Search

The ovary is one of the first organs to show overt signs of aging in the human body, and ovarian aging is associated with a loss of gamete quality and quantity. The age-dependent decline in ovarian function contributes to infertility and an altered endocrine milieu, which has ramifications for overall health. The aging ovarian microenvironment becomes fibro-inflammatory and stiff with age, and this has implications for ovarian physiology and pathology, including follicle growth, gamete quality, ovulation dynamics, and ovarian cancer. Thus, developing a non-invasive tool to measure and monitor the stiffness of the human ovary would represent a major advance for female reproductive health and longevity. Shear wave elastography is a quantitative ultrasound imaging method for evaluation of soft tissue stiffness. Shear wave elastography has been used clinically in assessment of liver fibrosis and characterization of tendinopathies and various neoplasms in thyroid, breast, prostate, and lymph nodes as a non-invasive diagnostic and prognostic tool. In this study, we review the underlying principles of shear wave elastography and its current clinical uses outside the reproductive tract as well as its successful application of shear wave elastography to reproductive tissues, including the uterus and cervix. We also describe an emerging use of this technology in evaluation of human ovarian stiffness via transvaginal ultrasound. Establishing ovarian stiffness as a clinical biomarker of ovarian aging may have implications for predicting the ovarian reserve and outcomes of Assisted Reproductive Technologies as well as for the assessment of the efficacy of emerging therapeutics to extend reproductive longevity. This parameter may also have broad relevance in other conditions where ovarian stiffness and fibrosis may be implicated, such as polycystic ovarian syndrome, late off target effects of chemotherapy and radiation, premature ovarian insufficiency, conditions of differences of sexual development, and ovarian cancer.Summary sentence: Shear Wave Elastography is a non-invasive technique to study human tissue stiffness, and here we review its clinical applications and implications for reproductive health and disease.

The female reproductive system is one of the first to age in humans, resulting in infertility and endocrine disruptions. The aging ovary assumes a fibro-inflammatory milieu which negatively impacts gamete quantity and quality as well as ovulation. Here, we tested whether the systemic delivery of anti-inflammatory (Etanercept) or anti-fibrotic (Pirfenidone) drugs attenuates ovarian aging in mice. We first evaluated the ability of these drugs to decrease the expression of fibro-inflammatory genes in primary ovarian stromal cells treated with a pro-fibrotic or a pro-inflammatory stimulus. Whereas Etanercept did not block Tnfexpression in ovarian stromal cells, Pirfenidone significantly reduced Col1a1expression. We then tested Pirfenidone in vivo where the drug was delivered systemically via mini-osmotic pumps for 6 weeks. Pirfenidone mitigated the age-dependent increase in ovarian fibrosis without impacting overall health parameters. Ovarian function was improved in Pirfenidone-treated mice as evidenced by increased follicle and corpora lutea number, AMH levels, and improved estrous cyclicity. Transcriptomic analysis revealed that Pirfenidone treatment resulted in an upregulation of reproductive function-related genes at 8.5 months and a downregulation of inflammatory genes at 12 months of age. These findings demonstrate that reducing the fibroinflammatory ovarian microenvironment improves ovarian function, thereby supporting modulating the ovarian environment as a therapeutic avenue to extend reproductive longevity.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Ovulation is an integral part of women’s menstrual cycle and fertility. Understanding the mechanisms of ovulation has broad implications for the treatment of anovulatory diseases and the development of novel contraceptives. Now, few studies have developed effective models that both faithfully recapitulate the hallmarks of ovulation and possess scalability. We established a three-dimensional encapsulated in vitrofollicle growth (eIVFG) system that recapitulates folliculogenesis and produces follicles that undergo ovulation in a controlled manner. Here, we determined whether ex vivoovulation preserves molecular signatures of ovulation and demonstrated its use in discovering novel ovulatory pathways and nonhormonal contraceptive candidates through a high-throughput ovulation screening. Mature murine follicles from eIVFG were induced to ovulate ex vivousing human chorionic gonadotropin and collected at 0, 1, 4, and 8 hours post-induction. Phenotypic analyses confirmed key ovulatory events, including cumulus expansion, oocyte maturation, follicle rupture, and luteinization. Single-follicle RNA-sequencing analysis revealed the preservation of ovulatory genes and dynamic transcriptomic profiles and signaling. Soft clustering identified distinct gene expression patterns and new pathways that may critically regulate ovulation. We further used this ex vivoovulation system to screen 21 compounds targeting established and newly identified ovulatory pathways. We discovered that proprotein convertases activate gelatinases to sustain follicle rupture and do not regulate luteinization and progesterone secretion. Together, our ex vivoovulation system preserves molecular signatures of ovulation, presenting a new powerful tool for studying ovulation and anovulatory diseases as well as for establishing a high-throughput ovulation screening to identify novel nonhormonal contraceptives for women.Ex vivoovulation enables novel contraception discovery for women.Graphical Abstract

