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1.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802633

RESUMO

The current study was designed to investigate the protective role of diosmin against cyclophosphamide-induced premature ovarian insufficiency (POI). Female Swiss albino rats received a single intraperitoneal dose of cyclophosphamide (200 mg/kg) followed by 8 mg/kg/day for the next 15 consecutive days either alone or in combination with oral diosmin at 50 or 100 mg/kg. Histopathological examination of ovarian tissues, hormonal assays for follicle stimulating hormone (FSH), estradiol (E2), and anti-Mullerian hormone (AMH), assessment of the oxidative stress status, as well as measurement of the relative expression of miRNA-145 and its target genes [vascular endothelial growth factor B (VEGF-B) and regulator of cell cycle (RGC32)] were performed. Diosmin treatment ameliorated the levels of E2, AMH, and oxidative stress markers. Additionally, both low and high diosmin doses significantly reduced the histopathological alterations and nearly preserved the normal ovarian reserve. MiRNA-145 expression was upregulated after treatment with diosmin high dose. miRNA-145 target genes were over-expressed after both low and high diosmin administration. Based on our findings, diosmin has a dose-dependent protective effect against cyclophosphamide-induced ovarian toxicity in rats.


Assuntos
Diosmina/uso terapêutico , Insuficiência Ovariana Primária/induzido quimicamente , Insuficiência Ovariana Primária/tratamento farmacológico , Animais , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Caspase 3/metabolismo , Catalase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Colágeno/metabolismo , Ciclofosfamida , Diosmina/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/sangue , Malondialdeído/sangue , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/patologia , Estresse Oxidativo/efeitos dos fármacos , Insuficiência Ovariana Primária/sangue , Insuficiência Ovariana Primária/patologia , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ratos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
2.
Medicine (Baltimore) ; 100(17): e25361, 2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33907092

RESUMO

ABSTRACT: We aimed to investigate ovarian reserve status, and explore differences in ovarian reserve between fertile and infertile healthy Chinese women of reproductive age.We recruited 442 fertile women aged 23 to 49 years (mean: 35.22 ±â€Š4.91 years) as subjects, and 196 infertile women aged 23 to 46 years (mean: 32.34 ±â€Š4.34 years) as controls. For all participants, a number of parameters were tested on days 2 to 4 of a spontaneous cycle, including basal serum follicle-stimulating hormone (FSH), estradiol (E2), luteinizing hormone (LH), total testosterone, anti-Müllerian hormone (AMH), ovarian response prediction index (ORPI), and antral follicle count (AFC).There were significant differences in terms of AFC, serum AMH levels, and ORPI among subject subgroups (10.58 ±â€Š5.80; 2.533 ±â€Š2.146 ng/mL; 1.28 ±â€Š1.87; respectively), and among control subgroups (12.44 ±â€Š5.69; 3.189 ±â€Š2.551 ng/mL; 1.88 ±â€Š2.68; respectively) (P < .01 for all). For both subjects and controls, AFC, AMH levels, and ORPI decreased gradually with increasing age, and presented with similar age-related trends; there were positive correlations between AMH and AFC (P < .001), and negative correlations between age and AFC, AMH, ORPI (P < .05 for all). There was a significant difference in age (P < .001), serum E2 (P < .01), and AMH (P < .01) levels between subjects and controls; however, when controlling for confounding factors (age, body mass index, total testosterone, and LH), we found no differences between the 2 groups with regards to the serum levels of AMH, FSH, E2, and AFC (P > .05 for all). Moreover, receiver operating characteristic curve analysis indicated that the significant variables of subjects and controls for evaluating ovarian reserve included age, AMH and ORPI, and ORPI was more valuable than other variables.A diminished ovarian reserve was one of the manifestations caused by female aging. When confounding factors were controlled for, we found no differences in ovarian reserve when compared between fertile and infertile women, and no correlation with infertility.


Assuntos
Envelhecimento/fisiologia , Fertilidade/fisiologia , Infertilidade Feminina/fisiopatologia , Reserva Ovariana/fisiologia , Adulto , Fatores Etários , Hormônio Antimülleriano/sangue , China , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Infertilidade Feminina/sangue , Hormônio Luteinizante/sangue , Pessoa de Meia-Idade , Folículo Ovariano/crescimento & desenvolvimento , Curva ROC , Testosterona/sangue , Adulto Jovem
3.
Methods Mol Biol ; 2273: 75-84, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33604845

RESUMO

The technological revolution in reproductive biology that started with artificial insemination procedures and embryo transfer led to the development of assisted reproduction techniques such as in vitro fertilization or even cloning of domestic animals by nuclear transfer from somatic cells. Currently, procedures of isolated immature ovarian follicles in vitro culture are becoming the prominent technology aimed to preserve or restore fertility especially of young oncological patients or those at risk of premature ovarian failure.Here, we describe a protocol that can be applied for in vitro growth of porcine, preantral ovarian follicles in three-dimensional (3D) culture conditions. After enzymatic isolation from the ovarian cortex, preantral follicles are suspended in a drop of medium and enclosed with fluorinated ethylene propylene (FEP) powder particles (microbioreactors). Such microbioreactors maintain the 3D structure of the follicles during the whole process of in vitro growth what is crucial to ensure proper folliculogenesis progression and their ability to survive.


