Early Stage Ovarian Follicles Research Articles

3D culture of ovarian follicles in granular and nanofibrillar hydrogels

3D culture of ovarian follicles in hydrogel matrices is an important emerging tool for basic scientific studies as well as clinical applications such as fertility preservation. For optimizing and scaling 3D culture of preantral follicles, there is a need for identifying biomaterial matrices that simplifies and improves the current culture procedures. At present, microencapsulation of follicles in alginate beads is the most commonly used approach. However, this technique involves notable manual handling and is best suited for encapsulation of single or several follicles. As a potential alternative, we here explore the suitability of different particle-based hydrogel matrices, where follicles can easily be introduced in tunable 3D environments, in large numbers. Specifically, we study the growth of secondary murine follicles in microgranular alginate and nanofibrillar cellulose matrices, with and without cell-binding cues, and map follicle growth against the viscoelastic properties of the matrices. We cultured follicles within the particle-based hydrogels for 10 days and continuously monitored their size, survival, and tendency to extrude oocytes. Interestingly, we observed that the diameter of the growing follicles increased significantly in the particle-based matrices, as compared to state-of-the-art alginate micro-encapsulation. On the other hand, the follicles displayed an increased tendency for early oocyte extrusion oocyte extrusion in the granular matrices, leading to a notable reduction in the number of intact follicles. We propose that this may be caused by impaired diffusion of nutrients and oxygen through thicker matrices, attributable to our experimental setup. We propose that this may be caused by impaired diffusion of nutrients and oxygen through thicker matrices, attributable to our experimental setup. Still, our findings suggest that viscoelastic, granular hydrogels represent promising matrices for 3D culture of early-stage ovarian follicles. In particular, these materials may easily be implemented in advanced culturing devices such as micro-perfusion systems.

Androgens improve ovarian follicle function impaired by glucocorticoids through an androgen-IGF1-FSH synergistic effect.

High concentrations of glucocorticoids caused by chronic stress are known to affect ovarian function and cause diminished ovarian reserve. Androgens are essential for early-stage ovarian follicle development, but the effects and mechanisms of androgens on follicle development under chronic stress remain unclear. In this study, we aim to investigate the effects of high concentrations of glucocorticoids on the function of in vitro cultured ovarian cells and mouse early-stage ovarian follicles and to validate the hypothesis that androgen-insulin-like growth factor 1 (IGF1)-follicle-stimulating hormone (FSH) synergistic signaling helps to ameliorate the damage caused by high concentrations of glucocorticoids. KGN cells (human granulosa cell line) and mouse primary cells were treated with different concentrations of glucocorticoids, and the cell proliferation, apoptosis, and sex hormone secretion were detected. The effects of glucocorticoid and androgens on IGF1 receptor (IGF1R) and FSH receptor (FSHR) expression in KGN cells were detected by Western blot. Steroidogenic synthase expressions under androgens and androgen-IGF1-FSH combination treatment were examined by qPCR after manipulation using low and high concentrations of glucocorticoids. The mechanism of androgen regulation of IGF1R and FSHR was explored by small interfering RNA (siRNA) and chromatin immunoprecipitation (ChIP)-qPCR. Damage of glucocorticoids and the treatment effects of androgens were further validated in mouse ovarian follicles cultured in vitro. The results demonstrated that prolonged treatment with high-dose glucocorticoids reduced cell viability of granulosa cells, inhibited their sex hormone secretion, and impaired their sensitivity to IGF1 and FSH signaling by affecting IGF1R and FSHR functions. Androgens at an appropriate dose range improved early-stage follicle development and their hormone secretion under high-dose glucocorticoid treatment, which was related to increased transcription of Igf1r and Fshr. This work showed that excessive glucocorticoids impaired ovarian function and validated that balanced concentrations of androgens synergized with IGF1 and FSH to improve the function of early-stage ovarian follicles under conditions of chronic stress.

Vasopressin Promoter Transgenic and Vasopressin Gene-Edited Ascidian, Ciona intestinalis Type A (Ciona robusta): Innervation, Gene Expression Profiles, and Phenotypes.

