted. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that some potential target genes were involved in follicular development and hormone secretion-related biological processes or pathways, such as progesterone biosynthetic process, estrogen metabolic process, ovarian steroidogenesis, and PI3K-Akt signaling pathway. Furthermore, we also screened 19 differentially expressed lncRNAs in the PI3K-Akt signaling pathway as candidates. This study provides new insights into the roles of lncRNAs in follicular growth and development in pigs.

Impact of porcine follicular fluid during folliculogenesis as a supplement on the primary cell culture of oviductal epithelial cells

The impact of varying concentrations of porcine follicular fluid (pFF) derived from three ovarian follicle types and produced during folliculogenesis on porcine oviductal epithelial cell (pOEC) culture was studied for 24 h using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cells treated with pFF at a protein concentration of 500 µg/mL (from small- and large-sized ovarian follicles) and 600 µg/mL (from medium-sized ovarian follicles) showed the highest viability, which was significantly different from that observed in the control group (p&lt;0.05) and not significantly higher than that observed in the positive control group.This study demonstrated that the impact of pFF on pOECs can be used as a model for biotechnological studies. Further, the study showed that instead of costly fetal calf serum, pFF produced during folliculogenesis can be used as a supplement in culture media to promote porcine oviductal epithelium cell growth and development.

Supraphysiological leptin levels shift the profile of steroidogenesis in porcine ovarian follicles toward progesterone and testosterone secretion through increased expressions of CYP11A1 and 17β-HSD: a tissue culture approach

Evidence from both clinical and animal studies suggests that exposure to excess androgens results in cyst formation. The present in vitro study assessed the effects of supraphysiological concentrations of leptin (20 and 40 ng/ml) on progesterone (P(4)), androstenedione androstendione (A(4)), testosterone and estradiol (E(2)) secretion by ELISA and the expression of CYP11A1, CYP17, 17b-hydroxysteroid dehydrogenase (17b-HSD) and CYP19 by western blot to answer the question of whether leptin could be independent risk factor for cystformation in pigs. Small- and medium-sized ovarian follicles were collected from prepubertal and cycling pigs. Increased P(4) and testosterone secretions were observed in both small- and medium-sized follicles in prepubertal and cycling animals whereas there was no change in E2 secretion. Leptin treatment resulted in an increase in CYP11A1 and 17b-HSD protein expression but had no effect on CYP17 and CYP19 expression in follicles of either size from prepubertal and cycling pigs. Results of presented data suggest that leptin in elevated doses, by stimulatory effect on CYP11A1 and 17b-HSD protein expression resulting in elevated P(4) and testosterone secretions could be an independent risk factor for cyst formation in both prepubertal and cycling pigs.

Effect of ammonium chloride on the growth and metabolism of bovine ovarian granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride

Three experiments determined first, the effect of increasing ammonium chloride (NH 4Cl) concentrations on the growth and metabolism of bovine granulosa cells isolated from small and medium-sized bovine ovarian follicles; secondly, whether the changes in granulosa cell growth and metabolism induced by NH 4Cl were reversible; and thirdly, whether granulosa cells, previously conditioned with NH 4Cl, were able to support maturation of oocytes in vitro. In Experiment 1, using a 2 ( follicle size class )×5 ( NH 4 Cl concentration ) factorial design, granulosa cells from small or medium-sized ovarian follicles were incubated for 96 h with 0, 0.2, 0.4, 0.8 or 1.6 μmol NH 4Cl/ml. Experiment 2 used a split plot factorial design where granulosa cells were incubated for 96 h in the presence or absence of 1 μmol/ml NH 4Cl and then incubated in the absence or presence of 1 μmol/ml NH 4Cl for a further 48 h. Finally in Experiment 3, ovine oocytes were matured on layers of bovine granulosa cells which had not been conditioned with NH 4Cl or conditioned with 0.5 or 1.0 μmol/ml NH 4Cl and development of embryos to the blastocyst stage followed and blastocyst quality assessed. In Experiment 1, incubation of granulosa cells in increasing concentrations of NH 4Cl reduced cell growth, increased cell protein concentrations and increased the amounts of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) oxidised and oestradiol and progesterone produced per 10 5 cells. Cells from medium-sized follicles were more sensitive to NH 4Cl concentration and oxidised more MTT and produced less progesterone at high NH 4Cl concentrations than cells from small-sized follicles. When, in Experiment 2, NH 4Cl was removed from cell culture after 96 h incubation, cells previously exposed to NH 4Cl grew at a slower rate during the subsequent 48 h, contained more cellular protein, oxidised more MTT and produced more oestradiol and progesterone than cells not previously exposed to NH 4Cl. Maturation of ovine oocytes in coculture with bovine granulosa cells not exposed to NH 4Cl (Experiment 3) increased egg cleavage rate and the proportion of cleaved eggs which developed to the blastocyst stage. Conditioning of granulosa cells with NH 4Cl supported egg cleavage and development to the blastocyst stage at rates similar to those observed in the absence of granulosa cells. In conclusion, these experiments showed that the in vitro growth and metabolism of granulosa cells were altered by concentrations of NH 4Cl similar to ammonium ion concentrations measured in follicular fluid and that these effects were not immediately reversible. Furthermore, the ability of granulosa cells conditioned with NH 4Cl to support in vitro maturation of oocytes was impaired.