Assuntos
Técnicas de Cultura de Células/métodos , Fertilização In Vitro/métodos , Folículo Ovariano/crescimento & desenvolvimento , Animais , Meios de Cultura/química , Transferência Embrionária/métodos , Feminino , Humanos , Oócitos/citologia , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/citologia , Suínos
4.
Poult Sci ; 100(1): 325-333, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33357697

RESUMO

Avian reproductive behavior is regulated through the neuroendocrine system. The transition from laying to brooding is strictly controlled by the hypothalamus-pituitary-gonadal (HPG) axis. Cross talk on the HPG axis relies on the circulatory system, where the dynamics of serum proteins can be observed during different reproductive phases. Some canonical hormones, such as prolactin and luteinizing hormone, play important roles in the transition through reproductive phases. However, little is known at the whole-proteome level. To discover novel serum proteins, we employed isobaric tags for relative and absolute quantification to assay the serum proteome during different reproductive phases in chicken. We identified a total of 1,235 proteins from chicken serum; 239 of these proteins showed differential expression between the laying and brooding stages, including a low concentration of steroid metabolism-related proteins and a high concentration of calcium signaling-related proteins (fold change ≥1.5 or ≤0.66; P < 0.05). Pathway analysis and protein-protein interaction networks predicated the difference in follicle development between the brooding stage and laying stages and were related to the 14-3-3 protein family, which is associated with oocyte meiosis and maturation. Together, these results provided a proteomics foundation for investigating the dynamic changes taking place in the circulatory system during reproductive phase transition, and also uncovered new insights regarding follicle development that underlie the avian reproductive cycle.


Assuntos
Proteínas Aviárias , Galinhas , Folículo Ovariano , Proteoma , Reprodução , Animais , Proteínas Aviárias/genética , Galinhas/genética , Feminino , Folículo Ovariano/crescimento & desenvolvimento , Proteômica , Reprodução/genética
5.
PLoS Biol ; 18(12): e3001025, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33351795

RESUMO

Primordial follicle assembly in the mouse occurs during perinatal ages and largely determines the ovarian reserve that will be available to support the reproductive life span. The development of primordial follicles is controlled by a complex network of interactions between oocytes and ovarian somatic cells that remain poorly understood. In the present research, using single-cell RNA sequencing performed over a time series on murine ovaries, coupled with several bioinformatics analyses, the complete dynamic genetic programs of germ and granulosa cells from E16.5 to postnatal day (PD) 3 were reported. Along with confirming the previously reported expression of genes by germ cells and granulosa cells, our analyses identified 5 distinct cell clusters associated with germ cells and 6 with granulosa cells. Consequently, several new genes expressed at significant levels at each investigated stage were assigned. By building single-cell pseudotemporal trajectories, 3 states and 1 branch point of fate transition for the germ cells were revealed, as well as for the granulosa cells. Moreover, Gene Ontology (GO) term enrichment enabled identification of the biological process most represented in germ cells and granulosa cells or common to both cell types at each specific stage, and the interactions of germ cells and granulosa cells basing on known and novel pathway were presented. Finally, by using single-cell regulatory network inference and clustering (SCENIC) algorithm, we were able to establish a network of regulons that can be postulated as likely candidates for sustaining germ cell-specific transcription programs throughout the period of investigation. Above all, this study provides the whole transcriptome landscape of ovarian cells and unearths new insights during primordial follicle assembly in mice.


Assuntos
Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ovário/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas , Células da Granulosa/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Folículo Ovariano/fisiologia , Ovário/citologia , Gravidez , Análise de Célula Única/métodos , Transcriptoma/genética
6.
Ecotoxicol Environ Saf ; 204: 110973, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32781346