Folliculogenesis is a tightly coordinated process essential for generating a fertilization-competent gamete while also producing gonadal hormones that sustain endocrine function. In vitro follicle growth systems have been critical to our understanding of key events in folliculogenesis, such as gonadotropin-independent and dependent growth, steroid hormone production, and oocyte growth and maturation (cytoplasmic and meiotic). Although there are several successful follicle culture strategies, the following protocol details an encapsulated in vitro follicle growth (eIVFG) system for use with mouse ovarian follicles. Encapsulated IVFG is performed with alginate hydrogels, which are biologically inert, maintains cell-to-cell interactions between granulosa cells and the oocyte, and preserves follicle architecture as found in the ovary. The system supports follicle growth, development, and differentiation from the early primary follicle to the antral follicle stage. Moreover, post-folliculogenesis events including meiotic maturation, ovulation, and luteinization are also supported. Importantly, the culture of secondary follicles has successfully resulted in viable pups after blastocyst transfer. This alginate-based eIVFG system is versatile and has broad applications as a tool for interrogating the fundamental biology of the ovarian follicle in a controlled manner, a screening platform for toxicity and bioactivity, and a potential fertility preservation method for endangered species as well as humans.This is a detailed protocol for culture of mouse ovarian follicles utilizing an alginate hydrogel-based encapsulated in vitro growth system.

The ovary is the first organ to age in humans with functional decline evident already in women in their early 30s. Reproductive aging is characterized by a decrease in oocyte quantity and quality, which is associated with an increase in infertility, spontaneous abortions, and birth defects. Reproductive aging also has implications for overall health due to decreased endocrinological output. Understanding the mechanisms underlying reproductive aging has significant societal implications as women globally are delaying childbearing and medical interventions have greatly increased the interval between menopause and total lifespan. Age-related changes inherent to the female gamete are well-characterized and include defects in chromosome and mitochondria structure, function, and regulation. More recently, it has been appreciated that the extra-follicular ovarian environment may have important direct or indirect impacts on the developing gamete, and age-dependent changes include increased fibrosis, inflammation, stiffness, and oxidative damage. The cumulus cells and follicular fluid that directly surround the oocyte during its final growth phase within the antral follicle represent additional critical local microenvironments. Here we systematically review the literature and evaluate the studies that investigated the age-related changes in cumulus cells and follicular fluid. Our findings demonstrate unique genetic, epigenetic, transcriptomic, and proteomic changes with associated metabolomic alterations, redox status imbalance, and increased apoptosis in the local oocyte microenvironment. We propose a model of how these changes interact, which may explain the rapid decline in gamete quality with age. We also review the limitations of published studies and highlight future research frontiers.Aging is associated with genetic, epigenetic, transcriptomic, proteomic, metabolomic, and redox status changes in the local oocyte microenvironment: cumulus cells and follicular fluid.

