β-Galactosidase (β-gal) which is overexpressed in primary ovarian cancer can be employed as a valuable biomarker for ovarian cancer. Thus, monitoring and imaging endogenous β-gal in living cells is ...of great importance. Herein, a dicyanoisophorone-based near-infrared (NIR) fluorescent probe 2-(5,5-dimethyl-3-((E)-4-(((2R,3S,4R,5S,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)styryl)cyclohex-2-en-1-ylidene)malononitrile named DP-βgal, was rationally designed and synthesized for the monitoring of β-gal activity in living cells. In the presence of β-gal, with the breaking of the glycosidic bond, the NIR fluorescence of the dicyanoisophorone derivative gradually recovered, enabling the fluorescence “off-on” quantitative determination of β-gal activity. DP-βgal has the advantages of good selectivity and high sensitivity for the detection of β-gal, with the limit of detection (LOD) of 3.2 × 10−3 U. Furthermore, based on its advantages of long-wavelength emission and excellent biocompatibility, the practical applications of DP-βgal in NIR imaging of β-gal in living ovarian cancer cells (SKOV-3) were demonstrated.
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•An “off-on” NIR fluorescent probe (DP-βgal) was rationally constructed for the detection of β-galactosidase (β-gal).•DP-βgal shows high sensitivity, compatibility and selectivity toward β-gal with large Stokes shift and NIR emission.•A method for diagnosis by imaging of endogenous β-gal in living ovarian cancer cells was developed.
To create an artificial ovary to provide an alternative way of restoring fertility in patients who cannot benefit from transplantation of cryopreserved ovarian tissue due to the threat of ...reintroducing malignant cells.
In vivo experimental study.
Gynecology research unit in a university hospital.
Six-week-old female NMRI mice.
Autografting of isolated preantral follicles and ovarian cells (OCs) encapsulated in two fibrin matrices containing low concentrations of fibrinogen (F; mg/mL) and thrombin (T; IU/mL): F12.5/T1 and F25/T4.
Follicular density and development, OC survival and proliferation, inflammatory response, and vascularization.
After 1 week, the follicle recovery rate ranged from 30.8% (F25/T4) to 31.8% (F12.5/T1). With both fibrin formulations, all follicles were found to be alive or minimally damaged, as demonstrated by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and at the growing stage (primary, secondary, and antral follicles), confirmed by Ki67 immunostaining. Isolated OCs also survived and proliferated after grafting, as evidenced by <1% apoptotic cells and a high proportion of Ki67-positive cells. Vessels were found in both fibrin formulations, and the global vascular surface area varied from 1.35% (F25/T4) to 1.88% (F12.5/T1). Numerous CD45-positive cells were also observed in both F25/T4 and F12.5/T1 combinations.
The present study is the first to show survival and growth of isolated murine ovarian follicles 1 week after autotransplantation of isolated OCs in a fibrin scaffold. The results indicate that fibrin is a promising candidate as a matrix for the construction of an artificial ovary. Xenotransplantation of isolated human follicles and OCs is the necessary next step to validate these findings.
Premature ovarian failure (POF) is characterized by amenorrhea, hypoestrogenism, elevated gonadotropin levels, and infertility. Although some of previous studies reported that Mesenchymal stem cells ...(MSC) transplantation could rescue the ovary function of POF animal models through the paracrine pathways, these mechanisms require further investigation. Here, we aimed to investigate the possible mechanisms of therapeutic effects of human embryonic stem cells derived MSC (ES-MSC) in a mice model of chemotherapy-induced POF. For this purpose, Cyclophosphamide (Cy) was injected intraperitoneally into female mice to induce POF. 10 days after Cy injection, we evaluated follicle count, follicle-stimulating hormone (FSH) and estradiol (E2) hormone concentrations, and TUNEL assay. Then, ES-MSC was transplanted into mice and the expression of Anti-müllerian hormone (AMH) and apoptosis was evaluated in ovary. Results indicated that ES-MSC reduced apoptosis in the follicles and increased the expression of AMH protein in the ovary of POF mice. So, ES-MSC may inhibit the apoptosis of ovarian granulosa cells. Then, to investigate the potential mechanisms of therapeutic effects of ES-MSC and their fate in the ovary, MSC were labeled with green fluorescent protein (GFP) before transplantation. Immunofluorescence staining indicated that although GFP-labeled ES-MSC was located in the ovarian stroma, they did not express granulosa cell markers: AMH and Follicle-stimulating hormone receptor (FSHR), theca cell marker: luteinizing hormone receptor (LHR), and oocyte marker: Growth/differentiation factor 9 (GDF9). Therefore, ES-MSC may not differentiate into ovarian cells directly and they might restore ovarian function in chemotherapy-induced POF mice by paracrine mechanisms.
