Bo Pan

MicroRNA-378 regulates oocyte maturation via the suppression of aromatase in porcine cumulus cells

We sought to investigate whether miR-378 plays a role in cumulus cells and whether the manipulation of miRNA levels in cumulus cells influences oocyte maturation in vitro. Cumulus-oocyte complexes (COCs) from ovarian follicles had significantly lower levels of precursor and mature miR-378 in cumulus cells surrounding metaphase II (MII) oocytes than cumulus cells surrounding germinal vesicle (GV) oocytes, suggesting a possible role of miR-378 during COC maturation. Overexpression of miR-378 in cumulus cells impaired expansion and decreased expression of genes associated with expansion (HAS2, PTGS2) and oocyte maturation (CX43, ADAMTS1, PGR). Cumulus cell expression of miR-378 also suppressed oocyte progression from the GV to MII stage (from 54 ± 2.7 to 31 ± 5.1%), accompanied by a decrease of growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), zona pellucida 3 (ZP3), and CX37 in the oocytes. Subsequent in vitro fertilization resulted in fewer oocytes from COCs overexpressing miR-378 reaching the blastocyst stage (7.3 ± 0.7 vs. 16.6 ± 0.5%). miR-378 knockdown led to increased cumulus expansion and oocyte progression to MII, confirming a specific effect of miR-378 in suppressing COC maturation. Aromatase (CYP19A1) expression in cumulus cells was also inhibited by miR-378, leading to a significant decrease in estradiol production. The addition of estradiol to IVM culture medium reversed the effect of miR-378 on cumulus expansion and oocyte meiotic progression, suggesting that decreased estradiol production via suppression of aromatase may be one of the mechanisms by which miR-378 regulates the maturation of COCs. Our data suggest that miR-378 alters gene expression and function in cumulus cells and influences oocyte maturation, possibly via oocyte-cumulus interaction and paracrine regulation.

Notch pathway regulates female germ cell meiosis progression and early oogenesis events in fetal mouse

A critical process of early oogenesis is the entry of mitotic oogonia into meiosis, a cell cycle switch regulated by a complex gene regulatory network. Although Notch pathway is involved in numerous important aspects of oogenesis in invertebrate species, whether it plays roles in early oogenesis events in mammals is unknown. Therefore, the rationale of the present study was to investigate the roles of Notch signaling in crucial processes of early oogenesis, such as meiosis entry and early oocyte growth. Notch receptors and ligands were localized in mouse embryonic female gonads and 2 Notch inhibitors, namely DAPT and L-685,458, were used to attenuate its signaling in an in vitro culture system of ovarian tissues from 12.5 days post coitum (dpc) fetus. The results demonstrated that the expression of Stra8, a master gene for germ cell meiosis, and its stimulation by retinoic acid (RA) were reduced after suppression of Notch signaling, and the other meiotic genes, Dazl, Dmc1, and Rec8, were abolished or markedly decreased. Furthermore, RNAi of Notch1 also markedly inhibited the expression of Stra8 and SCP3 in cultured female germ cells. The increased methylation status of CpG islands within the Stra8 promoter of the oocytes was observed in the presence of DAPT, indicating that Notch signaling is probably necessary for maintaining the epigenetic state of this gene in a way suitable for RA stimulation. Furthermore, in the presence of Notch inhibitors, progression of oocytes through meiosis I was markedly delayed. At later culture periods, the rate of oocyte growth was decreased, which impaired subsequent primordial follicle assembly in cultured ovarian tissues. Taken together, these results suggested new roles of the Notch signaling pathway in female germ cell meiosis progression and early oogenesis events in mammals.

Regulation of primordial follicle recruitment by cross-talk between the Notch and phosphatase and tensin homologue (PTEN)/AKT pathways

The growth of oocytes and the development of follicles require certain pathways involved in cell proliferation and survival, such as the phosphatidylinositol 3-kinase (PI3K) pathway and the Notch signalling pathway. The aim of the present study was to investigate the interaction between Notch and the PI3K/AKT signalling pathways and their effects on primordial follicle recruitment. When the Notch pathway was inhibited by L-685,458 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester (DAPT) in vitro, the expression of genes in the pathway and the percentage of oocytes in growing follicles decreased significantly in mouse ovaries. By 2 days postpartum, ovaries exposed to DAPT, short interference (si) RNA against Notch1 or siRNA against Hairy and enhancer of split-1 (Hes1) had significantly decreased expression of HES1, the target protein of the Notch signalling pathway. In contrast, expression of phosphatase and tensin homologue (Pten), a negative regulator of the AKT signalling pathway, was increased significantly. Co immunoprecipitation (Co-IP) revealed an interaction between HES1 and PTEN. In addition, inhibition of the Notch signalling pathway suppressed AKT phosphorylation and the proliferation of granulosa cells. In conclusion, the recruitment of primordial follicles was affected by the proliferation of granulosa cells and regulation of the interaction between the Notch and PI3K/AKT signalling pathways.

