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1.
Genes Dev ; 34(3-4): 239-249, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31919193

RESUMO

Addressing the complexity of organogenesis at a system-wide level requires a complete understanding of adult cell types, their origin, and precursor relationships. The Drosophila ovary has been a model to study how coordinated stem cell units, germline, and somatic follicle stem cells maintain and renew an organ. However, lack of cell type-specific tools have limited our ability to study the origin of individual cell types and stem cell units. Here, we used a single-cell RNA sequencing approach to uncover all known cell types of the developing ovary, reveal transcriptional signatures, and identify cell type-specific markers for lineage tracing. Our study identifies a novel cell type corresponding to the elusive follicle stem cell precursors and predicts subtypes of known cell types. Altogether, we reveal a previously unanticipated complexity of the developing ovary and provide a comprehensive resource for the systematic analysis of ovary morphogenesis.


Assuntos
Drosophila/citologia , Folículo Ovariano/citologia , Células-Tronco/citologia , Animais , Drosophila/genética , Drosophila/metabolismo , Feminino , Modelos Animais , Ovário/citologia , Análise de Sequência de RNA , Análise de Célula Única , Transcrição Genética
2.
Endocrinology ; 161(1)2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31865385

RESUMO

Follicle-stimulating hormone (FSH) is a dimeric glycoprotein secreted by the anterior pituitary gonadotrope that is necessary for reproductive function in mammals. FSH primarily regulates granulosa cells and follicular growth in females, and Sertoli cell function in males. Since its identification in the 1930s and sequencing in the 1970s, significant progress has been made in elucidating its regulation and downstream function. Recent advances provide deeper insight into FSH synthesis, and effects in the gonads suggest potential roles in extragonadal tissues and examine pharmacological approaches and clinical applications in infertility treatment that now affect 18% of couples. These advances were discussed in detail in a number of reviews published in the last 2 years in Endocrinology. In this brief commentary, we summarize these reviews and point to the outstanding questions that should be answered in the near future to bridge a gap in our understanding of this hormone.


Assuntos
Hormônio Foliculoestimulante/genética , Regulação da Expressão Gênica , Células da Granulosa/metabolismo , Processamento de Proteína Pós-Traducional , Células de Sertoli/metabolismo , Animais , Feminino , Hormônio Foliculoestimulante/metabolismo , Humanos , Masculino , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores do FSH/metabolismo , Literatura de Revisão como Assunto , Transdução de Sinais , Espermatogênese/genética
3.
Elife ; 82019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31850843

RESUMO

The follicle stem cells (FSCs) in the Drosophila ovary are an important experimental model for the study of epithelial stem cell biology. Although decades of research support the conclusion that there are two FSCs per ovariole, a recent study used a novel clonal marking system to conclude that there are 15-16 FSCs per ovariole. We performed clonal analysis using both this novel clonal marking system and standard clonal marking systems, and identified several problems that may have contributed to the overestimate of FSC number. In addition, we developed new methods for accurately measuring clone size, and found that FSC clones produce, on average, half of the follicle cells in each ovariole. Our findings provide strong independent support for the conclusion that there are typically two active FSCs per ovariole, though they are consistent with up to four FSCs per germarium.


Assuntos
Drosophila melanogaster/citologia , Células Epiteliais/citologia , Folículo Ovariano/citologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Animais , Feminino , Ovário
4.
Int J Mol Sci ; 20(19)2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31623386

RESUMO

This study aimed to examine the effect of follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and growth hormone (GH) on Aquaporin 5 (AQP5) expression in granulosa (Gc) and theca cells (Tc) from medium (MF) and large (LF) ovarian follicles of pigs. The results showed that GH significantly decreased the expression of AQP5 in Gc from MF in relation to the control. In the Gc of large follicles, PRL stimulated the expression of AQP5. However, the increased expression of AQP5 in the Tc of LF was indicated by GH and PRL in relation to the control. A significantly higher expression of the AQP5 protein in the Gc from MF and LF was indicated by FSH and PRL. In co-cultures, an increased expression of AQP5 was observed in the Gc from LF incubated with LH, PRL, and GH. A significantly increased expression of AQP5 was also observed in co-cultures of Tc from all type of follicles incubated with LH, whereas PRL stimulated the expression of AQP5 in Tc from MF. Moreover, AQP5 protein expression increased in the co-culture isolated from MF and LF after treatment with FSH, LH, PRL, and GH. AQP5 immunoreactivity was observed in the cytoplasm, mainly in the perinuclear region and endosomes, as well as in the cell membranes of Gc and Tc from the LF and MF.