RESUMO

Arsenic (As) exerts a wide range of adverse effects on biological systems, including the reproductive organs in males and females. However, the mechanisms of As-induced reproductive toxicity are mostly obscure. Recently, we showed that autophagy is an essential route for As2O3-induced reprotoxicity through the hypothalamic-pituitary-gonadal-sperm (HPG-S) axis in pubertal and matured F1-male mice. However, the role of autophagy in As2O3- induced ovarian toxicity is mostly unknown. Hence, this study aimed to elucidate the role of oxidative stress, mitochondrial impairment, and autophagic processes in the ovary of As-exposed female mice. For this purpose, mature female mice were challenged with 0, low (0.2), medium (2), and high (20 ppm) As2O3 from 35-days before mating till weaning their pups, and the F1- females from weaning until maturity. Then, all the mice were sacrificed, and oxidative stress parameters, mitochondrial indices, electron microscopic evaluation of the ovaries, expression of autophagic-related genes and proteins, and autophagosome formation were assessed. It was shown that medium and high As2O3 doses were a potent inducer of oxidative stress, mitochondrial dysfunction, and autophagy in the ovary of F1-generation. A dose-dependent increment in the gene expression of PDK1, PI3K, TSC2, AMPK, ULK1, ATG13, Beclin1, ATG12, ATG5, LC3, P62, ATG3, ATG7, and p62, as well as protein expression of Beclin1, and LC3- I, II, was evident in the ovaries of the As-treated animals. Moreover, a dose-dependent decrease in the expression of mTOR and Bcl-2 genes, and mTOR protein was detected with increasing doses of As, suggesting that As treatment-induced autophagy. Along with a dose-dependent increase in the number of MDC-labeled autophagic vacuoles, transmission electron microscopy also confirmed more autophagosomes and injured mitochondria in medium and high As2O3 doses groups. As2O3 also negatively affected the mean body weight, litter size, organ coefficient, and stereological indices in female mice. Finally, in physiological conditions, arsenic trioxide (As2O3) leads to an increased level of autophagy in the oocyte when many oocytes were being lost. These findings indicated that an imbalance in the oxidant-antioxidant system, mitochondrial impairment, and the autophagic process, through inhibition of mTOR, dependent and independent pathways, and Bcl-2, as well as activation of AMPK/PI3K/Beclin1/LC3 routes, could play a pivotal role in As-induced reproductive toxicity through ovarian dysfunction in females.


Assuntos
Arsênico/toxicidade , Autofagia/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Folículo Ovariano/crescimento & desenvolvimento , Ovário/ultraestrutura , Distribuição Aleatória
7.
Arq. bras. med. vet. zootec. (Online) ; 72(3): 879-888, May-June, 2020. tab, graf
Artigo em Português | LILACS, VETINDEX | ID: biblio-1129561

RESUMO

Dez éguas, sem raça definida, foram submetidas a avaliações ultrassonográficas durante o intervalo interovulatório, avaliando-se folículos ≥ 5mm. Cinco éguas foram tratadas com 500mg de r-bST no primeiro e no 14º dia pós-ovulação (grupo GT), e as demais com soro fisiológico (grupo GC). Quando o folículo dominante atingiu diâmetro ≥ 40mm, foram induzidas com hCG e inseminadas 24 horas após, sendo submetidas à coleta de embrião seis dias após a ovulação. Os dados foram agrupados de acordo com o diâmetro do folículo dominante nas fases de emergência, divergência, dominância, pré-ovulatória, indução, inseminação e ovulação. Todas as éguas foram usadas duas vezes, no mesmo grupo. O GT apresentou crescimento folicular precoce para as fases de emergência, divergência, dominância e pré-ovulatória, assim como para o seu maior folículo subordinado, que cresceu mais cedo. As taxas de recuperação foram de 90% (GC) e 70% (GT), em 16 estruturas coletadas, obtendo-se uma não fecundada e um blastocisto inicial para o grupo GC; os demais, no estágio de mórula, apresentaram comportamento semelhante entre os grupos. Conclui-se que a r-bST influencia a dinâmica folicular de éguas, levando a uma antecipação do desenvolvimento folicular, que pode ser utilizada para encurtar o ciclo estral.(AU)


Ten undefined mare breeds were submitted to ultrasonographic evaluations during the interovulatory interval, evaluating follicles measuring ≥ 5mm. Five mares were treated with 500mg r-bST on the first and the 14th day after ovulation (TG group), and the others with saline (CG group). When the dominant follicle reached a diameter ≥ 40mm the ovulation was induced with hCG, and the mares were inseminated 24 hours later and submitted to embryo collection six days after ovulation. The data were grouped according to the diameter of the dominant follicle in the emergence, divergence, dominance, preovulatory, induction, insemination and ovulation phases. All mares were used twice, in the same group. The GT showed early follicular growth for the emergence, divergence, dominance and pre-ovulatory phases, as well as for its greater subordinate follicle, growing earlier. The recovery rates were 90% (CG) and 70% (TG), and 16 structures were collected, obtaining an unfertilized embryo and an initial blastocyst for the CG group, the others in the morula stage behaved similarly between the groups. It can be concluded that r-bST influences the follicular dynamics of the mares, leading to an anticipation of the follicular development that can be used to shorten the estrous cycle.(AU)


Assuntos
Animais , Feminino , Gravidez , Proteínas Recombinantes/análise , Hormônio do Crescimento/análise , Desenvolvimento Embrionário , Folículo Ovariano/crescimento & desenvolvimento , Cavalos/embriologia , Ultrassonografia/veterinária
8.
Poult Sci ; 99(5): 2757-2765, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32359613