Oxytocin (OT) and vasopressin (VP) superfamily neuropeptides are distributed in not only vertebrates but also diverse invertebrates. However, no VPergic innervation of invertebrates has ever been documented. In the ascidian, Ciona intestinalis Type A (Ciona robusta), an OT/VP superfamily peptide was identified, and the Ciona vasopressin (CiVP) induces oocyte maturation and ovulation. In the present study, we characterize the innervation and phenotypes of genetically modified Ciona: CiVP promoter-Venus transgenic and CiVP mutants. CiVP promoter-Venus transgenic Ciona demonstrated that CiVP gene was highly expressed in the cerebral ganglion and several nerves. Fluorescence was also detected in the ovary of young CiVP promoter-Venus transgenic ascidians, suggesting that the CiVP gene is also expressed temporarily in the ovary of young ascidians. Furthermore, a marked decrease of post-vitellogenic (stage III) follicles was observed in the ovary of CiVP mutants, whereas pre-vitellogenic (stage I) and vitellogenic (stage II) follicles were increased in the mutant ovary, compared with that of wildtype Ciona. Gene expression profiles showed that the expression of various genes, including genes related to ovarian follicle growth, was altered in the ovary of CiVP mutants. Altogether, these results indicated that CiVP, mainly as a neuropeptide, plays pivotal roles in diverse biological functions, including growth of early-stage ovarian follicles via regulation of the expression of a wide variety of genes. This is the first report describing a VP gene promoter-transgenic and VP gene-edited invertebrate and also on its gene expression profiles and phenotypes.

Synergy of Paracrine Signaling During Early-Stage Mouse Ovarian Follicle Development In Vitro.

Paracrine signals, such as soluble cytokines and extracellular matrix cues, are essential for the survival and development of multicellular ovarian follicles. While it is well established that hydrogel-based culture systems successfully support the growth of late-stage follicles for fertility preservation, growing small, early-stage ovarian follicles still proves to be challenging. We hypothesized that paracrine factors secreted from neighboring follicles may be crucial for improving the survival of early-stage follicles in vitro. To test our hypothesis, we investigated the bi-directional crosstalk of the paracrine signals, such as cell-secreted cytokines, sex hormones and transcription factors (TFs), in follicles encapsulated and cultured for 12days in alginate in groups of five (5×) and ten (10×). The differential profiles of TF activity and secretome during folliculogenesis were analyzed using TRanscriptional Activity CEllular aRray (TRACER) and data-driven multivariate modeling approach. The mechano- and oxygen-responsive TFs, NF-κB and HIF1, exhibited a unique upregulation signature in 10× follicles. Consistently, levels of proangiogenic factors, such as VEGF-A and angiopoietin-2, were significantly higher in 10× follicles than those in 5× follicles, reaching 269.77 and 242.82pg/mL on the last day of culture. The analysis of TRACER and secreted cytokines also revealed critical early interactions between cytokines and TFs, correlating with the observed phenotypical and functional differences between conditions. We identified unique signatures of synergism during successful early-stage ovarian follicle development. These findings bring us closer to understanding of mechanisms underlying the downstream effects of interactions between the extracellular microenvironment and early-stage folliculogenesis in vitro.

Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in-vitro culture in cryopreservation studies

Development of in vitro culture protocol for early stage ovarian follicles of zebrafish is important since cryopreserved early stage ovarian follicles would need to be matured in vitro following cryopreservation before they can be fertilised. Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in vitro culture of early stage zebrafish ovarian follicles in ovarian tissue fragments is reported here for the first time although some work has been reported for in vitro culture of isolated early stage zebrafish ovarian follicles. The main aim of the present study was to develop molecular markers in an optimised in vitro culture protocol for stage I and stage II zebrafish ovarian follicles in ovarian tissue fragments. The effect of concentration of the hormones human chorionic gonadotropin and follicle stimulating hormones, and additives such as Foetal Bovine Serum and Bovine Serum Albumin were studied. The results showed that early stage zebrafish ovarian fragments containing stage I and stage II follicles which are cultured in vitro for 24 h in 20% FBS and 100mIU/ml FSH in 90% L-15 medium at 28 °C can grow to the size of stage II and stage III ovarian follicles respectively. More importantly the follicle growth from stage I to stage II and from stage II to stage III were confirmed using molecular markers such as cyp19a1a (also known as P450aromA) and vtg1 genes respectively. However, no follicle growth was observed following cryopreservation and in vitro culture.