Assuntos
Aquaporina 5/genética , Regulação da Expressão Gênica de Plantas , Folículo Ovariano/metabolismo , Hormônios Hipofisários/metabolismo , Animais , Biomarcadores , Técnicas de Cocultura , Feminino , Hormônio Foliculoestimulante/metabolismo , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Hormônio do Crescimento/metabolismo , Hormônio Luteinizante/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Hormônios Hipofisários/farmacologia , Prolactina/metabolismo , Suínos , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo
5.
PLoS Biol ; 17(10): e3000509, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613895

RESUMO

The Hippo signalling pathway restricts cell proliferation in animal tissues by inhibiting Yes-associated protein (YAP or YAP1) and Transcriptional Activator with a PDZ domain (TAZ or WW-domain-containing transcriptional activator [WWTR1]), coactivators of the Scalloped (Sd or TEAD) DNA-binding transcription factor. Drosophila has a single YAP/TAZ homolog named Yorkie (Yki) that is regulated by Hippo pathway signalling in response to epithelial polarity and tissue mechanics during development. Here, we show that Yki translocates to the nucleus to drive Sd-mediated cell proliferation in the ovarian follicle cell epithelium in response to mechanical stretching caused by the growth of the germline. Importantly, mechanically induced Yki nuclear localisation also requires nutritionally induced insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) via phosphatidyl inositol-3-kinase (PI3K), phosphoinositide-dependent kinase 1 (PDK1 or PDPK1), and protein kinase B (Akt or PKB) in the follicular epithelium. We find similar results in the developing Drosophila wing, where Yki becomes nuclear in the mechanically stretched cells of the wing pouch during larval feeding, which induces IIS, but translocates to the cytoplasm upon cessation of feeding in the third instar stage. Inactivating Akt prevents nuclear Yki localisation in the wing disc, while ectopic activation of the insulin receptor, PI3K, or Akt/PKB is sufficient to maintain nuclear Yki in mechanically stimulated cells of the wing pouch even after feeding ceases. Finally, IIS also promotes YAP nuclear localisation in response to mechanical cues in mammalian skin epithelia. Thus, the Hippo pathway has a physiological function as an integrator of epithelial cell polarity, tissue mechanics, and nutritional cues to control cell proliferation and tissue growth in both Drosophila and mammals.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Fosfatidilinositol 3-Quinase/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transativadores/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Animais , Fenômenos Biomecânicos , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Polaridade Celular , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Células Epiteliais/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mecanotransdução Celular , Camundongos , Proteínas Nucleares/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transativadores/metabolismo , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
6.
Adv Exp Med Biol ; 1169: 213-223, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31487026

RESUMO

Every organ in the body is thought to harbor two populations of stem cells, including the quiescent and the actively dividing, that leads to heterogeneity among them. It is generally believed that the ovary harbors a fixed number of follicles at birth that differentiate during fetal development from the primordial germ cells. The numbers of follicles decrease by age, leading to menopause. However, in 2004, it was suggested that ovary may harbor stem cells that are possibly involved in the formation of new follicles throughout reproductive life. Research over little more than a decade shows that ovarian stem cells include a quiescent population of very small embryonic-like stem cells (VSELs) and slightly bigger, actively dividing ovarian stem cells (OSCs). This heterogeneity among ovarian stem cells is similar to the presence of VSELs along with spermatogonial stem cells (SSCs) in the testis or hematopoietic stem cells (HSCs) in the hematopoietic system. VSELs express embryonic markers, including nuclear OCT-4, and are lodged in the ovary surface epithelium (OSE). Ovarian VSELs undergo asymmetric cell division to self-renew and give rise to OSCs that in turn undergo symmetric cell divisions and clonal expansion (germ cell nest) followed by meiosis to form an oocyte that gets assembled as a primordial follicle. Both VSELs and OSCs also express receptors for follicle-stimulating hormone (FSHR) and are directly activated by FSH to undergo neo-oogenesis and primordial follicle assembly. Whether stimulation of ovaries by FSH in Infertility Clinics activates the stem cells leading to the formation of multiple follicles needs further investigation. Epithelial cells lining the surface of ovary provide a niche to the stem cells under normal circumstances and undergo epithelial-mesenchymal transition (EMT) to form granulosa cells for primordial follicle assembly. Compromised function of the epithelial cells with age possibly leads to inability of stem cells to form follicles, leading to menopause. More than 90% of ovarian cancers arise in the OSE, possibly due to excessive self-renewal of VSELs. Altered biology of the OSE cells results in the formation of myofibroblasts by EMT and may provide a cancerous niche that supports excessive expansion of the stem cells lodged in the OSE, leading to ovarian cancer. Ovarian cancer cells express markers like OCT-4 and FSHR, which are also expressed by the VSELs lodged in the OSE, whereas the epithelial cells are distinctly negative for the same. Lot more research is required in the field to gain further understanding of ovarian stem cell biology.


