Entropy and mechanistic concepts after intraovarian platelet-rich plasma: Experimental considerations for local tissue responses mediated by NF-κB and TNF-α

Female germline or oogonial stem cells transiently residing in fetal ovaries are analogous to the spermatogonial stem cells or germline stem cells (GSCs) in adult testes where GSCs and meiosis continuously renew. Oocytes can be generated in vitro from embryonic stem cells and induced pluripotent stem cells, but the existence of GSCs and neo-oogenesis in adult mammalian ovaries is less clear. Preliminary findings of GSCs and neo-oogenesis in mice and humans have not been consistently reproducible. Monkeys provide the most relevant model of human ovarian biology. We searched for GSCs and neo-meiosis in ovaries of adult monkeys at various ages, and compared them with GSCs from adult monkey testis, which are characterized by cytoplasmic staining for the germ cell marker DAZL and nuclear expression of the proliferative markers PCNA and KI67, and pluripotency-associated genes LIN28 and SOX2, and lack of nuclear LAMIN A, a marker for cell differentiation. Early meiocytes undergo homologous pairing at prophase I distinguished by synaptonemal complex lateral filaments with telomere perinuclear distribution. By exhaustive searching using comprehensive experimental approaches, we show that proliferative GSCs and neo-meiocytes by these specific criteria were undetectable in adult mouse and monkey ovaries. However, we found proliferative nongermline somatic stem cells that do not express LAMIN A and germ cell markers in the adult ovaries, notably in the cortex and granulosa cells of growing follicles. These data support the paradigm that adult ovaries do not undergo germ cell renewal, which may contribute significantly to ovarian senescence that occurs with age.

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Many studies have focused on the mechanisms of stem cell maintenance via their interaction with a particular niche or microenvironment in adult tissues, but how formation of a functional niche is initiated, including how stem cells within a niche are established, is less well understood. Adult Drosophila melanogaster ovary Germline Stem Cell (GSC) niches are comprised of somatic cells forming a stack called a Terminal Filament (TF) and associated Cap and Escort Cells (CCs and ECs, respectively), which are in direct contact with GSCs. In the adult ovary, the transcription factor Engrailed is specifically expressed in niche cells where it directly controls expression of the decapentaplegic (dpp) gene encoding a member of the Bone Morphogenetic Protein (BMP) family of secreted signaling molecules, which are key factors for GSC maintenance. In larval ovaries, in response to BMP signaling from newly formed niches, adjacent primordial germ cells become GSCs. The bric-à-brac paralogs (bab1 and bab2) encode BTB/POZ domain-containing transcription factors that are expressed in developing niches of larval ovaries. We show here that their functions are necessary specifically within precursor cells for TF formation during these stages. We also identify a new function for Bab1 and Bab2 within developing niches for GSC establishment in the larval ovary and for robust GSC maintenance in the adult. Moreover, we show that the presence of Bab proteins in niche cells is necessary for activation of transgenes reporting dpp expression as of larval stages in otherwise correctly specified Cap Cells, independently of Engrailed and its paralog Invected (En/Inv). Moreover, strong reduction of engrailed/invected expression during larval stages does not impair TF formation and only partially reduces GSC numbers. In the adult ovary, Bab proteins are also required for dpp reporter expression in CCs. Finally, when bab2 was overexpressed at this stage in somatic cells outside of the niche, there were no detectable levels of ectopic En/Inv, but ectopic expression of a dpp transgene was found in these cells and BMP signaling activation was induced in adjacent germ cells, which produced GSC-like tumors. Together, these results indicate that Bab transcription factors are positive regulators of BMP signaling in niche cells for establishment and homeostasis of GSCs in the Drosophila ovary.

Companion Blood Cells Control Ovarian Stem Cell Niche Microenvironment and Homeostasis.