RESUMO

In the present study, for the purpose of investigating the effects of the total flavonoids of Epimedium (TFE) in regard to preventing the development of atrophied oviducts and follicles induced by forced molting, 300-day-old Hy-Line Brown layer hens were divided into 3 study groups as follows: the control (CON) group was the normal group, without forced molting and TFE treatments; the TFE1 group was treated by adding a 1‰ TFE treatment after forced molting; and the TFE0 group was not treated by TFE after forced molting. During this study's experimental process, the egg production rates were recorded each day. In addition, the hens were randomly chosen to be weighed every 4 D and also randomly selected to be sacrificed every 7 D. Then, sample tissues of albumen-secreting part and uterus from the fallopian tube of the layer hens were collected for PCR and hematoxylin-eosin staining tests. The results showed that the body weights, number of follicles, and weights and sizes of the fallopian tube for the TFE1 and TFE0 groups were significantly reduced when compared with those of the control group on the 15th D of the experiment. Furthermore, at the end of study, it was found that the egg production rates, weights of the fallopian tube, and ovarian follicles of TFE1 had recovered to normal levels. At the same time, the serum estrogen and the expressions of the progesterone receptor and estrogen receptor mRNA in fallopian tube were higher than those observed for the TFE0 group. The results of this study provided valuable evidence that TFE could improve the development of atrophied oviducts and increase the egg laying rates, thereby making it a potential multicomponent natural drug for egg production in the future.


Assuntos
Proteínas Aviárias/metabolismo , Galinhas/metabolismo , Epimedium/química , Tubas Uterinas/efeitos dos fármacos , Flavonoides/metabolismo , Folículo Ovariano/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Animais , Estrogênios/metabolismo , Tubas Uterinas/crescimento & desenvolvimento , Feminino , Flavonoides/administração & dosagem , Muda , Folículo Ovariano/crescimento & desenvolvimento , RNA Mensageiro/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo
9.
PLoS One ; 15(5): e0232819, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469908

RESUMO

Follicular atresia is a cell death event that occurs in the great majority of follicles before ovulation in the mature mammalian ovary. Germ cell loss has been mainly associated to apoptosis although autophagy also seems to be at play. Aimed to increase our understanding on the possible cooperating role of autophagy and apoptosis in follicular atresia and/or follicular survival, we analyzed both programmed cell death mechanisms in a rodent model, the South American plains vizcacha, Lagostomus maximus. Female vizcacha shows highly suppressed apoptosis-dependent follicular atresia in the adult ovary, with continuous folliculogenesis and massive polyovulation. This strategy of massive ovulation requires a permanent remodeling of the ovarian architecture to maintain the availability of quiescent primordial follicles throughout the individual's reproductive lifespan. We report here our analysis of autophagy (BECN1, LAMP1 and LC3B-I/II) and apoptosis (BCL2 and ACTIVE CASPASE-3) markers which revealed interactive behaviors between both processes, with autophagy promoting survival or cell death depending on the ovarian structure. Strong BECN1, LC3B-II and LAMP1 staining was observed in atretic follicles and degenerating corpora lutea that also expressed nuclear ACTIVE CASPASE-3. Healthy follicles showed a slight expression of autophagy proteins but a strong expression of BCL2 and no detectable ACTIVE CASPASE-3. Transmission electron microscopy revealed a high formation of autophagosomes, autolysosomes and lysosomes in atretic follicles and degenerating corpora lutea and a low number of autophagic vesicles in normal follicles. The co-expression of LC3B-BECN1, LC3B-LAMP1 and LC3B-ACTIVE CASPASE-3 was only detected in atretic follicles and degenerating corpora lutea, while co-expression of BCL2-BECN1 was only observed in normal follicles. We propose that autophagy could act as a mechanism to eliminate altered follicles and remnant corpora lutea providing the necessary space for maturation of primordial follicles that continuously enter the growing follicular pool to sustain massive ovulation.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Apoptose/genética , Autofagia/genética , Roedores/genética , Animais , Autofagossomos/metabolismo , Corpo Lúteo/crescimento & desenvolvimento , Corpo Lúteo/metabolismo , Feminino , Atresia Folicular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , Roedores/crescimento & desenvolvimento
10.
Poult Sci ; 99(4): 2185-2195, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32241504

RESUMO

The signal pathway of target of rapamycin (TOR) plays an important role in regulating cell growth and proliferation, follicular development, and ovulation. Melatonin (N-acetyl-5-methoxytryptamine) (MT) is involved in the regulation of many physiological functions in animals. Recent studies have shown that MT affects the number and the degree of maturation of follicles in the ovary, but there are few studies concerning its mechanism. Therefore, the aim of this study was to investigate the mechanism of TOR signal pathway in the regulation of ovarian function by MT in aging laying hens. In the present study, a total of 60 hens (70-week-old) were randomly divided into 2 groups: control group and melatonin group (M). Melatonin was administered intraperitoneally at a dose of 20 mg/kg/D for 28 D in the M group. The results showed that MT significantly increased the levels of the antioxidant enzymes superoxide dismutase and total antioxidant capacity (P < 0.01) as well as levels of immunoglobulin (IgA, IgG, and IgM) (P < 0.05) and the reproductive hormones estradiol and luteinizing hormone (P < 0.01) in the plasma and also increased the numbers of middle white follicles and small white follicles (P < 0.05) and decreased the level of reactive oxygen species in plasma (P < 0.01) in laying hens. There were higher expression levels in MT receptor A (P < 0.05), melatonin receptor B (P < 0.01), and tuberous sclerosis complex 2 (P < 0.01). Activation of TOR, 4E binding protein-l (4E-BP1), and ribosomal protein 6 kinase (P < 0.01) was found in the M. The levels of mTOR and p-mTOR protein were increased in the M (P < 0.05). The mTORC1-dependent 4E-BP1 and p-4E-BP1 were increased in the M (P < 0.05). This study indicated that MT may enhance follicle growth by increasing levels of antioxidant enzymes and reproductive hormones and by activating the mTOR and downstream components in aging laying hens.


