RESUMEN
Perfluorooctanoic acid (PFOA) is a synthetic chemical resistant to biodegradation and is environmentally persistent. PFOA is found in many consumer products and is a major source of water contamination. While PFOA has been identified as a contaminant of concern for reproductive health, little is known about the effects of PFOA on ovarian follicular development and growth. Recent evidence indicates that the Hippo pathway is an important regulator of ovarian physiology. Here, we investigated the effects of PFOA on ovarian folliculogenesis during the neonatal period of development and potential impacts on the Hippo signaling pathway. Post-natal day 4 (PND4) neonatal ovaries from CD-1 mice were cultured with control medium (DMSO <0.01% final concentration) or PFOA (50 µM or 100 µM). After 96 h, ovaries were collected for histological analysis of folliculogenesis, gene and protein expression, and immunostaining. Results revealed that PFOA (50 µM) increased the number of secondary follicles, which was accompanied by increases in mRNA transcripts and protein of marker of proliferation marker Ki67 with no impacts on apoptosis markers Bax, Bcl2, or cleaved caspase-3. PFOA treatment (50 µM and 100 µM) stimulated an upregulation of transcripts for cell cycle regulators Ccna2, Ccnb2, Ccne1, Ccnd1, Ccnd2, and Ccnd3. PFOA also increased abundance of transcripts of Hippo pathway components Mst1/2, Lats1, Mob1b, Yap1, and Taz, as well as downstream Hippo pathway targets Areg, Amotl2, and Cyr61, although it decreased transcripts for anti-apoptotic Birc5. Inhibition of the Hippo pathway effector YAP1 with Verteporfin resulted in the attenuation of PFOA-induced follicular growth and proliferation. Together, these findings suggest that occupationally relevant levels of PFOA (50 µM) can stimulate follicular activation in neonatal ovaries potentially through activation of the Hippo pathway.
Asunto(s)
Vía de Señalización Hippo , Ovario , Proteínas Adaptadoras Transductoras de Señales , Animales , Animales Recién Nacidos , Caprilatos , Caspasa 3/metabolismo , División Celular , Dimetilsulfóxido , Femenino , Fluorocarburos , Antígeno Ki-67/metabolismo , Ratones , Ovario/metabolismo , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismo , Transducción de Señal , Verteporfina/farmacología , Agua/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
STUDY QUESTION: Does hypo-glycosylated human recombinant FSH (hFSH18/21) have greater in vivo bioactivity that drives follicle development in vivo compared to fully-glycosylated human recombinant FSH (hFSH24)? SUMMARY ANSWER: Compared with fully-glycosylated hFSH, hypo-glycosylated hFSH has greater bioactivity, enabling greater follicular health and growth in vivo, with enhanced transcriptional activity, greater activation of receptor tyrosine kinases (RTKs) and elevated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling. WHAT IS KNOWN ALREADY: Glycosylation of FSH is necessary for FSH to effectively activate the FSH receptor (FSHR) and promote preantral follicular growth and formation of antral follicles. In vitro studies demonstrate that compared to fully-glycosylated recombinant human FSH, hypo-glycosylated FSH has greater activity in receptor binding studies, and more effectively stimulates the PKA pathway and steroidogenesis in human granulosa cells. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study evaluating the actions of purified recombinant human FSH glycoforms on parameters of follicular development, gene expression and cell signaling in immature postnatal day (PND) 17 female CD-1 mice. To stimulate follicle development in vivo, PND 17 female CD-1 mice (n = 8-10/group) were treated with PBS (150 µl), hFSH18/21 (1 µg/150 µl PBS) or hFSH24 (1 µg/150 µl PBS) by intraperitoneal injection (i.p.) twice daily (8:00 a.m. and 6:00 p.m.) for 2 days. Follicle numbers, serum anti-Müllerian hormone (AMH) and estradiol levels, and follicle health were quantified. PND 17 female CD-1 mice were also treated acutely (2 h) in vivo with PBS, hFSH18/21 (1 µg) or hFSH24 (1 µg) (n = 3-4/group). One ovary from each mouse was processed for RNA sequencing analysis and