Assuntos
Células-Tronco Embrionárias , Folículo Ovariano , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Masculino , Oogênese , Folículo Ovariano/citologia
7.
J Assist Reprod Genet ; 36(9): 1823-1835, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31376104

RESUMO

PURPOSE: Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. METHODS: Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9 days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. RESULTS: No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. CONCLUSIONS: Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.


Assuntos
Criopreservação/métodos , Folículo Ovariano , Ovário/fisiologia , Animais , Estradiol/metabolismo , Feminino , Preservação da Fertilidade/métodos , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Progesterona/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Puberdade , Ovinos , Técnicas de Cultura de Tecidos , Vitrificação
8.
Nat Commun ; 10(1): 3164, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320652

RESUMO

The ovary is perhaps the most dynamic organ in the human body, only rivaled by the uterus. The molecular mechanisms that regulate follicular growth and regression, ensuring ovarian tissue homeostasis, remain elusive. We have performed single-cell RNA-sequencing using human adult ovaries to provide a map of the molecular signature of growing and regressing follicular populations. We have identified different types of granulosa and theca cells and detected local production of components of the complement system by (atretic) theca cells and stromal cells. We also have detected a mixture of adaptive and innate immune cells, as well as several types of endothelial and smooth muscle cells to aid the remodeling process. Our results highlight the relevance of mapping whole adult organs at the single-cell level and reflect ongoing efforts to map the human body. The association between complement system and follicular remodeling may provide key insights in reproductive biology and (in)fertility.


Assuntos
Células Endoteliais/classificação , Células da Granulosa/classificação , Miócitos de Músculo Liso/classificação , Folículo Ovariano/crescimento & desenvolvimento , Células Tecais/classificação , Adulto , Sequência de Bases , Feminino , Humanos , Folículo Ovariano/anatomia & histologia , Folículo Ovariano/citologia , Ovulação/fisiologia , Análise de Sequência de RNA , Útero/anatomia & histologia , Útero/citologia , Útero/crescimento & desenvolvimento
9.
Reprod Biol Endocrinol ; 17(1): 51, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31272468

RESUMO

BACKGROUND: In general, anti-Müllerian hormone (AMH) is positively associated with antral follicle count (AFC). However, there is often discordance between the AMH level and AFC in clinical practice. In cases of discordance, which indicator should be chosen to predict ovarian response and subsequently develop an ovulation induction protocol? The objective of this study was to investigate which indicator was more accurate in predicting ovarian response and pregnancy outcomes when the AMH level and AFC were discordant. METHODS: A total of 1121 infertile women undergoing IVF/ICSI were recruited in this study. During the study period, patients were subjected to individualized controlled ovarian hyperstimulation (COH) protocols according to specific characteristics. The AMH levels and AFCs were measured on days 2-3 of the menstrual cycle. Serum samples were obtained to determine AMH levels. Transvaginal ultrasound was performed to determine the AFC. All patients were divided into four groups: Group A had AFCs and AMH levels in the normal range; Group B had normal AFCs and low AMH levels; Group C had low AFCs and normal AMH levels; and Group D had low AFCs and AMH levels. RESULTS: Two hundred three women (18.11%) showed discordant AFCs and AMH levels. In the two groups with discordant AFCs and AMH levels, namely, Group B and Group C, the oocyte yield, good-quality embryo rate and clinical pregnancy rate were significantly higher in Group B than in Group C. The incidence of poor ovarian response (POR) was significantly lower in Group B than in Group C. According to the stratified analysis of age, for the three categories above the age of 30, oocyte yield was higher in Group B than in Group C. In all age categories, the clinical pregnancy rate was higher in Group B than in Group C. CONCLUSIONS: Our study demonstrated that approximately one in five patients in clinical practice showed discordance between AFCs and AMH levels. In view of the AFC being better than AMH for predicting POR, the AFC should be the preferred indicator for predicting ovarian response to subsequently develop an optimal individualized COH protocol.