Studies suggest a renewable source of eggs and stir more controversy, especially about the origin of female germline stem cells (FGSCs). It should be elucidated whether or not neo-oogenesis continues in the ovaries of mammalian female during postnatal life. Therefore, the establishment of FGSCs is very important for many applications. Here, using adult pig ovary, we isolate, identify, characterize FGSCs to elucidate their origin, then examined the proliferation, growth and differentiation of them. These cells were heterogeneous, depending on both of c-kit expression and cell size, and also express stem cell and germ cell markers. Importantly, we show clearly that the cells with the characteristics of early primordial germ cells are present in the adult pig ovary. Once FGSCs were established, they could be expanded in vitro for months without loss of the identifying markers and proliferative potential. Under appropriate conditions, the FGSCs differentiated into primordial oocyte-like cells and grow close to full-sized oocytes. These may assist in therapeutic strategies in human with their potential to make new oocytes and support ovarian function and fertility. Our results support the theory that the ovary contains a small number of undifferentiated cells with stem cell characteristics. These might remain in the postnatal and adult ovary and under certain conditions could resume mitosis, enter meiosis and give rise to oocytes. Given the existence of these FGSCs in mammalian ovaries and the depletion in ovarian reserve during female reproductive aging, one can hypothesize that such “neo-oogenesis” was present in ancestral forms, is still present in insects, some fish and mollusks, but has been lost in land vertebrates through evolution. FGSCs cannot proliferate in the ovary normally because of inhibitory factors, but under appropriate conditions, they can undergo proliferation and differentiation, and provide a potential mechanism for the self-renewal of germline stem cells.

In clinical infertility practice, one intractable problem is low (or absent) ovarian reserve which in turn reflects the natural oocyte depletion associated with advancing maternal age. The number of available eggs has been generally thought to be finite and strictly limited, an entrenched and largely unchallenged tenet dating back more than 50 years. In the past decade, it has been suggested that renewable ovarian germline stem cells (GSCs) exist in adults, and that such cells may be utilized as an oocyte source for women seeking to extend fertility. Currently, the issue of whether mammalian females possess such a population of renewable GSCs remains unsettled. The topic is complex and even agreement on a definitive approach to verify the process of ‘ovarian rescue’ or ‘re-potentiation’ has been elusive. Similarities have been noted between wound healing and ovarian tissue repair following capsule rupture at ovulation. In addition, molecular signaling events which might be necessary to reverse the effects of reproductive ageing seem congruent with changes occurring in tissue injury responses elsewhere. Recently, clinical experience with such a technique based on autologous activated platelet-rich plasma (PRP) treatment of the adult human ovary has been reported. This review summarizes the present state of understanding of the interaction of platelet-derived growth factors with adult ovarian tissue, and the outcome of human reproductive potential following PRP treatment.

Stem cell culture for regenerative medicine needs platelet rich plasma (PRP) as fetal bovine/calf serum (FBS/FCS) substitute. However, the various studies used various protocols in preparing and processing the PRP. This study aimed to compare and conclude the most effective and efficient protocol. we searched in vitro studies that used human PRP as FBS/FCS substitute to culture human cells, and compared the various available protocols to identify the easiest and effective protocols for the preparation of PRP and the release of the growth factors (GFs) to support the highest cell growth in stem cell culture. ten studies fulfilled the selection criteria and were included in the analysis. Almost all studies on bone marrow mesenchymal stem cell (BM-MSC) and adipose stem cell (AT-SC) showed that platelet lysate and/or activated platelet releasate were superior or at least the same as either FBS or FCS, except for one study that got different results on human AT-SC. Several studies showed that either 5% activated PRP (aPRP) or platelet lysate (PL) was sufficient to support cell growth, or even better when they were compared to 10% FBS, while higher concentrations were counterproductive. However, some studies showed that 10% aPRP or PL was needed. The difference between studies was due to the difference in either the PRP preparation from blood and in the PRP processing to release the GFs, which yield various GF concentrations. In conclusion, studies are needed to reveal the optimal final platelet counts for the various PRP processing methods for various kinds of cells. The easiest PRP processing is freezing to -20°C followed by thawing, or thrombin activation using a final concentration of 100U/mL.