Resveratrol has dose-dependent effects on DNA fragmentation and mitochondrial activity of ovine secondary follicles cultured in vitro.

The worldwide consumption of red wine, nuts and grapes has resulted in increased human exposure to resveratrol, which could affect reproductive function. However, the effect of resveratrol on in vitro culture of early-stage ovarian follicles has never been investigated. The aims of the present study were to evaluate the effect of resveratrol on sheep secondary follicle morphology, growth, DNA fragmentation, intracellular levels of glutathione (GSH) and active mitochondria. Secondary follicles were isolated from the ovaries and cultured for 18 days in supplemented α-MEM+ (control medium) or in control medium containing resveratrol (2, 10 or 30 µM). The parameters analyzed were morphology, antrum formation, follicle diameter, DNA fragmentation, GSH levels and mitochondrial activity. After 18 days, all resveratrol groups significantly decreased the percentages of morphologically normal follicles compared with the control group (α-MEM+). Antrum formation was higher in both α-MEM+ and 2 µM resveratrol groups than in the 10 µM resveratrol group. In addition, 30 µM resveratrol increased the percentage of oocytes with DNA damage compared with the control. Oocytes from follicles treated with 10 or 30 µM resveratrol significantly decreased intracellular GSH levels compared with the 2 µM resveratrol group. Moreover, follicles in α-MEM+ (control) showed more active mitochondria than those in 10 or 30 µM resveratrol. In conclusion, ovine isolated secondary follicles are able to grow to the antral stage after in vitro culture in medium containing 2 µM resveratrol, maintaining the same rates of DNA damage, GSH levels and mitochondrial function as the control medium. However, the addition of 30 µM resveratrol increased DNA fragmentation and oxidative stress through decreasing mitochondrial activity.

137 DYNAMIC SHIFT IN CYTOPLASMIC LIPIDS IN CAT OOCYTES DURING OVARIAN FOLLICLE DEVELOPMENT

Cytoplasmic lipids play key roles during oocyte and embryo development. However, there is little information on the changes in lipid types during intraovarian follicular and oocyte growth. Here, we used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to understand lipid composition in the cat oocyte during differing stages of folliculogenesis. Follicles at different developmental stages were mechanically isolated from cat ovaries within 4 h after routine ovariohysterectomy. Oocytes with granulosa cells were recovered from secondary (preantral stage, n = 387), early antral (<0.5 mm diameter, n = 177), small antral (0.5–1 mm, n = 144), and antral (>1 mm, n = 120) follicles, then subjected to lipid extraction and MALDI-TOF analysis (n = 3 replicates). Resulting mass spectra data were evaluated using MALDIquant in R package to eliminate baseline (background) and to identify peak values. The latter maximal values for each follicle stage were selected and subjected to principal component analysis to identify similarities and differences in mass spectra profile among oocytes from varying developmental stages. Peaks were compared to those calculated molecular formulae available in the Lipid MAP database. Error estimates were calculated using Excel (Microsoft Corp., Redmond, WA), and lipid species identification performed using Lipid MS Predict. Twenty-nine lipid species from six glycerophospholipids groups (glycerophosphates, PA; glycerophosphoserines, PS; glycerophosphoinositols, PI; glycerophosphoglycerols, PG; glycerophosphoinositol monophosphates, PIP; and glycerophosphoethanolamines, PE) were identified, 15 being found in more than 1 developmental stage. Two species in particular, [PI(29:4)+Na]+ and [PIP(23:0)+Na]+, were the most abundant lipids and were identified in oocytes from all developmental stages. There were dynamic shifts in lipid species expressed at different follicle stages. Oocytes from secondary and antral follicles contained more lipid types (15 and 22, respectively) than early (10) and small antral (4) counterparts. Four (PA, PS, PI, and PIP) of the 6 glycerophospholipids were found only in oocytes from secondary and antral follicles. Oocytes from small antral follicles also lacked PA, PS, and PG, whereas PG was not found in early antral stage oocytes. In summary, we showed for the first time in the cat that, similar to goats and rats, there are temporal changes in lipid types within the oocyte during folliculogenesis. We suspect that these changing dynamics, including shifts in presence or absence of lipid species with follicle stage, may be playing key roles in oocyte growth and viability. Our findings also serve as in vivo benchmarks for parallel studies focused on enhancing an in vitro culture system for early-stage ovarian follicles to preserve fertility of genetically valuable domestic and wild felids.