Assuntos
Hormônio Antimülleriano/metabolismo , Fertilização In Vitro , Oócitos/citologia , Folículo Ovariano/citologia , Adulto , Contagem de Células , Feminino , Humanos , Infertilidade Feminina , Reserva Ovariana , Gravidez , Taxa de Gravidez
10.
Acta Histochem ; 121(7): 776-783, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31307787

RESUMO

Sodium Valproate (VPA) is known to have deleterious consequences on ovarian function and folliculogenesis. Folic acid (FA) is associated with the quality of many parameters in folliculogenesis. Therefore, we aimed to investigate the effects of chronic Valproate administration on ovarian morphology, folliculogenesis, reproductive hormones, and the possible protective effect of Folic acid supplementation. Forty adult female albino rats were divided into four groups and treated orally for 90 days as follows: Control group received distilled water; FA group received (folic acid 400 µg/day); VPA group received (Na Valproate 200 mg/kg/day) and VPA + FA group received (Na Valproate 200 mg/kg/day + folic acid 400 µg/day). In addition, ovaries were processed for routine histology and immunohistochemistry (TGFß1 and PCNA) and reproductive hormones levels were measured. Results showed a significant decrease in number of follicles in VPA group, while atretic follicles increased compared with control group (P < 0.001). Interestingly, the number of follicles significantly increased in VPA + FA group compared with VPA group (P < 0.001). Also, number of atretic follicles significantly decreased in the VPA + FA group compared to the VPA group. Histochemistry score decreased for TGFß1 and PCNA staining in VPA group compared with control group (P < 0.01). Moreover, Valproate demonstrated a significant increase in testosterone levels in VPA group than control group (P < 0.001). However, VPA group demonstrated a significant decrease in levels of estradiol, progesterone, FSH and LH levels compared with control group. These changes were partially improved in VPA + FA group. In conclusion, FA co-treatment can modulate ovarian follicular and hormonal disturbances induced by valproate, which needs further investigations to identify the precise mechanisms.


Assuntos
Ácido Fólico/farmacologia , Hormônios Esteroides Gonadais/metabolismo , Folículo Ovariano , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ácido Valproico/farmacologia , Animais , Feminino , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ratos , Ratos Wistar
11.
Gen Comp Endocrinol ; 282: 113216, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31278920

RESUMO

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.


Assuntos
Reprodução/fisiologia , Estações do Ano , Atum/fisiologia , Animais , Feminino , Hormônio Foliculoestimulante/sangue , Gametogênese/genética , Regulação da Expressão Gênica , Hormônios Esteroides Gonadais/metabolismo , Masculino , Folículo Ovariano/citologia , Ovário/citologia , Ovário/metabolismo , Testículo/citologia , Testículo/metabolismo , Atum/sangue , Atum/genética , Vitelogeninas/sangue
12.
In Vivo ; 33(4): 1095-1102, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31280197

RESUMO

BACKGROUND/AIM: Perinatal diethylstilbestrol (DES) treatment induces the polyovular follicle containing two or more oocytes in a follicle of mouse ovary through estrogen receptor (ER) ß. The aim of the study was to investigate the direct effects of DES on the neonatal mouse ovary and the gene expression of activins. MATERIALS AND METHODS: Ovaries from neonatal wild-type (WT) or ERß- knockout (ERßKO) mice were organ-cultured in a serum-free medium with or without DES, and polyovular follicle induction and expression of activin signaling related genes were examined. RESULTS: The polyovular follicle and cyst incidence in DES-treated organ-cultured ovaries from WT mice, but not from ERßKO mice, was significantly higher than that of control non-treated cultures. DES altered inhibin (Inh) a, Inhba and Inhbb expression in organ-cultured ovaries from C57BL/6J mice, while no change in Inha and an increase of Inhbb were observed by DES, in both WT and ERßKO mice. CONCLUSION: Alterations in activin signaling are involved in the polyovular follicle induction by DES.


