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1.
J Assist Reprod Genet ; 37(9): 2137-2150, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32671735

RESUMO

PURPOSE: Our purpose was to identify human ovarian extracellular matrix (ECM) components that would support in vitro culture of human ovarian tissue and be compatible with possible future clinical applications. We characterized ovarian expression of laminins and selected three laminin tripeptides for culture experiments to be compared with Matrigel, an undefined and animal-based mixture of ECM components. METHODS: Expression of the 12 laminin genes was determined on transcript and protein levels using cortical tissue samples (n = 6), commercial ovary RNA (n = 1), follicular fluid granulosa cells (n = 20), and single-cell RNA-sequencing data. Laminin 221 (LN221), LN521, LN511, and their mixture were chosen for a 7-day culture experiment along with Matrigel using tissue from 17 patients. At the end of the culture, follicles were evaluated by scoring and counting from serial tissue sections, apoptosis measured using in situ TUNEL assay, proliferation by Ki67 staining, and endocrine function by quantifying steroids in culture media using UPLC-MS/MS. RESULTS: Approximately half of the cells in ovarian cortex expressed at least one laminin gene. The overall most expressed laminin α-chains were LAMA2 and LAMA5, ß-chains LAMB1 and LAMB2, and γ-chain LAMC1. In culture experiments, LN221 enhanced follicular survival compared with Matrigel (p < 0.001), whereas tissue cultured on LN521 had higher proportion of secondary follicles (p < 0.001). LN511 and mixture of laminins did not support the cultures leading to lower follicle densities and higher apoptosis. All cultures produced steroids and contained proliferating cells. CONCLUSIONS: LN221 and LN521 show promise in providing xeno-free growth substrates for human ovarian tissue cultures, which may help in further development of folliculogenesis in vitro for clinical practices. The system could also be used for identification of adverse effects of chemicals in ovaries.


Assuntos
Matriz Extracelular/química , Laminina/farmacologia , Ovário/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos , Adulto , Cromatografia Líquida , Colágeno/química , Colágeno/farmacologia , Meios de Cultura/farmacologia , Combinação de Medicamentos , Matriz Extracelular/genética , Feminino , Células da Granulosa , Humanos , Laminina/química , Pessoa de Meia-Idade , Folículo Ovariano , Ovário/efeitos dos fármacos , Proteoglicanas/química , Proteoglicanas/farmacologia , RNA-Seq , Análise de Célula Única , Espectrometria de Massas em Tandem
2.
Placenta ; 35(4): 260-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24559727

RESUMO

INTRODUCTION: We have previously identified sex-specific differences in the fetal-placental response to cortisol. Our recent studies suggest that this differential response to cortisol is driven by differences in glucocorticoid receptor (GR) protein function rather than through changes in gene transcription or protein expression. METHODS: This study was designed to define whether the human placenta expresses different isoforms of the GR and whether expression was altered by fetal sex and maternal asthma. Asthma and non-asthma pregnant women were prospectively recruited at their first antenatal visit and placentae collected at delivery. Placental GR expression was examined in relation to maternal asthma, fetal sex and birthweight. RESULTS: Twelve specific bands for the GR were identified at molecular weights of 94, 91, 81, 74, 69, 68, 65, 60, 55, 50, 48 and 38 kDa. The 12 isoforms were localised to the placental trophoblast and expression varied in relation to cellular location in either the cytoplasm or nucleus, fetal sex, fetal size and the presence and absence of maternal asthma. CONCLUSION: This is the first study to identify the presence of several protein isoforms of the GR in the human placenta. The data suggest glucocorticoid resistance observed in male placentae may be mediated through increased GRß, GR A and GR P localisation to the nucleus. While female placentae may be more sensitive to cortisol in the presence of maternal asthma through a decrease in GRß and an enhancement GRα activity via an interaction with GRα D3 and GRα C.


Assuntos
Asma/metabolismo , Placenta/metabolismo , Complicações na Gravidez/metabolismo , Receptores de Glucocorticoides/metabolismo , Caracteres Sexuais , Corticosteroides/uso terapêutico , Adulto , Asma/tratamento farmacológico , Estudos de Casos e Controles , Feminino , Retardo do Crescimento Fetal/metabolismo , Células HEK293 , Humanos , Hidrocortisona/sangue , Recém-Nascido , Masculino , Fosforilação , Gravidez , Complicações na Gravidez/tratamento farmacológico , Isoformas de Proteínas/metabolismo
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