Follicle Culture Research Articles

Dynamic maternal synthesis and segregation of the germ plasm organizer, Bucky ball, in chicken oocytes and follicles.

Maternal germ plasm determines the germline in birds. Previously, we proposed the chicken-specific Bucky ball (cBuc) as a functional equivalent of the zebrafish germ plasm organizer. This study demonstrated the maternal cBuc synthesis, and verified a highly dynamic distribution of Bucky ball from oocyte nests to maturing follicles using specific antibodies. The dynamic re-localization of cBuc from the ovarian stroma to the granulosa cells, and the Balbiani structure of the oocyte was revealed. Following the accumulation of cBuc in the Balbiani body, an increased signal of chicken vasa homolog (CVH) in close contact to cBuc could be detected. Highest transcription of cBuc was recorded in follicles with diameters up to 500µm. First RNA-interference experiments in an in-vivo follicle culture assay revealed inhibiting effects on cBuc in small follicles. These data demonstrate the maternal origin of cBuc, and underpin its role as germ plasm organizer.

Anti-Hair Loss Effects of the DP2 Antagonist in Human Follicle Dermal Papilla Cells

Prostaglandin D2 (PGD2) levels are high in the balding areas of human scalps, and PGD2 has been found to inhibit hair growth. It is known that the inhibition of the PGD2 receptor can promote hair growth by preventing hair follicles from entering the catagen phase. Thus, we identified an antagonist of DP2, the receptor for PGD2, as a potential treatment for hair loss using an AI-based DeepZema® drug development program. In this study, we identified that the DP2 antagonist (DP2A) could ameliorate alopecia in human follicle dermal papilla cells (HFDPCs) that were stimulated by dihydrotestosterone (DHT), a known molecule related to hair loss. We observed that the DP2A promoted wound healing efficiency and increased alkaline phosphatase levels in the HFDPCs that were damaged with DHT. In addition, we found that the DP2A diminished the reactive oxygen species (ROS) levels generated in the DHT-damaged HFDPCs. We confirmed that the DP2A effectively recovered the membrane potential of mitochondria in these cells. We also demonstrated that the DP2A enhanced the phosphorylation levels of both Akt and ERK in the HFDPCs that were damaged with DHT. Notably, we revealed that the DP2A slightly enlarged the three-dimensional spheroid size in these cells and confirmed that the DP2A improved hair growth in the organ culture of human hair follicles. Taken together, we suggest that DP2A has therapeutic effects on HFDPCs that are damaged by DHT and holds promise as a potential treatment for treating hair loss.

A spotlight on factors influencing the in vitro folliculogenesis of isolated preantral follicles.

Female fertility preservation via complete in vitro folliculogenesis is still chimerical. Due to many factors affecting the efficiency of isolation and culture of preantral follicles, the improvement of techniques geared to fertility preservation in higher mammals seems to be at an impasse. We need an objective view of the current stand to understand how to progress further. As such, a survey was conducted to analyze the relative distribution of studies performed in ten mammalian species on preantral follicle culture available on PubMed. Using the bovine as a reference model, we explore some factors influencing data variation that contribute to the difficulty in reproducing studies. While years of research have enabled the recapitulation of folliculogenesis from as modest as the early antral follicle stage ex vivo, in vitro preantral folliculogenesis remains elusive. Herein, we revisit the classical evidence that laid the foundations for understanding preantral folliculogenesis and review the length, breadth, and depth of information that the era of big data has currently levied. Moving forward, we recognize the urgency of synthesizing the multi-disciplinary approaches to mimic folliculogenesis in vitro to achieve a translational landscape of infertility at individual and large-scale conservation levels.

Culture of Immature Ovarian Follicles within Decellularized Ovary Enhances Oocyte Maturation and Improves In vitro Fertilization Results