Ovarian-Cell-Like Cells from Skin Stem Cells Restored Estradiol Production and Estrus Cycling in Ovariectomized Mice

Reduction of estradiol production and high serum concentrations of follicular stimulating hormone (FSH) are endocrine disorders associated with premature ovarian failure. Here, we report that transplantation of ovarian-like cells differentiated from stem cells restored endogenous serum estradiol levels. Stem cells were isolated from postnatal mouse skin and differentiated into ovarian-cell-like cells that are consistent with female germ, and ovarian follicle somatic cells. The ovarian-cell-like cells were transplanted into ovariectomized mice (Cell Trans), whereas control mice were subjected to bilateral ovariectomies without cell transplantation (OVX). Using vaginal cytology analysis, it was revealed that in 13 out of 19 Cell Trans mice, estrus cycles were restored around 8 weeks after cell transplantation and were maintained until 16 weeks post-transplantation, whereas in the OVX group, all mice were arrested at metestrus/diestrus of the estrus cycle. The uterine weight in the Cell Trans group was similar to sham operation mice (Sham OP), while severe uterine atrophy and a decreased uterine weight were observed in the OVX group. Histologically, ectopic follicle-like structures and blood vessels were found within and around the transplants. At 12-14 weeks after cell transplantation, mean serum estradiol level in Cell Trans mice (178.0±35 pg/mL) was comparable to that of the Sham OP group (188.9±29 pg/mL), whereas it was lower in the OVX group (59.0±4 pg/mL). Serum FSH concentration increased in the OVX group (1.62±0.32 ng/mL) compared with the Sham OP group (0.39±0.34 ng/mL). Cell Trans mice had a similar FSH level (0.94±0.23 ng/mL; P<0.05) to Sham OP mice. Our results suggest that ovarian somatic cells differentiated from stem cells are functional in vivo. In addition to providing insights into the function of ovarian somatic cells derived from stem cells, our study may offer potential therapeutic means for patients with hypo-estradiol levels like those encountered in premature ovarian failure.

Antimicrobial peptide expression in swine granulosa cells in response to lipopolysaccharide

Antimicrobial peptides (AMP) are host defense peptides present in all species examined. The objective of the current study was to characterize the expression of a group of antimicrobial peptides in ovarian cells, and to investigate their expression response to pathogen ligands. It was found that while PG1 transcript was not detected in the ovary, the expression of BD2 is the highest in small follicle derived granulosa cells (SGC), and its expression decreases during follicular development to large follicle stage (LGC; p < 0.05). The expression of BD2 in cumulus cells also decreased from GV to MII stage of oocyte maturation. ANG4 expression increased in granulosa cells during follicular development from SGC to LGC stage (p < 0.05), although no significant difference was observed in cumulus cells from different stages of oocyte maturation. We further examined AMP expression in follicle cells treated with different toll-like receptor (TLR) ligands which mimic pathogen exposure in the ovary. Of the four TLR ligands examined, lipopolysaccharide (LPS) exposure resulted in a 11.5 fold increase of BD2 expression, and a significant decrease of LYZ in LGC. A similar response pattern in BD2 and LYZ expression was also observed in SGC. These responses of AMP expression to LPS are associated with increased TLR4 signaling pathway component in mRNA and protein level, such as MyD88 and NFkB, and pro-inflammatory cytokines/chemokines, such as IL-6, TNFα and IL-8 (p < 0.05). Our data suggest that AMPs may play a role in innate defense as well as other physiological functions during ovarian follicular development and oocyte maturation.

MicroRNA-21 up-regulates metalloprotease by down-regulating TIMP3 during cumulus cell-oocyte complex in vitro maturation