Abstract ID 101237

Abstract ID 97333

Oocytes are highly radiosensitive, so agents that prevent radiation-induced ovarian follicle destruction are important fertility preservation strategies. A previous study in rhesus macaques demonstrated that ovarian treatment with antiapoptotic agents, sphingosine-1-phosphate (S1P) and FTY720, its long-acting mimetic, preserved follicles following a single dose of 15 Gy X-ray radiation, and live offspring were obtained from FTY720-treated animals. However, it is unknown whether these antiapoptotic agents also protected the ovarian stroma from late effects of radiation, including vascular damage and fibrosis. Using ovarian histological sections from this study, we evaluated the vasculature and extracellular matrix in the following cohorts: vehicle + sham irradiation, vehicle + irradiation (OXI), S1P + irradiation (S1P), and FTY720 + irradiation (FTY720). One ovary from each animal was harvested prior to radiation whereas the contralateral ovary was harvested 10 months post-treatment. We assessed vasculature by immunohistochemistry with a PECAM1 antibody, hyaluronan by a hyaluronan binding protein assay, and collagen by picrosirius red and Masson’s trichrome staining. Disorganized vessels were observed in the medulla in the OXI and S1P cohorts relative to the sham, but the vasculature in the FTY720 cohort appeared intact, which may partially explain fertoprotection. There were no differences in the hyaluronan matrix among the cohorts, but there was thickening of the tunica albuginea and fibrosis in the OXI cohort relative to the sham, which was not mitigated by either S1P or FTY720 treatment. Thus, the fertoprotective properties of S1P and FTY720 may be limited given their inability to protect the ovarian stroma against the late effects of radiation-induced fibrosis.Although the fertoprotective agents S1P and FTY720 preserve the ovarian reserve, they do not protect the ovarian stroma from radiation-induced fibrosis in the nonhuman primate, underscoring the need to prevent damage in the ovarian microenvironment.

Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980-2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

Cancer survivorship has increased considerably, but common cancer treatments may threaten female reproductive health and fertility. In females, standard fertility preservation techniques include egg and embryo banking and ovarian tissue cryopreservation, but these methods are not suitable for all individuals. Emerging fertility preservation technologies include in vitrofollicle growth and ovarian bioprosthetics. Although these platforms hold tremendous promise, they remain in the preclinical phase likely because of our inability to adequately phenocopy the complexity of the in vivoovarian environment. The goal of this study was to use an established research archive of fixed human ovarian tissue established through the Oncofertility Consortium to better understand the dynamics and milieu of growing follicles within the human ovary. We performed a histological analysis of the immediate surroundings of primary and secondary stage follicles. We evaluated oocyte and follicle diameters of these growing follicles, analyzed their growth trajectories, and mapped their precise relationships to other stage follicles within a defined area. We also stratified our findings according to participant age and previous treatment history. Our results serve as in vivobenchmarks for follicles grown in vitroand provide insight into how follicles should be seeded spatially within bioprosthetic ovaries, potentially improving the efficacy and clinical translation of these emerging technologies.Impact statementLife-preserving cancer treatments have greatly increased survivorship. However, treatments often have off-target health consequences that threaten female reproductive health and fertility. Although several standard fertility preservation options exist, there is a constant need to explore and expand options for all populations. In vitrofollicle growth and ovarian bioprosthetics are new experimental procedures, which are currently limited to proof of concept. In this study, we analyzed human ovarian tissue from a deidentified biospecimen repository to characterize the growing follicle landscape with the ultimate goal of informing bioengineering practices. This spatial analysis pinpoints the geometry of growing follicles within the human ovary and provides a framework for paralleling this environment in ex vivoplatforms.

Radiation induces ovarian damage and accelerates reproductive aging. Inbred mouse strains exhibit differential sensitivity to lethality induced by total body irradiation (TBI), with the BALB/cAnNCrl (BALB/c) strain being more sensitive than the 129S2/SvPasCrl (129) strain. However, whether TBI-induced ovarian damage follows a similar pattern of strain sensitivity is unknown. To examine this possibility, female BALB/c and 129 mice were exposed to a single dose of 1 Gy (cesium-137 γ) TBI at 5 weeks of age, and ovarian tissue was harvested for histological and gene expression analyses 2 weeks post exposure. Sham-treated mice served as controls. 1 Gy radiation nearly eradicated the primordial follicles and dramatically decreased the primary follicles in both strains. In contrast, larger growing follicles were less affected in the 129 relative to BALB/c strain. Although this TBI paradigm did not induce detectable ovarian fibrosis in either of the strains, we did observe strain-dependent changes in osteopontin (Spp1) expression, a gene involved in wound healing, inflammation, and fibrosis. Ovaries from BALB/c mice exhibited higher baseline Spp1 expression that underwent a significant decrease in response to radiation relative to ovaries from the 129 strain. A correspondingly greater change in the ovarian matrix, as evidenced by reduced ovarian hyaluronan content, was also observed following TBI in BALB/c mice relative to 129 mice. These early changes in the ovary may predispose BALB/c mice to more pronounced late effects of TBI. Taken together, our results demonstrate that aspects of ovarian damage mirror other organ systems with respect to overall strain-dependent radiation sensitivity.