The goal of this study is to improve methodologies that define the maturation of ovarian follicles and enhance in vitro fertilization by employing decellularized ovaries. Preantral follicles of mice were cultured for 14 days in both the decellularized ovary and two- dimensional (2D) conditions. The oocyte maturation rate, fertilization rate, and the subsequent embryo development rate were assessed in 2D and the decellularized ovary and compared to in vivo condition. Additionally, the gene expression profile of IGF1R, integrin αvβ3, Cox2, Caspase-3, Bax, and Bcl2 l1 was determined in blastocysts. The culture in the decellularized ovary showed a significantly higher number of MII oocytes in comparison to the 2D culture (P < 0.05). Compared to in vivo, both the 2D and the decellularized ovary cultures exhibited significantly lower percentages of MII oocytes, 2PN, two-cell, cleavage, and blastocyst (P < 0.05). In the decellularized ovary culture, significantly higher percentages of 2PN and blastocyst were observed (P < 0.05) compared to the 2D culture. The gene expression level of IGF1R and Cox2 in blastocysts from both the 2D and the decellularized ovary cultures was markedly lower compared to in vivo. However, the gene expression levels of Integrin αv and β3 were comparable in blastocysts derived from in vivo and decellularized ovary-matured oocytes. Blastocysts derived from decellularized ovary-matured oocytes showed a higher bcl211 expression level compared to the blastocysts from 2D (P < 0.05). Employing decellularized ovarian tissues methodologies for in vitro maturation of oocytes provides a promising avenue towards generating embryos with improved implantation potential.

Three-dimensional culture in a bioengineered matrix and somatic cell complementation to improve growth and survival of bovine preantral follicles.

Here we explored poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture and examined the potential for differentiation of bovine embryonic stem cells (bESCs) towards gonadal somatic cells to develop a system more similar to the ovarian microenvironment. Bovine preantral follicles were first cultured in two-dimensional (2D) control or within PEG hydrogels (3D) and then co-cultured within PEG hydrogels with bovine ovarian cells (BOCs) to determine growth and viability. Finally, we tested conditions to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad fate. Primary follicles grew over the 10-day culture period in PEG hydrogels compared to 2D control. Early secondary follicles maintained a similar diameter within the PEG while control follicles decreased in size. Follicles lost viability after co-encapsulation with BOCs; BOCs lost stromal cell signature over the culture period within hydrogels. Induction of bESCs towards gonadal somatic fate under WNT signaling was sufficient to upregulate intermediate mesoderm ( LHX1 ) and early coelomic epithelium/bipotential gonad markers ( OSR1 , GATA4 , WT1 ). Higher BMP4 concentrations upregulated the lateral plate mesoderm marker FOXF1 . PAX3 expression was not induced, indicating absence of the paraxial mesoderm lineage. Culture of primary stage preantral follicles in PEG hydrogels promoted growth compared to controls; BOCs did not maintain identity in the PEG hydrogels. Collectively, we demonstrate that PEG hydrogels can be a potential culture system for early preantral follicles pending refinements, which could include addition of ESC-derived ovarian somatic cells using the protocol described here. We demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.

Liraglutide improves follicle development in polycystic ovary syndrome by inhibiting CXCL10 secretion

BackgroundAt present, a number of clinical trials have been carried out on GLP-1 receptor agonist liraglutide in the treatment of polycystic ovary syndrome (PCOS). However, the effect of liraglutide on follicle development and its specific mechanism are still unclear.MethodsRNA sequencing was used to explore the molecular characteristics of granulosa cells from patients with PCOS treated with liraglutide. The levels of C-X-C motif chemokine ligand 10 (CXCL10) in follicular fluid were detected by ELISA, the expression levels of ovulation related genes and inflammatory factor genes in follicles and granulosa cells were detected by qPCR and the protein levels of connexin 43 (Cx43), Janus Kinase 2 (JAK2) and phosphorylated JAK2 were detected by Western blot. The mouse ovarian follicles culture system in vitro was used to detect the status of follicle development and ovulation.ResultsIn the present study, we found that liraglutide inhibited the secretion of inflammatory factors in PCOS granulosa cells, among which CXCL10 was the most significant. In addition, CXCL10 was significantly higher in granulosa cells and follicular fluid in PCOS patients than in non-PCOS patients. We applied in vitro follicle culture and other techniques to carry out the mechanism exploration which revealed that CXCL10 disrupted the homeostasis of gap junction protein alpha 1 (GJA1) between oocyte and granulosa cells before physiological ovulation, thus inhibiting follicular development and ovulation. Liraglutide inhibited CXCL10 secretion in PCOS granulosa cells by inhibiting the JAK signaling pathway and can improved dehydroepiandrosterone (DHEA)-induced follicle development disorders, which is reversed by CXCL10 supplementation.ConclusionsThe present study suggests that liraglutide inhibits CXCL10 secretion in granulosa cells through JAK signaling pathway, thereby improving the homeostasis of GJA1 between oocyte and granulosa cells before physiological ovulation and ultimately improving the follicular development and ovulation of PCOS, which provides more supportive evidence for the clinical application of liraglutide in the treatment of ovulatory disorders in PCOS.Trial registrationNot applicable.

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 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. 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.