the other ovary processed for signal transduction analysis. An in vitro ovary culture system was used to confirm the relative signaling pathways. PARTICIPANTS/MATERIALS, SETTING, METHODS: The purity of different recombinant hFSH glycoforms was analyzed using an automated western blot system. Follicle numbers were determined by counting serial sections of the mouse ovary. Real-time quantitative RT-PCR, western blot and immunofluorescence staining were used to determine growth and apoptosis markers related with follicle health. RNA sequencing and bioinformatics were used to identify pathways and processes associated with gene expression profiles induced by acute FSH glycoform treatment. Analysis of RTKs was used to determine potential FSH downstream signaling pathways in vivo. Western blot and in vitro ovarian culture system were used to validate the relative signaling pathways. MAIN RESULTS AND THE ROLE OF CHANCE: Our present study shows that both hypo- and fully-glycosylated recombinant human FSH can drive follicular growth in vivo. However, hFSH18/21 promoted development of significantly more large antral follicles compared to hFSH24 (P < 0.01). In addition, compared with hFSH24, hFSH18/21 also promoted greater indices of follicular health, as defined by lower BAX/BCL2 ratios and reduced cleaved Caspase 3. Following acute in vivo treatment with FSH glycoforms RNA-sequencing data revealed that both FSH glycoforms rapidly induced ovarian transcription in vivo, but hypo-glycosylated FSH more robustly stimulated Gαs and cAMP-mediated signaling and members of the AP-1 transcription factor complex. Moreover, hFSH18/21 treatment induced significantly greater activation of RTKs, PI3K/AKT and MAPK/ERK signaling compared to hFSH24. FSH-induced indices of follicle growth in vitro were blocked by inhibition of PI3K and MAPK. LARGE SCALE DATA: RNA sequencing of mouse ovaries. Data will be shared upon reasonable request to the corresponding author. LIMITATIONS, REASONS FOR CAUTION: The observations that hFSH glycoforms have different bioactivities in the present study employing a mouse model of follicle development should be verified in nonhuman primates. The gene expression studies reflect transcriptomes of whole ovaries. WIDER IMPLICATIONS OF THE FINDINGS: Commercially prepared recombinant human FSH used for ovarian stimulation in human ART is fully-glycosylated FSH. Our findings that hypo-glycosylated hFSH has greater bioactivity enabling greater follicular health and growth without exaggerated estradiol production in vivo, demonstrate the potential for its development for application in human ART. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by NIH 1P01 AG029531, NIH 1R01 HD 092263, VA I01 BX004272, and the Olson Center for Women's Health. JSD is the recipient of a VA Senior Research Career Scientist Award (1IK6 BX005797). This work was also partially supported by National Natural Science Foundation of China (No. 31872352). The authors declared there are no conflicts of interest.
Asunto(s)
Hormona Folículo Estimulante Humana , Proteínas Quinasas Activadas por Mitógenos , Folículo Ovárico/crecimiento & desarrollo , Fosfatidilinositol 3-Quinasas , Transducción de Señal , Animales , China , Estudios Transversales , Femenino , Glicosilación , Ratones , Proteínas RecombinantesRESUMEN
Ataxia-telangiectasia-mutated (ATM) protein recognizes and repairs DNA double strand breaks through activation of cell cycle checkpoints and DNA repair proteins. Atm gene mutations increase female reproductive cancer risk. Phosphoramide mustard (PM) induces ovarian DNA damage and destroys primordial follicles, and pharmacological ATM inhibition prevents PM-induced follicular depletion. Wild-type (WT) C57BL/6 or Atm+/- mice were dosed once intraperitoneally with sesame oil (95%) or PM (25 mg/kg) in the proestrus phase of the estrous cycle and ovaries harvested 3 days thereafter. Atm+/- mice spent ~25% more time in diestrus phase than WT. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) on ovarian protein was performed and bioinformatically analyzed. Relative to WT, Atm+/- mice had 64 and 243 proteins increased or decreased in abundance, respectively. In WT mice, PM increased 162 and decreased 20 proteins. In Atm+/- mice, 173 and 37 proteins were increased and decreased, respectively, by PM. Exportin-2 (XPO2) was localized to granulosa cells of all follicle stages and was 7.2-fold greater in Atm+/- than WT mice. Cytoplasmic FMR1-interacting protein 1 was 6.8-fold lower in Atm+/- mice and was located in the surface epithelium with apparent translocation to the ovarian medulla post-PM exposure. PM induced γH2AX, but fewer γH2AX-positive foci were identified in Atm+/- ovaries. Similarly, cleaved caspase-3 was lower in the Atm+/- PM-treated, relative to WT mice. These findings support ATM involvement in ovarian DNA repair and suggest that ATM functions to regulate ovarian atresia.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Reparación del ADN/fisiología , Ovario/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Femenino , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Ratones , Ratones Noqueados , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Ovario/efectos de los fármacos , Mostazas de Fosforamida/farmacologíaRESUMEN
In the overweight or obese female, reproductive complications include poor oocyte quality, decreased fecundity, gestational diabetes, and higher risk of reproductive cancers. Using lean and hyperphagia-induced obese female mice aged 10 weeks, we determined that the ovary from obese female mice had elevated (P < 0.10) levels of ataxia telangiectasia mutated (ATM) protein in oocytes of both small and large follicles. Phosphorylated ATM at serine 1981 was greater (P < 0.05) in large relative to small follicles with no additional impact of obesity. Obesity increased (P < 0.05) γH2AX in small follicles in obese relative to lean ovaries, while large follicles of both lean and obese mice had detectable levels of γH2AX. Cleaved caspase 3 was reduced (P < 0.05) in the small follicles of obese relative to lean ovaries. In large follicles of lean mice, cleaved caspase 3 was increased in large compared to small follicles (P < 0.05) but this pattern was absent in obese mice. Breast cancer type 1 susceptibility protein (BRCA1) or the phosphorylated BRCA1 proteins were observably altered by obesity. These data demonstrate that markers of DNA damage and repair have a follicle-dependent stage location and that obesity alters ATM and cleaved caspase 3 in a follicular stage dependent manner.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Daño del ADN , Reparación del ADN , Obesidad/fisiopatología , Ovario/patología , Delgadez/fisiopatología , Animales , Femenino , Ratones , Ovario/metabolismoRESUMEN
Gestational diabetes mellitus (GDM) is an obstetric disorder affecting approximately 10% of pregnancies. The four high-fat, high-sucrose (HFHS) mouse model emulates GDM in lean women. Dams are fed a HFHS diet 1 week prior to mating and throughout gestation resulting in inadequate insulin response to glucose in mid-late pregnancy. The offspring of HFHS dams have increased adiposity, thus, we hypothesized that maternal metabolic alterations during lean GDM would compromise ovarian function in offspring both basally and in response to a control or HFHS diet in adulthood. Briefly, DLPL were lean dams and control diet pups; DLPH were lean dams and HFHS pups; DHPL were HFHS dams and control diet pups; and DHPH were HFHS dams and HFHS pups. A HFHS challenge in the absence of maternal GDM (DLPL vs. DLPH) increased 3 and decreased 30 ovarian proteins. Maternal GDM in the absence of a dietary stress (DLPL vs. DHPL) increased abundance of 4 proteins and decreased abundance of 85 proteins in the offspring ovary. Finally, 87 proteins increased, and 4 proteins decreased in offspring ovaries due to dietary challenge and exposure to maternal GDM in utero (DLPL vs. DHPH). Canopy FGF signaling regulator 2, deleted in azoospermia-associated protein 1, septin 7, and serine/arginine-rich splicing factor 2 were altered across multiple offspring groups. Together, these findings suggest a possible impact on fertility and oocyte quality in relation to GDM exposure in utero as well as in response to a western diet in later life.