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix. The spatial-temporal localization of HA can be visualized in situ using biotinylated HA binding proteins (HABPs). This assay is sensitive to fixation conditions, and there are currently no best practices for HA detection. Thus, the goal of this study was to optimize fixation conditions for visualizing HA in the ovary, kidney, and liver through analysis of six commonly used fixatives for HA detection: Bouin’s Solution, Carnoy’s Solution, Ethanol-Formalin-Glacial Acetic Acid (EFG), Histochoice, Modified Davidson’s Solution, and 10% Neutral Buffered Formalin. Organs were harvested from CB6F1 mice and fixed with one of the identified fixatives. Fixed organs were sectioned, and the HABP assay was performed on sections in parallel. Hematoxylin and eosin staining was also performed to visualize tissue architecture. HABP signal localization and intensity varied between fixatives. EFG and Carnoy’s Solution best preserved the HA signal intensity in the ovary and liver, showing HA localization in various sub-organ structures. In the kidney, only Modified Davidson’s Solution was less than optimal. Our findings demonstrate that fixation can alter the ability to detect HA in tissue macro- and microstructures, as well as localization in a tissue-specific manner, in situ.

Immunohistochemistry (IHC) is a robust scientific tool whereby cellular components are visualized within a tissue, and this method has been and continues to be a mainstay for many reproductive biologists. IHC is highly informative if performed and interpreted correctly, but studies have shown that the general use and reporting of appropriate controls in IHC experiments is low. This omission of the scientific method can result in data that lack rigor and reproducibility. In this editorial, we highlight key concepts in IHC controls and describe an opportunity for our field to partner with the Histochemical Society to adopt their IHC guidelines broadly as researchers, authors, ad hoc reviewers, editorial board members, and editors-in-chief. Such cross-professional society interactions will ensure that we produce the highest quality data as new technologies emerge that still rely upon the foundations of classic histological and immunohistochemical principles.Immunohistochemistry is a critical tool in the reproductive sciences, and we suggest that our field adopts the guidelines set forth by the Histochemical Society to maximize the rigor and reproducibility of our data.

Advanced maternal age is unequivocally associated with increased aneuploidy in human eggs and infertility, but the molecular basis for this phenomenon is unknown. An age-dependent deterioration of the spindle assembly checkpoint (SAC) has been proposed as a probable cause of aneuploidy. Accurate chromosome segregation depends on correct chromosome attachment to spindle microtubules, and the SAC provides time for this process by delaying anaphase onset until all chromosomes are stably attached. If SAC function decreases with age, oocytes from reproductively old mice would enter anaphase of meiosis I (AI) prematurely, leading to chromosome segregation errors and aneuploid eggs. Although intuitively appealing, this hypothesis is largely untested. We used a natural reproductive aging mouse model to determine if a defective SAC is the primary cause of aneuploidy in eggs. We tracked the progress of individual oocytes from young and old mice through meiosis I by time-lapse microscopy and counted chromosomes in the resulting eggs. This data set allowed us to correlate the timing of AI onset with aneuploidy in individual oocytes. We found that oocytes from old mice do not enter AI prematurely compared to young counterparts despite a 4-fold increase in the incidence of aneuploidy. Moreover, we did not observe a correlation between the timing of AI onset and aneuploidy in individual oocytes. When SAC function was challenged with a low concentration of the spindle toxin nocodazole, oocytes from both young and old mice arrested at meiosis I, which is indicative of a functional checkpoint. These findings indicate that a defective SAC is unlikely the primary cause of aneuploidy associated with maternal age.

In mammalian oocytes, cyclic AMP‐dependent protein kinase (PKA) is responsible for maintaining meiotic arrest. We examined the role of the predominant regulatory subunit, RIα in regulating PKA activity during mouse oocyte maturation by knocking down the protein levels using an RNA interference approach. In oocytes in which RIα protein was reduced to non‐detectable levels, compensatory decreases were also observed in the RIIα and catalytic (Cα) subunit levels. These oocytes resumed meiosis, despite culture under conditions that maintain elevated intracellular cAMP levels, suggesting that the remaining Cα was not sufficient to maintain meiotic arrest. The resulting eggs, however, displayed meiotic spindle abnormalities and abnormal cleavage planes leading to extrusion of large polar bodies. These results demonstrate that RIα is required for regulating PKA activity in maturing oocytes and that compensatory upregulation of RII does not occur. Furthermore, we implicate PKA as a modulator of spindle morphology and function during meiosis. Developmental Dynamics 235:2961–2968, 2006. © 2006 Wiley‐Liss, Inc.