Resveratrol attenuates doxorubicin-induced toxicity during in vitro culture of mouse-isolated preantral follicles.

The aims of this study were to evaluate the doxorubicin concentration that induces toxic effects on in vitro culture of isolated mouse secondary follicles and to investigate whether resveratrol can inhibit or reduce this toxicity. Secondary follicles were isolated and cultured for 12 days in control medium (α-MEM+) or in α-MEM+ supplemented with doxorubicin (0.1 µg/ml) or different concentrations of resveratrol (0.5, 2, or 5 µM) associated with doxorubicin (0.1 µg/ml) (experiment 1). For experiment 2, follicles were cultured in α-MEM+ alone or supplemented with doxorubicin (0.3 µg/ml) or different concentrations of resveratrol (5 or 10 µM) associated or not with doxorubicin (0.3 µg/ml) (experiment 2). The endpoints analyzed were morphology (survival), antrum formation, follicular diameter, mitochondrial activity, glutathione (GSH) levels and DNA fragmentation. In the first experiment, doxorubicin (0.1 µg/ml) maintained survival and antrum formation similar to the control, while 5 µM resveratrol showed increased parameters, maintained mitochondrial activity and increased GSH levels compared to the control. In the second experiment, doxorubicin (0.3 µg/ml) reduced survival, antrum formation and follicular diameter compared to the control. Resveratrol at a concentration of 10 µM attenuated the damage caused by doxorubicin by improving follicular survival and did not present DNA fragmentation. In conclusion, supplementation of the in vitro culture medium with 0.3 µg/ml doxorubicin reduced the survival and impaired the development of mouse-isolated preantral follicles. Resveratrol at 10 µM reduced doxorubicin-induced follicular atresia, without DNA fragmentation in the follicles.

Three-Dimensionally Printed Agarose Micromold Supports Scaffold-Free Mouse Ex Vivo Follicle Growth, Ovulation, and Luteinization.

Ex vivo follicle growth is an essential tool, enabling interrogation of folliculogenesis, ovulation, and luteinization. Though significant advancements have been made, existing follicle culture strategies can be technically challenging and laborious. In this study, we advanced the field through development of a custom agarose micromold, which enables scaffold-free follicle culture. We established an accessible and economical manufacturing method using 3D printing and silicone molding that generates biocompatible hydrogel molds without the risk of cytotoxicity from leachates. Each mold supports simultaneous culture of multiple multilayer secondary follicles in a single focal plane, allowing for constant timelapse monitoring and automated analysis. Mouse follicles cultured using this novel system exhibit significantly improved growth and ovulation outcomes with comparable survival, oocyte maturation, and hormone production profiles as established three-dimensional encapsulated in vitro follicle growth (eIVFG) systems. Additionally, follicles recapitulated aspects of in vivo ovulation physiology with respect to their architecture and spatial polarization, which has not been observed in eIVFG systems. This system offers simplicity, scalability, integration with morphokinetic analyses of follicle growth and ovulation, and compatibility with existing microphysiological platforms. This culture strategy has implications for fundamental follicle biology, fertility preservation strategies, reproductive toxicology, and contraceptive drug discovery.

P-370 The age-associated increase in ovarian stiffness compromises follicle development and induces changes in the follicles’ transcriptome, resulting in poor-quality oocytes