Asunto(s)
Diabetes Gestacional , Enfermedades del Ovario/etiología , Ovario/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Proteoma/metabolismo , Animales , Recuento de Células , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Gestacional/metabolismo , Diabetes Gestacional/patología , Dieta , Femenino , Ratones , Ratones Endogámicos C57BL , Enfermedades del Ovario/metabolismo , Folículo Ovárico/metabolismo , Folículo Ovárico/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/patología , Proteoma/análisis , Delgadez/complicaciones , Delgadez/patologíaRESUMEN
Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals known for their environmental persistence and resistance to biodegradation. This study investigated the impact of adolescent exposure to a PFAS mixture on adult ovarian function. Female CD-1 mice were orally exposed to vehicle control or a PFAS mixture (comprised of perfluorooctanoic acid [PFOA], perfluorooctanesulfonic acid [PFOS], undecafluoro-2-methyl-3-oxahexanoic acid [GenX/HFPO-DA], and perfluorobutanesulfonic acid [PFBS]) for 15 days. After a 42-day recovery period, reproductive hormones, ovarian fibrosis, and ovarian gene and protein expression were analyzed using ELISA, Picrosirius red (PSR) staining, qPCR, and immunoblotting, respectively. Results revealed that PFAS exposure did not affect adult body or organ weight, although ovarian weight slightly decreased. PFAS exposed mice exhibited a disturbed estrous cycle, with less time spent in proestrus than control mice. Follicle counting indicated a reduction in primordial and primary follicles. Serum analysis revealed no changes in steroid hormones, follicle-stimulating hormone, or anti-Müllerian hormone, but a significant increase in luteinizing hormone was observed in PFAS-treated mice. Ovaries collected from PFAS treated mice had increased mRNA transcripts for steroidogenic enzymes and fatty acid synthesis-related genes. PFAS exposure also increased collagen content in the ovary. Additionally, serum tumor necrosis factor-α levels were higher in PFAS treated mice. Finally, transcripts and protein abundance for Hippo pathway components were upregulated in the ovaries of the PFAS treated mice. Overall, these findings suggest that adolescent exposure to PFAS can disrupt ovarian function in adulthood.
RESUMEN
Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals that are resistant to biodegradation and are environmentally persistent. PFAS are found in many consumer products and are a major source of water and soil contamination. This study investigated the effects of an environmentally relevant PFAS mixture (perfluorooctanoic acid [PFOA], perfluorooctanesulfonic acid [PFOS], perfluorohexanesulfonic acid [PFHxS]) on the transcriptome and function of human granulosa cells (hGCs). Primary hGCs were harvested from follicular aspirates of healthy, reproductive-age women who were undergoing oocyte retrieval for in vitro fertilization. Liquid Chromatography with tandem mass spectrometry (LC/MS-MS) was performed to identify PFAS compounds in pure follicular fluid. Cells were cultured with vehicle control or a PFAS mixture (2 nM PFHxS, 7 nM PFOA, 10 nM PFOS) for 96 h. Analyses of cell proliferation/apoptosis, steroidogenesis, and gene expression were measured via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays/immunofluorescence, ELISA/western blotting, and RNA sequencing/bioinformatics, respectively. PFOA, PFOS, and PFHxS were detected in 100% of follicle fluid samples. Increased cell proliferation was observed in hGCs treated with the PFAS mixture with no impacts on cellular apoptosis. The PFAS mixture also altered steroid hormone synthesis, increasing both follicle-stimulating hormone-stimulated and basal progesterone secretion and concomitant upregulation of STAR protein. RNA sequencing revealed inherent differences in transcriptomic profiles in hGCs after PFAS exposure. This study demonstrates functional and transcriptomic changes in hGCs after exposure to a PFAS mixture, improving our knowledge about the impacts of PFAS exposures and female reproductive health. These findings suggest that PFAS compounds can disrupt normal granulosa cell function with possible long-term consequences on overall reproductive health.
