Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification

Bus, A.; Szymanska, Katarzyna; Pintelon, Isabel; Leroy, Jo; Leybaert, Luc; Bols, P.E.J.

doi:10.1007/s10815-020-01993-2

Cited by 8 publications

(6 citation statements)

References 55 publications

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“…The EPR29 regulates the connexin 43 in the Golgi complex, stabilizing these monomers ( 33 ). Connexins are membrane proteins that play a role in intercellular communication, allowing the transfer of ions, such as AMPc ( 34 , 35 ). Tokuhiro et al ( 36 ) demonstrated that calreticulin regulates the folding of the proteins Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) in the ER.…”

Section: Discussionmentioning

confidence: 99%

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue

et al. 2022

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This study evaluated the effects of different concentrations (10, 20, or 40 μM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 μg/mL; AA) or anethole (300 μg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.

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Section: Discussionmentioning

confidence: 99%

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue

et al. 2022

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“…Hence, for evaluating cell–cell communications, including granulosa cell–oocyte, follicles were cultured for only 4 days. A total of 22 follicles were analyzed for the Cx43 and TZPs staining, and the categorization of Cx43 and TZPs was performed based on Bus et al (2021) . The results showed that all follicles were stained positive for Cx43.…”

Section: Resultsmentioning

confidence: 99%

“…Follicle growth and cell–cell communication in follicles were evaluated by Ki67, connexin 43 (Cx43), and TZP staining on Days 4 and 7, by adapting the protocols from Ouni et al (2022) and Bus et al (2021) . Briefly, at the end of each culture period (4 or 7 days), the hydrogels were washed with PBS.…”

Section: Methodsmentioning

confidence: 99%

“…The images of follicles in the Ki67 and Cx43 staining groups were captured using 20×- and 40×-oil immersion objectives, respectively. The categorization of Cx43 and TZPs was performed based on the Bus et al (2021) protocol, where Cx43 was classified as positive or negative in case of the presence or absence of this staining. Moreover, TZPs were considered a total absence, partial absence, or complete when no physical connection was observed between oocyte and granulosa cells, gaps existed between oocyte and granulosa cells, or no contact was lost between oocyte and granulosa cells, respectively ( Bus et al , 2021 ).…”

Section: Methodsmentioning

confidence: 99%

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Mind the mechanical strength: tailoring a 3D matrix to encapsulate isolated human preantral follicles