Perchlorate disrupts embryonic androgen synthesis and reproductive development in threespine stickleback without changing whole-body levels of thyroid hormone

Perchlorate, an environmental contaminant, disrupts normal functioning of the thyroid. We previously showed that perchlorate disrupts behavior and gonad development, and induces external morphological changes in a vertebrate model organism, the threespine stickleback. Whether perchlorate alters these phenotypes via a thyroid-mediated mechanism, and the extent to which the effects depend on dose, are unknown. To address these questions, we chronically exposed stickleback to control conditions and to three concentrations of perchlorate (10, 30 and 100ppm) at various developmental stages from fertilization to reproductive maturity. Adults chronically exposed to perchlorate had increased numbers of thyroid follicles and decreased numbers of thyrocytes. Surprisingly, T4 and T3 levels in larval, juvenile, and adult whole fish chronically exposed to perchlorate did not differ from controls, except at the lowest perchlorate dose, suggesting a non-monotonic dose response curve. We found no detectable abnormalities in external phenotype at any dose of perchlorate, indicating that the increased number of thyroid follicles compensated for the disruptive effects of these doses. In contrast to external morphology, gonadal development was altered substantially, with the highest dose of perchlorate causing the largest effects. Perchlorate increased the number both of early stage ovarian follicles in females and of advanced spermatogenic stages in males. Perchlorate also disrupted embryonic androgen levels. We conclude that chronic perchlorate exposure may not result in lasting adult gross morphological changes but can produce lasting modifications to gonads when compensation of T3 and T4 levels occurs by thyroid follicle hyperplasia. Perchlorate may therefore affect vertebrate development via both thyroidal and non-thyroidal mechanisms.

65. Development of molecular markers for stage II and stage III zebrafish ovarian follicles after in vitro culture

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but had not been successful in fish so far. Fish oocytes offers several advantages over embryos due to smaller size of the oocytes, low water content and absence of fully formed chorion. Studies in our lab have showed that early stage ovarian follicles are more suitable for cryopreservation than late stage oocytes. Hence ovarian tissue containing stage I and stage II ovarian follicles have been used in this study. Development of in vitro culture protocol for early stage ovarain follicles is important since cryopreserved early stage ovarian follicles would need to be matured in vitro. The results obtained from the in vitro culture study carried out in our lab demonstrated that the early stage ovarian fragments containing stage I and stage II follicles can be cultured in 90% L-15 medium (pH 9.0) with 20% feotal bovine serum (FBS) and 100 mIU/ml follicle stimulating hormone (FSH) for 24 h at 28 °C. The study demonstrated follicle growth in size from stage I to stage II or from stage II to stage III, respectively. Follicle viability following culture was not significantly different when compared to the cultured fresh fragments. Although follicle growth can be assessed by measuring the diameter of the follicles, molecular markers would provide more important information on follicle development. The aim of the present study was to develop a molecular marker to identify stage II and stage III ovarian follicles after in vitro culture. Studies on the levels of vtg1 and p450aromA genes in stage I, II and II ovarian follicles of zebrafish before and after culture were carried out. Ovarian follicles of zebrafish were collected from mature zebrafish females and the ovaries were placed in 90% L-15 medium. Ovarian follicles of different stages (I, II and III) were separated using syringe needles. Total RNA was extracted from ovarian follicles using trizol method. The quality of RNA of each sample was determined by the level of absorbance at 260 nm. A quantitative RT-PCR approach was used to investigate the level of gene expression. Preliminary results from the present study showed that prior to culture of the ovarian follicles the level of expression of vtg 1 was higher in stage III when compared to stage I and stage II follicles; and the level of expression in p450aromA was higher in stage II when compared to stage I and stage III ovarian follicles. Analysis on the level of expression of genes after in vitro culture of ovarian fragments are in progress. The development of molecular markers would provide a reliable method for assessing the growth of ovarian follicles in vitro.

