Tubulin Complexity in Cancer and Metastasis.

Tubulin plays a fundamental role in cellular function and as the subject for microtubule-active agents in the treatment of ovarian cancer. Microtubule-binding proteins (e.g., tau, MAP1/2/4, EB1, CLIP, TOG, survivin, stathmin) and posttranslational modifications (e.g., tyrosination, deglutamylation, acetylation, glycation, phosphorylation, polyamination) further diversify tubulin functionality and may permit additional opportunities to understand microtubule behavior in disease and to develop microtubule-modifying approaches to combat ovarian cancer. Tubulin-based structures that project from suspended ovarian cancer cells known as microtentacles may contribute to metastatic potential of ovarian cancer cells and could represent an exciting novel therapeutic target.

Microtubule-Targeting Agents: Disruption of the Cellular Cytoskeleton as a Backbone of Ovarian Cancer Therapy.

Microtubules are dynamic polymers composed of α- and ß-tubulin heterodimers. Microtubules are universally conserved among eukaryotes and participate in nearly every cellular process, including intracellular trafficking, replication, polarity, cytoskeletal shape, and motility. Due to their fundamental role in mitosis, they represent a classic target of anti-cancer therapy. Microtubule-stabilizing agents currently constitute a component of the most effective regimens for ovarian cancer therapy in both primary and recurrent settings. Unfortunately, the development of resistance continues to present a therapeutic challenge. An understanding of the underlying mechanisms of resistance to microtubule-active agents may facilitate the development of novel and improved approaches to this disease.

"On-Off" Control for On-Demand H2 Release upon Dimethylamineborane Hydrolysis over Ru0.8Ni0.2/MoS2 Nanohybrids.

In spite of the fact that remarkable developments are achieved in the design and development of novel nanocatalysts for H2 release upon dimethylamineborane hydrolysis, the development of an "on-off" switch for demand-based H2 evolution upon dimethylamineborane hydrolysis is still a matter of supreme importance, however. Herein, we synthesized a string of MoS2 nanosheet-supported RuNi bimetallic nanohybrids (RuxNi1-x/MoS2), by fixation of RuNi nanoparticles at the MoS2 surface, for the H2 evolution upon the hydrolysis of dimethylamineborane at 30 °C. For safely and effectively generating, transporting, and storing H2 gas, the selective "on-off" switch for on-demand H2 evolution upon dimethylamineborane hydrolysis over the Ru0.8Ni0.2/MoS2 nanohybrid has been successfully realized by the Zn2+/EDTA-2Na system. In particular, the H2 evolution is totally switched off by adding Zn(NO3)2. It seems that Zn2+ ions are attached and anchored at the Ru0.8Ni0.2/MoS2 surface, inhibiting their surface-active sites, leading to the termination of H2 evolution. Then, the H2 generation is subsequently reactivated by adding the EDTA-2Na solution because of its excellent coordination ability with Zn2+ ions. This study not only offers a new and efficient RuNi nanocatalyst for dimethylamineborane hydrolysis but also proposes a new method for the demand-based H2 production.

Insulin-like growth factor 2 is produced by antral follicles and promotes preantral follicle development in macaques†.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Matrix-free 3D culture supports human follicular development from the unilaminar to the antral stage in vitro yielding morphologically normal metaphase II oocytes.

