Enhancing differentiation of menstrual blood-derived stem cells into female germ cells using a bilayer amniotic membrane and nano-fibrous fibroin scaffold.

Three-dimensional nanofiber scaffolds offer a promising method for simulating in vivo conditions within the laboratory. This study aims to investigate the influence of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold on the differentiation of human menstrual blood mesenchymal stromal/stem cells (MenSCs) into female germ cells. MenSCs were isolated and assigned to four culture groups: (i) MenSCs co-cultured with granulosa cells (GCs) using the scaffold (3D-T group), (ii) MenSCs using the scaffold alone (3D-C group), (iii) MenSCs co-cultured only with GCs (2D-T group), and (iv) MenSCs without co-culture or scaffold (2D-C group). Both MenSCs and GCs were independently cultured for two weeks before co-culturing was initiated. Flow cytometry was employed to characterize MenSCs based on positive markers (CD73, CD90, and CD105) and negative markers (CD45 and CD133). Additionally, flow cytometry and immunocytochemistry were used to characterize the GCs. Differentiated MenSCs were analyzed using real-time PCR and immunostaining. The real-time PCR results demonstrated significantly higher levels of VASA expression in the 3D-T group compared to the 3D-C, 2D-T, and 2D-C groups. Similarly, the SCP3 mRNA level in the 3D-T group was notably elevated compared to the 3D-C, 2D-T, and 2D-C groups. Moreover, the expression of GDF9 was significantly higher in the 3D-T group when compared to the 3D-C, 2D-T, and 2D-C groups. Immunostaining results revealed a lack of signal for VASA, SCP3, or GDF9 markers in the 2D-T group, while some cells in the 3D-T group exhibited positive staining for all these proteins. These findings suggest that the combination of a bilayer amniochorionic membrane/nanofibrous fibroin scaffold with co-culturing GCs facilitates the differentiation of MenSCs into female germ cells.

Evaluation of mutagenic susceptibility of different stages in germ cell development of Caenorhabditis elegans using whole genome sequencing.

In contrast to somatic mutations, mutations in germ cells affect every cell of any organism derived from the germ cell and therefore are related to numerous genetic diseases. However, there is no suitable assay to evaluate the mutagenic sensitivities of both male and female germ cells. The main type of Caenorhabditis elegans (C. elegans) is hermaphroditic, where spermatogenesis and oogenesis occur chronologically at specific stages, allowing induction of mutations in either sperm or eggs exclusively. In this study, we used the alkylating agent ethyl methanesulfonate and N-ethyl-N-nitrosourea to induce germline mutations in C. elegans at different developmental stages and analyzed mutation frequency and mutational spectrum from data gathered using next-generation sequencing (NGS) technology. Our results revealed low spontaneous mutation rates of C. elegans, along with distinct mutagenic effects elicited by the two mutagens. Our data show that the parental worms treated during germ cell mitosis, spermatogenesis, and oogenesis resulted in different mutation frequencies in their offspring, and female germ cells could be very susceptible to mutagen exposure during oogenesis. In summary, our study indicates that the use of C. elegans and its specific chronological hermaphroditism would be a promising way to explore the sensitivities of both male and female germ cells to mutagens.

From in vivo to in vitro: exploring the key molecular and cellular aspects of human female gametogenesis.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

Dynamic Autophagy Map in Mouse Female Germ Cells Throughout the Fetal to Postnatal Life.

Autophagy plays vital roles in mouse female germ cells, but the potential mechanism is largely unknown. In this study, by interrogating single-cell RNA-seq dataset, we investigated the dynamic expression of autophagy-related genes in seven types of germ cells (mitosis, pre-leptotene, leptotene, zygotene, pachytene, diplotene, and dictyate) and discovered stage-specific autophagy-related genes. Using immunofluorescence (IF) and transmission electron microscopy (TEM), autophagy activity and autophagosome numbers were revealed from mitosis to follicular assembly (E12.5 (embryonic day 12.5) to P5 (postnatal day 5)). Furthermore, single-sample gene set enrichment analysis (ssGSEA) was performed to validate the autophagy kinetics from E12.5 to P5. Our study proved that the mitosis, diplotene, and dictyate female germ cells had relatively higher autophagy activity among the seven subtypes. In summary, our work provided an autophagy map, suggesting that autophagy was complicated in mouse female germ cell development from the fetal to postnatal life, which paved a new insight for deciphering the autophagy regulatory networks for cell-fate transition and female infertility issues like primary ovarian insufficiency (POI).

Advanced bioengineering of female germ cells to preserve fertility.

