Follicle Culture Research Articles

IN VITRO MATURED OOCYTES DERIVED FROM THREE DIMENSIONAL CULTURE (3-D ) OF PRE-ANTRAL FOLLICLES IN A HYALURONAN BIOMATRIX ARE FUNCTIONALLY COMPETENT

In vitro culture systems that preserve 3-D follicle architecture and oocyte-granulosa cell interaction may be the key to in vitro oocyte maturation (IVM). Hyaluronan is one component of natural extracellular matrices that provide support to cells in vivo. This report describes the application of hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes.

Evaluating the Effects of Different Concentrations of Human Follicular Fluid on Growth, Development, and PCNA Gene Expression of Mouse Ovarian Follicles

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

The optimized research of the in vitro culture of preantral follicles in mice.

ObjectiveEfficiency of preantral follicle culture in vitro is low and is dependent on species, development stage, and follicle‐stimulating hormone (FSH) concentration. Here, we optimized the preantral follicle in vitro culture system in mice.MethodsThe primary follicles (PM follicles, 80‐100 μm diameter ) and early secondary follicles (ES follicles, 110‐130 μm diameter) isolated from 14‐day female mice were cultured in mediums containing 10 mIU/mL or 100 mIU/mL r‐FSH. The follicle growth and oocyte maturation were observed. Estradiol (E2) was detected by ELISA. FSH receptor (FSHR), Ki‐67, 3β‐HSD, CYP17, and CYP19 levels were detected by immunofluorescence and Western blot.ResultsThe antrum formation and oocyte maturation rates of ES follicles were significantly higher than those of PM follicles (P < .05). They were also significantly higher in ES follicles with 100 mIU/mL r‐FSH than with 10 mIU/mL r‐FSH (P < .05). A higher FSHR level was found in ES follicles. Meanwhile, with 10 mIU/mL r‐FSH, the ES follicles exhibited a pattern of flat growth, whereas a pattern of stereoscopic spatial growth was observed with 100 mIU/mL r‐FSH. The 100 mIU/mL r‐FSH stimulated granulosa cell proliferation more significantly than 10 mIU/mL r‐FSH. Moreover, FSH significantly promoted ES follicle granulosa cell proliferation compared to PM follicular granulosa cells. The secretion of E2 and the expressions of 3β‐HSD, CYP 17, and CYP 19 in ES follicles with 100 mIU/mL r‐FSH were significantly higher than those with 10 mIU/mL r‐FSH.ConclusionsThe 100 mIU/mL r‐FSH ideally promotes the development of ES follicles, whose growth pattern can more reasonably simulate the growth of follicles in vivo.

First pregnancy after in vitro culture of early antral follicles in goats: Positive effects of anethole on follicle development and steroidogenesis.

This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.

The effect of bioidentical nanostructured progesterone in the in vitro culture of preantral follicles and oocyte maturation.

This study evaluated the effects of bioidentical nanostructured progesterone alone or in association with human chorionic gonadotropin (hCG) on the in vitro survival and development of preantral follicles (experiment 1) and oocyte maturation (experiment 2). Bioidentical hormones have a molecular structure identical with that of endogenous hormones; nanostructured substances refer to those reduced to a nanoscale. In experiment 1, fragments of goat ovarian tissue were cultured for 7 days in α-MEM+ alone or supplemented with nanoprogesterone (MEM+ + P4) or P4 and hCG (MEM+ + P4 + hCG). In experiment 2, two mediums of oocyte in vitro maturation (IVM) were compared. Medium 1 consisted of TCM 199+ + LH, and medium 2 consisted of TCM 199+ with nanoprogesterone and hCG. The MEM+ + P4 + hCG treatment showed the lowest percentage of follicular survival after 7 days of culture. MEM+ + P4 and MEM+ + P4 + hCG treatments showed higher percentage of follicular activation than MEM+. In experiment 2, there were no differences between mediums 1 and 2 for all endpoints evaluated. In conclusion, the addition of nanoprogesterone is advisable for in vitro culture of preantral follicles and oocyte maturation. However, the association of nanoprogesterone with hCG causes the cellular death of initial follicles but shows efficacy in IVM.