STUDY QUESTION: Can group culture with stage-specific anti-Müllerian hormone (AMH) modulation support human follicular development and oocyte maturation in vitro? SUMMARY ANSWER: In the presence of FSH, AMH supplementation at the secondary-to-early antral stage followed by AMH depletion promotes the coordinated growth and function of human follicles during group culture, thereby yielding mature oocytes. WHAT IS KNOWN ALREADY: Stage-specific AMH modulation promotes in-vitro development of nonhuman primate follicles. The group culture method supports nonhuman primate follicle growth from the primary to antral stage, producing developmentally competent oocytes. STUDY DESIGN, SIZE, DURATION: Ovarian tissue samples were collected from 19 patients of reproductive age (22-47 years old having menstrual cycles) who underwent oophorectomy or hysterectomy for clinical purposes. Tissue pieces were cultured in a matrix-free system for 3 weeks followed by isolation of follicles for the subsequent 6-week individual or group culture. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pieces of ovarian cortical tissue were cultured to support primordial follicle activation and early-stage follicle growth. Secondary follicles isolated from cultured tissue were then randomly assigned to two groups for individual culture: control and AMH modulation, i.e., recombinant human AMH protein supplementation during the secondary-to-early antral stage followed by the addition of neutralizing anti-human AMH antibody. Secondary follicles were also cultured in groups with the same AMH modulation. Follicle survival, growth, steroid hormone and paracrine factor production, steroidogenic protein expression, as well as oocyte maturation and morphology were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Follicles grew to the secondary stage during 3 weeks of ovarian tissue culture. In-vitro-developed follicles expressed AMH and levels of secreted AMH increased (P < 0.05) in the culture media over time. Secondary follicles isolated from cultured ovarian tissue survived and grew to the antral stage during 6 weeks of individual follicle culture. In-vitro-developed antral follicles produced granulosa and theca cell-derived steroid hormones and paracrine factors, which were detectable in the culture media. Germinal vesicle oocytes obtained from cultured follicles exhibited a perinucleolar chromatin rim configuration. AMH modulation did not alter follicle survival or oocyte maturation relative to those of the control follicles. However, follicle diameters, as well as steroid hormone and paracrine factor production, increased (P < 0.05) in the AMH-modulation group compared with the control group. Secondary follicles isolated from cultured ovarian tissue formed aggregates and grew to the antral stage during 6 weeks of group culture. In-vitro-developed antral follicles expressed steroidogenic enzymes and secreted steroid hormones were detectable in the culture media. Oocytes obtained from cultured follicle aggregates with AMH-modulation progressed to the metaphase II stage after IVM, containing a normal-sized first polar body and meiotic spindle. Oocytes exhibited a typical ultrastructure. LIMITATIONS, REASONS FOR CAUTION: Follicles were obtained from fresh ovarian tissue of adult patients. Oocyte maturation rates were relatively low and oocytes were assessed by morphological evaluation. Owing to the lack of a control group, the beneficial effects of AMH modulation remained undetermined for the group culture in this study. WIDER IMPLICATIONS OF THE FINDINGS: Stage-specific AMH modulation supports human follicular development in the matrix-free group culture, which is consistent with previously reported AMH actions on growing follicles in nonhuman primates. Oocytes generated by in-vitro-developed follicles achieve meiotic maturation with a typical morphology and ultrastructure, which supports in-vitro follicle maturation as a potential approach for fertility preservation in women. STUDY FUNDING/COMPETING INTEREST(S): NICHD R01HD082208 and NIH Office of the Director P51OD011092. The authors have no competing interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Vitamin D in follicular development and oocyte maturation.

Vitamin D (VD) is a secosteroid hormone synthesized predominantly in the skin upon UV light exposure, which can also be obtained from dietary sources. In target cells, the bioactive VD binds to specific VD receptor to regulate downstream transcription of genes that are involved in a wide range of cellular processes. There is an increasing recognition that the proper physiological levels of VD are critical for optimizing reproductive potential in women. The direct VD action in the ovary was first suggested in the 1980s. Since then, research has attempted to determine the role of VD in follicular development and oocyte maturation in animal models and clinical settings. However, data published to date are inconclusive due to the complexity in VD metabolism and the fact that VD actions are pervasive in regulating physiological functions in various systems, including the reproductive, endocrine and nervous systems that control reproduction. This review summaries in vitro, in vivo, and clinical evidence regarding VD metabolism and signaling in the ovary, as well as VD-regulated or VD-associated ovarian follicular development, steroidogenic function, and oocyte maturation. It is suggested that adequate animal models are needed for well-controlled studies to unravel molecular mechanisms of VD action in the ovary. For clinical studies, follicular development and function may be evaluated more effectively in a relatively homogeneous patient population under a well-controlled experimental design. A comprehensive understanding of VD-regulated folliculogenesis and oogenesis will provide critical insight into the impact of VD in female reproductive health.

Rapid actions of anti-Müllerian hormone in regulating synaptic transmission and long-term synaptic plasticity in the hippocampus.