(Abstracted from Proc Natl Acad Sci U S A2017;114:3186–3191)The ovaries have a fixed pool of immature (primordial) follicles at birth known as the ovarian reserve. Activation or loss of follicles in this reserve causes an irreversible decline in reproductive function that culminates in menopause.

(Abstracted from Aging Cell2017;16:1381–1393)The growing oocyte is one of the most translationally active cells in the body and must accumulate high-quality, maternally derived proteins to support subsequent embryo development. Women of advanced reproductive age are at increased risk of infertility, miscarriages, and birth defects because of a marked decline in oocyte quality.

Advanced reproductive age is unequivocally associated with increased aneuploidy in human oocytes, which contributes to infertility, miscarriages, and birth defects. The frequency of meiotic chromosome segregation errors in oocytes derived from reproductively aged mice appears to be similar to that observed in humans, but a limitation of this important model system is our inability to accurately identify chromosome-specific aneuploidy. Here we report the validation and application of a new low-pass whole-genome sequencing approach to comprehensively screen chromosome aneuploidy in individual mouse oocytes and blastocysts. First, we validated this approach by using single mouse embryonic fibroblasts engineered to have stable trisomy 16. We further validated this method by identifying reciprocal chromosome segregation errors in the products of meiosis I (gamete and polar body) in oocytes from reproductively aged mice. Finally, we applied this technology to investigate the incidence of aneuploidy in blastocysts derived from in vitro- and in vivo-matured oocytes in both young and reproductively aged mice. Using this next generation sequencing approach, we quantitatively assessed meiotic and mitotic segregation errors at the single chromosome level, distinguished between errors due to premature separation of sister chromatids and classical nondisjunction of homologous chromosomes, and quantified mitochondrial DNA (mtDNA) segregation in individual cells. This whole-genome sequencing technique, therefore, greatly improves the utility of the mouse model system for the study of aneuploidy and is a powerful quantitative tool with which to examine the molecular underpinnings of mammalian gamete and early embryo chromosome segregation in the context of reproductive aging and beyond.

During embryonic development, mouse female germ cells enter meiosis in an anterior-to-posterior wave believed to be driven by retinoic acid. It has been proposed that ovarian follicle formation and activation follow the same general wave of meiotic progression; however, the precise anatomic specification of these processes has not been delineated. Here, we created a mouse line using Mvh, Gdf9,and Zp3promoters to drive distinct temporal expression of three fluorescent proteins in the oocytes and to identify where the first follicle cohort develops. The fluorescent profile revealed that the first growing follicles consistently appeared in a specific region of the ovary, the anterior-dorsal region, which led us to analyze if meiotic onset occurred earlier in the dorsal ovarian region. Surprisingly, in addition to the anterior-to-posterior wave, we observed an early meiotic entry in the ventral region of the ovary. This additional anatomic stratification of meiosis contrasts with the localization of the initial follicle formation and activation in the dorsal region of the ovary. Therefore, our study suggests that the specification of cortical and medullar areas in the ventral and dorsal regions on the ovary, rather than the onset of meiosis, impacts where the first follicle activation event occurs.

Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)—a maternal effect gene that is part of the subcortical maternal complex—as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation when added prior to GVBD but not after. Taken together, these results highlight the importance of oocyte-intrinsic PKA in regulating meiotic progression after the prophase I arrest and offer new insights into downstream targets of its activity.