Assuntos
Ativinas/genética , Dietilestilbestrol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ativinas/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Feminino , Inibinas/genética , Inibinas/metabolismo , Camundongos , Camundongos Knockout , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , RNA Mensageiro/genética
13.
Hereditas ; 156: 21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31293364

RESUMO

Background: Characterization of molecular factors regulating ovarian follicular development is critical to understanding its functional mechanism of controlling the estrous cycle, determining oocyte competency, and regulating ovulation. In previous studies, we performed next-gene sequencing to investigate the differentially expressed transcripts of bovine follicular granulosa cells (GCs) at the dominant follicle (DF) and subordinate follicle (SF) stages during the first follicular wave. This study aims to investigate the proteomic characterization of GCs of DF and SF in the bovine estrous cycle. Results: In total, 3409 proteins were identified from 30,321 peptides obtained from liquid chromatograph-mass spectrometer analysis. Two hundred fifty-nine of these proteins were found to be expressed differently in DF and SF. Out of 259, a total of 26 proteins were upregulated (fold change≥2) and 233 proteins were downregulated (fold change≤0.5) in DF. Gene Ontology (GO) analysis of proteome data revealed the biological process, cellular component and molecular function of expressed proteins in DF and SF, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed important signaling pathways associated with follicular development such as the PI3K-Akt, estrogen, and insulin signaling pathways. Immunoblotting results of OGN, ROR2, and HSPB1 confirmed the accuracy of the data. Bioinformatics analysis showed that 13 proteins may be linked to follicular development. Conclusions: Findings from this study will provide useful information for exploring follicular development and function.


Assuntos
Células da Granulosa/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Proteoma , Proteômica , Animais , Bovinos , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Espectrometria de Massas , Proteômica/métodos , Transdução de Sinais , Transcriptoma
14.
Int J Mol Sci ; 20(13)2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288388

RESUMO

Two methods for the cryopreservation of human ovarian tissue were compared using a xenotransplantation model to establish a safe and effective cryopreservation method. Ovarian tissues were obtained from women who underwent benign ovarian surgery in the gynecology research unit of a university hospital. The tissues were transplanted into 112 ovariectomized female severe combined immunodeficient mice 4 weeks after slow freezing or vitrification cryopreservation. Tissues were retrieved 4 weeks later. Primordial follicular counts decreased after cryopreservation and xenotransplantation, and were significantly higher in the slow freezing group than in the vitrification group (p < 0.001). Immunohistochemistry and TUNEL assay showed that the Ki-67 and CD31 markers of follicular proliferation and angiogenesis were higher in the slow freezing group (p < 0.001 and p = 0.006, respectively) and DNA damage was greater in the vitrification group (p < 0.001). Western blotting showed that vitrification increased cellular apoptosis. Anti-Müllerian hormone expression was low in transplanted samples subjected to both cryopreservation techniques. Electron microscopy revealed primordial follicle deformation in the vitrification group. Slow freezing for ovarian tissue cryopreservation is superior to vitrification in terms of follicle survival and growth after xenotransplantation. These results will be useful for fertility preservation in female cancer patients.


Assuntos
Criopreservação , Congelamento , Ovário , Vitrificação , Adulto , Animais , Biomarcadores , Contagem de Células , Proliferação de Células , Sobrevivência Celular , Criopreservação/métodos , Dano ao DNA , Feminino , Preservação da Fertilidade , Imunofluorescência , Xenoenxertos , Humanos , Camundongos , Neovascularização Fisiológica , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Ovário/citologia , Ovário/metabolismo , Ovário/transplante , Ovário/ultraestrutura , Adulto Jovem
15.
J Ovarian Res ; 12(1): 65, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324205

RESUMO

BACKGROUND: Premature ovarian insufficiency (POI) is characterized by early loss of ovarian function that affects women before the age of 40. We aim to explore the protective effects of transcutaneous electrical acupoint stimulation (TEAS) against irradiation-induced ovarian damage in mice. METHODS: C57BL6 mice were randomly divided into control and irradiation (IR) groups. Then, control group was divided into two treatment subgroups: mock TEAS treatment (control-) and TEAS treatment (control+). IR group was divided into four subgroups according to the time of treatment started: mock TEAS treatment initiated at 2 days after irradiation (IR 2D-), TEAS treatment initiated at 2 days after irradiation (IR 2D+), mock TEAS treatment initiated at 1 week after irradiation (IR 1 W-), and TEAS treatment initiated at 1 week after irradiation (IR 1 W+). The radiation model mice were exposed to single whole body X-ray irradiation (4 Gy), and the control mice received 0 Gy. TEAS stimulation (2 Hz, 1 mA, 30 min/day) was given once a day for six consecutive days per week for 2 weeks. Estrous cycle, ovarian weight, serum AMH level and follicle counts were evaluated. Then, proliferation markers, apoptotic markers and oxidative stress markers were examined. RESULTS: Compared with the control group, the estrous cycle was disordered, and the ovarian weight, serum AMH, and primordial, primary and secondary follicles counts decreased (all P < 0.01) in the IR 2D- and IR 1 W- groups. In the irradiation with early TEAS treatment group (IR 2D+), the estrous cycle improved, the AMH level and primordial follicular significantly increased compared to the irradiation with mock group (IR 2D-). However, there were no significant differences in the estrous cycle, AMH level and follicle counts between IR 1 W- and IR 1 W+ groups. Moreover, IR 2D+ mice reduced the expression of Bax protein and increased the levels of Bcl-2 and PCNA compared to the IR 2D- group. Furthermore, the early TEAS treated mice showed significantly lower levels of oxidative stress and number of TUNEL (+) granulosa cells than that in the IR 2D- group. CONCLUSION: This study is first to evaluate TEAS as a potential therapy to attenuate irradiation-induced ovarian failure through inhibiting primordial follicles loss, increasing serum AMH secretion, inducing antioxidant, and anti-apoptotic systems.