Anti-Müllerian hormone (Amh) is a peptide factor that is known to regulate sexual differentiation and gonadal function in mammals. Although Amh is also suggested to be associated with cognitive development and function in the postnatal brain, little is known about its expression or direct effects on neuronal activities in the hippocampus. Therefore, we assessed Amh and its receptor expression in the hippocampus of male and female mice using PCR, Western blot, and immunofluorescence staining. While Amh-specific receptor expression was comparable between males and females, mRNA and protein levels of Amh were higher in females than those of males. Electrophysiological recordings on acute hippocampal slices showed that exogenous Amh protein addition increased synaptic transmission and long-term synaptic plasticity at the Cornu Ammonis (CA) 3-CA1 synapses. Amh exposure also increased the excitatory postsynaptic potential at CA1 synapses. Our findings support direct and rapid actions of Amh as a paracrine and/or autocrine factor in regulating hippocampal neuronal activities. Data provide functional evidence of Amh-mediated postsynaptic modulation of synaptic transmission and Amh-regulated long-term synaptic plasticity in the hippocampus. These results suggest a potential role of Amh in learning and memory, and a possible cause of the sex differences in cognitive development and function.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector + recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Changes in immune cell distribution and their cytokine/chemokine production during regression of the rhesus macaque corpus luteum.

Our previous flow cytometry results demonstrated a significant increase in neutrophils, macrophages/monocytes, and natural killer (NK) cells in dispersed rhesus monkey corpora lutea (CL) after progesterone (P4) levels had fallen below 0.3 ng/ml for ≥3 days during the natural menstrual cycle. In this study, immunohistochemistry revealed the CD11b+ cells (neutrophils, macrophages/monocytes) present in the CL after luteal P4 synthesis ceased were distributed throughout the tissue. CD16+ cells (presumptive NK cells) were observed mainly near the vasculature in functional CL, until their numbers increased and they became widely distributed in regressing CL. To determine if the immune cells that enter luteal tissue during structural regression are functionally different from those that are present during peak function, CD11b+ or CD16+ populations were enriched from mid-late stage (functional) and regressing (days 1.8 ± 0.3 postmenses) CL using antibody-conjugated magnetic microbeads. Flow cytometry analyses revealed the majority of CD11b+ cells expressed CD14, a protein mainly produced by macrophages/monocytes. The antibody-enriched and depleted fractions were cultured for 24 h, and the media then analyzed for the production of 29 cytokines/chemokines. From the mid-late CL, the CD11b+-enriched fraction produced three cytokines/chemokines, whereas CD16+-enriched cells only produced the chemokine CCL2. However, CD11b +-enriched cells isolated from regressed CL produced eight cytokines/chemokines. The CD16+-enriched cells isolated from regressing CL produced significant levels of only three cytokines. Thus, the CD11b+ cells that appear in the rhesus macaque CL after functional regression produce several cytokines/chemokines that likely play a role in orchestrating structural regression.

Subcutaneous ovarian tissue transplantation in nonhuman primates: duration of endocrine function and normalcy of subsequent offspring as demonstrated by reproductive competence, oocyte production, and telomere length.

PURPOSE: The main purposes of the study were to investigate the endocrine function of ovarian tissue transplanted to heterotopic subcutaneous sites and the reproductive competence and telomere length of a nonhuman primate originating from transplanted tissue. METHODS: Ovarian cortex pieces were transplanted into the original rhesus macaques in the arm subcutaneously, in the abdomen next to muscles, or in the kidney. Serum estradiol (E2) and progesterone (P4) concentrations were measured weekly for up to 8 years following tissue transplantation. A monkey derived from an oocyte in transplanted ovarian tissue entered time-mated breeding and underwent controlled ovarian stimulation. Pregnancy and offspring were evaluated. Telomere lengths and oocytes obtained following controlled ovarian stimulation were assessed. RESULTS: Monkeys with transplants in the arm and abdomen had cyclic E2 of 100 pg/ml, while an animal with arm transplants had E2 of 50 pg/ml. One monkey with transplants in the abdomen and kidney had ovulatory cycles for 3 years. A monkey derived from an oocyte in transplanted tissue conceived and had a normal gestation until intrapartum fetal demise. She conceived again and delivered a healthy offspring at term. Controlled ovarian stimulations of this monkey yielded mature oocytes comparable to controls. Her telomere length was long relative to controls. CONCLUSIONS: Heterotopic ovarian tissue transplants yielded long-term endocrine function in macaques. A monkey derived from an oocyte in transplanted tissue was reproductively competent. Her telomere length did not show epigenetically induced premature cellular aging. Ovarian tissue transplantation to heterotopic sites for fertility preservation should move forward cautiously, yet optimistically.