Assuntos
Antioxidantes/farmacologia , Galinhas/fisiologia , Melatonina/farmacologia , Folículo Ovariano/crescimento & desenvolvimento , Ovário/fisiologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Antioxidantes/administração & dosagem , Proteínas Aviárias/metabolismo , Feminino , Injeções Intraperitoneais/veterinária , Melatonina/administração & dosagem , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Distribuição Aleatória
11.
Endocrinology ; 161(4)2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32141513

RESUMO

Follicle-stimulating hormone (FSH)-induced growth of ovarian follicles is independent of follicular vascularization. Recent evidence has indicated that follicular vascularization is critical to ovarian follicle development and survival. FSH, a gonadotropin that induces follicular growth and development, also acts as the major survival factor for antral follicles. FSH has been reported to stimulate angiogenesis in the theca layers mediated in part by the vascular endothelial growth factor A (VEGFA) and the transcription factor hypoxia inducible factor 1α (HIF-1α). However, it remains largely undetermined whether FSH-dependent growth and survival of antral follicles relies on FSH-induced vascularization. Here, we first demonstrated that induction of angiogenesis through the FSH-HIF-1α-VEGFA axis is not required for FSH-stimulated follicular growth in mouse ovary. FSH increased the total number of blood vessels in mouse ovarian follicles, which was correlated with elevated expression of VEGFA and HIF-1α in granulosa cells. In contrast, blocking of follicular angiogenesis using inhibitors against the HIF-1α-VEGFA pathway repressed vasculature formation in follicles despite FSH administration. Interestingly, by measuring follicular size and ovarian weight, we found that the suppression of angiogenesis via HIF-1α-VEGFA pathway did not influence FSH-mediated follicular growth. However, inhibition of FSH-induced follicular vascularization by PX-478, a small-molecule inhibitor that suppresses HIF-1α activity, blocked ovulation and triggered atresia in large follicles. On the other hand, PX-478 injection reduced oocyte quality via impairing the meiotic apparatus, showing a prominently defective spindle assembly and actin dynamics. Collectively, our findings unveiled a vascularization-independent effect of FSH on follicular growth, whereas follicular survival, ovulation, and oocyte development relies on FSH-mediated angiogenesis in the follicles.


Assuntos
Hormônio Foliculoestimulante/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/crescimento & desenvolvimento , Ovulação/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Feminino , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovulação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
12.
Sci Rep ; 10(1): 2300, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32042028

RESUMO

MicroRNAs (miRNAs) are known post-transcriptional regulators of various biological processes including ovarian follicle development. We have previously identified miRNAs from human pre-ovulatory ovarian granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. The present study aims to identify the target genes regulated by these miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were transfected into KGN cell line and the gene expression changes were analyzed by Affymetrix microarray. Potential miRNA-regulated genes were further filtered by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. LIFR, PTEN, NEO1 and SP110 were confirmed as targets for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM19, PXDN and FMNL3 also passed all verification steps. Additionally, the expression pattern of the miRNAs was studied in human primary cumulus granulosa cell culture in relation to the expression of their host genes and FSH stimulation. Based on our findings we propose the involvement of hsa-miR-548ba in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions by regulating the expression of its identified targets.


Assuntos
Células do Cúmulo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Adulto , Aromatase/genética , Linhagem Celular Tumoral , Feminino , Hormônio Foliculoestimulante/metabolismo , Perfilação da Expressão Gênica , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/genética , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Folículo Ovariano/citologia , PTEN Fosfo-Hidrolase/genética , Cultura Primária de Células , Receptores do FSH/genética , Adulto Jovem
13.
Anim Reprod Sci ; 213: 106277, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31987328

RESUMO

Although detrimental effects of heat stress on antral follicle development have been well studied, long-term effects - affecting the preantral follicle pool - are still largely unknown. The goal of this study was to evaluate effects of heat stress on growth, viability, gene expression and ATP production of preantral follicles of cattle. Follicles at the primary, early secondary and secondary stages were isolated from cattle ovaries and individually cultured while imposing physiological (CON; 38.5 °C) or intermittent heat stress (HS; 38.5 °C for 16 h and 41 °C for 8 h daily) conditions for 7 days. Individual follicles were subjected to real-time qPCR for determination of relative abundance of BAX, HSPA1A and SOD1 mRNA transcripts and evaluated for ATP production. Treatment for 7 days with intermittent HS decreased viability (P =  0.01) and diameter (P =  0.03) of preantral follicles. Relative abundances of BAX and HSPA1A mRNA transcripts were greater in follicles of the CON and HS groups that became non-viable during culture (P <  0.05); relative abundance of SOD1 mRNA transcript, however, was only greater in non-viable follicles of the HS group (P <  0.05), but not non-viable follicles of the CON group (P =  0.3). The ATP production was not different between viable follicles of the CON and HS group (P = 0.86). In conclusion, all stages of growing preantral follicles of cattle were susceptible to negative effects of heat stress. Follicles at the secondary stage of development were most sensitive, followed by early secondary and primary follicles.