Assuntos
Pontos de Acupuntura , Eletroacupuntura , Insuficiência Ovariana Primária/etiologia , Insuficiência Ovariana Primária/prevenção & controle , Radioterapia/efeitos adversos , Estimulação Elétrica Nervosa Transcutânea , Animais , Biomarcadores , Modelos Animais de Doenças , Eletroacupuntura/métodos , Feminino , Expressão Gênica , Camundongos , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Estresse Oxidativo , Insuficiência Ovariana Primária/metabolismo , Lesões Experimentais por Radiação , Estimulação Elétrica Nervosa Transcutânea/métodos , Raios X
16.
Theriogenology ; 138: 66-76, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31302433

RESUMO

Our goal was to develop an objective computer-assisted volumetric method of assessing vascular flow from colour Doppler ultrasound data of ovarian structures recorded by free-hand movement. We hypothesized that a vascularity index (ratio of the region of blood flood to the region of ovarian structure) obtained from the three-dimensional volumetric analysis would be more precise (less variable) than conventional two-dimensional analysis of single images in estimating the functional status of the preovulatory follicles and corpus luteum. Doppler ultrasound cineloops of water buffaloes (Bubalus bubalis; n = 22) ovaries were recorded daily from 12 h before GnRH treatment to four days after ovulation. Cineloops were processed using Fiji and Imaris software packages for segmenting the area (two-dimensional analysis) and the volume (three-dimensional analysis) occupied by the blood-flow and associated tissue to calculate the vascularity index. For volumetric measurement, all images in a cineloop were used (i.e., no a-priori selection of images) while for two-dimensional analysis, three images from the region with apparent maximum vascularity were selected. The volumetric method was verified with theoretical ellipsoidal volume of the follicle (r = 0.96 P < 0.01) or corpus luteum (r = 0.58 P = 0.02). The variability in the follicular vascularity index among animals was lower using the volumetric method than two-dimensional analysis (0.018 ±â€¯0.002 vs 0.030 ±â€¯0.005, P < 0.01), while the variability for CL vascularity was similar between methods (P = 0.23). An increase in the follicular vascularity index was detected at 12 h after GnRH treatment using both methods (two-dimensional: 0.030 ±â€¯0.008, P < 0.01; three-dimensional: 0.016 ±â€¯0.006, P < 0.02). Buffaloes that ovulated tended to have a greater increase in 3D vascularity index than non-responding buffaloes (P = 0.06); the two-dimensional method was not able to detect these changes. Using the three-dimensional method, a moderate positive correlation (r = 0.59; P = 0.02) was evident between the follicular vascularity index at 14-16 h after GnRH treatment and follicular diameter. In conclusion, an objective volumetric method for assessing relative ovarian blood flow changes was developed using Doppler ultrasound cineloops recorded by free-hand movement. The 3-dimensional method eliminates the need for a-priori selection of images and is more precise as a result of decreased technical variability.


