Gene Expression In Ovarian Tissue Research Articles

P-424 Effects of in vitro activation vs fragmentation on human ovarian tissue in culture

Abstract Study question What is the added value of in vitro activation (IVA) protocol compared with fragmentation only in human ovarian tissue culture? Summary answer Although histological assessment shows that the IVA increases follicle survival and growth, IVA and fragmentation stimulate extensive and nearly identical transcriptomic changes in cultured tissues. What is known already Treatments based on activation of the PTEN/PI3K pathway in ovarian tissue in vitro have been administrated to refractory primary ovarian insufficiency (POI) patients and resulted in live births following auto-transplantation of the tissue. However, some studies have shown that mere fragmentation of the tissue produces comparative effects, questioning the added value of chemical stimulation protocol which could potentially activate oncogenic pathways. Study design, size, duration Thirty-three ovarian cortical biopsies were obtained from consenting women undergoing elective caesarean section at Karolinska University Hospital Huddinge. The samples were fragmented for culture studies. Half of the fragments were exposed to bpV (HOpic) +740Y-P (IVA group) during the first 24 h of culture while half were exposed to vehicle only (fragmentation only group). Subsequently, both groups were cultured for additional 6 days. Media change was performed every other day. Participants/materials, setting, methods Effects on follicles were evaluated by counting and scoring follicles before and after the 7-day culture via HE-stained serial sections. Follicle function was assessed by quantification of steroids by UPLC-MS/MS at the end of the culture. Transcriptomic effects were measured by RNA-sequencing (RNA-seq) of the tissue after the 24-h initial culture step. Selected differentially expressed genes (DEGs) were validated by qPCR and immunofluorescence in independent culture experiments. Main results and the role of chance Compared to fragmentation only group, significantly higher follicle survival rate, increased secondary follicle number and sizes were found in IVA group. No significant differences were detected in levels of steroids in culture media on day 7 between the two groups. In our RNA-seq data, when comparing the IVA group to fragmentation only group, only 110 DEGs were found with the relaxed cutoff of FDR<0.1. The signaling pathways affected by IVA involved follicle growth but also inflammation and DNA damage. However, we found that the gene expression was profoundly affected in both IVA and fragmentation only groups compared to the fresh tissue: in total 3,676 and 4,223 DEGs were found (FDR<0.001), respectively. The top enriched gene sets in both groups included several pathways that are known to modulate follicle growth, e.g. PI3K/AKT, MTORC1. We also found that classical steroidogenesis genes (e.g. CYP11A1, CYP17A1) were significantly downregulated in both groups after 24-h culture. Moreover, upregulation of genes related to glycolysis, a process that was shown to promote activation of primordial follicles, and its upstream regulator were shown in the RNA-seq data. This effect was validated by qPCR (ENO1, PKM, LDHA, p < 0.0001). Future validation will be performed using western blot and immunofluorescence. Limitations, reasons for caution The study was performed in an in vitro model where tissue was isolated from the regulation of hypothalamic-pituitary-ovarian (HPO) axis. And thus, further experiments with xeno-transplantation may be needed to explore the effect of IVA in vivo in the future. Wider implications of the findings The impact of 24-h culture on gene expression in ovarian tissue far exceeds the effect of IVA. Yet, follicle growth was stimulated by IVA, which may suggest effects on specific cell populations that are diluted in bulk transcriptomics. Cell type-specific impacts need further studies to conclude about effectiveness of IVA. Trial registration number Not applicable

Dulaglutide, a long-acting GLP-1 receptor agonist, can improve hyperandrogenemia and ovarian function in DHEA-induced PCOS rats