Stimulation of Nitric Oxide Synthesis in Porcine Granulosa Cells by Phorbol Ester.

This study was designed to assess the release of nitric oxide (NO) from porcine granulosa cells. The cells obtained from medium-sized ovarian follicles (3-6 mm diameter) were cultured for 48 h with ovine FSH to be developed. A significant accumulation of nitrite was observed during development, showing an increase in NO synthesis. The mRNA of the endothelial NO synthase was detected in the granulosa cells, as revealed by RT-PCR. Furthermore, nitrite was accumulated in the culture medium after 1-h stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with 4α-phorbol, 4β-phorbol 13-monoacetate (4β-PMA) and forskolin. TPA led to an increase in the nitrite content in a dose-dependent manner. NO was measured directly in the cell suspension with an amperometric NO sensor. The NO sensor signal gradually increased to reach a plateau (5-15 pA) after exposure to TPA in the presence of L-arginine, but not D-arginine. The concentration of NO was approximately 5.5-11 nM, but no detectable change was observed after the addition of 4α-phorbol or 4β-PMA. These results suggest that the synthesis of NO is promoted at least in part through the activation of protein kinase C in granulosa cells.

Valproate-induced alterations in testosterone, estradiol and progesterone secretion from porcine follicular cells isolated from small- and medium-sized ovarian follicles

The aim of the present study was to investigate whether long-term exposure to valproate (VPA) alters follicular steroidogenesis and whether or not this effect is dependent on the degree of follicular development. Small- and medium-sized follicles were obtained from pig ovaries collected, respectively, at days 8–10 and 14–16 of oestrus cycle. Theca interna and granulosa cells were isolated from follicles and placed in the same well in the ratio 1 : 3 with or without the VPA in doses of 100, 300 and 500 μ g ml−1. The culture medium was changed after 2, 4, 6 and 8 days. In both types of follicles, VPA caused a significant and dose-dependent reduction in both testosterone and estradiol secretion from follicular cells. In small-sized follicles, the testosterone to oestrogen ratio increased at all doses used and after all lengths of time in culture. In medium-sized follicles, a significant increase in the testosterone to oestrogen ratio was only observed at the highest dose level. All doses of VPA caused a marked inhibition of progesterone secretion after 48 hours while during long-term VPA exposure progesterone gradually increased demonstrating luteinization of cells. In conclusion, the present study demonstrates a direct effect of VPA on steroidogenesis. The effect seems to differ to some extent depending on the follicular stage of development. The elevated ratio of testosterone to estradiol suggests that VPA inhibits the conversion of testosterone to estradiol.

Effects of growth hormone on FSH, insulin and triiodothyronine-mediated estradiol production by granulosa cells from prepubertal gilts in vitro

Three experiments were performed using porcine granulosa cells from medium-sized follicles (2–4 mm) cultured in serum-free medium at a density of 2 × 106 viable cells per well. Cells were cultured in the presence of combinations of FSH, insulin, growth hormone (GH) and triiodothyronine (T3), and subsequent estradiol (E2) production was monitored. In the presence of insulin, a dose-dependent E2 production response to FSH was observed (exp. 1). However, in all experiments, E2 production was reduced (P &lt; 0.05) by the inclusion of GH in the media. The inclusion of T3 augmented the E2 response to insulin, an effect attenuated by GH. We conclude that GH, at the dose employed, is inhibitory to E2 production in granulosa cells of medium-sized ovarian follicles. Key words: Granulosa cells, growth hormone, estradiol

Influence of follicular ablation during lactation on postweaning interval to estrus, ovulation rate, and endocrine function in sows

Duroc sows farrowed second litters in March and lactated 35 ± 2 days. At 36 hr before weaning, electrocautery of follicles ≥3 mm in diameter (n=8) or sham surgery (n=5) was performed to test the hypothesis that ablation of medium-sized follicles would prolong the duration of postweaning anestrus. Number of follicles and diameters at surgery were: 1.3 ± .6 (> 5 mm diameter), 26 ± 1 (3 to 5 mm) and > 20 (< 3 mm). Blood samples were collected at 15 min intervals for 3 hr beginning at −12, 0, 12, 60 and 96 hr from weaning. Interval to estrus was 3.4 ± .2 days in seven of eight cauterized sows and 3.6 ± .6 days for sham-surgery sows. The remaining cauterized sow was anestrus at slaughter, 40 days after weaning. Number of corpora lutea and pregnancy rate were 15.8 ± .6 and 92%, respectively, and were similar between sham-surgery and cauterized sows. Concentration of follicle stimulating hormone (FSH) at 12 hr before weaning was greater in sows subjected to electrocautery than for sham-surgery sows, but FSH values were similar at other sampling times. Concentrations of estradiol were similar at all times for both treatment groups. Luteinizing hormone (LH) was higher (P<.05) at 60 hr in cauterized sows because of the onset of the preovulatory LH surge in one sow. We conclude that destruction of medium-sized ovarian follicles before weaning did not influence postweaning reproductive performance.