Assuntos
Bovinos , Resposta ao Choque Térmico , Folículo Ovariano/crescimento & desenvolvimento , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Técnicas de Cultura de Tecidos
14.
BMC Cancer ; 20(1): 67, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996163

RESUMO

BACKGROUND: We previously reported that tamoxifen (TAM)-induced ovarian hyperstimulation (OHS) is associated with high serum concentrations of estradiol in premenopausal women with breast cancer. To investigate risk factors for TAM-induced OHS, we performed a retrospective multicenter study. METHODS: Premenopausal patients who received surgical therapy for endocrine-dependent breast cancer (n = 235) were recruited in this study and classified into 4 groups: group A, treated with TAM alone; group B, TAM treatment after 2-year-combined therapy with a gonadotropin-releasing hormone (Gn-RH) agonist; group C, TAM treatment after chemotherapy; group D, 5-year-combined therapy with TAM and a Gn-RH agonist. A serum estradiol value of more than 300 pg/mL or mean follicular diameter of more than 30 mm was defined as OHS. RESULTS: The incidence of OHS in group A (n = 13/26, 50.0%) was significantly higher than those in group B (n = 17/63, 27.0%), group C (n = 20/110, 18.2%), and group D (n = 0/36, 0%). The incidence of OHS was significantly correlated with aging, and the median serum concentration of estradiol in the presence of OHS was 823.0 pg/mL. The incidence of OHS (less than 47 years old) was 62.5% in group A, 48.6% in group B, and 28.2% in group C, respectively. Notably, the incidence rate of OHS following amenorrhea in group C (n = 13/20, 65.0%) was significantly higher than that in group B (n = 1/17, 5.9%). CONCLUSIONS: These findings indicate that the onset of OHS following amenorrhea was common in the post-chemotherapeutic group, while its ratio was low in the group after Gn-RH analog treatment, suggesting that combined treatment-based management involving TAM therapy is necessary for premenopausal patients with breast cancer.


Assuntos
Antineoplásicos Hormonais/efeitos adversos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovário/efeitos dos fármacos , Ovário/metabolismo , Pré-Menopausa , Tamoxifeno/efeitos adversos , Adulto , Fatores Etários , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/fisiopatologia , Esquema de Medicação , Estradiol/sangue , Feminino , Humanos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Estadiamento de Neoplasias , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , Estudos Retrospectivos , Tamoxifeno/administração & dosagem , Tamoxifeno/uso terapêutico
15.
Sci Rep ; 10(1): 242, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937877

RESUMO

Phloroglucinol (1,3,5 tri-hydroxy-benzene) (PGL), a natural phenolic substance, is a peroxidase inhibitor and has anti-oxidant, anti-diabetic, anti-inflammatory, anti-thrombotic, radio-protective, spasmolytic and anti-cancer activities. PGL, as a medicine, is administered to patients to control the symptoms of irritable bowel syndrome and acute renal colic, in clinical trials. PGL, as a phenolic substance, can cause cytotoxic effects. Administration of PGL up to 300 mg/kg (bw) is well tolerated by animals, while in cell lines its toxicity is developed at concentrations above the dose of 10 µg/ml. Furthermore, it seems that tumor or immortalized cells are more susceptible to the toxic power of PGL, than normal cells. However, studies of its cytotoxic potency, at the cellular level, in complex, differentiated and meta-mitotic biological systems, are still missing. In the present work, we have investigated the toxic activity of PGL in somatic epithelial cells, constituting the follicular compartment of a developing egg-chamber (or, follicle), which directs the choriogenesis (i.e. chorion assembly) process, during late oogenesis of Drosophila melanogaster. Our results reveal that treatment of in vitro growing Drosophila follicles with PGL, at a concentration of 0.2 mM (or, 25.2 µg/ml), does not lead to follicle-cell toxicity, since the protein-synthesis program and developmental pattern of choriogenesis are normally completed. Likewise, the 1 mM dose of PGL was also characterized by lack of toxicity, since the chorionic proteins were physiologically synthesized and the chorion structure appeared unaffected, except for a short developmental delay, being observed. In contrast, concentrations of 10, 20 or 40 mM of PGL unveiled a dose-dependent, increasing, toxic effect, being initiated by interruption of protein synthesis and disassembly of cell-secretory machinery, and, next, followed by fragmentation of the granular endoplasmic reticulum (ER) into vesicles, and formation of autophagic vacuoles. Follicle cells enter into an apoptotic process, with autophagosomes and large vacuoles being formed in the cytoplasm, and nucleus showing protrusions, granular nucleolus and condensed chromatin. PGL, also, proved able to induce disruption of nuclear envelope, activation of nucleus autophagy (nucleophagy) and formation of a syncytium-like pattern being produced by fusion of plasma membranes of two or more individual follicle cells. Altogether, follicle cell-dependent choriogenesis in Drosophila has been herein presented as an excellent, powerful and reliable multi-cellular, differentiated, model biological (animal) system for drug-cytotoxicity assessment, with the versatile compound PGL serving as a characteristic paradigm. In conclusion, PGL is a substance that may act beneficially for a variety of pathological conditions and can be safely used for differentiated somatic -epithelial- cells at clinically low concentrations. At relatively high doses, it could potentially induce apoptotic and autophagic cell death, thus being likely exploited as a therapeutic agent against a number of pathologies, including human malignancies.