Aneuploidy in human eggs increases with maternal age and can result in infertility, miscarriages, and birth defects. Recent findings in mouse suggest that deteriorating chromosome cohesion is a primary mechanism of maternal age-associated aneuploidy. Although cohesin proteins are conserved between humans and mouse, it is not known if a functional deterioration of these components also occurs in humans. Such studies have been hampered largely by the difficulty in obtaining oocytes from reproductively young and old women. Here we collected oocyte-cumulus-complexes directly from ovarian tissue from 15 subjects ranging in age from 16.4 to 49.7 years old. Ovarian tissue was being removed either for fertility preservation or for other medical indications. Oocytes within these complexes were meiotically-competent as 25.8% ± 5.9% reached metaphase of meiosis II (MII) following in vitro maturation. The MII eggs derived from in vitro maturation had characteristic morphology with a small first polar body, a bipolar spindle asymmetrically positioned in the cortex, and condensed chromosomes tightly aligned on the metaphase plate. A subset of these MII eggs was used to assess how chromosome cohesion is affected by maternal age. As a readout of chromosome cohesion, we measured the distance between kinetochores, or the inter-kinetochore distance, of sister chromatids using an in situ chromosome spreading technique. The average inter-kinetochore distance increased significantly from 0.82 μm ± 0.03 μm in the youngest subject (16.4 years) to 1.1 μm ± 0.03 μm in the oldest subject (37.3 years) (P<0.001). Moreover, we observed chromosome segregation errors in MII eggs from the two oldest subjects. Interestingly, the majority of these errors involved the premature separation of sister chromatids, a clear manifestation of a complete loss of cohesion. Taken together, these results demonstrate that a maternal-age associated deterioration of chromosome cohesion occurs in humans. In addition, this study constitutes the first direct examination of MII eggs from cancer patients and provides a baseline of the quality that can be expected from such retrievals. Grants from the National Institutes of Health supported this work (RL1HD058295, UL1DE19587, PL1CA133835, HD058730).

Three-dimensional in vitro follicle culture is an emerging technology that may be a viable fertility preservation option for patients who have cryopreserved ovarian tissue but are not eligible for ovarian transplant. In this in vitro culture system, immature ovarian follicles are grown in an alginate hydrogel that supports follicular architecture through the antral stages. Murine follicles, grown from the two layer secondary stage with this method, produce fertilization-competent female gametes capable of giving rise to live offspring. A significant obstacle to culturing smaller follicles in vitro is the dissociation that occurs between the somatic cells and the oocyte. We have recently demonstrated that co-culturing primary follicles with macrophage- and theca-enriched stromal cells supports follicle survival and growth. In this study, we tested the hypothesis that follicles themselves can exert a beneficial co-culture effect. We isolated follicles ranging in diameter from 85 μm to 115 μm from pre-pubertal mouse ovaries and encapsulated them in 0.5% alginate hydrogels either individually (x1) or in groups (x5 or x10) and cultured them in vitro for up to 12 days. Follicle survival and growth were tracked, and follicle integrity was determined by analysis of transzonal projections between the oocyte and the somatic cells. We found that follicle survival decreases dramatically on D4 of culture when primary follicles are encapsulated and cultured individually. In fact, less than 25% of the smallest size follicles examined (85-100 μm) survive past D4 because follicle integrity, defined as the connections between the oocyte and somatic cells, is compromised. In contrast, when size-matched follicles are cultured in groups, survival is enhanced to over 85% on D4 of culture. Co-cultured follicles not only survive, but also can grow to diameters larger than 300 μm in 12 days. This somatic cell proliferation is paralleled by an increase in oocyte diameter. The positive growth and survival effects of follicle co-culture are dependent on the size of the follicles and the number of follicles being co-cultured. Although follicle integrity was lost in a fraction of co-cultured follicles, more than 75% of them re-established somatic cell-oocyte contacts during the culture period. In contrast, less than 10% of follicles cultured individually were able to do so. These results suggest that signals inherent to the follicle, when present at high enough levels, have the ability to promote and maintain somatic cell-oocyte connections during the early stages of in vitro culture. This work was supported by Award Number U54HD041857 from the Eunice Kennedy Shriver National Institute of Child Health & Human Development.(poster)