Abstract Study question Does the age-associated increase in ovarian stiffness trigger the decline in oocyte quantity and quality observed in women of advanced maternal age? Summary answer An encapsulated in vitro follicle culture mimicking ovarian biomechanics revealed that age-associated increase in stiffness impacts follicle growth, modulates follicles’ transcriptome and reduces oocyte quality. What is known already Female reproductive aging is characterized by a progressive decline in oocyte quality and quantity, resulting in sub-fertility. We recently discovered that with aging the mouse ovary assumes a pro-fibrotic milieu which induces ovarian stiffness. Moreover, human ovaries also become stiffer with aging; however, it is not known whether this age-associated increase in stiffness impacts ovarian function. The present study aims to examine whether stiffness is a novel mechanism that mediates the age-associated progressive decline in oocyte quality and how stiffness affects the transcriptome of growing follicles, resulting in poor-quality oocytes. Study design, size, duration We used an in vitro alginate-encapsulated follicle culture to mimic young and old environments in which follicles grow. Alginate hydrogel synthesis replicated the soft (0.5% - 1.79±0.08 kPa) and stiff (2% - 4.56±2.03 kPa) environments of young and aged ovaries, respectively. Mouse secondary follicles were cultured in 0.5 or 2% alginate and used for i) long-term culture (day 0-10) to evaluate follicle development and oocyte quality; ii) short-term culture (hour 0-24) to investigate follicles’ transcriptome. Participants/materials, setting, methods During the long-term culture, markers of follicle development were evaluated (n = 31 follicles), including: follicle size, estradiol production (ELISA), cell death (cleaved-caspase3,CC3; immunohistochemistry). At D10, oocyte quality was inspected on isolated oocytes. For the short-term culture experiment, 32 follicles were analyzed by RNAsequencing at 3h (baseline) and 24h. We compared 3h and 24h gene expression (DESeq2,R/Bioconductor) in 0.5% and 2%. Significant differences reported at padj<0.05 and log2FoldChange±2. Two mice strains (CB6F1;CD1) were used in all experiments. Main results and the role of chance Follicles cultured in stiff environment showed a significant reduction in follicle size compared to follicles cultured in soft environment (0.5% 226.9±17.4um, 2% 160.8±9.9um; p < 0.0001). These size differences suggest that granulosa cells are not proliferating in a stiff environment. Estradiol production was reduced in follicles cultured at 2% (0.5% 11.3±15.9ng/ml, 2% 0.3±0.5ng/ml, p = 0.296), while CC3 intensity was increased (CC3 staining intensity/area, 0.5% 20.4±5.8a.u., 2% 27.1±4.3a.u., p < 0.05), suggesting that the stiff environment impacts granulosa cell viability. Oocyte quality significantly declined in follicles cultured in 2%, with 68.9±16.8% of the oocytes degenerated, compared to 23.6±9.2% in 0.5% alginate. The use of two different mice strains yielded comparable results. Using RNAsequencing analysis, we found 1033 differentially expressed genes (DEGs) between 3h and 24h of culture in 2% alginate, mostly involved in apoptosis, inflammation and collagen processes, biological pathways typically associated with aging. The 64% of those DEGs were upregulated at 24h. Conversely, the 1295 DEGs (61% upregulated at 24h) detected comparing 3h and 24h in 0.5% alginate were relate to metabolism, cell cycle and development pathways, biological processes associated to the normal behavior of growing follicles. These differences in gene expression suggest an early-response in the transcriptome of follicles cultured in a stiff environment. Limitations, reasons for caution The stiffness of the alginate gels was based on whole ovary biomechanics; future investigations are needed to measure each follicle-stage-specific stiffness. The changes in follicles’ transcriptome in the stiff environment were evaluated only in short-term culture. This study utilizes in vitro approaches, in vivo modulation of stiffness is currently undergoing. Wider implications of the findings Increased ovarian stiffness is observed in conditions such as aging or ovaries exposed to cancer treatments. Studying how stiffness impacts oocyte quality will allow the development of suitable in vitro systems to support human folliculogenesis and in vivo approaches to modulate biomechanics of the ovary, preserving female fertility. Trial registration number not applicable

AP collagen peptides (APCPs) promote hair growth by activating the GSK-3β/β-catenin pathway and improve hair condition.

AP collagen peptides (APCPs) are enzymatically decomposed collagen peptides that contain tri-peptides such as glycine-proline-hydroxyproline. We found that APCPs increased the proliferation of both human dermal papilla cells (hDPCs) and human outer root sheath cells (hORSCs). APCPs also stimulated the secretion of several growth factors, including IGFBP-6, PDGF-AB, PIGF and VEGF in hDPCs. Moreover, APCPs enhanced the phosphorylation of Akt(Ser473), GSK-3β(Ser9) and β-catenin(Ser675), indicating the activation of the GSK-3β/β-catenin signalling pathway. Exvivo culture of human hair follicles (hHFs) tissue and invivo patch assay revealed that APCPs promoted the elongation of hHFs and the induction of new hair shafts. In a mouse model, APCPs significantly promoted the transition from telogen to anagen phase and prolonged anagen phase, resulting in increased hair growth. APCPs also improved the thickness, amino acid content (cystine and methionine) and roughness of mouse hair. Taken together, these findings demonstrate that APCPs accelerate hair growth and contribute to overall hair health. Therefore, APCPs have the potential to be utilized as a food supplement and ingredient for preventing hair loss and maintaining hair health.