The Effects of Ischemia and Hyperoxygenation on Hair Growth and Cycle

ABSTRACT Alopecia has several causes, but its relationship with ischemia/hypoxia has not yet been investigated in detail. In this study, we studied the changes of hair follicles induced by ischemia and potential effects of normobaric hyperoxygenation (NBO) on the hair cycle and growth. We found that skin ischemia reduced hair growth rate, hair shaft size, and its pigmentation in the anagen phase of mice, which may reflect an aspect of pathophysiology of hair loss (alopecia) and depigmentation (gray/white hairs). Hyperoxygenation increased hair growth rate in organ culture of both human and murine hair follicles. Systemic NBO promoted hair growth in early anagen and mid-anagen, and delayed catagen onset in mice. However, telogen-to-anagen transition was not affected by NBO as far as non-ischemic skin is concerned. The results of this study indicated that the hair follicle is very sensitive to oxygen tension and oxygen tension affects the regulation of hair growth and cycle in vitro and in vivo. It was suggested that systemic NBO can be safely applied for a long period and can be a noninvasive therapeutic approach to alter hair growth and cycle by manipulating the microenvironment of hair follicles.

Optimization of Porcine Ovarian Follicle Isolation Methods for Better Developmental Potential.

In the present study, we present a comparative analysis among the outputs of porcine follicle isolation using either mechanical technique alone or in combination with enzymes, proposing an optimized protocol useful for all further applications related to follicle in vitro growth and reproductive tissue engineering. The porcine follicles were isolated using mechanical technique alone (hand blender and scalpels) or in combination with collagenase or Liberase Dispase High (DH) at different doses applying different protocols. Finally, the number, morphology, and stage of isolated follicles were compared between the protocols. Moreover, the follicle viability (live/dead assay) and morphology (rhodamine phalloidin and 4',6-diamidino-2-phenylindole staining and scanning electron microscopy analysis) were evaluated after 10 days of culture. We found an optimum protocol for intact follicle isolation using the mechanical technique in combination with enzymes at a concentration of 0.5 mg/mL. However, the number of total isolated follicles and primordial follicles was significantly higher when collagenase was used compared to Liberase DH (p < 0.05), while Liberase DH could isolate a significantly higher percentage of preantral follicles. After 10 days of culture, the morphology and health status of follicles were statistically higher when Liberase DH was used in comparison with collagenase. Moreover, on the follicles extracted with Liberase DH, it was possible to observe theca cells covering part of the follicle surface. In conclusion, we demonstrated that the intact primary or secondary follicles could not be obtained using only mechanical methods, which led to the isolation of denuded oocytes and dramatically damaged follicles. We concluded that the collagenase-based follicle isolation could negatively affect the morphology and developmental potential of the follicles. Moreover, the incubation of ovarian cortex tissues with Liberase DH solution is an optimized protocol for porcine ovarian follicle isolation with developmental competence. Impact statement Isolation and in vitro maturation of follicles can pave the way for activities on reproductive tissue engineering (REPROTEN) and developing an artificial ovary. In this regard, the standardization and optimization of the extraction methods are pivotal for the design of experiment of follicle in vitro growth. In the present study, we provided a comparative analysis among the outputs of porcine follicle isolation using either mechanical technique alone or in combination with collagenase or Liberase DH, proposing an optimized protocol useful for all further applications related to follicles' in vitro growth and REPROTEN.

Spatiotemporal expression of aquaporin 9 is critical for the antral growth of mouse ovarian follicles†.