Development of in vitro culture method for early stage zebrafish (Danio rerio) ovarian follicles for use in cryopreservation studies

There have been no reported methods for in vitro growth of early stage ovarian follicles for fish and their cryopreservation is still under investigation. If cryopreservation of early stage ovarian follicles can be achieved, in vitro procedures for ovarian follicle culture, development, ovulation and fertilisation after cryopreservation would be needed. The aim of the present study was to develop an in vitro culture method for early stage zebrafish ovarian follicles for use after their cryopreservation. Procedures for in vitro culture of stage I (primary growth) and stage II (cortical alveolus) ovarian follicles were developed. The effects of concentration of L-15 medium, pH and the concentration of human chorionic gonadotropin (hCG) and activin A were studied. The results demonstrated that early stage zebrafish ovarian follicles can be cultured in vitro for 24 h, stage I and II ovarian follicles can grow to the sizes of early stage II and stage III ovarian follicles after hCG treatment. The method developed here is effective for assessing early stage zebrafish ovarian follicles growth competence in vitro. The results from the present study indicated that in vitro culture is the most reliable method for assessing ovarian follicle viability when compared with vital dye staining methods.

A novel two-step strategy for in vitro culture of early-stage ovarian follicles in the mouse

To develop an in vitro strategy to support the growth of early-stage follicles and produce mature oocytes competent for fertilization. Whole ovaries from 8-day-old mice were cultured for 4 days, and then secondary follicles were isolated and cultured for 12 days in a three-dimensional alginate or fibrin-alginate (FA) hydrogel matrix. University-affiliated laboratory. Mice. None. Histologic evaluation of follicle development, steroid hormone production, and rates of oocyte maturation, oocyte fertilization, and embryo formation. Culture of 8-day-old mouse ovaries for 4 days resulted in transition of the follicle population from primordial and primary follicles to secondary follicles, similar to that seen in a 12-day-old ovary. Isolated secondary follicles cultured for 12 days showed larger increases in oocyte diameter and more frequent antrum formation and theca cell differentiation in the FA-hydrogel matrix compared with the alginate matrix. Steroid hormone secretion patterns were consistent with the changes in follicle morphology and cell differentiation observed in the cultured follicles. Compared with oocytes from alginate follicle cultures, a greater number of oocytes retrieved from the FA-based follicle cultures progressed to metaphase I, reached metaphase II, and could be fertilized and cleaved to two-cell embryos. The organ culture plus FA-hydrogel follicle culture strategy produced a very high rate of oocyte progression to metaphase II (88 +/- 8.7% [mean +/- SEM]) and formation of two-cell embryos (54 +/- 4%). A strategy combining whole ovary culture of early-stage follicles and subsequent FA hydrogel in vitro follicle culture produced a high percentage of oocytes competent for fertilization; this might provide new options for fertility preservation in women and prepubertal girls facing fertility-threatening diseases or treatments.

Studies on chilling sensitivity of early stage zebrafish ( Danio rerio) ovarian follicles

