Characterization of Oogonial Stem Cells in Adult Mouse Ovaries with Age and Comparison to In Silico Data on Human Ovarian Aging.

Many adult somatic stem cell lineages are comprised of subpopulations that differ in gene expression, mitotic activity, and differentiation status. In this study, we explored if cellular heterogeneity also exists within oogonial stem cells (OSCs), and how chronological aging impacts OSCs. In OSCs isolated from mouse ovaries by flow cytometry and established in culture, we identified subpopulations of OSCs that could be separated based on differential expression of stage-specific embryonic antigen 1 (SSEA1) and cluster of differentiation 61 (CD61). Levels of aldehyde dehydrogenase (ALDH) activity were inversely related to OSC differentiation, whereas commitment of OSCs to differentiation through transcriptional activation of stimulated by retinoic acid gene 8 was marked by a decline in ALDH activity and in SSEA1 expression. Analysis of OSCs freshly isolated from ovaries of mice between 3 and 20 months of age revealed that these subpopulations were present and persisted throughout adult life. However, expression of developmental pluripotency associated 3 (Dppa3), an epigenetic modifier that promotes OSC differentiation into oocytes, was lost as the mice transitioned from a time of reproductive compromise (10 months) to reproductive failure (15 months). Further analysis showed that OSCs from aged females could be established in culture, and that once established the cultured cells reactivated Dppa3 expression and the capacity for oogenesis. Analysis of single-nucleus RNA sequence data sets generated from ovaries of women in their 20s versus those in their late 40s to early 50s showed that the frequency of DPPA3-expressing cells decreased with advancing age, and this was paralleled by reduced expression of several key meiotic differentiation genes. These data support the existence of OSC subpopulations that differ in gene expression profiles and differentiation status. In addition, an age-related decrease in Dppa3/DPPA3 expression, which is conserved between mice and humans, may play a role in loss of the ability of OSCs to maintain oogenesis with age.

Biomechanical Strain Promotes the Differentiation of Murine Oogonial Stem Cells.

Cells within tissues are routinely subjected to physiological stress and strain, arising from direct interactions with neighboring cells as well as with extracellular matrix components. Accordingly, there is tremendous interest in deciphering how cells sense, and respond to, changes in biomechanical forces. In this study, we explored the effects of mechanostimulation on the differentiation of mouse female germline or oogonial stem cells (OSCs) as a model for adult stem cell function. We report that increasing levels, or repeated application of a subthreshold fixed level, of radial strain to OSCs in culture significantly increased rates of in vitro oocyte formation as a measure of stem cell differentiation. These responses involved changes in F-actin-mediated cytoskeletal tension as well as in activation of intracellular signaling by Rho-associated protein kinase (ROCK) and Yes-associated protein (YAP) phosphorylation. In addition, application of mechanical strain to OSCs enhanced association of YAP with muscle-specific cytidine-adenosine-thymidine (MCAT) response elements in the promoter stimulated by retinoic acid gene 8 (Stra8), the transcriptional activation of which is required for germline meiotic commitment. These data indicate that biomechanical strain directly promotes the differentiation of adult female germline stem cells through a signaling pathway involving F-actin, ROCK, YAP, and Stra8.

Estrogen regulation of germline stem cell differentiation as a mechanism contributing to female reproductive aging.

Progressive loss of ovarian estrogen (E2) production is a hallmark feature of, if not a driving force behind, reproductive aging and the menopause. Recent genetic studies in mice have shown that female germline or oogonial stem cells (OSCs) contribute to maintenance of adult ovarian function and fertility under physiological conditions through support of de-novo oogenesis. Here we show that mouse OSCs express E2 receptor-α (ERα). In the presence of E2, ERα interacts with the stimulated by retinoic acid gene 8 (Stra8) promoter to drive Stra8 expression followed by oogenesis. Treatment of mice with E2 in vivo increases Stra8 expression and oogenesis, and these effects are nullified by ERα (Esr1), but not ERß (Esr2), gene disruption. Although mice lacking ERα are born with a normal quota of oocytes, ERα-deficient females develop premature ovarian insufficiency in adulthood due to impaired oogenesis. Lastly, mice treated with reversible ER antagonists show a loss of Stra8 expression and oocyte numbers; however, both endpoints rebound to control levels after ceasing drug treatment. These findings establish a key physiological role for E2-ERα signaling in promoting OSC differentiation as a potential mechanism to maintain adequate numbers of ovarian follicles during reproductive life.

A nanoscale, multi-parametric flow cytometry-based platform to study mitochondrial heterogeneity and mitochondrial DNA dynamics.

Mitochondria are well-characterized regarding their function in both energy production and regulation of cell death; however, the heterogeneity that exists within mitochondrial populations is poorly understood. Typically analyzed as pooled samples comprised of millions of individual mitochondria, there is little information regarding potentially different functionality across subpopulations of mitochondria. Herein we present a new methodology to analyze mitochondria as individual components of a complex and heterogeneous network, using a nanoscale and multi-parametric flow cytometry-based platform. We validate the platform using multiple downstream assays, including electron microscopy, ATP generation, quantitative mass-spectrometry proteomic profiling, and mtDNA analysis at the level of single organelles. These strategies allow robust analysis and isolation of mitochondrial subpopulations to more broadly elucidate the underlying complexities of mitochondria as these organelles function collectively within a cell.

Extracellular matrix signaling activates differentiation of adult ovary-derived oogonial stem cells in a species-specific manner.