ObjectiveThe purpose of this study was to explore the effect of dulaglutide on DHEA induced PCOS rats and its mechanism, to provide new drugs and research directions for clinical treatment of PCOS. MethodsIn this study, the PCOS model was established by giving female SD rats subcutaneous injection of DHEA for 21 consecutive days. After modeling, the treatment group was injected subcutaneously with three doses of dulaglutide for 3 weeks. The model group was injected with sterile ultrapure water, and the normal group did not get any intervention. The body weight changes of rats in each group were recorded from the first day when rats received the administration of dulaglutide. Three weeks later, the rats were fasted the night after the last treatment, determined fasting insulin and fasting glucose the next day. After the rats were anesthetized by chloral hydrate, more blood was collected from the heart of the rat. The serum insulin, testosterone and sex hormone binding globulin (SHBG) levels were detected by the enzyme-linked immunoassay method. After removing the adipose tissue, the obtained rat ovary tissue was used for subsequent experimental detection, using HE staining for morphology and follicular development analysis; qRT-PCR for the detection of 3βHSD, CYP17α1, CYP19α1, and StAR gene expression in ovarian tissue; and western blotting analysis of CYP17α1, CYP19α1, StAR protein expression and insulin level to verify whether dulaglutide has a therapeutic effect on PCOS in rats. ResultsAfter treated with different concentrations of dulaglutide, we found that the body weight of rats in the treatment groups were reduced. Compared with the rats in PCOS group, the serum androgen level of rats in the treatment groups was significantly decreased, and the serum sex hormone binding protein content was significantly increased, and there was statistically significant difference between these groups and PCOS group. In terms of protein expression and gene regulation, the expression of 3βHSD, CYP19α1 and StAR in the ovarian tissue of rats in treatment groups were decreased significantly after received the treatment of dulaglutide, and there was statistically significant difference between these groups and PCOS group. In addition, dulaglutide reduced the insulin content in the ovarian tissue of PCOS rats. ConclusionDulaglutide may reduce the hyperandrogenemia of PCOS rats by regulating the content of serum SHBG and the expression of 3βHSD, CYP19α1, and StAR related genes and proteins, thereby inhibiting the excessive development of small follicles and the formation of cystic follicles in the ovaries of PCOS rats, thereby improving polycystic ovary in PCOS rats. In addition, dulaglutide may reduce the weight of PCOS rats, further reducing the level of high androgen in PCOS rats, and improving the morphology of their polycystic ovaries.

Effect of N-acetylcystein on ERK Gene Expression in Ovarian Tissue of Acrylamide-Treated Adult Rats

Acrylamide (AA) is a toxic and carcinogenic compound produced in cooking process. The purpose of this study is to evaluate the effect of N-acetylcysteine (NAC) on extracellular signal-regulated kinase (ERK) gene expression level and ovarian histopathological changes in AA-treated rats. Thirty-six female adult Wistar rats were randomly divided into 6 groups including control, positive control (+VE Con), negative control (-VE Con), experimental 1 (Exp1), experimental 2 (Exp2) and experimental 3 (Exp3). Twenty eight days after the treatment, ERK gene expression level was measured by real-time PCR method and ovarian histopathological changes were evaluated. The ERK gene expression level was significantly decreased in the +VE Con, Exp1 and Exp2 groups as compared to the control group (p˂0.05), but not in the -VE Con and Exp3 groups (p˃0.05). Histologically, the +VE Con group showed a significant decrease in the number of primary, secondary and Graafian follicles as well as corpus luteum as compared to the control group (p˂0.05), but not in the negative, Exp2 and Exp3 groups (p˃0.05). In the Exp1 group, the number of primary and secondary follicles as well as corpus luteum significantly decreased (p˂0.05), however, the numbers of Graafian follicle and the corpus luteum were significantly increased as compared to the +VE Con group (p˂0.05). The AA was supposed to increase the apoptosis and folliculogenesis degradation in the rat ovarian tissue by decreasing ERK gene expression. Administration of NAC ameliorated the deleterious effects of AA in a dose-dependent manner and improve folliculogenesis by reducing apoptosis level. Thus, the NAC supplement could be helpful in ameliorating animal fertility.

Effect of intra-ovarian injection of mesenchymal stem cells in aged mares.

This study aims to determine if intra-ovarian injection of bone marrow-derived mesenchymal stem cells (MSCs) improves or restores ovarian function in aged females. Prospective randomized study of eight aged mares and six young mares receiving intra-ovarian injection of MSCs or vehicle. Main outcome measures were antral follicle count and serum anti-Müllerian hormone (AMH) (aged and young mares), and for aged mares, oocyte meiotic and developmental competence; gross and histological ovarian assessment; evaluation of presence of chimerism in recovered granulosa cells and in ovarian tissue samples; and gene expression in ovarian tissue as assessed by RNA sequencing. Injection of MSCs was not associated with significant changes in follicle number, oocyte recovery rate on follicle aspiration, oocyte maturation rate, or blastocyst rate after ICSI in aged mares, or in changes in follicle number in young mares. There were no significant changes in peripheral AMH concentrations, indicating a lack of effect on growing follicles. MSC donor DNA was not recovered in granulosa cells or in ovarian tissue, indicating lack of persistence of injected MSC. RNA sequencing revealed significant differences in gene expression between MSC- and vehicle-injected ovaries. Intra-ovarian injection of bone marrow-derived MSCs altered gene expression but did not improve ovarian function in aged mares.