Cryopreservation of fish gametes is of great importance in aquaculture, conservation and human genomic research. The creation of gamete cryobanks allows the storage of genetic material of targeted species for almost unlimited time periods. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryos and oocytes. One of the obstacles to fish oocyte cryopreservation is their high chilling sensitivity and especially at subzero temperatures. Although studies on late stage oocyte cryopreservation has been carried out, there have been no reported studies on cryopreservation of early stage ovarian follicles. The aim of this study is to investigate the chilling sensitivity of early stage zebrafish ovarian follicles before developing protocols for their cryopreservation. Experiments were conducted with stage I (primary growth), stage II (cortical alveolus) and stage III (vetillogenesis) ovarian follicles, which were chilled in KCl buffer and L-15 medium for up to 144 h at −1 °C in a low temperature bath. Ovarian follicles were also exposed to 2 M methanol or 2 M DMSO in L-15 medium for up to 168 h at −1 and −5 °C, respectively. Control follicles were kept at 28 °C. Ovarian follicle viability was assessed using trypan blue staining. The results showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles. These results were also confirmed following in vitro maturation of the chilled ovarian follicles. The results also showed that L-15 medium is more beneficial than KCl buffer for ovarian follicles at all stages. The presence of both methanol and DMSO reduced chilling sensitivity of ovarian follicles at all stages with methanol being the most effective. The study indicated that stage I and II follicles are less sensitive to chilling than stage III follicles, and that early stage zebrafish ovarian follicles may be better candidates for cryopreservation.

Immunohistochemical Analysis of Tyrosine Phosphorylation and AP-1 Transcription Factors c-Jun, Jun D, and Fos Family During Early Ovarian Follicle Development in the Mouse

The growth control mechanism of early-stage ovarian follicles is unknown. Tyrosine phosphorylation of signaling molecules and changes in expression and activation of AP-1 transcription factors have been implicated in growth regulation of numerous cell types. In this study, we used immunohistochemistry to analyze tyrosine phosphorylation patterns and expression and activation of selected AP-1 transcription factors in mouse ovarian follicles. The ovaries were collected from B62F1/J mice in estrus. Representative sections were immunostained for phosphotyrosine, phospho-c-Jun, Jun D, and c-Fos. Phosphotyrosine staining was perioocytic from the transitional stage until approximately 5 to 7 layers of granulosa cells had formed. Perioocytic staining was then replaced by scattered stippled staining in granulosa cells of larger follicles. Phospho c-Jun was exclusively expressed in mitotic granulosa cells of follicles from transitional to antral stages. Jun D was expressed in the oocytes of primordial, primary, or transitional follicles and disappeared at the 2-layer preantral stage. Fos was present in corpora lutea and theca cells but not in granulosa cells. Collectively, these data indicate that phosphotyrosine signaling and AP-1 transcription factors are intimately involved in early stages of ovarian follicle growth.

Melatonin Effects on Steroid Production by Rainbow Trout Ovarian Follicles In Vitro: Influence of Stage of Follicle Maturation

Rainbow trout ovarian follicles at four different stages of maturation (very early vitellogenesis, early vitellogenesis, peak vitellogenesis, and pre-ovulatory) were incubated either in Cortland's medium alone, or medium containing melatonin at one of five concentrations (1 × 10-10, 1 × 10-8, 1 × 10-6, 1 × 10-4 or 1 × 10-2M) to assess the direct actions of melatonin on basal (non-gonadotrophin stimulated) secretion of 17β-estradiol and testosterone. Testosterone secretion by peak vitellogenic phase follicles was significantly (p < 0.01) reduced by melatonin at 1 × 10-4 or 1 × 10-2 M, but there were no other significant effects of melatonin on T secretion. There were no significant effects of melatonin at concentrations of 1 × 10-10 or 1 × 10-8 M on 17β-estradiol secretion for any stage of follicular maturation, although for follicles at the peak vitellogenesis stage, melatonin at 1 × 10-6 M had a significant (p < 0.05) stimulatory effect compared with the controls. Melatonin also stimulated 17β-estradiol secretion by very early vitellogenic stage follicles (1 × 10-2 M concentration; p < 0.05). At concentrations of 1 × 10-4 and 1 × 10-2 M, melatonin significantly (p < 0.01) inhibited 17β-estradiol secretion of peak vitellogenic and preovulatory stage follicles. The findings suggest a stimulatory action of melatonin on steroidogenesis of early stage ovarian follicles, a shift to an inhibitory action of the higher concentrations of melatonin in later stages of development, but with a bimodal action of melatonin (related to hormone concentration) being present during the time of maximum follicular growth.