1,25-(OH)2D3 promotes hair growth by inhibiting NLRP3/IL-1β and HIF-1α/IL-1β signaling pathways

Vitamin D is a crucial vitamin that participates in various biological processes through the Vitamin D Receptor (VDR). While there are studies suggesting that VDR might regulate hair growth through ligand-independent mechanisms, the efficacy of Vitamin D in treating hair loss disorders has also been reported. Here, through in vivo experiments in mice, in vitro organ culture of hair follicles, and cellular-level investigations, we demonstrate that 1,25-(OH)2D3 promotes mouse hair regeneration, prolongs the hair follicle anagen, and enhances the proliferation and migration capabilities of dermal papilla cells and outer root sheath keratinocytes in a VDR-dependent manner. Transcriptome analysis of VDR-knockout mouse skin reveals the involvement of HIF-1α, NLRP3, and IL-1β in these processes. Finally, we confirm that 1,25-(OH)2D3 can counteract the inhibitory effects of DHT on hair growth. These findings suggest that 1,25-(OH)2D3 has a positive impact on hair growth and may serve as a potential therapeutic agent for androgenetic alopecia (AGA).

In Vitro Development of Mouse Preantral Follicle with Using Amniotic Membrane Extract-Loaded Hydrogels.

An artificial ovary based on the alginate (ALG) hydrogel has been widely implemented to preserve prepubertal female fertility. However, this platform is not fully capable of successful an ovary microenvironment simulation for follicle development, holding great potential for its improvement. Therefore, this experimental study aimed to evaluate the effect of an amniotic membrane extract (AME) -loaded hydrogel on the mouse preantral follicles in vitro development. In order to have better follicle development, first, the impact of different concentrations of follicle-stimulating hormone (FSH) was evaluated on the mouse preantral follicles encapsulated in ALG. Later, the appropriate dose was adjusted for the follicles encapsulated in the ALG-AME hydrogel. Results demonstrated that 100 mIU/ml FSH showed a significant follicle survival rate compared with 10 mIU/ml FSH (P=0.005). According to MTT assay finding, the rate of weight loss, and rheology evaluations, ALG containing 1 mg/ml AME was identified as an optimal sample of follicle culture instead of other AME concentrations. Follicle diameter significantly increased in the ALG-AME 1 hydrogel compared with the ALG control group without AME (P=0.027). The storage modulus of ALG-AME 1 was 773 Pa and retained the follicle morphology for 13 days. No statistically substantial difference was seen in survival, antrum cavity formation, and competent oocyte in terms of the normal chromosomal arrangement and meiotic spindle rate in comparison with the control group. It can be concluded that ALG-AME 1 could not significantly impact the mouse preantral follicle.

A more natural follicle culture system: Detailed steps of In Vitro 3D follicle culture with alginate gel

Follicle culture is a process of dividing follicle unit structures from ovaries for continued culture in vitro in an incubator, which simulates the in vivo environment. Alginate gel is the most stable and most convenient 3D material currently used in follicle culture. We performed in vitro follicle culture following the standard operating procedure recommended by the Follicle Handbook and we have summarized our experience and skills in details. Through several experiments, we found only follicles tightly surrounded by theca cells can grow healthily until the preovulatory stage. In addition, the hardness of alginate gel is crucial for constructing the 3D culture system, and selecting appropriate tools can reduce damage to the alginate gel and shorten the time follicles are exposed to room temperature. Our detailed operation improves bioavailability and provides a more natural environment for the entire process of follicular growth.•Alginate gel is still the most suitable 3D material used for in vitro follicle culture.•Follicle integrity and the hardness of alginate gel are the keys for in vitro culture.•Detailed operation steps better protect the follicular microenvironment and improve bioavailability.

Bee Venom Stimulates Growth Factor Release from Adipose-Derived Stem Cells to Promote Hair Growth.

Limited evidence suggests that stimulating adipose-derived stem cells (ASCs) indirectly promotes hair growth. We examined whether bee venom (BV) activated ASCs and whether BV-induced hair growth was facilitated by enhanced growth factor release by ASCs. The induction of the telogen-to-anagen phase was studied in mice. The underlying mechanism was investigated using organ cultures of mouse vibrissa hair follicles. When BV-treated ASCs were injected subcutaneously into mice, the telogen-to-anagen transition was accelerated and, by day 14, the hair weight increased. Quantitative polymerase chain reaction (qPCR) revealed that BV influenced the expression of several molecules, including growth factors, chemokines, channels, transcription factors, and enzymes. Western blot analysis was employed to verify the protein expression levels of extracellular-signal-regulated kinase (ERK) and phospho-ERK. Both the Boyden chamber experiment and scratch assay confirmed the upregulation of cell migration by BV. Additionally, ASCs secreted higher levels of growth factors after exposure to BV. Following BV therapy, the gene expression levels of alkaline phosphatase (ALP), fibroblast growth factor (FGF)-1 and 6, endothelial cell growth factor, and platelet-derived growth factor (PDGF)-C were upregulated. The findings of this study suggest that bee venom can potentially be utilized as an ASC-preconditioning agent for hair regeneration.