Assuntos
Córion/efeitos dos fármacos , Córion/crescimento & desenvolvimento , Drosophila melanogaster/embriologia , Floroglucinol/toxicidade , Animais , Relação Dose-Resposta a Droga , Drosophila melanogaster/efeitos dos fármacos , Feminino , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Testes de Toxicidade
16.
Toxicol Appl Pharmacol ; 388: 114875, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31884101

RESUMO

Phthalates are used as solvents and plasticizers in a wide variety of consumer products. Most people are exposed to phthalates as parent compounds through ingestion, inhalation, and dermal contact. However, these parent compounds are quickly metabolized to more active compounds in several tissues. Although studies indicate that phthalate metabolites reach the ovary, little is known about whether they are ovarian toxicants. Thus, this study tested the hypothesis that phthalate metabolites influence the expression of genes involved in sex steroid synthesis, cell cycle regulation, cell death, oxidative stress, and key receptors, as well as production of sex steroid hormones by mouse antral follicles. The selected metabolite mixture consisted of 36.7% monoethyl phthalate (MEP), 19.4% mono(2-ethylhexyl) phthalate (MEHP), 15.3% monobutyl phthalate (MBP), 10.2% monoisobutyl phthalate (MiBP), 10.2% monoisononyl phthalate (MiNP), and 8.2% monobenzyl phthalate (MBzP). Antral follicles from adult CD-1 mice were cultured for 96 h with vehicle control (DMSO) or metabolite mixture (0.065-325 µg/mL). Growth of follicles in culture was monitored every 24 h. Total RNA was isolated after 24 and 96 h and used for gene expression analysis. Media were collected and subjected to hormone analysis. Exposure to the phthalate mixture inhibited follicle growth, decreased expression of steroidogenic enzymes, and altered the levels of sex steroids relative to control. The mixture, primarily at the two highest doses, also altered expression of cell cycle regulators, apoptotic factors, oxidative stress genes, and some receptors. Collectively, these data suggest that mixtures of phthalate metabolites can directly impact follicle health.


Assuntos
Exposição Ambiental/efeitos adversos , Hormônios Esteroides Gonadais/biossíntese , Folículo Ovariano/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Animais , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Feminino , Perfilação da Expressão Gênica , Camundongos , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Ácidos Ftálicos/metabolismo , Técnicas de Cultura de Tecidos , Testes de Toxicidade Aguda/métodos
17.
Environ Toxicol Chem ; 39(3): 667-677, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31877584

RESUMO

Monitoring the adverse effects of environmental contaminants on the reproduction of invertebrate species in the field remains a challenge in aquatic ecotoxicology. To meet the need for reliable tools for in situ toxicity assessment, we present the first part of a methodological study of the in situ implementation of a reproductive bioassay in Gammarus previously developed for screening the toxicity of chemical compounds during laboratory exposure. To ensure the correct interpretation of the modulation of reproductive markers (molting, fecundity, follicle growth, and embryonic development) in uncontrolled environmental conditions, we experimentally assessed and statistically modeled the variability in the female reproductive cycle during laboratory exposure under several temperature and water hardness conditions. Whereas water hardness did not influence the reproductive cycle, the significant accelerating effect of temperature on the dynamics of molting and marsupial development was finely modeled, by detailing the influence of temperature on the probability of transition between all molt and embryonic stages along the female cycle. In addition, no effect of temperature or water hardness was detected on the number of oocytes and embryos carried by females. Furthermore, the finding that the relative durations of the first 4 molt and embryonic stages are constant whatever the temperature makes it possible to predict the molting dynamics in fluctuating temperature conditions. Because this could allow us to take into account the confounding influence of temperature on the measurement of reproductive markers, the implications of these findings for an optimal in situ implementation of the reproductive bioassay with G. fossarum are discussed. The relevance of this modeling approach during in situ implementation is tested in a companion study. Environ Toxicol Chem 2020;39:667-677. © 2019 SETAC.


Assuntos
Anfípodes/efeitos dos fármacos , Bioensaio/métodos , Temperatura , Água/química , Anfípodes/embriologia , Anfípodes/crescimento & desenvolvimento , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Fertilidade/efeitos dos fármacos , Muda/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Reprodução/efeitos dos fármacos
18.
J Morphol ; 281(1): 47-54, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710388

RESUMO

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended "postfertilization" activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.