Assuntos
Búfalos , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/diagnóstico por imagem , Folículo Ovariano/irrigação sanguínea , Folículo Ovariano/diagnóstico por imagem , Ultrassonografia Doppler em Cores , Animais , Corpo Lúteo/citologia , Sincronização do Estro/métodos , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Hemodinâmica , Imageamento Tridimensional/veterinária , Células Lúteas/citologia , Células Lúteas/ultraestrutura , Folículo Ovariano/citologia , Ovário/irrigação sanguínea , Ovário/citologia , Ovário/diagnóstico por imagem , Ovulação/fisiologia , Detecção da Ovulação/métodos , Detecção da Ovulação/veterinária , Indução da Ovulação/métodos , Indução da Ovulação/veterinária , Fluxo Sanguíneo Regional , Ultrassonografia Doppler em Cores/métodos , Ultrassonografia Doppler em Cores/veterinária
17.
Anim Reprod Sci ; 207: 119-130, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31208845

RESUMO

Fertility preservation is not only a concern for humans with compromised fertility after cancer treatment. The preservation of genetic material from endangered animal species or animals with important genetic traits will also greatly benefit from the development of alternative fertility preservation strategies. In humans, embryo cryopreservation and mature-oocyte cryopreservation are currently the only approved methods for fertility preservation. Ovarian tissue cryopreservation is specifically indicated for prepubertal girls and women whose cancer treatment cannot be postponed. The cryopreservation of pre-antral follicles (PAFs) is a safer alternative for cancer patients who are at risk of the reintroduction of malignant cells. As PAFs account for the vast majority of follicles in the ovarian cortex, they represent an untapped potential, which could be cultivated for reproduction, preservation, or research purposes. Vitrification is being used more and more as it seems to yield better results compared to slow freezing, although protocols still need to be optimized for each specific cell type and species. Several methods can be used to assess follicle quality, ranging from simple viability stains to more complex xenografting procedures. In vitro development of PAFs to the pre-ovulatory stage has not yet been achieved in humans and larger animals. However, in vitro culture systems for PAFs are under development and are expected to become available in the near future. This review will focus on recent developments in (human) fertility preservation strategies, which are often accomplished by the use of in vitro animal models due to ethical considerations and the scarcity of human research material.


Assuntos
Criopreservação/métodos , Preservação da Fertilidade/métodos , Folículo Ovariano/citologia , Ovário , Vitrificação , Animais , Criopreservação/veterinária , Feminino , Preservação da Fertilidade/veterinária , Humanos
18.
BMC Dev Biol ; 19(1): 12, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226923

RESUMO

BACKGROUND: Alterations of bioelectrical properties of cells and tissues are known to function as wide-ranging signals during development, regeneration and wound-healing in several species. The Drosophila follicle-cell epithelium provides an appropriate model system for studying the potential role of electrochemical signals, like intracellular pH (pHi) and membrane potential (Vmem), during development. Therefore, we analysed stage-specific gradients of pHi and Vmem as well as their dependence on specific ion-transport mechanisms. RESULTS: Using fluorescent indicators, we found distinct alterations of pHi- and Vmem-patterns during stages 8 to 12 of oogenesis. To determine the roles of relevant ion-transport mechanisms in regulating pHi and Vmem and in establishing stage-specific antero-posterior and dorso-ventral gradients, we used inhibitors of Na+/H+-exchangers and Na+-channels (amiloride), V-ATPases (bafilomycin), ATP-sensitive K+-channels (glibenclamide), voltage-dependent L-type Ca2+-channels (verapamil), Cl--channels (9-anthroic acid) and Na+/K+/2Cl--cotransporters (furosemide). Either pHi or Vmem or both parameters were affected by each tested inhibitor. While the inhibition of Na+/H+-exchangers (NHE) and amiloride-sensitive Na+-channels or of V-ATPases resulted in relative acidification, inhibiting the other ion-transport mechanisms led to relative alkalisation. The most prominent effects on pHi were obtained by inhibiting Na+/K+/2Cl--cotransporters or ATP-sensitive K+-channels. Vmem was most efficiently hyperpolarised by inhibiting voltage-dependent L-type Ca2+-channels or ATP-sensitive K+-channels, whereas the impact of the other ion-transport mechanisms was smaller. In case of very prominent effects of inhibitors on pHi and/or Vmem, we also found strong influences on the antero-posterior and dorso-ventral pHi- and/or Vmem-gradients. For example, inhibiting ATP-sensitive K+-channels strongly enhanced both pHi-gradients (increasing alkalisation) and reduced both Vmem-gradients (increasing hyperpolarisation). Similarly, inhibiting Na+/K+/2Cl--cotransporters strongly enhanced both pHi-gradients and reduced the antero-posterior Vmem-gradient. To minor extents, both pHi-gradients were enhanced and both Vmem-gradients were reduced by inhibiting voltage-dependent L-type Ca2+-channels, whereas only both pHi-gradients were reduced (increasing acidification) by inhibiting V-ATPases or NHE and Na+-channels. CONCLUSIONS: Our data show that in the Drosophila follicle-cell epithelium stage-specific pHi- and Vmem-gradients develop which result from the activity of several ion-transport mechanisms. These gradients are supposed to represent important bioelectrical cues during oogenesis, e.g., by serving as electrochemical prepatterns in modifying cell polarity and cytoskeletal organisation.