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2.

Epigenetic control of gene expression is critical for normal fetal development. However, chromatin-related mechanisms that activate bone-specific programs during osteogenesis have remained underexplored. Therefore, we investigated the expression profiles of a large cohort of epigenetic regulators (>300) during osteogenic differentiation of human mesenchymal cells derived from the stromal vascular fraction of adipose tissue (AMSCs). Molecular analyses establish that the polycomb group protein EZH2 (enhancer of zeste homolog 2) is down-regulated during osteoblastic differentiation of AMSCs. Chemical inhibitor and siRNA knockdown studies show that EZH2, a histone methyltransferase that catalyzes trimethylation of histone 3 lysine 27 (H3K27me3), suppresses osteogenic differentiation. Blocking EZH2 activity promotes osteoblast differentiation and suppresses adipogenic differentiation of AMSCs. High throughput RNA sequence (mRNASeq) analysis reveals that EZH2 inhibition stimulates cell cycle inhibitory proteins and enhances the production of extracellular matrix proteins. Conditional genetic loss of Ezh2 in uncommitted mesenchymal cells (Prrx1-Cre) results in multiple defects in skeletal patterning and bone formation, including shortened forelimbs, craniosynostosis, and clinodactyly. Histological analysis and mRNASeq profiling suggest that these effects are attributable to growth plate abnormalities and premature cranial suture closure because of precocious maturation of osteoblasts. We conclude that the epigenetic activity of EZH2 is required for skeletal patterning and development, but EZH2 expression declines during terminal osteoblast differentiation and matrix production.

SWI/SNF-Mediated Lineage Determination in Mesenchymal Stem Cells Confers Resistance to Osteoporosis.

Redirecting the adipogenic potential of bone marrow-derived mesenchymal stem cells to other lineages, particularly osteoblasts, is a key goal in regenerative medicine. Controlling lineage selection through chromatin remodeling complexes such as SWI/SNF, which act coordinately to establish new patterns of gene expression, would be a desirable intervention point, but the requirement for the complex in essentially every lineage pathway has generally precluded selectivity. However, a novel approach now appears possible by targeting the subset of SWI/SNF powered by the alternative ATPase, mammalian brahma (BRM). BRM is not required for development, which has hindered understanding of its contributions, but knockdown genetics here, designed to explore the hypothesis that BRM-SWI/SNF has different regulatory roles in different mesenchymal stem cell lineages, shows that depleting BRM from mesenchymal stem cells has a dramatic effect on the balance of lineage selection between osteoblasts and adipocytes. BRM depletion enhances the proportion of cells expressing markers of osteoblast precursors at the expense of cells able to differentiate along the adipocyte lineage. This effect is evident in primary bone marrow stromal cells as well as in established cell culture models. The altered precursor balance has major physiological significance, which becomes apparent as protection against age-related osteoporosis and as reduced bone marrow adiposity in adult BRM-null mice.

Anti-Müllerian hormone is produced heterogeneously in primate preantral follicles and is a potential biomarker for follicle growth and oocyte maturation in vitro.