Although a few aquaporins (AQPs) expressed in granulosa cells have been postulated to mediate fluid passage into the antrum, the specific expression of AQPs in different follicle cell types and stages and their roles have not been evaluated extensively. The spatiotemporal expression of aquaporin (Aqp) 7, 8, and 9 and the functional roles of Aqp9 in antral growth and ovulation were examined using a superovulation model and 3-dimensional follicle culture. Aqp9 was expressed at a high level in the rapid growth phase (24-48h post equine chorionic gonadotropin (eCG) for superovulation induction) compared to Aqp7 (after human chorionic gonadotropin (hCG)) and Aqp8 (8-24h post eCG and 24h post hCG). A dramatic increase in the expression and localization of Aqp9 mRNA in theca cells was observed, as evaluated using quantitative reverse transcription-polymerase (RT-PCR) coupled with laser capture microdissection and immunohistochemistry. AQP9 was located primarily on the theca cells of the tertiary and preovulatory follicles but not on the ovulated follicles. In phloretin-treated mice, the diameter of the preovulatory follicles and the number of ovulated oocytes decreased. Consistent with these findings, knocking down Aqp9 expression with an Aqp9 siRNA inhibited follicle growth (0.28:1=siRNA:control) and decreased the number of ovulated follicles (0.36:1=siRNA:control) during in vitro growth and ovulation induction. Based on these results, the expression of AQPs is under the control of the physiological status, and AQP9 expression in theca during folliculogenesis is required for antral growth and ovulation in a tissue-specific and stage-dependent manner.

In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†.

Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980-2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

Whole-ovary decellularization generates an effective 3D bioscaffold for ovarian bioengineering.

To develop a new protocol for whole-ovary decellularization for the production of a 3D bioscaffold suitable for in vitro/ex vivo studies and for the reconstruction of a bioengineered ovary. Porcine ovaries were subjected to the decellularization process (DECELL; n = 20) that involved a freeze-thaw cycle, followed by sequential incubations in 0.5% SDS for 3h, 1% Triton X-100 for 9h, and 2% deoxycholate for 12h. Untreated ovaries were used as a control (CTR; n = 6). Both groups were analyzed to evaluate cell and DNA removal as well as ECM preservation. DECELL bioscaffolds were assessed for cytotoxicity and cell homing ability. DECELL ovaries maintained shape and homogeneity without any deformation, while their color turned from red to white. Histological staining and DNA quantification confirmed a decrease of 98.11% in DNA content, compared with the native tissue (CTR). Histochemical assessments demonstrated the preservation of intact ECM microarchitecture after the decellularization process. This was also confirmed by quantitative analysis of collagen, elastin, and GAG contents. DECELL bioscaffold showed no cytotoxic effects in co-culture and, when re-seeded with homologous fibroblasts, encouraged a rapid cell adhesion and migration, with repopulating cells increasing in number and aggregating in cluster-like structures, consistent with its ability to sustain cell adherence, proliferation, and differentiation. The protocol described allows for the generation of a 3D bioscaffold that may constitute a suitable model for ex vivo culture of ovarian cells and follicles, as well as a promising tool for the reconstruction of a bioengineered ovary.

Intraovarian regulation of folliculogenesis in the dog: A review.

Dog reproductive cycle is unique among other mammals in that females experience long and variable periods of ovarian inactivity. Neuroendocrine controls of the reproductive cycle have been thoroughly studied in the dog. However, there is little information regarding endocrine, paracrine and autocrine controls of dog ovarian folliculogenesis. Advancements in the understanding of mechanisms regulating dog ovarian follicle development will be helpful in the establishment of an approach to control cyclicity in this species. Furthermore, such information will likely be useful for the establishment of an in vitro follicle culture system to preserve fertility of genetically valuable disease models or endangered canids. This review highlights current knowledge on dog folliculogenesis with emphasis on endocrine, paracrine and autocrine controls of follicular development.