Assuntos
Epitélio/fisiologia , Transporte de Íons/fisiologia , Folículo Ovariano/citologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Membrana Celular/metabolismo , Drosophila melanogaster , Eletroquímica , Feminino , Concentração de Íons de Hidrogênio , Potenciais da Membrana/fisiologia , Oogênese , Canais de Potássio/fisiologia , Canais de Sódio/fisiologia
19.
BMC Bioinformatics ; 20(1): 307, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182013

RESUMO

BACKGROUND: The maturation of the female germ cell, the oocyte, requires the synthesis and storing of all the necessary metabolites to support multiple divisions after fertilization. Oocyte maturation is only possible in the presence of surrounding, diverse, and changing layers of somatic cells. Our understanding of metabolic interactions between the oocyte and somatic cells has been limited due to dynamic nature of ovarian follicle development, thus warranting a systems approach. RESULTS: Here, we developed a genome-scale metabolic model of the mouse ovarian follicle. This model was constructed using an updated mouse general metabolic model (Mouse Recon 2) and contains several key ovarian follicle development metabolic pathways. We used this model to characterize the changes in the metabolism of each follicular cell type (i.e., oocyte, granulosa cells, including cumulus and mural cells), during ovarian follicle development in vivo. Using this model, we predicted major metabolic pathways that are differentially active across multiple follicle stages. We identified a set of possible secreted and consumed metabolites that could potentially serve as biomarkers for monitoring follicle development, as well as metabolites for addition to in vitro culture media that support the growth and maturation of primordial follicles. CONCLUSIONS: Our systems approach to model follicle metabolism can guide future experimental studies to validate the model results and improve oocyte maturation approaches and support growth of primordial follicles in vitro.


Assuntos
Comunicação Celular , Genoma , Modelos Biológicos , Folículo Ovariano/metabolismo , Animais , Diferenciação Celular , Feminino , Redes e Vias Metabólicas , Camundongos , Folículo Ovariano/citologia
20.
Reprod Biol Endocrinol ; 17(1): 46, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189477

RESUMO

BACKGROUND: Reproductive aging is a robust phenotype that occurs in all females and is characterized by a significant reduction in gamete quantity and quality, which can have negative consequences on both endocrine function and fertility. Age-associated differences in the oocyte, follicle, and ovary have been well-documented, but how the broader environment changes with age is less well understood. Fat is one of the largest organs in the body, and peri-gonadal adipose tissue surrounds the rodent ovary and comprises a local ovarian environment. The goal of this study was to characterize how peri-ovarian adipose tissue changes with advanced reproductive age. METHODS: We isolated peri-gonadal adipose tissue from two cohorts of CB6F1 mice: reproductively young (6-12 weeks) and reproductively old (14-17 months). A comparative histological analysis was performed to evaluate adipocyte architecture. We then extracted lipids from the tissue and performed multiple reaction monitoring (MRM)-profiling, a mass spectrometry-based method of metabolite profiling, to compare the lipid profiles of peri-gonadal adipose tissue in these age cohorts. RESULTS: We found that advanced reproductive age was associated with adipocyte hypertrophy and a corresponding decrease in the number of adipocytes per area. Of the 10 lipid classes examined, triacylglycerols (TAGs) had significantly different profiles between young and old cohorts, despite quantitative analysis revealing a decrease in the total amount of TAGs per weight of peri-gonadal adipose tissue with age. CONCLUSIONS: These findings pinpoint age-associated physiological changes in peri-gonadal adipose tissue with respect to adipocyte morphology and lipid profiles and lay the foundation for future studies to examine how these alterations may influence both adipocyte and ovarian function.


Assuntos
Tecido Adiposo/metabolismo , Envelhecimento/fisiologia , Lipídeos/análise , Ovário/metabolismo , Reprodução/fisiologia , Adipócitos/citologia , Adipócitos/metabolismo , Tecido Adiposo/citologia , Fatores Etários , Animais , Feminino , Camundongos , Oócitos/citologia , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ovário/citologia
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