Asunto(s)
Ácidos Alcanesulfónicos , Caprilatos , Proliferación Celular , Fluorocarburos , Células de la Granulosa , Humanos , Femenino , Fluorocarburos/toxicidad , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Proliferación Celular/efectos de los fármacos , Ácidos Alcanesulfónicos/toxicidad , Caprilatos/toxicidad , Células Cultivadas , Contaminantes Ambientales/toxicidad , Transcripción Genética/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Ácidos Sulfónicos/toxicidad , Líquido Folicular/metabolismo , Adulto , Hormonas Esteroides Gonadales/biosíntesis , Hormonas Esteroides Gonadales/metabolismoRESUMEN
Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Due to the ubiquitous presence of PFOA in the environment, the impacts of PFOA exposure not only affect human reproductive health but may also affect livestock reproductive health. The focus of this study was to determine the effects of PFOA on the physiological functions of bovine granulosa cells in vitro. Primary bovine granulosa cells were exposed to 0, 4, and 40 µM PFOA for 48 and 96 h followed by analysis of granulosa cell function including cell viability, steroidogenesis, and mitochondrial activity. Results revealed that PFOA inhibited steroid hormone secretion and altered the expression of key enzymes required for steroidogenesis. Gene expression analysis revealed decreases in mRNA transcripts for CYP11A1, HSD3B, and CYP19A1 and an increase in STAR expression after PFOA exposure. Similarly, PFOA decreased levels of CYP11A1 and CYP19A1 protein. PFOA did not impact live cell number, alter the cell cycle, or induce apoptosis, although it reduced metabolic activity, indicative of mitochondrial dysfunction. We observed that PFOA treatment caused a loss of mitochondrial membrane potential and increases in PINK protein expression, suggestive of mitophagy and mitochondrial damage. Further analysis revealed that these changes were associated with increased levels of reactive oxygen species. Expression of autophagy related proteins phosphoULK1 and LAMP2 were increased after PFOA exposure, in addition to an increased abundance of lysosomes, characteristic of increased autophagy. Taken together, these findings suggest that PFOA can negatively impact granulosa cell steroidogenesis via mitochondrial dysfunction.
Asunto(s)
Caprilatos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol , Femenino , Humanos , Animales , Bovinos , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Caprilatos/toxicidad , Caprilatos/metabolismo , Células de la Granulosa , MitocondriasRESUMEN
Perfluorooctanoic acid (PFOA) is a common environmental contaminant that belongs to a group of manmade fluorinated chemicals called per- and polyfluoroalkyl substances (PFAS). Due to the pervasive nature of PFOA, the environmental health risks of PFOA contamination and exposure on reproductive health have increasing concern. In the present study, we exposed HGrC1 cells, an immortalized human granulosa cell line, to environmentally relevant (1-10 µM) concentrations of PFOA. Results indicated that HGrC1 cells treated with PFOA had increased proliferation and migration relative to vehicle treated controls. No differences in cell apoptosis were observed with 1-10 µM PFOA. Gene expression analysis revealed increases in mRNA transcripts for cell cycle regulators CCND1, CCNA2, and CCNB1. Upregulation of YAP1 protein and downstream target CTGF protein was also observed, suggesting that the Hippo pathway is involved in the proliferation and migratory effects of PFOA on HGrC1 cells. Further, the YAP1 inhibitor Verteporfin prevented the stimulatory effects of PFOA on HGrC1 cells. Together, these findings support a role for the Hippo pathway effector YAP1 in response to PFOA exposure in human granulosa cells.
Asunto(s)
Fluorocarburos , Caprilatos/farmacología , Proliferación Celular , Femenino , Fluorocarburos/farmacología , Genes cdc , Células de la Granulosa/metabolismo , Vía de Señalización Hippo , Humanos , Proteínas Señalizadoras YAPRESUMEN
Emerging studies indicate that the Hippo pathway, a highly conserved pathway that regulates organ size control, plays an important role in governing ovarian physiology, fertility, and pathology. Specific to the ovary, the spatiotemporal expression of the major components of the Hippo signaling cascade are observed throughout the reproductive lifespan. Observations from multiple species begin to elucidate the functional diversity and molecular mechanisms of Hippo signaling in the ovary in addition to the identification of interactions with other signaling pathways and responses to various external stimuli. Hippo pathway components play important roles in follicle growth and activation, as well as steroidogenesis, by regulating several key biological processes through mechanisms of cell proliferation, migration, differentiation, and cell fate determination. Given the importance of these processes, dysregulation of the Hippo pathway contributes to loss of follicular homeostasis and reproductive disorders such as polycystic ovary syndrome (PCOS), premature ovarian insufficiency, and ovarian cancers. This review highlights what is currently known about the Hippo pathway core components in ovarian physiology, including ovarian development, follicle development, and oocyte maturation, while identifying areas for future research to better understand Hippo signaling as a multifunctional pathway in reproductive health and biology.