•Mesenchymal stem cells (MSC) are a promising tool for cell therapy of diseases.•Embryonic stem cells derived MSC (ES-MSC) increased AMH expression in POF mice.•ES-MSC transplantation reduced cell apoptosis in the ovary of POF mice.•GFP-labeled ES-MSC was found in the ovary but did not express ovarian cells markers.
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•Cu exposure hinders FSH binding to FSHR.•Cu induces ovarian steroidogenesis disorders via the FSHR/CYP19A1 pathway.•Hypomethylation on SF-1 promoter is involved in Cu-induced ...steroidogenesis disorders.
Information on the effects of copper on reproduction is limited. Our previous study indicated that copper induces abnormal steroidogenesis in human ovarian granulosa cells, but the underlying mechanism remains unclear. In this study, human ovarian granulosa cells were treated with multiple concentrations of copper for 24 h. After treatment, the 17-estradiol levels were significantly increased (29.83 % and 45.12 %, respectively) in the 1.0 and 2.0 μg/mL groups but decreased (23.06 % and 31.56 %, respectively) in the 20.0 and 40.0 μg/mL groups (P < 0.05). Similar changes in the levels of FSHR, StAR, CYP11A1, CYP19A1, HSD3β1, and SF-1 were observed. The protein levels of FSHR were increased in the 2.0 μg/mL group but decreased in the 20.0 and 40.0 μg/mL groups (P < 0.05). Moreover, copper partially reversed the FSH-induced increase in FSHR, CYP19A1 and 17-estradiol levels, and the decreased effect of the FSH receptor binding inhibitor fragment on FSHR, CYP19A1, and 17-estradiol became more apparent after adding copper. Additionally, the total methylation levels of the SF-1 promoter and DNMTs expression were significantly decreased following copper treatment. Overall, our results indicate that copper exposure induces steroidogenesis disorders via the FSHR/CYP19A1 pathway and changes DNA methylation on the SF-1 promoter in human ovarian granulosa cells.
Abstract For women diagnosed with leukemia, transplantation of cryopreserved ovarian tissue after disease remission is not advisable. Therefore, to restore fertility in these patients, we aim to ...develop a biodegradable artificial ovary that offers an environment where isolated follicles and ovarian cells (OCs) can survive and grow. Four NMRI mice were ovariectomized and their ovaries used to isolate OCs. Groups of 50,000 OCs were embedded in an alginate–matrigel matrix for further fixation (fresh controls), one week of in vitro culture (IVC) or heterotopic autografting. OC proliferation (Ki67), apoptosis (TUNEL), scaffold degradation, vessel formation (CD34) and inflammation (CD45) were analyzed. Ki67-positive OCs were found in 2.3%, 9.0% and 15.5% cells of cases in fresh, IVC and grafted beads respectively, while cells were TUNEL-positive in 0%, 1.5% and 6.9% of cases. After IVC or grafting, the beads degraded, losing their original round aspect, and infiltrating blood capillaries could be observed in the grafted beads. CD34-positive cells and 22% CD45-positive cells were found around and inside the matrix. In conclusion, our results demonstrate that an alginate-based matrix is a promising proposition to graft isolated OCs. After transplantation, this matrix was able to degrade, allowed vascularization and elicited a low inflammatory response.
Curcumin is a natural molecule with proved anticancer efficacy on several human cancer cell lines. However, its clinical application has been limited due to its poor bioavailability. ...Nanocarrier-based drug delivery approaches could make curcumin dispersible in aqueous media, thus overtaking the limits of its low solubility. The aim of this study was to increase the bioavailability and the antitumoral activity of curcumin, by entrapping it into nanostructured lipid carriers (NLCs). For this purpose here we describe the preparation and characterization of three kinds of curcumin-loaded NLCs. The nanosystems allowed the achievement of a controlled release of curcumin, the amounts of curcumin released after 24 h from Compritol–Captex, Compritol–Miglyol, and Compritol NLCs being, respectively, equal to 33, 28, and 18% w/w on the total entrapped curcumin. Considering the slower curcumin release profile, Compritol NLCs were chosen to perform successive in vitro studies on ovarian cancer cell lines. The results show that curcumin-loaded NLCs maintain anticancer activity, and reduce cell colony survival more effectively than free curcumin. As an example, the ability of A2780S cells to form colonies was decreased after treatment with 5 μM free curcumin by 50% ± 6, whereas, at the same concentration, the delivery of curcumin with NLC significantly (p < 0.05) inhibited colony formation to approximately 88% ± 1, therefore potentiating the activity of curcumin to inhibit A2780S cell growth. The obtained results clearly suggest that the entrapment of curcumin into NLCs increases curcumin efficacy in vitro, indicating the potential use of NLCs as curcumin delivery systems.