Aneuploidy arising early in development is the leading genetic cause of birth defects and developmental disabilities in humans. It is now well established that the probability of a trisomic pregnancy (i.e., an embryo with an extra copy of a chromosome) increases dramatically with age: from only 2% for a woman in her 20s to about 35% by age 40. Most trisomies and all monosomies are inviable, and the few that are viable result in severe developmental disabilities (e.g., Down syndrome). Aneuploidies in the embryo usually originate from the egg instead of sperm, and more specifically, from chromosome segregation errors in Meiosis I of oocyte maturation. The frequency of these errors increases with maternal age, which is likely related to the long meiotic arrest that occurs during female meiosis in mammals. Primary oocytes enter meiosis during fetal development and remain arrested until ovulation in the adult, which in humans can be as long as 40 years. Although the phenomenon of age-related aneuploidy is well known, the molecular basis for the chromosome segregation errors in female meiosis is unclear. Here we show that weakened chromosome cohesion is the primary cause of aneuploidy in oocytes in a natural aging mouse model. In oocytes from old mice, we find that sister kinetochores are farther apart at both Metaphase I and II, indicating reduced centromere cohesion. Moreover, levels of the meiotic cohesin protein REC8 are severely reduced on chromosomes in these oocytes. In mice at different ages, REC8 levels gradually decrease while inter-kinetochore distances increase with age (from 3 to 15 month-old mice); yet despite this gradual loss of cohesion, chromosome segregation errors remain low until 12 months and then increase sharply between 12 and 15 months. These results suggest that cohesins are initially loaded in large excess, and errors arise only after cohesin levels drop below ~10% of the starting amount. To test whether cohesion defects lead to the observed aneuploidies, we monitored chromosome segregation dynamics at Anaphase I in live oocytes and assessed ploidy in the resulting Metaphase II eggs. We found that about 90% of age-related aneuploidies are best explained by weakened centromere cohesion. Together, these results demonstrate that the maternal age-associated increase in aneuploidy is primarily due to a failure to effectively replace cohesin proteins that are lost from chromosomes during aging. Because of the prolonged time interval in female meiosis, this may create a selective pressure to load an excess of cohesins in pre-meiosis S-phase to protect oocytes against impending age-related meiotic chromosome segregation errors, and thus enhance a mother's reproductive fitness by extending her period of fertility.(platform)

Advanced maternal age is unequivocally associated with increased aneuploidy in human eggs and with infertility, but the molecular basis for this phenomenon is unknown. An age-dependent deterioration of the spindle assembly checkpoint (SAC) has been proposed as a probable cause of aneuploidy. Accurate chromosome segregation depends on correct chromosome attachments to spindle microtubules, and the SAC provides time for this process by delaying anaphase onset until all chromosomes are stably attached. If SAC function decreases with age, oocytes from reproductively old mice should enter Anaphase I (AnaI) prematurely, leading to chromosome segregation errors and aneuploid eggs. Although intuitively appealing, this hypothesis is largely untested. We used a natural reproductive aging mouse model to determine if a defective SAC is a leading cause of aneuploidy in eggs. We tracked the progress of individual oocytes from young and old mice through meiosis I (MI) by timelapse microscopy and counted chromosomes in the resulting eggs. This data set allowed us to correlate the timing of AnaI onset with aneuploidy in individual oocytes. We found that oocytes from old mice do not enter AnaI prematurely compared to young counterparts despite a 24% higher incidence of aneuploidy. Moreover, we did not observe a correlation between the timing of AnaI onset and aneuploidy. When SAC function was challenged with low doses of the spindle toxin, Nocodazole, oocytes from both young and old mice arrested at MI, indicative of a functional checkpoint. These data suggest that a defective SAC is unlikely the primary cause of aneuploidy associated with maternal age. This research was supported by the Bingham Trust (MAL and RMS) and the NIH (T32 HD 007305 22, FED).(platform)

Sperm-egg interaction at fertilization results in a rise in the intracellular concentration of Ca2+that triggers a series of events known as egg activation. Several calcium-dependent proteins have been postulated to mediate the different events. One of them is calcium/calmodulin-dependent protein kinase II (CaMKII). In Xenopuseggs, the involvement of CaMKII in metaphase II exit has been recently demonstrated. However, whether the same signaling cascade operates in mice is unknown. Moreover, CaMKII's role in triggering other events of egg activation and the identity of the isoform/s present in mouse eggs are still unclear. Here we show that CaMKIIγ is the only isoform present in mouse eggs. We generated CaMKIIγ-deficient mice and report that they are viable, but CaMKIIγ-/-females are infertile. While CaMKIIγ-/-eggs exhibit a normal pattern of calcium oscillations, they fail to get fertilized or parthenogenetically activated. Instead, they remain arrested at metaphase II with high mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) activities. Maternal mRNA recruitment does not occur in CaMKIIγ-deficient eggs. On the other hand, null eggs are able to mount a post-fertilization block to polyspermy, as revealed by their ability to proteolytically cleave the zona pellucidaprotein ZP2. This demonstrates that while cell cycle resumption and mRNA recruitment are CaMKII-dependent, the block to polyspermy is not. Additionally, we report that the egg activation defects observed in CaMKIIγ-/-eggs can be reversed by introduction of either CaMKIIγ or CaMKIIδ mRNA into mouse oocytes, suggesting that CaMKII's role during egg activation is not isoform-specific.(platform)