Pituitary porcine FSH, and recombinant bovine and human FSH differentially affect growth and relative abundances of mRNA transcripts of preantral and early developing antral follicles in goats

Three different sources of FSH (porcine pituitary, pFSH; recombinant bovine, rbFSH; and recombinant human, rhFSH) were compared during in vitro culture of preantral and early antral follicles of goats for 18 days. Treatments were: base medium supplemented with no FSH (control), 10, 50, or 100 mIU/mL pFSH (pFSH10, pFSH50, and pFSH100, respectively), 100 ng/mL rbFSH (rbFSH), and 50 mIU/mL rhFSH (rhFSH). There were evaluations of follicle morphology, antrum formation, growth rate, estradiol production, oocyte viability and chromatin configuration, and follicle wall relative abundance of mRNA transcript for MMP-9, TIMP-2, CYP17, CYP19A1, FSHR, Insulin-R, and BAX/BCL-2 ratio. Follicle degeneration rates were similar among all treatment groups at the end of culturing. When there were treatments with pFSH, however, there was a lesser (P < 0.05) percentage of intact follicles and estradiol production, and greater (P < 0.05) extrusion rates. Furthermore, with only pFSH10 (antral follicles) and pFSH100 (preantral and antral follicles) treatments, there was a lesser (P < 0.05) follicle growth. For preantral follicles, when there was addition of pFSH10, pFSH100, and rhFSH there was lesser (P < 0.05) oocyte meiotic resumption compared to control and rbFSH treatments. For antral follicles, when there were treatments with rhFSH and pFSH10 there was greater (P = 0.08 – P < 0.05) oocyte maturation. In conclusion, the source of FSH differentially affected gene expression, as indicated by mRNA abundances, and follicular dynamics of preantral and antral follicles in vitro. Addition of FSH during the in vitro culture improved the developmental outcomes only for antral follicles.

Decreased microRNA-125b-5p disrupts follicle steroidogenesis through targeting PAK3/ERK1/2 signalling in mouse preantral follicles

BackgroundHyperandrogenism is one of the major characteristics of polycystic ovary syndrome (PCOS). Abnormal miR-125b-5p expression has been documented in multiple diseases, but whether miR-125b-5p is associated with aberrant steroidogenesis in preantral follicles remains unknown. MethodsSteriod hormone concentrations and miR-125b-5p expression were measured in clinical serum samples from PCOS patients. Using a mouse preantral follicle culture model and a letrozole-induced PCOS mouse model, we investigated the mechanism underlying miR-125b-5p regulation of androgen and oestrogen secretion. ResultsThe decreased miR-125b-5p expression was observed in the sera from hyperandrogenic PCOS (HA-PCOS) patients. In mouse preantral follicles, inhibiting miR-125b-5p increased the expression of androgen synthesis-related genes and stimulated the secretion of testosterone, while simultaneously downregulating oestrogen synthesis-related genes and decreasing oestradiol release. Ectopically expressed miR-125b-5p reversed the effects on steroidogenesis-related gene expression and hormone release. Mechanistic studies identified Pak3 as a direct target of miR-125b-5p. Furthermore, inhibiting miR-125b-5p facilitated the activation of ERK1/2 in mouse preantral follicles, while inhibiting Pak3 abrogated this activating effect. These results were recapitulated in letrozole-induced PCOS mouse ovaries. Of note, inhibiting PAK3 antagonised the positive effect of miR-125b-5p siRNA on the expressions of androgen synthesis-related enzymes and testosterone secretion. Luteinizing hormone (LH) inhibited miR-125b-5p expression, and stimulated Pak3 expression. ConclusionHigh serum LH concentrations in PCOS patients repress miR-125b-5p expression, which further increases Pak3 expression, leading to activation of ERK1/2 signalling, thus stimulating the expression of androgen synthesis-related enzymes and testosterone secretion in HA-PCOS.

