scholarly journals Establishment and Mechanism Study of a Primary Ovarian Insufficiency Mouse Model Using Lipopolysaccharide

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Si-Ji Lv ◽  
Shu-Hui Hou ◽  
Lei Gan ◽  
Jing Sun

This study is aimed at establishing a lipopolysaccharide- (LPS-) induced primary ovarian insufficiency (POI) mouse model and investigating the underlying mechanism. C57BL/6N female mice were intraperitoneally injected with low-dose LPS (0.5 mg/kg) once daily for 14 days, high-dose LPS (2.5 mg/kg) twice weekly for 2 weeks, or cyclophosphamide (CTX; 150 mg/kg) once weekly for 2 weeks. Ovarian function was assessed by measuring the length of estrous cycle, the number of primordial follicles, and the levels of serum hormones. Expression and production of interleukin 1β (IL-1β) were determined to evaluate ovarian inflammation. Histopathological examination was performed to examine ovarian fibrosis. TUNEL assay was carried out to evaluate granulosa cell apoptosis. Western blotting was performed to measure the levels of inflammation-, fibrosis-, and apoptosis-related proteins in the mouse ovaries. Like CTX, both low- and high-dose LPS significantly impaired ovarian functions in mice, as evidenced by extended lengths of estrous cycles, reduced counts of primordial follicles, and alterations in the levels of serum hormones. Also, LPS promoted granulosa cell apoptosis and ovarian fibrosis in mice. However, LPS but not CTX promoted IL-1β expression and production in mice. Moreover, LPS but not CTX enhanced TLR, p-p65, p65, and MyD88 expression in mouse ovaries, suggesting that LPS differs from CTX in triggering ovarian inflammation. In general, continuous low-dose LPS stimulation was less potent than high-dose LPS to affect the ovarian functions. In conclusion, LPS may induce ovarian inflammation, fibrosis, and granulosa cell apoptosis and can be used to establish a POI model in mice.

2021 ◽  
Author(s):  
Si-Ji Lv ◽  
Shu-Hui Hou ◽  
Lei Gan ◽  
Jing Sun

Abstract Background: This study aimed to establish a lipopolysaccharide (LPS)-induced primary ovarian insufficiency (POI) mouse model and to investigate the underlying mechanism.Methods: C57BL/6N female mice were intraperitoneally injected with low-dose LPS (0.5 mg/kg) once daily for 14 days, high-dose LPS (2.5 mg/kg) twice weekly for 2 weeks, and cyclophosphamide (CTX; 150 mg/kg) once weekly for 2 weeks. Ovarian function was assessed by measuring the length of the estrous cycle, the number of primordial follicles, and the levels of serum pituitary/ovarian hormones. Expression and production of interleukin 1β (IL-1β) were determined to evaluate ovarian inflammation. Histopathological examination was performed to examine ovarian fibrosis. TUNEL assay was carried out to evaluate granulosa cell apoptosis. Western blotting was performed to measure the levels of inflammation-, fibrosis-, and apoptosis-related proteins in mouse ovaries.Results: Like CTX, both low- and high-dose LPS administration significantly impaired ovarian functions in mice, as evidenced by extended lengths of estrous cycles, reduced counts of primordial follicles, and alterations in the levels of serum hormones. Also, LPS administration promoted granulosa cell apoptosis and ovarian fibrosis in mice. However, LPS but not CTX significantly promoted IL-1β expression and production in mice. Moreover, LPS treatment but not CTX significantly enhanced TLR, p-p65, p65, and MyD88 protein expression in mouse ovaries, suggesting that LPS differs from CTX in triggering ovarian inflammation. In general, continuous low-dose LPS stimulation was less potent than high-dose LPS stimulation in the above-mentioned effects.Conclusions: LPS induces ovarian inflammation, fibrosis, and granulosa cell apoptosis and can be used to establish a POI model in mice.


2016 ◽  
Vol 22 (6) ◽  
pp. 384-396 ◽  
Author(s):  
Carola Conca Dioguardi ◽  
Bahar Uslu ◽  
Monique Haynes ◽  
Meltem Kurus ◽  
Mehmet Gul ◽  
...  