PURPOSE: The main goals of this study were to investigate the expression of anti-Müllerian hormone (AMH) and its receptor (AMHR2) during follicular development in primates, and to evaluate the potential of AMH as a biomarker for follicle growth and oocyte maturation in vitro. METHODS: The mRNA and protein expression of AMH and AMHR2 were determined using isolated follicles and ovarian sections from rhesus macaques (n = 4) by real-time PCR and immunohistochemistry, respectively. Isolated secondary follicles were cultured individually. Follicle growth and media AMH concentrations were assessed by ELISA. The mRNA expression profiles, obtained from RNA sequencing, of in vitro- and in vivo-developed antral follicles were compared. Secondary follicles from additional animals (n = 35) were cultured. Follicle growth, oocyte maturation, and media AMH concentrations were evaluated for forecasting follicular development in vitro by AMH levels. RESULTS: AMH immunostaining was heterogeneous in the population of preantral follicles that were also stained for AMHR2. The mRNA expression profiles were comparable between in vivo- and in vitro-developed follicles. AMH levels produced by growing follicles were higher than those of nongrowing follicles in culture. With a cutoff value of 1.40 ng/ml, 85 % of nongrowing follicles could be identified while eliminating only 5 % of growing follicles. Growing follicles that generated metaphase II-stage oocytes secreted greater amounts of AMH than did those yielding immature germinal vesicle-stage oocytes. CONCLUSIONS: AMH, co-expressed with AMHR2, was produced heterogeneously by preantral follicles in macaques with levels correlated positively with follicle growth and oocyte maturation. AMH may serve as a biomarker for primate follicular development in vitro.

Dynamics of Immune Cell Types Within the Macaque Corpus Luteum During the Menstrual Cycle: Role of Progesterone.

The goal of the current study was to characterize the immune cell types within the primate corpus luteum (CL). Luteal tissue was collected from rhesus females at discrete intervals during the luteal phase of the natural menstrual cycle. Dispersed cells were incubated with fluorescently labeled antibodies specific for the immune cell surface proteins CD11b (neutrophils and monocytes/macrophages), CD14 (monocytes/macrophages), CD16 (natural killer [NK] cells), CD20 (B-lymphocytes), and CD3epsilon (T-lymphocytes) for analysis by flow cytometry. Numbers of CD11b-positive (CD11b(+)) and CD14(+) cells increased significantly 3 to 4 days after serum progesterone (P4) concentrations declined below 0.3 ng/ml. CD16(+) cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases and were 3-fold increased 3 to 4 days after serum P4 decreased to baseline levels. CD3epsilon(+) cells tended to increase 3 to 4 days after P4 decline. To determine whether immune cells were upregulated by the loss of luteotropic (LH) support or through loss of LH-dependent steroid milieu, monkeys were assigned to 4 groups: control (no treatment), the GnRH antagonist Antide, Antide plus synthetic progestin (R5020), or Antide plus the estrogen receptor agonists diarylpropionitrile (DPN)/propyl-pyrazole-triol (PPT) during the mid-late luteal phase. Antide treatment increased the numbers of CD11b(+) and CD14(+) cells, whereas progestin, but not estrogen, replacement suppressed the numbers of CD11b(+), CD14(+), and CD16(+) cells. Neither Antide nor steroid replacement altered numbers of CD3epsilon(+) cells. These data suggest that increased numbers of innate immune cells in primate CL after P4 synthesis declines play a role in onset of structural regression of primate CL.

Synovial joints: from development to homeostasis.

Synovial joint morphogenesis occurs through the condensation of mesenchymal cells into a non-cartilaginous region known as the interzone and the specification of progenitor cells that commit to the articular fate. Although several signaling molecules are expressed by the interzone, the mechanism is poorly understood. For treatments of cartilage injuries, it is critical to discover the presence of joint progenitor cells in adult tissues and their expression gene pattern. Potential stem cell niches have been found in different joint regions, such as the surface zone of articular cartilage, synovium, and groove of Ranvier. Inherited joint malformations as well as joint-degenerating conditions are often associated with other skeletal defects and may be seen as the failure of morphogenic factors to establish the correct microenvironment in cartilage and bone. Therefore, exploring how joints form can help us understand how cartilage and bone are damaged and develop drugs to reactivate this developing mechanism.

Quantification of dynamic changes to blood volume and vascular flow in the primate corpus luteum during the menstrual cycle.