Optimization of Novel Follicle Culture System to Generate Developmentally Competent Oocytes

This study aimed to develop a novel culture system for porcine ovarian follicles that yields developmentally competent oocytes. We mechanically isolated ovarian follicles of various sizes 325–500 mm and treated them with ovine follicle stimulating hormone OFSH at different concentrations 0–400 mIU. Follicle diameter, antrum formation and cumulus oocyte complex COC recovery rate were significantly higher p andlt; 0.05 under the 0 and 50 mIU OFSH treatments compared with the remaining concentrations 100, 200 and 400 mIU. Additionally, follicles cultured for 3 and 4 d differed significantly p andlt; 0.05 in follicle diameter, antrum formation rate and COC recovery from those cultured for 5 and 6 d. Follicle characteristics did not differ across diameter: those at 250–300, 301–400 and 401–500 mm in vitro had antrum formation rates of 90%, 92% and 90%, along with COC recovery of 78%, 82% and 85%, respectively. Furthermore, nuclear maturation percentages for oocytes that experienced germinal vesicle breakdown (GVBD) were 10%, 13% and 14%, depending on the size of the originating follicle (250–300, 301–400 and 401–500 mm). Nuclear maturation for metaphase II (MII) oocytes derived from follicles of those three sizes were 1%, 2% and 1%, respectively. After 3 d of culture, the 250–300 mm group differed significantly from other size groups in follicle diameter and COC recovery. This study provides insight into establishing effective protocols of ovarian follicle culture, thus improving efforts to preserve large-mammal fertility.

Applicability of Hyaluronic Acid-Alginate Hydrogel and Ovarian Cells for In Vitro Development of Mouse Preantral Follicles.

Objective In the present study, the applicability of hyaluronic acid-alginate (HAA) hydrogel and ovarian cells (OCs) for the culture of mouse ovarian follicles were investigated and compared with those of alginate (ALG) and fibrin-alginate (FA) hydrogels. Materials and Methods In the first step of this experimental study, mechanically isolated preantral follicles from the ovaries of two-week-old mice were encapsulated in the absence or presence of OCs in ALG, HAA, and FA hydrogels and cultured for 14 days. The morphology, diameter, survival and antrum formation rates of the follicles and the maturation and quality of the oocytes were evaluated during culture. In the second step, preantral follicles were cultured similar to the first step, but for 13 days, and their gene expressions and hormonal secretion were assessed on the last day of culture. Results In the absence of OCs, higher numbers of ALG- and HAA-encapsulated follicles reached the antral stage compared to FA-encapsulated follicles (P<0.05). However, a higher percentage of HAA-developed oocytes resumed meiosis up to the germinal vesicle breakdown (GVBD)/metaphase II (MII) stages in comparison with ALG-developed oocytes (P<0.05). HAA-encapsulated follicles had significant overexpression of most of the growth and differentiation genes, and secreted higher levels of estradiol (E2) compared to ALG- and FA-encapsulated follicles (P<0.05). The co-culture condition increased the diameter of ALG-encapsulated follicles on day 13 of culture (P<0.05). It also increased the survival and maturation rates of ALG- and FA-encapsulated follicles, respectively (P<0.05). The co-culture condition improved cortical granule distribution in all groups, increased E2 and progesterone (P4) secretions in the ALG and FA groups, and androstenedione (A4) secretion in the FA group (P<0.05). ConclusionThe present study results show that HAA hydrogel is a promising hydrogel for follicle culture. OCs utilization could ameliorate the culture conditions regardless of the type of hydrogel.

Methods to Study Human Hair Follicle Growth Ex Vivo: Human Microdissected Hair Follicle and Human Full Thickness Skin Organ Culture.

The culture of microdissected hair follicles (HFs) and scalp skin enriched in terminal HFs are the best currently available preclinical assays for studying hair and skin biology/pathology in the human system. While microdissected HF organ culture only allows the testing of compounds added into the culture medium, mimicking a systemic application, the scalp skin organ culture also is suitable to test topical and intradermal applications. Here, we describe different methods for isolation of human scalp HFs, the procedures for culturing the scalp skin and microdissected HFs and we also outline different delivery techniques (e.g., topical, systemic) to test active and control substances.