Oogenesis and folliculogenesis are considered as complex and species-specific cellular differentiation processes, which depend on the in vivo ovarian follicular environment and endocrine cues. Considerable efforts have been devoted to driving the differentiation of female primordial germ cells toward mature oocytes outside of the body. The recent experimental attempts have laid stress on offering a suitable microenvironment to assist the in vitro folliculogenesis and oogenesis. Despite developing a variety of bioengineering techniques and generating functional mature gametes through in vitro oogenesis in earlier studies, we still lack knowledge of appropriate microenvironment conditions for building biomimetic culture systems for female fertility preservation. Therefore, this review paper can provide a source for a large body of scientists developing cutting-edge in vitro culture systems for female germ cells or setting up the next generation of reproductive medicine as feasible options for female infertility treatment. The focal point of this review outlines advanced bioengineering technologies such as 3D biofabricated hydrogels/scaffolds and microfluidic systems utilized with female germlines for fertility preservation through in vitro folliculogenesis and oogenesis.

Dissecting the initiation of female meiosis in the mouse at single-cell resolution.

Meiosis is one of the most finely orchestrated events during gametogenesis with distinct developmental patterns in males and females. However, the molecular mechanisms involved in this process remain not well known. Here, we report detailed transcriptome analyses of cell populations present in the mouse female gonadal ridges (E11.5) and the embryonic ovaries from E12.5 to E14.5 using single-cell RNA sequencing (scRNA seq). These periods correspond with the initiation and progression of meiosis throughout the first stage of prophase I. We identified 13 transcriptionally distinct cell populations and 7 transcriptionally distinct germ cell subclusters that correspond to mitotic (3 clusters) and meiotic (4 clusters) germ cells. By analysing cluster-specific gene expression profiles, we found four cell clusters correspond to different cell stages en route to meiosis and characterized their detailed transcriptome dynamics. Our scRNA seq analysis here represents a new important resource for deciphering the molecular pathways driving female meiosis initiation.

Critical roles of mRNA m6A modification and YTHDC2 expression for meiotic initiation and progression in female germ cells.

Meiotic entry and progression require dynamic regulation of germline gene expression. m6A on mRNAs and recognition by YTHDC2 has been known as post-transcriptional regulatory complex, but the roles of this regulator remain unclear for meiotic initiation and progression in female germ cells (FGCs). This study showed that m6A modification occurred mainly in FGCs rather than ovarian somatic cells (SOMAs), and m6A levels in FGCs increased significantly with meiotic initiation. m6A inhibition suppressed expression of the meiotic markers and affected the percent of FGCs at zygotene, pachytene and diplotene stage respectively. YTHDC2 expression also increased in the same pattern with m6A. Ythdc2 knockdown decreased the percent of STRA8-positive FGCs and altered the percent of FGCs at zygotene and pachytene stage respectively. Taken together, these results suggest that mRNA m6A modification and YTHDC2 expression are essential for meiotic initiation and progression in FGCs.

Mitochondria and Female Germline Stem Cells-A Mitochondrial DNA Perspective.

Mitochondria and mitochondrial DNA have important roles to play in development. In primordial germ cells, they progress from small numbers to populate the maturing oocyte with high numbers to support post-fertilization events. These processes take place under the control of significant changes in DNA methylation and other epigenetic modifiers, as well as changes to the DNA methylation status of the nuclear-encoded mitochondrial DNA replication factors. Consequently, the differentiating germ cell requires significant synchrony between the two genomes in order to ensure that they are fit for purpose. In this review, I examine these processes in the context of female germline stem cells that are isolated from the ovary and those derived from embryonic stem cells and reprogrammed somatic cells. Although our knowledge is limited in this respect, I provide predictions based on other cellular systems of what is expected and provide insight into how these cells could be used in clinical medicine.

The timeline development of female canine germ cells.

During the sex differentiation, the primordial germ cells (PGCs) pass through a differentiation, becoming spermatogonial cells in males and oocytes in females. In this phase, there is difference in gene expression and differentiation potency between males and females. Specific cell markers have been essential in the PGC meiosis beginning and become oocyte cells. However, there are few studies about germline in domestic animals. The domestic dog (Canis lupus familiaris) is an interesting animal model to be used in the investigation about the mammal development because it has several biochemical and physiological similarities to humans. In addition, some additional investigations about dogs may contribute to a better understanding of the biology and genetic components, improving clinical veterinary and zoological sciences. Here, we elucidated by immunofluorescence and quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), the dynamics of the expression of pluripotent (POU5F1 and NANOG) and germline (DDX4, DAZL and DPPA3) markers that are very important in the development of female canine germ cells during 35-50 days post-fertilization (dpf). The female canine germ cells were positive for pluripotent markers during middle developmental period. The number of DDX4, DAZL and DPPA3 cells increased along the germ cell maturation from 45 to 50 dpf. We provided an expression analysis of the pluripotent and germline markers in paraffin sections using the middle and later periods in female canine germ cells. The results can contribute the understanding about the timeline of each marker along the maturation of female canine germ cells. These results have a great significance to demonstrate the germ cell profile changes because it may allow the development of protocols about in vitro germ cell derivation.

