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Ovarian follicle Disaggregation to Assess Granulosa Cell Viability

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International Journal of Clinical Medicine 2018年第5期9卷 377-399页

作者： Mohammad Asaduzzaman Daniela Figueroa Gonzalez Fiona Young Medical Biotechnology Flinders University Adelaide South Australia

Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 μL, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 ±87) GC per antral follicle of which 82% ±5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 ±16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.

关键词： Ovarian follicle Granulosa Cells Viability Collagenase Hyaluronidase

Vitamin B12 Activates the Wnt-Pathway in Human Hair follicle Cells by Induction of <i>beta</i>-Catenin and Inhibition of Glycogensynthase Kinase-3 Transcription

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Journal of Cosmetics, Dermatological Sciences and Applications 2011年第2期1卷 25-29页

作者： Walter Krugluger Karl Stiefsohn Katharina Laciak Karl Moser Claudia Moser 不详

Background and Objectives: Micrograft transplantation is accompanied by a transient induction of telogen in transplanted hair follicles (HF), which might be avoided by supporting the metabolic pathways of the micrograft during the ex vivo period. Vitamin B12 (cobalamin) has been suggested to influence HF growth and cycling in humans, but the mechanisms are unclear. Method: HFs were obtained from patients undergoing routine micrograft transplantation and were cultured for 5 days in Dulbecco’s modified Eagles Medium, supplemented with different amounts of vitamin B12. Hair shaft elongation (HSE) of the isolated HFs as well as quantitative changes of mRNA for beta-catenin, glykogensynthase kinase-3 (GSK-3) and TCF/Lef-1 in HF cells were determined. Results: In vitro HSE demonstrated a dose dependent induction of HSE after stimulation with 2.5 ug/ml and 25 ug/ml vitamin B12 (6.2 +/- 2.1% and 15.4 +/- 3.8% respectively). A dose dependent induction of beta-catenin-mRNA could be demonstrated in cultured HFs after stimulation with 2.5 ug/ml and 25 ug/ml vitamin B12 (fold change compared to DMEM: 9.5 +/- 2.7, p < 0.05 and 23.1 +/- 7.4, p < 0.01;respectively). Concomitantly the amounts of GSK-3 were significantly reduced after stimulation with 25 ug/ml vitamin B12 (fold change compared to DMEM: 0.76 +/- 0.12, p < 0.05). Conclusions: Our data demonstrate a hair growth promoting effect of vitamin B12 in vitro. This effect is accompanied by the modulation of intracellular signal transduction molecules of the wnt-pathway and might promote hair growth after micrograft transplantation.

关键词： Hair follicle Growth Factors Cell Signaling

Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer

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Science China(Life Sciences) 2009年第4期52卷 390-397页

作者： BOU ShorGan Key Laboratory for Mammalian Reproductive Biology and Biotechnology Ministry of Education Inner Mongolia University

In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO- Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%,P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P<0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly ex- pressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neor genes were correctly expressed indicating that transgenic somatic cell lines and positive trans- genic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and optim

关键词： cashmere goat insulin like growth factor 1 hair follicle fetal fibroblasts cells transgene somatic cell nuclear transfer

Hair follicle stem cells: In vitro and in vivo neural differentiation

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World Journal of Stem Cells 2015年第5期7卷 866-872页

作者： Nowruz Najafzadeh Banafshe Esmaeilzade Maryam Dastan Imcheh Research Laboratory for Embryology and Stem Cells Department of Anatomical Sciences and Pathology School of Medicine Ardabil University of Medical Sciences Department of Anatomical Sciences School of Medicine Bushehr University of Medical Sciences Department of Biology Faculty of Sciences Urmia University

Hair follicle stem cells(HFSCs) normally give rise to keratinocytes, sebocytes, and transient amplifying progenitor cells. Along with the capacity to proliferate rapidly, HFSCs provide the basis for establishing a putative source of stem cells for cell therapy. HFSCs are multipotent stem cells originating from the bulge area. The importance of these cells arises from two important characteristics, distinguishing them from all other adult stem cells. First, they are accessible and proliferate for long periods. Second, they are multipotent, possessing the ability to differentiate into mesodermal and ectodermal cell types. In addition to a developmental capacity in vitro, HFSCs display an ability to form differentiated cells in vivo. During the last two decades, numerous studies have led to the development of an appropriate culture condition for producing various cell lineages from HFSCs. Therefore, these stem cells are considered as a novel source for cell therapy of a broad spectrum of neurodegenerative disorders. This review presents the current status of human, rat, and mouse HFSCs from both the cellular and molecular biology and cell therapy perspectives. The first section of this review highlights the importance of HFSCs and in vitro differentiation, while the final section emphasizes the significance of cell differentiation in vivo.