Comparison of Bovine Small Antral Follicle Development in Two- and Three-Dimensional Culture Systems.

To compare the effects of two-(2D, microplate) and three-dimensional (3D, alginate) culture systems on the in vitro growth of small antral follicles in cattle, individual follicles were separately cultured in the two culture systems for 8 days. Half of the culture medium was replaced by fresh medium every 2 days; the former medium was used to assess the amount of follicular hormone secretion using ELISA. Individual follicle morphology, diameter, and survival rate were recorded every alternate day. The results showed that in 4 days, there was no significant difference between the two systems, except that the growth rate of follicles in 2D system was relatively faster. After 4 days, estradiol concentration in 3D system was higher than that in 2D system. However, progesterone concentration was lower than that in the 2D system. The survival rate and oocyte quality of follicles in 2D system were significantly lower than those in 3D system on day 8. The follicle diameter slightly increased (30-60 μm) in the entire process. Taken together, for in vitro culture of follicles within 4 days, the 2D culture system is more suitable. However, when the culture duration is >4 days, the 3D culture system is more suitable.

Progesterone Affects Clinic Oocyte Yields by Coordinating with FSH &lt;i&gt;via&lt;/i&gt; the PI3K/AKT and MAPK Pathways

Background : Over recent years, progestin has been incorporated into the novel ovary stimulation regimens as an alternative for GnRH analogs to prevent premature luteinizing hormone surge, and has become widely used in treatment for infertility. However, the influence of progesterone administration in terms of clinical outcomes and the mechanisms involved remains poorly understood. Methods: The clinic outcomes from patients undergoing ovary stimulation for in vitro fertilization were analyzed. The mouse ovary stimulation model and follicle culture system were used to evaluate the effect of progesterone on oocyte yield, follicle development, granulosa cell proliferation, hormone secretion. Phospho-Specific Protein Microarray, western blot and RT-PCR were employed to explored the signal mechanism. Findings: We present the evidence from patients suggesting that progesterone decreased the oocyte yields, that was rescued by the higher dose of human menopausal gonadotropin. Using both in vivo and in vitro mouse models, we demonstrated that the administration of progesterone inhibited the proliferation of granulosa cells and therefore reduced the growth rate of follicles. The action of progesterone was mediated by progesterone receptor membrane component-1 and rescued by FSH. Using a phosphorylation array, we identified the PI3K/AKT and MAPK are pivotal pathways that integrated the action of progesterone into the FSH signaling network via the downregulation of cyclin D2 and c-fos in granulosa cells. Interpretation: Our findings highlight the mechanism by which progesterone coordinates with FSH to regulate follicle growth and subsequently influence oocyte outcomes in clinic. These findings advance our understanding of the alterations of ovary response to gonadotrophins during progestin primed ovarian stimulation and gives rise to the opportunity for manipulating individual oocyte yields by signaling regulation. Funding Statement: This study was financially supported by the Natural Science Foundation of Shanghai (Grant number 19ZR1429300, awarded to HL), research grants from the National Natural Science Foundation of China (Grant number 81871163, awarded to QL; Grant number 81771533, awarded to YK; Grant number 81671520, awarded to QC), and the National Key Project (Grant number 2018YFC1003000). Declaration of Interests: The authors have declared that no conflict of interest exists. Ethics Approval Statement: Approval for human retrospective analysis was obtained from the institutional Ethics Committee of Shanghai Ninth People’s Hospital affiliated to Shanghai JiaoTong University’s School of Medicine. Animal studies were approved by the institutional Ethics Committees of Care and Use of Experimental Animals of Shanghai Jiaotong University.