Effects of nutrient restriction and arginine treatment on oxidative stress in the ovarian tissue of ewes during the luteal phase

The aim of this study was to determine whether nutrient restriction and arginine treatment affect energy metabolism changes and oxidative stress through the mitochondrial pathway in the ovarian tissue of ewes during the luteal phase. On days 6–15 of the estrous cycle, 24 multiparous Hu sheep (BW = 43.56 ± 1.53 kg) were randomly assigned to three groups: control group (CG; n = 6), restriction group (RG; n = 9), and l-arginine group (AG; n = 9) administered Arg treatment (or vehicle) three times per day. The ewes were slaughtered at the end of treatment, and blood samples and ovaries were collected for analysis. In this study, the expression levels of antioxidase enzymes (SOD2, CAT and GPX1) and mitochondrial biogenesis-related genes (ESRRA and TFAM), as well as antioxidase activity and mitochondrial function were examined in ovarian tissue. Nutrient restriction resulted in activation of ESRRA and TFAM and an increase in relative mtDNA copy number, whereas arginine treatment led to a pronounced recovery of ovarian tissue. In addition, we observed increased AMPK phosphorylation at Thr172 and SIRT3 levels in nutrient restricted ewes, and these effects decreased with arginine treatment. In conclusion, the present results indicated that short-term nutritional restriction led to changes in energy metabolism and oxidative stress. These changes disrupted the redox balance, thus leading to apoptosis through the mitochondria-dependent apoptosis pathway. Arginine treatment altered gene expression in ovarian tissue and increased the resistance to oxidative stress and the anti-apoptosis capacity. The results presented here suggest a potential method to increase agricultural productivity and economic benefits in the sheep industry by using dietary supplementation with arginine to decrease temporary undernutrition of ewes.

Ovarian Transcriptome Analysis of Vitellogenic and Non-Vitellogenic Female Banana Shrimp (Fenneropenaeus merguiensis).

The banana shrimp (Fenneropenaeus merguiensis) is one of the most commercially important penaeid species in the world. Its numbers are declining in the wild, leading to a loss of broodstock for farmers of the shrimp and a need for more successful breeding programs. However, the molecular mechanism of the genes involved in this shrimp’s ovarian maturation is still unclear. Consequently, we compared transcriptomic profiles of ovarian tissue from females in both the vitellogenic stage and the non-vitellogenic stage. Using RNA-Seq technology to prepare the transcriptome libraries, a total of 12,187,412 and 11,694,326 sequencing reads were acquired from the non-vitellogenic and vitellogenic stages respectively. The analysis of the differentially expressed genes identified 1,025 which were significantly differentially expressed between the two stages, of which 694 were up-regulated and 331 down-regulated. Four genes putatively involved in the ovarian maturation pathway were chosen for validation by quantitative real-time PCR (RT-qPCR). The data from this study provided information about gene expression in ovarian tissue of the banana shrimp which could be useful for a better understanding of the regulation of this species’ reproductive cycle.

BMP15 c.-9C>G promoter sequence variant may contribute to the cause of non-syndromic premature ovarian failure

BMP15 has drawn particular attention in the pathophysiology of reproduction, as its mutations in mammalian species have been related to different reproductive phenotypes. In humans, BMP15 coding regions have been sequenced in large panels of women with premature ovarian failure (POF), but only some mutations have been definitely validated as causing the phenotype. A functional association between the BMP15 c.-9C>G promoter polymorphism and cause of POF have been reported. The aim of this study was to determine the potential functional effect of this sequence variant on specific BMP15 promoter transactivation disturbances. Bioinformatics was used to identify transcription factor binding sites located on the promoter region of BMP15. Reverse transcription polymerase chain reaction was used to study specific gene expression in ovarian tissue. Luciferase reporter assays were used to establish transactivation disturbances caused by the BMP15 c.-9C>G variant. The c.-9C>G variant was found to modify the PITX1 transcription factor binding site. PITX1 and BMP15 co-expressed in human and mouse ovarian tissue, and PITX1 transactivated both BMP15 promoter versions (-9C and -9G). It was found that the BMP15 c.-9G allele was related to BMP15 increased transcription, supporting c.-9C>G as a causal agent of POF.