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hang-soo Park ◽  
Rishi Man Chugh ◽  
Abdeljabar El Andaloussi ◽  
Elie Hobeika ◽  
Sahar Esfandyari ◽  
...  

AbstractPrimary ovarian insufficiency (POI) is defined as the loss of ovarian function before 40 years of age. It clinically manifests as amenorrhea, infertility, and signs of estrogen insufficiency. POI is frequently induced by chemotherapy. Gonadotoxic chemotherapy reagents damage granulosa cells, which are essential for follicular function and development. Our recently published studies demonstrated that intraovarian transplantation of human mesenchymal stem cells (hMSCs) can restore fertility in a chemotherapy-induced POI mouse model. However, the regenerative mechanism underlying the hMSC effect in POI mice is not fully understood. Here, we report that the hMSC secretome increased the proliferation of human granulosa cells (HGrC1). We showed by FACS analysis that treatment of HGrC1 cells with hMSC-conditioned media (hMSC CM) stimulates cellular proliferation. We also demonstrated that the expression of steroidogenic enzymes involved in the production of estrogen, CYP19A1 and StAR, are significantly elevated in hMSC CM-treated HGrC1 cells. Our data suggest that hMSC CM stimulates granulosa cell proliferation and function, which may explain the therapeutic effect of hMSCs in our chemotherapy-induced POI animal model. Our findings indicate that the hMSC secretome may be a novel treatment approach for restoring granulosa cell and ovarian function in patients with POI.


2018 ◽  
Vol 51 (5) ◽  
pp. 2341-2358 ◽  
Author(s):  
Xiaowei Nie ◽  
Youjin Dai ◽  
Yuan Zheng ◽  
Dan Bao ◽  
Qin Chen ◽  
...  

Background/Aims: This study investigated the effect of consecutive superovulation on the ovaries and established a premature ovarian failure (POF) model in mice. Methods: The mouse POF model was induced by 5-15 consecutive superovulation treatments with pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (HCG) and prostaglandin F2α (PGF2α). Normal adult mice were compared with mice displaying natural ovarian aging. The following serum biochemical parameters were measured: including follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P), estradiol (E2), inhibin B (INH B), malondialdehyde (MDA), total superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. Follicles were counted using H&E staining. Levels of 8-hydroxyguanosine (8-OhdG), 4-hydroxynonenal (4-HNE), nitrotyrosine (NTY), anti-Mullerian hormone (AMH) and CDKN2A/ p16 (p16) were detected using immunohistochemical staining. Reactive oxygen species (ROS) levels were measured using dihydroethidium (DHE) staining. Cell apoptosis was detected using an in situ TUNEL fluorescence staining assay. Levels of proteins involved in ROS-related pathways and the p16 protein were detected using Western blotting. Sod1, Sod2 and Sod3 mRNA levels were detected using quantitative polymerase chain reaction (Q-PCR). Oocyte quality was evaluated using in vitro fertilization (IVF) and zygote culture. Results: Consecutive superovulation groups presented lower P, E2, SOD, GSH-Px and INH B levels, significantly higher FSH, LH, MDA and ROS levels, and significantly fewer primordial follicles compared with the control group. Consecutive superovulation groups presented significantly increased levels of Sod2, 8-OhdG, 4-HNE, NTY, significantly increased levels of the SIRT1 and FOXO1 proteins, significantly increased levels of the senescence-associated protein p16, as well as decreased AMH, Sod1 and Sod3 levels and increased granulosa cell apoptosis compared with the control group. Conclusion: Consecutive superovulation significantly decreased ovarian function and oocyte quality and increased oxidative stress and apoptosis in the ovary via a mechanism involving the p16 and SIRT1/FOXO1 signaling pathways. These findings suggest that consecutive superovulation may be used to establish a mouse model of ovarian aging.


PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e96622 ◽  
Author(s):  
Karen Tse ◽  
Sreekanth Puttachary ◽  
Edward Beamer ◽  
Graeme J. Sills ◽  
Thimmasettappa Thippeswamy

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Fangui Zhao ◽  
Wenjun Wang

Diminished ovarian reserve (DOR) refers to a decrease in the number and quality of oocytes. Western treatment of DOR does not improve the ovarian reserve fundamentally, and the effect is limited. Gengnianchun recipe (GNC) is a traditional Chinese medicine formula originally applied to treat menopausal syndrome but is also found to be effective in treating clinical DOR patients. Here we aim to examine the effect of GNC in a DOR rat model induced by 4-vinylcyclohexene diepoxide (VCD), a chemical that selectively destroys ovarian small preantral follicles, and further investigate the possible mechanisms. Female SD rats were randomly divided into four groups: control group (C), model group (M), high-dose GNC group (H), and low-dose GNC group (L). Rats in M, H, and L were administered with VCD and normal saline, high-dose GNC, and low-dose GNC separately. Rat ovaries were harvested either to conduct HE staining for follicle count, immunohistochemistry, or western blot. We found that high dose of GNC significantly increased the ovarian index and sustained the number of primordial follicles and primary follicles in VCD treated rats. Moreover, high dose of GNC significantly increased the ovarian protein expression of mouse vasa homologue (MVH), anti-Müllerian hormone (AMH), follicle-stimulating hormone receptor (FSHR), and estrogen receptor β (ERβ) compared with that in the model group. Besides, high-dose GNC significantly increased ovarian AKT phosphorylation and the expression of downstream forkhead box O3 (FOXO3a). Proapoptosis proteins of Bax, cleaved caspase-3, and poly ADP-ribose polymerase (PARP) were significantly decreased after high-dose GNC treatment compared with those in the model group. Taken together, these findings suggest that high-dose GNC could protect ovarian reserve against VCD-induced toxicity via the activation of the AKT signaling pathway and reduced cell apoptosis in SD Rats. This effect could either be induced by the increased FSHR signaling or by the nontranscriptional activation of ERβ, which requires further investigation.


2020 ◽  
Vol 26 (7) ◽  
pp. 485-497
Author(s):  
Diego Hernández-López ◽  
Adriana Geisinger ◽  
María Fernanda Trovero ◽  
Federico F Santiñaque ◽  
Mónica Brauer ◽  
...  

Abstract More than 50% of cases of primary ovarian insufficiency (POI) and nonobstructive azoospermia in humans are classified as idiopathic infertility. Meiotic defects may relate to at least some of these cases. Mutations in genes coding for synaptonemal complex (SC) components have been identified in humans, and hypothesized to be causative for the observed infertile phenotype. Mutation SYCE1 c.721C>T (former c.613C>T)—a familial mutation reported in two sisters with primary amenorrhea—was the first such mutation found in an SC central element component-coding gene. Most fundamental mammalian oogenesis events occur during the embryonic phase, and eventual defects are identified many years later, thus leaving few possibilities to study the condition’s etiology and pathogenesis. Aiming to validate an approach to circumvent this difficulty, we have used the CRISPR/Cas9 technology to generate a mouse model with an SYCE1 c.721C>T equivalent genome alteration. We hereby present the characterization of the homozygous mutant mice phenotype, compared to their wild type and heterozygous littermates. Our results strongly support a causative role of this mutation for the POI phenotype in human patients, and the mechanisms involved would relate to defects in homologous chromosome synapsis. No SYCE1 protein was detected in homozygous mutants and Syce1 transcript level was highly diminished, suggesting transcript degradation as the basis of the infertility mechanism. This is the first report on the generation of a humanized mouse model line for the study of an infertility-related human mutation in an SC component-coding gene, thus representing a proof of principle.


2015 ◽  
Vol 309 (3) ◽  
pp. R223-R234 ◽  
Author(s):  
Asada Leelahavanichkul ◽  
Poorichaya Somparn ◽  
Tanabodee Bootprapan ◽  
Hongbin Tu ◽  
Pattarin Tangtanatakul ◽  
...  