Asunto(s)
Vía de Señalización Hippo , Insuficiencia Ovárica Primaria , Femenino , Humanos , Folículo Ovárico/fisiología , FertilidadRESUMEN
Previous studies have reported hypo-glycosylated FSH and fully-glycosylated FSH to be naturally occurring in humans, and these glycoforms exist in changing ratios over a woman's lifespan. The precise cellular and molecular effects of recombinant human FSH (hFSH) glycoforms, FSH21 and FSH24, have not been documented in primary granulosa cells. Herein, biological responses to FSH21 and FSH24 were compared in primary porcine granulosa cells. Hypo-glycosylated hFSH21 was significantly more effective than fully-glycosylated hFSH24 at stimulating cAMP accumulation and protein kinase A (PKA) activity, leading to the higher phosphorylation of CREB and ß-Catenin. Compared to fully-glycosylated hFSH24, hypo-glycosylated hFSH21 also induced greater levels of transcripts for HSD3B, STAR and INHA, and higher progesterone production. Our results demonstrate that hypo-glycosylated hFSH21 exerts more robust activation of intracellular signals associated with steroidogenesis than fully-glycosylated hFSH24 in primary porcine granulosa cells, and furthers our understanding of the differing bioactivities of FSH glycoforms in the ovary.
Asunto(s)
Hormona Folículo Estimulante Humana/farmacología , Células de la Granulosa/efectos de los fármacos , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Hormona Folículo Estimulante Humana/química , Hormona Folículo Estimulante Humana/metabolismo , Glicosilación , Células de la Granulosa/citología , Células de la Granulosa/fisiología , Ovario/efectos de los fármacos , Ovario/metabolismo , Cultivo Primario de Células/veterinaria , Progesterona/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Porcinos , beta Catenina/metabolismoRESUMEN
BACKGROUND: Primary cell culture is a valuable tool to utilize in parallel with in vivo studies in order to maximize our understanding of the mechanisms surrounding neurogenesis and central nervous system (CNS) regeneration and plasticity. The zebrafish is an important model for biomedical research and primary neural cells are readily obtainable from their embryonic stages viatissue dissociation. Further, transgenic reporter lines with cell type-specific expression allows for observation of distinct cell populations within the dissociated tissue. NEW METHOD: Here, we define an efficient method for ex vivo quantification and characterization of neuronal and glial tissue dissociated from embryonic zebrafish. RESULTS: Zebrafish brain dissociated cells have been documented to survive in culture for at least 9 days in vitro (div). Anti-HuC/D and anti-Acetylated Tubulin antibodies were used to identify neurons in culture; at 3 div approximately 48% of cells were HuC/D positive and 85% expressed serotonin, suggesting our protocol can efficiently isolate neurons from whole embryonic zebrafish brains. Live time-lapse imaging was also carried out to analyze cell migration in vitro. COMPARISON WITH EXISTING METHODS: Primary cultures of zebrafish neural cells typically have low rates of survivability in vitro. We have developed a culture system that has long term cell viability, enabling direct analysis of cell-cell and cell-extracellular matrix interactions. CONCLUSIONS: These results demonstrate a practical method for isolating, dissociating and culturing of embryonic zebrafish neural tissue. This approach could further be utilized to better understand zebrafish regeneration in vitro.
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Neuroglía , Neuronas , Neurociencias/métodos , Cultivo Primario de Células/métodos , Pez Cebra , Animales , Animales Modificados Genéticamente , Embrión no Mamífero , Neuroglía/citología , Neuroglía/fisiología , Neuronas/citología , Neuronas/fisiologíaRESUMEN
Ovarian gap junctions function to provide intercellular communication between ovarian cell types and are critical for proper ovarian function. Connexons are communication channels that are comprised of connexin (CX) proteins. Connexins can be regulated through endocrine signals, thus have dynamic expression throughout the estrous cycle. Not surprisingly, ovarian function is negatively affected in mouse models deficient in Cx genes; loss of Gja4 impairs folliculogenesis while ovaries devoid of Gja1 have reductions in oocyte growth. Chemicals that negatively affect ovarian function, termed ovotoxicants, can directly target Cx mRNA or protein abundance. Endocrine disrupting chemicals, medicinal drugs, pesticides, industrial chemicals, polycyclic aromatic hydrocarbons, recreational drugs and dietary components can affect Cx levels and/or function. Also, aging and obesity can impact ovarian Cx's. This review highlights what is currently known about ovotoxicant exposures that impact ovarian gap junction function as well as identifies the many gaps in our knowledge in this area of ovarian biology.