关键词： Hair follicle Stem cells Bulge area Neuron Differentiation

Proteo-transcriptomic profiles reveal key regulatory pathways and functions of LDHA in the ovulation of domestic chickens(Gallus gallus)

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Transplantation of UC-MSCs on collagen scaffold activates follicles in dormant ovaries of POF patients with long history of infertility

畜牧与生物技术杂志（英文版） 2024年第4期15卷 1449-1465页

作者： Ruixue Nie Wenhui Zhang Haoyu Tian Junying Li Yao Ling Bo Zhang Hao Zhang Changxin Wu State Key Laboratory of Animal Biotech Breeding Beijing Key Laboratory for Animal Genetic ImprovementCollege of Animal Science and TechnologyChina Agricultural UniversityBeijing 100193China

Background In poultry,the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry ***,the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear.Results Critical biochemical events involved in ovulation in domestic chickens(Gallus gallus)were evaluated by transcriptomics,proteomics,and in vitro *** transcriptome analyses of the largest preovulatory follicle(F1)and postovulatory follicle(POF1)in continuous laying(CL)and intermittent laying(IL)chickens indicated the greatest difference between CL_F1 and IL_F1,with 950 differentially expressed genes(DEGs),and the smallest difference between CL_POF1 and IL_POF1,with 14 ***,data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_*** membrane synthesis,steroid biosynthesis,lysosomes,and oxidative phosphorylation were identifiied as pivotal pathways contributing to ovulation *** particular,the regulation of zona pellucida sperm-binding protein 3,plasminogen activator,cathepsin A,and lactate dehydrogenase A(LDHA)was shown to be essential for ***,the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro.Conclusions This study reveals several important biochemical events involved in the process of ovulation,as well as crucial role of *** findings improve our understanding of ovulation and its regulatory mechanisms in avian species.

关键词： Chicken Data-independent acquisition proteomics LDHA Ovulation Regulatory mechanism Transcriptome ZONA-PELLUCIDA PROTEINS EGG-ENVELOPE LUTEINIZING-HORMONE LACTATE-DEHYDROGENASE GENE-EXPRESSION MESSENGER-RNA follicle GRANULOSA PROGESTERONE HENS

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Science China(Life Sciences) 2018年第12期61卷 1554-1565页

作者： Lijun Ding Guijun Yan Bin Wang Lu Xu Yan Gu Tong Ru Xiaoying Cui Lei Lei Jingyu Liu Xiaoqiang Sheng Bin Wang Chunxue Zhang Yanjun Yang Ruiwei Jiang Jianjun Zhou Na Kong Feifei Lu Huaijun Zhou Yannan Zhao Bing Chen Yali Hu Jianwu Dai Haixiang Sun Center for Reproductive Medicine Department of Obstetrics and Gynecologythe Affiliated Drum Tower Hospital of Nanjing University Medieal SchoolNanjing 210008China Clinical Center for Stem Cell Research the Affiliated Drum Tower Hospital of Nanjing University Medical SchoolNanjing 210008China Department of Obstetrics and Gynecology the Affiliated Drum Tower Hospital of Nanjing University Medical SchoolNanjing 210008China State Key Laboratory of Molecular Development Biology Institute of Genetics and Developmental BiologyChinese Academy of Sciences Beijing 100190China

Premature ovarian failure(POF) is a refractory disease for clinical treatment with the goal of restoring fertility. In this study,umbilical cord mesenchymal stem cells on a collagen scaffold(collagen/UC-MSCs) can activate primordial follicles in vitro via phosphorylation of FOXO3 a and FOXO1. Transplantation of collagen/UC-MSCs to the ovaries of POF patients rescued overall ovarian function, evidenced by elevated estradiol concentrations, improved follicular development, and increased number of antral follicles. Successful clinical pregnancy was achieved in women with POF after transplantation of collagen/UC-MSCs or UC-MSCs. In summary, collagen/UC-MSC transplantation may provide an effective treatment for POF.

关键词： premature ovarian failure collagen scaffold UC-MSCs primordial follicle activation granulosa cells FOXO3a FOXO1

Sources of cumulus expansion enabling factor (CEEF) in porcine follicles

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Chinese Science Bulletin 2002年第19期47卷 1619-1622页

作者： SUN Xingshen YUE Kuizhong MA Suofeng YI Yaling ZOU Yi TAN Jinghe Faculty of Life Sciences Northeast Agricultural UniversityHarbin 150030China College of Animal and Veterinary Science Shandong Agricultural UniversityTai’an 271018China

It was shown that expansion of porcine cumulus did not depend on oocyte-secreted factor(s), and it is therefore presumed that porcine CEEF may not be produced exclusively by the oocyte. In this experiment, we used mouse oocytectomized complexes (OOX), which were incapable of CEEF production, to assess the secretion of CEEF by evacuated zona, oocytes of different quality and somatic cells in the porcine follicles. The results showed that: (i) Evacuated zonae from both porcine and mouse oocytes did not produce CEEF. (ii) Porcine oocytes of A, B and C types from 3-6 mm follicles were not significantly different in both production and activity of CEEF. (iii) Both porcine OOX from 3-6 mm follicles and granulose cells from follicles secreted CEEF in a large quantity, independent of gonadotro-pins; mural granulose cells from 3-6 mm follicles, however, produced neglectable amount of CEEF. (iv) The follicular fluid from 3-6 mm porcine follicles contained CEEF activity that was concentration-dependent,

关键词： cumulus expansion enabling factor cumulus expansion follicle oocyte porcine.