It has long been established that germline stem cells (GSCs) are responsible for lifelong gametogenesis in males, and some female invertebrates (for example, Drosophila) and lower vertebrates (for example, teleost fish and some prosimians) also appear to rely on GSCs to replenish their oocyte reserve in adulthood. However, the presence of such cells in the majority of female mammals is controversial, and the idea of a fixed ovarian reserve determined at birth is the prevailing belief among reproductive biologists. However, accumulating evidence demonstrates the isolation and culture of putative GSCs from the ovaries of adult mice and humans. Live offspring have been reportedly produced from the culture of adult mouse GSCs, and human GSCs formed primordial follicles using a mouse xenograft model. If GSCs were present in adult female ovaries, it could be postulated that the occurrence of menopause is not due to the exhaustion of a fixed supply of oocytes but instead is a result of GSC and somatic cell aging. Alternatively, they may be benign under normal physiological conditions. If their existence were confirmed, female GSCs could have many potential applications in both basic science and clinical therapies. GSCs not only may provide a valuable model for germ cell development and maturation but may have a role in the field of fertility preservation, with women potentially being able to store GSCs or GSC-derived oocytes from their own ovaries prior to infertility-inducing treatments. Essential future work in this field will include further independent corroboration of the existence of GSCs in female mammals and the demonstration of the production of mature competent oocytes from GSCs cultured entirely in vitro.

Female meiosis and beyond: more questions than answers?

According to classical knowledge of reproductive biology, in the ovary of female mammals there is a limited number of oocytes and there is no possibility of renewal if the oocytes are lost due to disease or injury. However, in recent years, the results of some studies on renewal and formation of oocytes and follicles in the adult mammalian ovary have led to the questioning of this opinion. The aim of our study is to demonstrate the presence of putative germline and pluripotent stem cells in the adult mouse ovary and their differentiation potential into germ and somatic cells. In ovary tissues and cells harvested from pre-differentiation step, the expression of pluripotent and germline stem cell markers was analysed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and western blotting. Embryoid bodies that formed in this step were analysed using immunofluorescence staining and transmission electron microscopy. Ovarian stem cells were induced to differentiate into oocyte, osteoblast, chondrocyte and neural cells. Besides morphological observation, differentiated cells were analysed by RT-PCR, histochemical and immunofluorescence staining. Expression of germline and pluripotent stem cell markers both in mRNA and at the protein level were detected in the pre-differentiated cells and ovary tissues. As a result of the differentiation process, the formation of oocyte-like cells, osteoblasts, chondrocytes and neural cells was observed and characteristics of differentiated cells were confirmed using the methods mentioned above. Our study results revealed that the adult mouse ovary contains germline and pluripotent stem cells with the capacity to differentiate into oocyte-like cells, osteoblasts, chondrocytes and neural cells.

Drosophila oogenesis starts when a germline stem cell divides asymmetrically to generate a daughter germline stem cell and a cystoblast that will develop into a mature egg. We show that the fs(1)Yb gene is essential for the maintenance of germline stem cells during oogenesis. We delineate fs(1)Yb within a 6.4 kb genomic region by transgenic rescue experiments. fs(1)Yb encodes a 4.1 kb RNA that is present in the third instar larval, pupal and adult stages, consistent with its role in regulating germline stem cells during oogenesis. Germline clonal analysis shows that all fs(1)Yb mutations are soma-dependent. In the adult ovary, fs(1)Yb is specifically expressed in the terminal filament cells, suggesting that fs(1)Yb acts in these signaling cells to maintain germline stem cells. fs(1)Yb encodes a novel hydrophilic protein with no potential signal peptide or transmembrane domains, suggesting that this protein is not itself a signal but a key component of the signaling machinery for germline stem cell maintenance.

A fundamental yet unexplored question in stem cell biology is how the fate of tissue stem cells is initially determined during development. In Drosophila, germline stem cells (GSCs) descend from a subset of primordial germ cells (PGCs) at the onset of oogenesis. GSC determination may occur at the onset of oogenesis when a subset of PGCs is induced to become GSCs by contacting niche cells. Alternatively, the GSC fate could be predetermined for a subset of PGCs before oogenesis, due to either their interaction with specific somatic cells in the embryonic/larval gonads, or their inherently heterogeneous potential in becoming GSCs, or both. Here, we show that anterior somatic cells in the embryonic gonad already differ from posterior somatic cells and are likely to be the precursors of niche cells in the adult ovary. Furthermore, only pole cells in the anterior half of the embryonic gonad give rise to the PGCs that frequently acquire contact with nascent niche cells in the late larval ovary. Eventually, only these contacting PGCs become GSCs, whereas non-contacting PGCs directly differentiate into cystoblasts. The strong preference of these 'anterior PGCs' towards contacting niche cells does not require DE-cadherin-mediated adhesion and is not correlated with either orientation or rate of their divisions. These data suggest that the GSC fate is predetermined before oogenesis. The predetermination probably involves soma/pole-cell interaction in the anterior half of the embryonic gonad, followed by an active homing mechanism during PGC proliferation to maintain the contact between the 'anterior PGCs' and anterior somatic cells.