Dadashzadeh

Moghassemi

Peaucelle

et al. 2023

Human Reproduction Open

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STUDY QUESTION Would a hydrogel with similar mechanical properties to the human ovarian cortex support preantral follicle development? SUMMARY ANSWER Yes, our tailored PEGylated fibrin hydrogel was shown to significantly improve follicle growth in vitro. WHAT IS KNOWN ALREADY One of the main challenges in developing an engineered ovary is to provide a 3D matrix that supports the follicle architecture and the interaction between granulosa cells and the oocyte as they are essential for folliculogenesis. Thanks to its biocompatibility and bioactivity, fibrin has been employed to fabricate a 3D matrix to encapsulate ovarian follicles. However, follicles lose their physical support within a few days owing to rapid fibrin degradation. Therefore, different strategies, including physical and chemical modifications, have been developed to enhance the stability of fibrin. STUDY DESIGN, SIZE, DURATION By developing a matrix made of a synthetic (polyethylene glycol: PEG) and natural polymer (fibrin), we aimed to overcome fibrin degradation by the chemical reaction of PEGylation and tailor a PEGylated fibrin hydrogel formulation with mechanical strength similar to the ovarian cortex in women of reproductive age. To this end, response surface methodology was employed to obtain a tailored formulation of PEGylated fibrin. This hydrogel was then tested to encapsulate and support isolated human preantral follicles in vitro. PARTICIPANTS/MATERIALS, SETTING, METHODS A PEGylated fibrin formulation was tailored using mathematical modeling software to mimic the mechanical properties of human ovarian tissue at reproductive age. Human preantral follicles were isolated from 11 patients of reproductive age and encapsulated in the tailored hydrogels, which were cultured in vitro for 4 or 7 days. Follicle survival and diameter were assessed on days 1 and 7. Furthermore, the follicles were subjected to confocal microscopy to evaluate their growth (Ki67 staining) on day 7 and analyze cell-cell communication (connexin 43 and transzonal projection staining) on day 4. MAIN RESULTS AND THE ROLE OF CHANCE In this study, mathematical modeling was applied to achieve the biomechanically tailored PEGylated fibrin formulation by targeting the specific goal of 3178 ± 245 Pascal, Young’s modulus of ovarian cortical tissue in reproductive-age women. Our results demonstrated that the PEGylated fibrin hydrogel consisting of 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin was the optimum condition with the desirability of 97.5%. This tailored hydrogel yielded a high follicle survival rate (83%) after 7 days of in vitro culture and supported its development up to the secondary stage. Follicle growth was confirmed by the presence of Ki67-positive granulosa cells on day 7. Additionally, connexin 43 and Phalloidin staining indicated the retention of connections between granulosa cells and the oocyte. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION In this study, our tailored hydrogel was only tested in vitro, which is not the same as the physiological environment. It is crucial to conduct a study assessing the follicles following their encapsulation in the tailored hydrogel and transplantation, which will be the next step of our investigation. WIDER IMPLICATIONS OF THE FINDINGS The findings from this study introduced a suitable biomaterial similar to the ovarian cortex in reproductive-age women in terms of biomechanical properties for encapsulating human preantral follicles. This biomaterial allowed the radial growth of follicles and preserved their viability. Furthermore, PEGylation improved the stability of fibrin and the physical support of follicles. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by a grant from the Fondation Louvain (awarded to C.A.A.). The authors declare no competing interests. WHAT DOES THIS MEAN FOR PATIENTS? Survival rates of leukemia patients have been steadily increasing but, owing to cancer treatment, some survivors will face infertility at a very young age. One of the strategies under development to restore fertility in these patients is the transplantable ‘engineered ovary’, which should mimic the normal ovary as closely as possible. To assemble the engineered ovary, it is necessary to encase isolated preantral follicles (follicles at an early stage of growth that contain one immature egg) and ovarian cells in a 3D matrix. Developing this matrix is one of the main challenges in creating an engineered ovary, as it should support the follicle architecture as well as the interaction between granulosa cells (which produce estrogen, progesterone) and the oocyte (egg), as they are essential for follicle development. This study demonstrates that a matrix made of a synthetic (polyethylene glycol) and natural polymer (fibrin), with mechanical strength similar to that of the outer layer of the ovary in women of reproductive age, can successfully be used to encapsulate human preantral follicles. Indeed, we have shown that our 3D matrix yielded a high follicle survival rate and supported follicle growth to the secondary stage.

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“…Based on the available studies, what we can affirm is the facilitative role of intercellular communications in folliculogenesis, and using this can also help us develop assisted reproduction techniques (ART). Fresh follicles were found to have higher levels of CX43 expression than vitrified follicles, and CX43 expression levels were also significantly lower in follicles cultured in vitro for 4 days ( Bus et al, 2021 ), therefore, preservation and promotion of communications in cultured follicles may increase their quality in IVM. Kawai and Shimada found that during vitrification, the attachment of the oocyte to CCs was impaired because of the loss of cadherin adhesion molecules, which was critical for AJs ( Kawai and Shimada, 2020 ).…”

Section: Applications Of Intercellular Communications and Future Pers...mentioning

confidence: 99%

Intercellular communication in the cumulus–oocyte complex during folliculogenesis: A review

Xie

Liu

2023

Front. Cell Dev. Biol.

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During folliculogenesis, the oocyte and surrounding cumulus cells form an ensemble called the cumulus-oocyte complex (COC). Due to their interdependence, research on the COC has been a hot issue in the past few decades. A growing body of literature has revealed that intercellular communication is critical in determining oocyte quality and ovulation. This review provides an update on the current knowledge of COC intercellular communication, morphology, and functions. Transzonal projections (TZPs) and gap junctions are the most described structures of the COC. They provide basic metabolic and nutrient support, and abundant molecules for signaling pathways and regulations. Oocyte-secreted factors (OSFs) such as growth differentiation factor 9 and bone morphogenetic protein 15 have been linked with follicular homeostasis, suggesting that the communications are bidirectional. Using advanced techniques, new evidence has highlighted the existence of other structures that participate in intercellular communication. Extracellular vesicles can carry transcripts and signaling molecules. Microvilli on the oocyte can induce the formation of TZPs and secrete OSFs. Cell membrane fusion between the oocyte and cumulus cells can lead to sharing of cytoplasm, in a way making the COC a true whole. These findings give us new insights into related reproductive diseases like polycystic ovary syndrome and primary ovarian insufficiency and how to improve the outcomes of assisted reproduction.

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Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification

Cited by 8 publications

References 55 publications

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue

Mind the mechanical strength: tailoring a 3D matrix to encapsulate isolated human preantral follicles

Intercellular communication in the cumulus–oocyte complex during folliculogenesis: A review

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