The new biocompatible material for mouse ovarian follicle development in three-dimensional in vitro culture systems

To establish a protocol of optimized three-dimensional (3D) culture of ovarian follicles, various biomaterials have been investigated with regard to their properties and functions on in vitro follicle growth. The present study aims to compare the new biomaterial, extracellular matrix-derived soft hydrogel (ES-hydrogel) and alginate, and evaluate the effects of biomaterials on further in vitro 3D culture growth of ovarian follicle and oocyte maturation. The isolated follicles from mouse ovaries were randomly divided into two-dimensional (2D) culture, alginate and ES-hydrogel, and just seeded on culture wells (2D culture) or encapsulated with alginate or ES-hydrogel (3D culture). Culture media from each group were collected on days 4, 8 and 10 or 11 for 17ß-oestradiol (E2) and progesterone (P4) measurement. On day 10 of in vitro culture, follicular survival and pseudo-antrum formation rate were examined, and oocyte maturation was induced by adding human chorionic gonadotropin and epidermal growth factor. After 17 h, ovulated mature oocytes collected and analyzed for oocyte diameter, normal spindle and chromosome alignment configuration, reactive oxygen species (ROS) level, and mitochondrial membrane potential level. To compare mechanical properties of two biomaterials, storage modulus was measured with the advanced rheometric expansion system. Our results showed that follicles cultured in ES-hydrogel, were significantly superior to those cultured 2D or alginate in the pseudo-antrum formation rate, cumulus-oocyte complexes (COCs) rate, MII oocyte rate, normal spindle rate, and E2 production. The ES-hydrogel and alginate groups were not significantly different in follicle survival rate, oocyte diameter, P4 production, ROS, and mitochondrial membrane potential levels. The storage modulus of ES-hydrogel was lower than that of alginate, suggesting that the improved follicular physiology and oocyte maturation in the ES-hydrogel group was due to better hormone exchange through a less stiff encapsulating material. This study shows that 3D culture system using ES-hydrogel effectively improve the outcome of in vitro ovarian follicle culture, supporting follicle morphology and growth and enhancing oocyte maturation. It means one of the most important factors for 3D culture of ovarian follicle was the selection of appropriate and effective biomaterial that can preserve the structure and morphology of ovarian follicle and facilitate nutrition and hormone exchange.

Melatonin improves development, mitochondrial function and promotes the meiotic resumption of sheep oocytes from in vitro grown secondary follicles

The aim of this study was to evaluate follicular survival and development of ovine isolated secondary follicles cultured in medium containing fixed or sequential concentrations of melatonin and further oocyte maturation. Isolated secondary follicles were cultured for 18 days in α-MEM+ alone (control) or with different concentrations of melatonin (100, 500 or 1000 pg/mL) or sequential concentrations of melatonin (Mel Seq: Day 6 = 100; Day 12 = 500; Day 18 = 1000 pg/mL). The percentages of morphologically normal follicles and antral cavity formation increased significantly in 1000 pg/mL melatonin compared to the other treatments. After 18 days, 1000 pg/mL melatonin (Mel 100) showed a greater (P < 0.05) follicular diameter than α-MEM+, 100 and 500 pg/mL melatonin. In addition, the concentration of 500 pg/mL melatonin showed a higher (P < 0.05) percentage of fully grown oocytes than α-MEM+, Mel 100 and Mel Seq treatments. After oocyte maturation, the levels of ROS were lower (P < 0.05) in 1000 pg/mL melatonin (Mel 1000) than in other treatments. Both Mel 1000 and Mel Seq treatments showed significantly higher levels of mitochondrial activity than other treatments. There were no significant differences between 500 and 1000 pg/mL melatonin regarding meiotic stages. In conclusion, the concentration of 1000 pg/mL melatonin maintains survival, promotes follicular development and increases the levels of active mitochondria after in vitro culture of sheep secondary follicles. Moreover, this concentration promotes the meiotic competence of oocytes and decreases the production of ROS during oocyte maturation.