Previous studies with perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) indicate that they act as endocrine disruptors, in addition to inducing alterations and damaging ...reproductive health; however, the biological mechanisms by which these disorders are produced are not yet understood. The aim of this study was to analyze the effect of PFOS and PFOA on in vitro steroidogenic secretion in porcine theca and granulosa cells, with or without gonadotropic stimulation. Granulosa and theca cells were isolated and cultured. Cell nature was performed by immunocytochemistry. PFOS and PFOA effect on steroid secretion was analyzed by chemiluminescence. In the present study, alterations in steroidogenic secretion were found when administering PFOS (0.12, 1.2, 12, 120 or 240μM) or PFOA (0.012, 0.12, 1.2, 12 or 24μM) to theca and granulosa cells. When theca and granulosa cells were stimulated with 500ng/mL LH or 500ng/mL FHS, respectively and immediately followed with 1.2μM of PFOS or PFOA, the perfluorinated compounds inhibited the secretion of steroid hormones in both stimulated cell types. The results indicate that PFOS and PFOA act on steroidogenic ovarian cells as endocrine disruptors, which could affect the dependent functions of sexual steroids.
•In stimulated theca cells, PFCs inhibit progesterone and androstenedione secretion.•In stimulated granulose cells, PFCs inhibit progesterone and estradiol secretion.•PFOS and PFOA act as endocrine disruptors in steroidogenic ovaric cells.
STUDY QUESTION
What is the best source of ovarian cells for the artificial ovary: medulla or cortex, cryopreserved or fresh?
SUMMARY ANSWER
Ovarian cells from fresh medullary tissue, which can be ...isolated in larger numbers, show higher viability and are able to improve graft vascularization.
WHAT IS KNOWN ALREADY
In a previous study, addition of endothelial cells along with ovarian cells was found to be crucial for formation of a well-vascularized ovary-like structure. This study is the first to evaluate both the effect of cryopreservation and the source of ovarian tissue on isolated ovarian cells.
STUDY DESIGN, SIZE, DURATION
Prospective experimental study in an academic research unit using ovarian tissue from seven patients undergoing surgery for benign gynecologic disease.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Ovarian tissue was retrieved from seven patients, with one half processed as fresh (fresh group) and the other half frozen and thawed before processing (frozen group). In each group, ovarian cells from the cortex and medulla were isolated separately, and their viability was tested using a calcein AM/ethidium homodimer viability assay. Fifty thousand cells were then encapsulated in fibrin and grafted to peritoneal pockets in nude mice (14 in all). Grafts recovered after 7 days were analyzed by immunohistochemistry for the presence of ovarian cells (vimentin), proliferation (Ki67) and graft vascularization (double CD34). Cell apoptosis was analyzed by TUNEL assay.
MAIN RESULTS AND THE ROLE OF CHANCE
Cryopreservation decreased ovarian cell yield (−2804 cells/mg, P = 0.015) and viability (−9.72%, P = 0.052) before grafting and had a considerable (5-fold, P = 0.2) but non-significant negative impact on ovarian cell presence in grafts. The medulla yielded many more cells (+3841 cells/mg, P < 0.001) with higher viability (+18.23%, P < 0.001) than did the cortex. Moreover, grafts with cells from the medulla exhibited a statistically significant 6.44- and 2.47-fold increase in human and total vascular surface area, respectively. P-values were adjusted for multiple testing using the Benjamini–Hochberg method to achieve a 10% false discovery rate and adjusted P-values < 0.1 were therefore considered significant.
LIMITATIONS, REASONS FOR CAUTION
Pilot study involving a limited number of experiments.
WIDER IMPLICATIONS OF THE FINDINGS
Knowing that fresh medullary tissue is the best source of stromal cells is important for construction of the artificial ovary, as isolated follicles require structural support and a rich vascular network for their survival and development.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (5/4/150/5 and 7.4518.12F), Fonds Spéciaux de Recherche, Fondation Saint Luc and Foundation Against Cancer, and donations from Mr Pietro Ferrero, Baron Frère and Viscount Philippe de Spoelberch. None of the authors have any conflicting interests to declare.
Human primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, ...initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (
COX4
,
OPA1, TOMM20
), steroidogenic (
CYP11A1
,
CYP19A1)
or cell–cell contact genes (
GJA1
) between the two groups in cells cultured for 1–5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.
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