nanos3 maintains germline stem cells and expression of the conserved germline stem cell gene nanos2 in the zebrafish ovary

Simple SummaryCyromazine, an insect growth regulator, is used to control the Dipteran pest population. Previous findings observed that treatment with cyromazine increased the larval mortality, by interfering with the ecdysone signaling. In addition, the application of exogenous 20E significantly reduced the mortality caused by cyromazine. Many studies have also supported the role of ecdysone signaling in the maintenance of germline stem cells (GSCs), where mutations in ecdysone signaling-related genes significantly decreased the number of GSCs. However, to date, no study has reported the effect of cyromazine on the GSCs of Drosophila melanogaster. In the present study, we observed that cyromazine significantly reduced the number of both GSCs and cystoblasts (CBs) in the ovary of adult female. To further understand the effect of cyromazine on germ cells, we selected some key genes related to the ecdysone signaling pathway and evaluated their expression through RT-qPCR. Additionally, we measured the ecdysone titer from the cyromazine-treated ovaries. Our results indicated a significant decrease in the expression of ecdysone signaling-related genes and also in the ecdysone titer. These results further supported our findings that cyromazine reduced the number of germ cells by interfering with the ecdysone signaling pathway.In the present study, we observed a 58% decrease in the fecundity of Drosophila melanogaster, after treatment with the cyromazine. To further elucidate the effects of cyromazine on reproduction, we counted the number of both germline stem cells (GSCs) and cystoblasts (CBs) in the ovary of a 3-day-old adult female. The results showed a significant decrease in the number of GSCs and CBs as compared to the control group. The mode of action of cyromazine is believed to be through the ecdysone signaling pathway. To further support this postulate, we observed the expression of key genes involved in the ecdysone signaling pathway and also determined the ecdysone titer from cyromazine-treated ovaries. Results indicated a significant decrease in the expression of ecdysone signaling-related genes as compared to the control group. Furthermore, the titer of the ecdysone hormone was also markedly reduced (90%) in cyromazine-treated adult ovaries, suggesting that ecdysone signaling was directly related to the decrease in the number of GSCs and CBs. However, further studies are required to understand the mechanism by which cyromazine affects the GSCs and CBs in female adult ovaries.

Tissue homeostasis is maintained by balancing stem cell self-renewal and differentiation. How surrounding cells support this process has not been entirely resolved. Here we show that the chromatin and telomere-binding factor Without children (Woc) is required for maintaining the association of escort cells (ECs) with germ cells in adult ovaries. This tight association is essential for germline stem cell (GSC) differentiation into cysts. Woc is also required in larval ovaries for the association of intermingled cells (ICs) with primordial germ cells. Reduction in the levels of two other proteins, Stat92E and its target Zfh1, produce phenotypes similar to woc in both larval and adult ovaries, suggesting a molecular connection between these three proteins. Antibody staining and RT-qPCR demonstrate that Zfh1 levels are increased in somatic cells that contact germ cells, and that Woc is required for a Stat92E-mediated upregulation of zfh1 transcription. Our results further demonstrate that overexpression of Zfh1 in ECs can rescue GSC differentiation in woc-deficient ovaries. Thus, Zfh1 is a major Woc target in ECs. Stat signalling in niche cells has been previously shown to maintain GSCs non-autonomously. We now show that Stat92E also promotes GSC differentiation. Our results highlight the Woc-Stat-Zfh1 module as promoting somatic encapsulation of germ cells throughout their development. Each somatic cell type can then provide the germline with the support it requires at that particular stage. Stat is thus a permissive factor, which explains its apparently opposite roles in GSC maintenance and differentiation.