Exploring the potential use of platelet rich plasma (PRP) from adult and umbilical cord blood in murine follicle culture

Ovarian follicle culture is a powerful tool to study follicular physiology and has potential applications in clinical and commercial settings. Despite remarkable progress, recreating folliculogenesis in vitro remains challenging for many mammalian species. This study investigates the impact of platelet-rich plasma (PRP) derived from adult blood (human platelet lysate, hPL) and umbilical cord blood (Umbilical cord plasma, UCP) on murine pre-antral follicle culture and oocyte maturation. Pre-antral follicles were cultured individually for 10 days with fetal bovine serum (FBS) serving as the control and two PRP sources (hPL and UCP) and their activated forms (Ac-hPL and Ac-UCP). The results suggest that neither hPL nor UCP, regardless of activation status, improved follicle culture outcomes compared to FBS. Interestingly, activation did not significantly impact the main functional outcomes such as maturation rates, survival, and growth. Oestradiol secretion and oocyte diameter, often considered hallmarks of follicle quality, did not show significant differences between matured and non-matured oocytes across the treatment groups. However, gene expression analysis revealed a significant upregulation of Gdf-9 and Bmp-15 mRNA levels in oocytes from the Ac-UCP group, regardless of maturation stage, suggesting that the accumulation of the mRNA could be due to potential challenges in translation in the Ac-UCP group. In conclusion, this study challenges the hypothesis that PRP, as a serum source, could improve follicle culture outcomes compared to FBS, the gold standard in murine follicle culture. Further research is needed to understand the species-specific effects of PRP and explore other potential factors affecting follicle culture and oocyte quality.

Effectiveness of a Novel Compound HAIR & SCALP COMPLEX on Hair Follicle Regeneration

Background: People lose between 50 and 100 hairs a day and generate new ones from stem cells in hair follicles, but in those suffering from baldness, the stem cells remain inactive and are unable to regenerate new hair. Although 9% of hair follicles remain in telogen at any time, a variety of factors, including growth factors and cytokines, promote the transition from telogen to anagen and the subsequent stimulation of hair growth. Methods: We compared in vitro, on cultures of human hair follicles, the effect on hair growth and regeneration of the dermal papilla of plant-derived nanovesicles, exosomes from cord blood stem cells and bovine colostrum, a mixture of growth factors and cytokines purified from bovine colostrum, called GF20, and a new compound called HAIR & SCALP COMPLEX obtained by adding exosomes isolated from colostrum to GF20. Results: The analyses demonstrated a significant increase in the growth of the bulb and the regeneration of the dermal papilla in the samples treated with HAIR & SCALP COMPLEX compared to the other elements tested. Conclusions: In this research, we propose a possible new treatment that could help significantly slow down hair loss and encourage new hair growth: HAIR & SCALP COMPLEX.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

Pronuclear transfer rescues poor embryo development of in vitro-grown secondary mouse follicles.

3D culture applied to reproduction in females: possibilities and perspectives.

In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction.

In vitro culture of mechanically isolated murine primary follicles in the presence of human platelet lysate PLTMax.

To develop a system for the culture of murine preantral ovarian follicles using Human Serum Albumin (HSA) and Human Platelet Lysate (PLTMax). Mechanically isolated preantral follicles (N=146) were obtained from Swiss mice and cultured in DMEM:F12 medium for ten days in a 96-well plate with conical bottom. The medium was supplemented with penicillin, streptomycin, and equine chorionic gonadotropin. Additional proteins were tested in 4 test groups: G1: human serum albumin (HSA), G2: human platelet lysate (PLTM), and G3 and G4: HSA + PLTMax at lower and higher concentrations, respectively. Cellular vitality and oocyte morphology were evaluated on day 11 of culture. The highest follicular growth (3.4 fold) was achieved in HSA (G1), while a significantly lower (1.8 fold) growth was achieved in the presence of PLTM (G2, G4) and even further reduced (1.2 fold) when HSA and PLTM were combined (G3). Cellular vitality was close to 70-80% among the four groups, and the highest number of intact oocytes were found in G1. PLTM did not improve follicular development and oocyte maturation compared to HSA but preserved cell vitality.