The Mussel Mytilus galloprovincialis in the Bay of Naples: New Insights on Oogenic Cycle and Its Hormonal Control.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17ß-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3ß-HSD, 17ß-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

Molecular characteristics of early-stage female germ cells revealed by RNA sequencing of low-input cells and analysis of genome-wide DNA methylation.

High-throughput stage-specific transcriptomics provides an unbiased approach for understanding the process of cell development. Here, we report transcriptome analysis of primordial germ cell, female germline stem cell (FGSC), germinal vesicle and mature oocyte by performing RNA sequencing of freshly isolated cells in mice. As expected, these stages and gene-expression profiles are consistent with developmental timing. Analysis of genome-wide DNA methylation during female germline development was used for confirmation. By pathway analysis and blocking experiments, we demonstrate PI3K-AKT pathway is critical for FGSC maintenance. We also identify functional modules with hub genes and lncRNAs, which represent candidates for regulating FGSC self-renewal and differentiation. Remarkably, we note alternative splicing patterns change dramatically during female germline development, with the highest occurring in FGSCs. These findings are invaluable resource for dissecting the molecular pathways and processes into oogenesis and will be wider applications for other types of stem cell research.

Deriving cells expressing markers of female germ cells from premature ovarian failure patient-specific induced pluripotent stem cells.

AIM: We proposed a two-step protocol for deriving cells expressing markers of female germ cells (FGCs) from premature ovarian failure patient-specific induced pluripotent stem cells (POF-iPSCs). MATERIAL & METHODS: We cultured POF-iPSCs in suspension and pretreated them with TGFß-1 (1 ng/ml) for 2 days and continued with both TGFß-1 and BMP4 (50 ng/ml) for 5 more days. Then changed to media containing retinoic acid (1 µM) and 5% follicular fluid for another 7 days. Expression of markers of different stages of FGCs were detected. RESULTS: c-KIT, STELLA/DPPA3, VASA/DDX4, SCP3, GDF9 and ZP3 were positively detected and statistically significant different when compared with control groups. CONCLUSION: Our in vitro system was beneficial for POF-iPSCs differentiated cells to express STELLA, VASA and SCP3, which were the markers of meiosis initiation of FGCs.

The adverse outcome pathway (AOP) for chemical binding to tubulin in oocytes leading to aneuploid offspring.

The Organisation for Economic Co-operation and Development (OECD) has launched the Adverse Outcome Pathway (AOP) Programme to advance knowledge of pathways of toxicity and improve the use of mechanistic information in risk assessment. An AOP links a molecular initiating event (MIE) to an adverse outcome (AO) through intermediate key events (KE). Here, we present the scientific evidence in support of an AOP whereby chemicals that bind to tubulin cause microtubule depolymerization resulting in spindle disorganization followed by altered chromosome alignment and segregation and the generation of aneuploidy in female germ cells, ultimately leading to aneuploidy in the offspring. Aneuploidy, an abnormal number of chromosomes that is not an exact multiple of the haploid number, is a well-known cause of human disease and represents a major cause of infertility, pregnancy failure, and serious genetic disorders in the offspring. Among chemicals that induce aneuploidy in female germ cells, a large majority impairs microtubule dynamics and spindle function. Colchicine, a prototypical chemical that binds to tubulin and causes microtubule depolymerization, is used here to illustrate the AOP. This AOP is specific to female germ cells exposed during the periovulation period. Although the majority of the data come from rodent studies, the available evidence suggests that the MIE and KEs are conserved across species and would occur in human oocytes. The development of AOPs related to mutagenicity in germ cells is expected to aid the identification of potential hazards to germ cell genomic integrity and support regulatory efforts to protect population health.

Regulation of Fertility by the p53 Family Members.

The p53 family members, which consist of 3 transcription factors-p53, p63, and p73-are conserved during evolution. The p53 family proteins are involved in many important cellular functions, including tumor suppression (p53 and p73), the development of epithelial cell layers (p63), and the development of central nervous system and immune system (p73). Studies on p53-like proteins in low organisms have demonstrated that their primordial functions are to maintain the genomic integrity of germ cells and ensure faithful development and reproduction. In vertebrates, the p53 family proteins retain these functions in reproduction and at the same time have developed additional important functions in reproduction, such as the regulation of embryonic implantation (p53). p53 regulates embryonic implantation through transcriptional regulation of leukemia inhibitory factor (LIF). p63, in particular TAp63, is a main regulator to protect the fidelity of female germ cells during meiotic arrest. p73, in particular TAp73, regulates the ovary function and the quality of oocytes. Loss of p53, p63, or p73 genes in female mice leads to a significant decrease in fertility. These functions of the p53 family proteins in reproduction provide a plausible explanation for positive evolutionary selection observed in a group of single nucleotide polymorphisms and haplotypes in the p53 family genes. A better understanding of the functions of the p53 family proteins in reproduction may lead to new strategies for fertility treatment.