Assuntos
Desenvolvimento Embrionário , Epitélio/embriologia , Insetos/embriologia , Morfogênese , Folículo Ovariano/crescimento & desenvolvimento , Viviparidade não Mamífera , Animais , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/ultraestrutura , Epitélio/ultraestrutura , Feminino , Insetos/ultraestrutura , Folículo Ovariano/ultraestrutura
19.
Domest Anim Endocrinol ; 70: 106398, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31677486

RESUMO

The aim of this study was to investigate some of the growth and transcriptional factors originating from oocytes and granulosa cells in follicular fluid and to identify the relationships between the basic blood metabolite-metabolic hormones and intrafollicular lipopolysaccharide (LPS) concentrations. Thirty cows included in the study were allocated into 2 groups comprising 15 cows with healthy preovulatory follicles (cyclic cows) and 15 cows with confirmed cystic follicles. The ovaries and uteri of all cows were assessed by transrectal ultrasonographic examination. Blood serum samples were collected at 15, 25, 35, 45, and 55 d after calving for analysis of nonesterified fatty acids, ß-hydroxybutyrate, insulin, glucose, IGF-I, ACTH, and cortisol. Ovaries and uteri were examined using transrectal ultrasound. Vaginal discharge was evaluated on the same days. Follicular fluid was also aspirated on days 35-55 from the healthy preovulatory follicles and cystic follicles using a transvaginal ovum pickup method. The densitometric levels of inhibin-α, growth and differentiation factor (GDF-9), bone morphogenetic protein (BMP-6), and GATA-4 and GATA-6 proteins were analyzed by the Western blotting technique; the concentrations of antimullerian hormone (AMH), IGF-I, estradiol-17 beta (E2), and progesterone (P4) were determined by ELISA; and the concentrations of LPS in the follicular fluid were measured by the Limulus amebocyte lysate test. The serum insulin, ACTH, and cortisol concentrations were higher in cystic cows than cyclic cows, but serum IGF-I concentrations were lower in cystic cows. The IGF-I concentrations of cystic follicular fluids were lower, whereas AMH levels were significantly greater than those of healthy preovulatory follicular fluids. The cystic follicles had significantly lower expression levels of GDF-9, BMP-6, GATA-4, and GATA-6; in contrast, inhibin-α expression and LPS concentrations were significantly higher than in healthy preovulatory follicles. The proportion of pathologic vaginal discharge within 25 d postpartum in cystic cows were higher than in the cyclic group. In conclusion, it is suggested that intrafollicular dysregulation of the transforming growth factor-ß superfamily, growth, and transcriptional factors is affected by high intrafollicular LPS concentrations and systemic metabolic changes and these disturbances may be responsible for the generation of ovarian cysts.


Assuntos
Doenças dos Bovinos/metabolismo , Lipopolissacarídeos/metabolismo , Cistos Ovarianos/veterinária , Folículo Ovariano/crescimento & desenvolvimento , Proteínas da Superfamília de TGF-beta/metabolismo , Fatores de Transcrição/metabolismo , Animais , Glicemia , Bovinos , Doenças dos Bovinos/sangue , Feminino , Regulação da Expressão Gênica , Cistos Ovarianos/metabolismo , Proteínas da Superfamília de TGF-beta/genética , Fatores de Transcrição/genética
20.
Cell Mol Life Sci ; 77(6): 1177-1196, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31327046

RESUMO

In mammalian ovaries, the theca layers of growing follicles are critical for maintaining their structural integrity and supporting androgen synthesis. Through combining the postnatal monitoring of ovaries by abdominal magnetic resonance imaging, endocrine profiling, hormonal analysis of the follicular fluid of growing follicles, and transcriptomic analysis of follicular theca cells, we provide evidence that the exposure of ovine fetuses to testosterone excess activates postnatal follicular growth and strongly affects the functions of follicular theca in adulthood. Prenatal exposure to testosterone impaired androgen synthesis in the small antral follicles of adults and affected the expression in their theca cells of a wide array of genes encoding extracellular matrix components, their membrane receptors, and signaling pathways. Most expression changes were uncorrelated with the concentrations of gonadotropins, steroids, and anti-Müllerian hormone in the recent hormonal environment of theca cells, suggesting that these changes rather result from the long-term developmental effects of testosterone on theca cell precursors in fetal ovaries. Disruptions of the extracellular matrix structure and signaling in the follicular theca and ovarian cortex can explain the acceleration of follicle growth through altering the stiffness of ovarian tissue. We propose that these mechanisms participate in the etiology of the polycystic ovarian syndrome, a major reproductive pathology in woman.


Assuntos
Síndrome do Ovário Policístico/metabolismo , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Testosterona/metabolismo , Células Tecais/metabolismo , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Síndrome do Ovário Policístico/genética , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Ovinos , Células Tecais/citologia , Células Tecais/ultraestrutura
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