BACKGROUND: The objective of the current study was to determine changes to vascular parameters of nonhuman primate dominant ovarian structures by dynamic contrast-enhanced ultrasound (DCE-US). MATERIALS AND METHODS: Dynamic contrast-enhanced ultrasound with intravenous microbubble infusion was performed on the rhesus macaque ovary bearing the pre-ovulatory follicle and corpus luteum (CL) sequentially during the natural luteal phase (n = 8) and GnRH antagonist (antide)-induced luteal regression (n = 6). RESULTS: Changes in luteal blood volume (BV) and vascular flow (VF) were observed between stages of the luteal phase Luteal BV was highest in early stage CL, before decreasing 2.5-fold in late stage CL (P < 0.06); in contrast, luteal VF peaked at mid luteal stage (P < 0.01). Two females identified with luteal insufficiency trended toward lower peak BV, compared to typical CLs. Another female was identified with a luteal cyst on the contralateral ovary, and a CL that regressed before P levels declined. After 72 hours of antide exposure, BV was reduced 2.3-fold (P = 0.03). CONCLUSIONS: DCE-US provides a sensitive, non-invasive measurement of the dynamics of blood volume and flow in dominant ovarian structures.

Reduced anti-Müllerian hormone action in cumulus-oocyte complexes is beneficial for oocyte maturation without affecting oocyte competence.

Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.

Essential role of ARID2 protein-containing SWI/SNF complex in tissue-specific gene expression.

Unfolding of the gene expression program that converts precursor cells to their terminally differentiated counterparts is critically dependent on the nucleosome-remodeling activity of the mammalian SWI/SNF complex. The complex can be powered by either of two alternative ATPases, BRM or BRG1. BRG1 is critical for development and the activation of tissue specific genes and is found in two major stable configurations. The complex of BRG1-associated factors termed BAF is the originally characterized form of mammalian SWI/SNF. A more recently recognized configuration shares many of the same subunits but is termed PBAF in recognition of a unique subunit, the polybromo protein (PBRM1). Two other unique subunits, BRD7 and ARID2, are also diagnostic of PBAF. PBAF plays an essential role in development, apparent from the embryonic lethality of Pbmr1-null mice, but very little is known about the role of PBAF, or its signature subunits, in tissue-specific gene expression in individual differentiation programs. Osteoblast differentiation is an attractive model for tissue-specific gene expression because the process is highly regulated and remains tightly synchronized over a period of several weeks. This model was used here, with a stable shRNA-mediated depletion approach, to examine the role of the signature PBAF subunit, ARID2, during differentiation. This analysis identifies a critical role for ARID2-containing complexes in promoting osteoblast differentiation and supports a view that the PBAF subset of SWI/SNF contributes importantly to maintaining cellular identity and activating tissue-specific gene expression.

Mechanistic insight into efficient H2 generation upon HCOONa hydrolysis.

Sodium formate (SF) is regarded as a technological additive to improve H2 generation upon HCOOH dehydrogenation. The development of SF as a hydrogen storage material is still a challenge. Herein, we report the design and synthesis of carbon nanosphere-stabilized Pd nanoparticles (Pd/CNS) for the direct H2 generation upon SF hydrolysis in the presence of Fe3+. The tandem reaction, isotopic mass spectrometry, and gas chromatograms result confirmed that SF hydrolysis generates H2 with one H atom provided by SF and other H by H2O. The kinetic study and detailed mechanistic investigations have demonstrated that the concerted process between the cleavage of O-H bond in H2O and -O2C-H bond oxidative addition is the rate-controlling step in SF hydrolysis. This work offers a new chemical hydrogen storage material (HCOONa) for the high-efficiency generation, transport, and storage of H2.

Haploidy in somatic cells is induced by mature oocytes in mice.

Haploidy is naturally observed in gametes; however, attempts of experimentally inducing haploidy in somatic cells have not been successful. Here, we demonstrate that the replacement of meiotic spindles in mature metaphases II (MII) arrested oocytes with nuclei of somatic cells in the G0/G1 stage of cell cycle results in the formation of de novo spindles consisting of somatic homologous chromosomes comprising of single chromatids. Fertilization of such oocytes with sperm triggers the extrusion of one set of homologous chromosomes into the pseudo-polar body (PPB), resulting in a zygote with haploid somatic and sperm pronuclei (PN). Upon culture, 18% of somatic-sperm zygotes reach the blastocyst stage, and 16% of them possess heterozygous diploid genomes consisting of somatic haploid and sperm homologs across all chromosomes. We also generate embryonic stem cells and live offspring from somatic-sperm embryos. Our finding may offer an alternative strategy for generating oocytes carrying somatic genomes.