Amphotericin B (Ampho B) is a fungicidal drug that causes cell wall injury. Pharmacological ascorbate induces the extracellular prooxidants, which might enter the Ampho B-induced cell wall porosity and act synergistically. We tested low-dose Ampho B with a short course of pharmacological ascorbate using a mouse model of sepsis preconditioned with an injection of Candida albicans 6 h prior to cecal ligation and puncture (CLP). In this model, candidemia reappeared as early as 6 h after CLP with a predictably high mortality rate. This characteristic mimics sepsis in the phase of immunosuppression in patients. Using the model, at 12- and 18-h post-CLP, we administered isotonic (pH neutralized) pharmacological ascorbate intravenously with low-dose Ampho B or sodium deoxycholate, vehicle-controlled, administered IP. The survival rate of low-dose Ampho B plus ascorbate was 53%, compared with <11% for low-dose Ampho B or high-dose Ampho B alone. In addition, a beneficial effect was demonstrated in terms of kidney damage, liver injury, spleen histopathology, and serum markers at 24 h after CLP. Kidney injury was less severe in low-dose Ampho B plus ascorbate combination therapy due to less severe sepsis. Moreover, ascorbate enhanced the effectiveness of phagocytosis against C. albicans in human phagocytic cells. Taken together, the data indicate that the new mouse model simulates sepsis-induced immunosuppression and that the combination of pharmacological ascorbate with an antifungal drug is a potentially effective treatment that may reduce nephrotoxicity, and perhaps also increase fungicidal activity in patients with systemic candidiasis caused by Candida albicans.


2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A484-A484
Author(s):  
Reem Sabry ◽  
Charlotte Apps ◽  
Cassidy Van Den Diepstraten ◽  
Laura A Favetta

Abstract Bisphenols are Endocrine Disrupting Compounds (EDCs) linked to negative fertility outcomes in humans. The most common one, bisphenol A (BPA) is a plasticizer that acts as an estrogen agonist. Alternatives such as BPS and BPF are becoming widespread and possess similar activities. The oocyte and nearby granulosa cells are particularly vulnerable to EDCs as they are dependent on hormone signaling. Granulosa-Granulosa cell communications are pivotal for proper development and are mediated by gap junction molecules, called connexins (Cxs). Cx37, Cx26, and Cx43 are crucial in follicular communication and are the focus of this study. Bisphenols can alter these genes which directly affects cell-cell communication and in turn proper development. This study uses an in vitro cell culture model of bovine granulosa cells as the bovine species is an excellent translational model for human reproductive toxicology. To determine optimal doses and exposure times, we performed a cytotoxic assay (CCK8) and treated cells with a low dose (0.5 μg/mL), a medium dose (5 μg/mL), the current reported LOAEL (50 μg/mL), and a high dose (0.5 mg/mL) of bisphenols for 1, 6, 12, 24, 48, and 72 hrs. The high dose was lethal at all time points for BPA/BPF, while BPS was toxic from 1 hrs (P=0.0016; n=4). The LOAEL dose was toxic for BPA at 6, 12, 24 hrs (P=0.018, 0.0021, 0.0042 respectively; n=7) and was lethal at 48/72 hrs. BPF at the LOAEL dose showed an increase in cell viability at 6 hrs which was significant at 12hrs (P=0.0119; n=5). This trend declined at 48/72 hrs but was not significant. BPS did not affect cell viability at any other dose and the medium dose did not affect viability for all bisphenols; however, the low dose of BPA/BPF showed a trend towards decreased viability at 48 hrs of treatment. Therefore, we looked at the effects of BPA/S/F at the low and LOAEL doses for 12/48 hrs. We used qPCR and western blotting to quantify transcripts and proteins of the three connexins. Our data show that Cx43 mRNA was significantly increased at the LOAEL dose for BPA/BPF at 12 hrs (p=0.02; n=6). Preliminary data on proteins indicate a decrease and increase in expression at 12hrs for BPA/BPF treatment, respectively, at the LOAEL dose. BPS shows no effect on Cx43. BPS low dose and BPA/BPF LOAEL doses at 12 and 48 hrs increase Cx37 mRNA. Preliminary data on the protein levels show a significant decrease of Cx37 protein at BPA low dose, but no changes after exposure to the other bisphenols. Lastly, Cx26 mRNA is significantly increased after BPA treatment at the LOAEL dose at 12 hrs (P&lt;0.0001; n=5). This novel study investigates the effects of low/high doses and short-term/chronic exposure of bisphenols on cell-cell communication in granulosa cells and provides the basis for future studies on bisphenol toxicity in early development.


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