OBJECTIVE: To test if ovarian microenvironmental cues affect oogonial stem cell (OSC) function in a species-specific manner. DESIGN: Animal and human study. SETTING: Research laboratory. PATIENT(S)/ANIMAL(S): Human ovarian cells obtained from cryopreserved ovarian cortical tissue of reproductive-age women, and ovarian cells and tissues from female C57BL/6 mice. INTERVENTION(S): Mouse ovarian tissue, mouse OSCs (mOSCs) and human OSCs (hOSCs) were analyzed for extracellular matrix (ECM) protein expression, and OSCs isolated from adult mouse and human ovaries were cultured in the absence or presence of ECM proteins without or with an integrin signaling inhibitor. MAIN OUTCOME MEASURE(S): Gene expression and in vitro derived (IVD) oocyte formation. RESULT(S): Culture of mOSCs on a collagen-based ECM significantly elevated the rate of differentiation of the cells into IVD oocytes. Mouse OSCs expressed many integrins, including Arg-Gly-Asp (RGD)-binding subunits, and ECM-mediated increases in mOSC differentiation were blocked by addition of integrin-antagonizing RGD peptides. In comparison, hOSCs expressed a different pattern of integrin subunits compared with mOSCs, and hOSCs were unresponsive to a collagen-based ECM; however, hOSCs exhibited increased differentiation into IVD oocytes when cultured on laminin. CONCLUSION(S): These data, along with in silico analysis of ECM protein profiles in human ovaries, indicate that ovarian ECM-based niche components function in a species-specific manner to control OSC differentiation.

Mitochondrial membrane depolarization enhances TRAIL-induced cell death in adult human granulosa tumor cells, KGN, through inhibition of BIRC5.

BACKGROUND: Cellular metabolic changes that accompany malignant transformation have been heralded as hallmark features of cancer. However, metabolic signatures between neoplasms can be unique, allowing for distinctions in malignancy, invasion and chemoresistance between cancer types and subtypes. Mitochondria are central metabolic mediators, as cellular bioenergetics veers from oxidative phosphorylation to glycolysis. Herein, we evaluate the role of mitochondria in maintenance of cellular metabolism, proliferation, and survival in the adult granulosa tumor cell line, KGN, as well as three epithelial ovarian cancer cell lines to determine distinctions in specific features. RESULTS: Notably, KGN cells were susceptible to TRAIL- and cisplatin-induced death following pretreatment with the metabolic inhibitor FCCP, but not oligomycin A. Collapse of mitochondrial membrane potential was found concomitant with cell death via apoptosis, independent from extrinsic canonical apoptotic routes. Rather, treatment with FCCP resulted in elevated cytochrome c release from mitochondria and decreased responsiveness to BIRC5. Following knockdown of BIRC5, mitochondrial membrane depolarization further sensitized KGN cells to induction of apoptosis via TRAIL. CONCLUSIONS: These results indicate an essential role, distinct from metabolism, for mitochondrial membrane potential in KGN cells to sense and respond to external mediators of apoptotic induction.

A method for freeze-fracture and scanning electron microscopy of isolated mitochondria.

Electron microscopy as a methodology for the study of mitochondria based on morphological features is a standard technique that has experienced little evolution over the course of several decades. This technology has identified heterogeneity of mitochondria populations across both whole tissues, as well between individual cells, using primarily ultrathin sections for transmission electron microscopy (TEM). However, this technique constrains the evaluation of a sample to a single two-dimensional plane. To overcome this limitation, scanning electron microscopy (SEM) has been successfully utilized to observe three-dimensional mitochondria structures within the complex microenvironment containing total cellular components. In response to these dual technical caveats of existing electron microscopy protocols, we developed a methodology to evaluate the three-dimensional ultrastructure of isolated mitochondria, utilizing a freeze-fracture step and rigorous preservation of sample morphology. This protocol allows for a more high-throughput analysis of mitochondria populations from a specimen of interest, as the sample has been previously purified, as well as a finer resolution of complex intra-mitochondrial structures, using the depth of field created by SEM. •Protocol designed for SEM of isolated mitochondria samples.•SEM visualizes mitochondria ultrastructure in 3-D.•Freeze-fracture creates cross-sectional plane for view of interior organelle structures.

New insights on mitochondrial heterogeneity observed in prepared mitochondrial samples following a method for freeze-fracture and scanning electron microscopy.

Mitochondria are dynamic intracellular organelles with diverse roles in tissue- and cell type-specific processes, extending beyond bioenergetics. In keeping with this array of functions, mitochondria are described as heterogeneous both between and within tissue types based on multiple parameters, including a broad spectrum of morphological features, and new research points toward a need for the evaluation of mitochondria as isolated organelles. Although transmission electron microscopy (TEM) is commonly used for the evaluation of mitochondria in tissues and renders mitochondrial structures in ultra-thin sections in two-dimensions, additional information regarding complex features within these organelles can be ascertained using scanning electron microscopy (SEM), which allows for analysis of phenotypic differences in three-dimensions. One challenge in producing mitochondrial images for evaluation of ultrastructure using SEM has been the ability to reliably visualize important intramitochondrial features, the inner membrane and cristae structures, on a large-scale (e.g. multiple mitochondria) within a sample preparation, as mitochondria are enclosed within a double membrane. This can be overcome using a 'freeze-fracture' technique in which mitochondrial preparations are snap-frozen followed by application of intense pressure to break open the organelles, revealing the intact components within. Previously published reports using freeze-fracture strategies for mitochondrial SEM have demonstrated feasibility in whole tissue specimens, but a detailed methodology for SEM analysis on isolated mitochondrial fractions has not been reported. By combining previously reported tissue freeze-fracture strategies, along with utilizing the depth of field created by SEM, herein we present a complete method reliant on the freeze-fracture of mitochondrial fractions prepared by differential centrifugation to produce a comprehensive and direct evaluation of three-dimensional mitochondrial ultrastructure by SEM. Image analysis of internal mitochondrial features demonstrates heterogeneity in mitochondrial ultrastructure from a single sample preparation.