The ghrelinergic system in zebrafish gonads is suppressed during food unavailability

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Aquaculture and Fisheries 2022年第5期7卷 494-499页

作者： Azadeh Hatef Jithine Jayakumar Rajeswari Suraj Unniappan Laboratory of Integrative Neuroendocrinology Department of Veterinary Biomedical SciencesWestern College of Veterinary MedicineUniversity of SaskatchewanSaskatoonSaskatchewanS7N 5B4Canada Toxicology Centre University of SaskatchewanSaskatoonSaskatchewanS7N 5B3Canada

Ghrelin is an appetite stimulatory peptide that regulates reproduction in *** acylation of ghrelin by ghrelin-O-acyltransferase(Goat)is essential for most of its *** ghrelinergic system(ghrelin,ghrelin receptor[Ghsr]and Goat)is present in teleost fish ovary and *** this research,we determined the abundance of the ghrelinergic system mRNAs in different stages of follicle(oocyte)development in zebrafish *** highest levels of preproghrelin mRNA were observed in the first stage of follicular development(previtellogenesis).Significantly low levels of preproghrelin mRNA were observed in stages 3II,4 and 5 of the follicle *** mRNA abundance remained unchanged at early stages of follicle development and decreased in stage 5 compared to other developmental stages.A significant decrease in preproghrelin and Ghsr mRNAs in the testis,and ghsr mRNA in the ovary of zebrafish deprived of food for 3 days were *** 7 days food deprivation,preproghrelin,ghsr and goat mRNA in the ovary and preproghrelin and goat in the testis of zebrafish were significantly *** results show that metabolic status modulates gonadal ghrelinergic system in zebrafish and provide support for a role for ghrelin in fish reproduction.

关键词： Ghrelin Ghrelin receptor Ghrelin-O-acyltransferase Fasting Ovary Testis follicle Zebrafish

Three Dimensional <i>In Vitro</i>Culture of Murine Secondary follicles in a Defined Synthetic Matrix

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Journal of Biomaterials and Nanobiotechnology 2018年第3期9卷 244-262页

作者： Mohammad Asaduzzman Xiaolin Cui Hu Zhang Fiona Young Medical Biotechnology Flinders University Adelaide Australia School of Chemical Engineering The University of Adelaide Adelaide Australia

Ovarian follicle growth in three dimensional (3D) matrices in vitro has limitations: a) matrices don’t expand as follicles grow, b) requirements for enzyme-mediated retrieval, and c) animal-derived components prevent clinical application. Therefore, we evaluated N-Isopropylacrylamide (SFX-1), a novel synthetic 3D culture matrix, for follicle culture. Groups of three murine secondary follicles were encapsulated in 50 μL of DMEM/F12-1%ITS-10%FCS (DMEM/F12) or SFX-1 (3:2 v/v DMEM/F12) or Matrigel (1:1 DMEM/F12) and cultured for 48 h. Matrigel contains growth factors but SFX-1 has no animal-derived factors. Each culture condition was examined in 6 wells containing 18 follicles, in four replicate experiments (n = 4). Photomicrographs were used to determine follicle diameters and morphological integrity. follicles were Live-Dead (LD) stained or disaggregated to generate cells for viability assessment using Trypan Blue (TB). Estradiol, progesterone and anti-mullerian hormone (AMH) in conditioned media were measured using Enzyme-linked Immunoassay. All culture conditions supported similar increases in follicle diameter. DMEM/F12 did not maintain morphological integrity which prevented follicle retrieval after 48 h;25% were retrieved from DMEM/F12, but 44% and 41% follicles were retrieved from SFX-1 and Matrigel respectively. follicles retrieved from Matrigel could not be disaggregated, which prevented TB viability assessment. LD estimations of viable cells/follicle were lower than TB, but culture conditions had no effect on viability;SFX-1 64% ± 8% and DMEM/F12 69% ± 9%. SFX-1 and Matrigel supported similar levels of progesterone synthesis, only Matrigel supported estrogen synthesis, but none of the culture conditions supported AMH production. SFX-1 was not cytotoxic and was comparable to Matrigel. Further development of SFX-1 for use with human follicles is supported.

关键词： Ovarian follicle Synthetic Matrix Matrigel Estradiol Progesterone Anti-Mullerian Hormone