Cumulus cells and the remodeling of their extracellular matrix around oocytes are essential for oocyte maturation and ovulation in the ovary. An important extracellular metalloprotease, ADAMTS1, and its partner VERSICAN, mediate essential cumulus-oocyte-complex (COC) structural remodeling. However, how the expression of these proteinases is regulated during oocyte maturation is unclear. Here we report that both ADAMTS1 and VERSICAN significantly increased in porcine cumulus cells during COC in vitro maturation (IVM). Interestingly, one of the tissue inhibitors of the metalloproteinase family member, TIMP3, was found to be significantly decreased in cumulus cells during this process. Down-regulation of TIMP3 using specific small interfering RNA decreased TIMP3 expression, while increased the levels of ADAMTS1 and VERSICAN, suggesting an inverse relationship between TIMP3, the metalloprotease, and the breakdown product of its substrate. MiR-21 significantly increased in cumulus cells during COC maturation. Knockdown of miR-21 in cumulus cells during COC maturation resulted in increased TIMP3 and decreased ADAMTS1 and VERSICAN expression, which is accompanied by a decrease in cumulus cell expansion and the ratio of oocytes that reached MII stage. In contrast, over-expression of miR-21 decreased TIMP3 and increased ADAMTS1, and enhanced cumulus cell expansion and oocyte maturation. Moreover, in silico prediction revealed that a miR-21 binding site is present at the 3-untranslated region (3-UTR) of the TIMP3 mRNA, which was further confirmed to be the target site of miR-21 by luciferase gene reporter assays. Our findings revealed that miR-21 promotes cumulus expansion and oocyte maturation via down-regulating TIMP3, and subsequent increase of ADAMTS1 and VERSICAN during in vitro COC maturation.Cumulus cells and the remodeling of their extracellular matrix around oocytes are essential for oocyte maturation and ovulation in the ovary. An important extracellular metalloprotease, ADAMTS1, and its partner VERSICAN, mediate essential cumulus-oocyte-complex (COC) structural remodeling. However, how the expression of these proteinases is regulated during oocyte maturation is unclear. Here we report that both ADAMTS1 and VERSICAN significantly increased in porcine cumulus cells during COC in vitro maturation (IVM). Interestingly, one of the tissue inhibitors of the metalloproteinase family member, TIMP3, was found to be significantly decreased in cumulus cells during this process. Down-regulation of TIMP3 using specific small interfering RNA decreased TIMP3 expression, while increased the levels of ADAMTS1 and VERSICAN, suggesting an inverse relationship between TIMP3, the metalloprotease, and the breakdown product of its substrate. MiR-21 significantly increased in cumulus cells during COC maturation. Knockdown of miR-21 in cumulus cells during COC maturation resulted in increased TIMP3 and decreased ADAMTS1 and VERSICAN expression, which is accompanied by a decrease in cumulus cell expansion and the ratio of oocytes that reached MII stage. In contrast, over-expression of miR-21 decreased TIMP3 and increased ADAMTS1, and enhanced cumulus cell expansion and oocyte maturation. Moreover, in silico prediction revealed that a miR-21 binding site is present at the 3-untranslated region (3-UTR) of the TIMP3 mRNA, which was further confirmed to be the target site of miR-21 by luciferase gene reporter assays. Our findings revealed that miR-21 promotes cumulus expansion and oocyte maturation via down-regulating TIMP3, and subsequent increase of ADAMTS1 and VERSICAN during in vitro COC maturation.

Endocrine Regulation in the Ovary by MicroRNA during the Estrous Cycle

Hormonal control of the estrous cycle that occurs in therian mammals is essential for the production of a functional egg. Supporting somatic cell types found within the ovary, such as granulosa and theca cells, respond to endocrine signals to support oocyte maturation and ovulation. Following the release of the egg, now available for fertilization, coordinated hormonal signaling between the mother and putative embryo are required for the establishment of pregnancy. If no conception occurs, both the ovary and uterus are “reset” in preparation for another cycle. The complex molecular changes that occur within cells in response to hormone signaling include a network of non-coding microRNAs (miRNAs) that posttranscriptionally regulate gene expression. They are thus able to fine-tune cellular responses to hormones and confer robustness in gene regulation. In this review, we outline the important roles established for miRNAs in regulating female reproductive hormone signaling during estrus, with a particular focus on signaling pathways in the ovary. Understanding this miRNA network can provide important insights to improving assisted reproductive technologies and may be useful in the diagnosis of female reproductive disorders.

Molecular regulation of miR-378 on the development of mouse follicle and the maturation of oocyte in vivo

ABSTRACT MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs which can bind to completely or partially complementary sequences in the 3’UTR of target mRNAs, therefore degrading the mRNA or repressing translation. We previously reported that miR-378 played a role in estradiol production via suppression of aromatase translation in porcine granulosa cells and could affect oocyte maturation in vitro by inhibiting cumulus cell expansion. However, the role of miR-378 on ovary development in vivo is unknown. The current study aimed to uncover the molecular mechanism of miR-378 in regulating mouse follicular development via micro-injection of CMV-miR-378 lentivirus into the bursa of mouse ovary. The results showed that CMV-miR-378 lentivirus transduction in the mouse ovaries resulted in reduced ovary size, extended oestrous cycle (6-7 d in miR-378 overexpression group and 4–5 dyas in GFP control group) due to continuous oestrum, decreased percentage of oocytes in vitro maturation rate (IVM 60.8% vs. 89.4% in GFP control), increased apoptosis rate (Bax/Bcl2 in mRNA and protein level), decreased expression of genes associated with gap junction, such as connexin 43 (Cx-43) and connexin (Cx-37) and decreased expression of genes associated with follicular development, such as BMP15 and GDF9. Moreover, the number of pups/litter was consistently lower in the miR-378 group in each batch of the paired breeding. Our data suggest that miR-378 alters gene expression in cumulus cells and indirectly influences oocyte maturation competency, possibly via inhibition of oocyte-cumulus interaction or induction of apoptosis.