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1.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502315

RESUMO

Cluster of differentiation 73 (CD73, also known as ecto-5'-nucleotidase) is an enzyme that converts AMP into adenosine. CD73 is a surface enzyme bound to the outside of the plasma membrane expressed in several cells and regulates immunity and inflammation. In particular, it is known to inhibit T cell-mediated immune responses. However, the regulation of CD73 expression by hormones in the uterus is not yet clearly known. In this study, we investigated the expression of CD73 in ovariectomized mice treated with estrogen or progesterone and its regulation in the mouse uterus during the estrous cycle. The level of CD73 expression was dynamically regulated in the uterus during the estrous cycle. CD73 protein expression was high in proestrus, estrus, and diestrus, whereas it was relatively low in the metestrus stage. Immunofluorescence revealed that CD73 was predominantly expressed in the cytoplasm of the luminal and glandular epithelium and the stroma of the endometrium. The expression of CD73 in ovariectomized mice was gradually increased by progesterone treatment. However, estrogen injection did not affect its expression. Moreover, CD73 expression was increased when estrogen and progesterone were co-administered and was inhibited by the pretreatment of the progesterone receptor antagonist RU486. These findings suggest that the expression of CD73 is dynamically regulated by estrogen and progesterone in the uterine environment, and that there may be a synergistic effect of estrogen and progesterone.


Assuntos
5'-Nucleotidase/metabolismo , Estrogênios/farmacologia , Ciclo Estral/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Progesterona/farmacologia , Útero/metabolismo , 5'-Nucleotidase/genética , Animais , Ciclo Estral/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos ICR , Progestinas/farmacologia , Útero/efeitos dos fármacos
2.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360784

RESUMO

In human spermatozoa, calcium dynamics control most of fertilization events. Progesterone, present in the female reproductive system, can trigger several types of calcium responses, such as low-frequency oscillations. Here we aimed to identify the mechanisms of progesterone-induced calcium signaling in human spermatozoa. Progesterone-induced activation of fluorophore-loaded spermatozoa was studied by fluorescent microscopy. Two computational models were developed to describe the spermatozoa calcium responses: a homogeneous one based on a system of ordinary differential equations and a three-dimensional one with added space dimensions and diffusion for the cytosolic species. In response to progesterone, three types of calcium responses were observed in human spermatozoa: a single transient rise of calcium concentration in cytosol, a steady elevation, or low-frequency oscillations. The homogenous model provided qualitative description of the oscillatory and the single spike responses, while the three-dimensional model captured the calcium peak shape and the frequency of calcium oscillations. The model analysis demonstrated that an increase in the calcium diffusion coefficient resulted in the disappearance of the calcium oscillations. Additionally, in silico analysis suggested that the spatial distribution of calcium signaling enzymes governs the appearance of calcium oscillations in progesterone-activated human spermatozoa.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Simulação por Computador , Modelos Biológicos , Progesterona/farmacologia , Espermatozoides/enzimologia , Humanos , Masculino , Microscopia de Fluorescência , Espermatozoides/citologia
3.
Plant Cell Rep ; 40(9): 1631-1646, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34146141

RESUMO

KEY MESSAGE: Studying RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes) in 5ß-cardenolide formation. Progesterone 5ß-reductases (P5ßR) are assumed to catalyze the reduction of progesterone to 5ß-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5ß-cardenolides. P5ßRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5ßRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5ßR1 (AY585867.1) and DlP5ßR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5ß-cardenolide formation, we here established several RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5ßR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5ßR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5ßR2-RNAi lines but not in Dl P5ßR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5ßR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).


Assuntos
Cardenolídeos/metabolismo , Digitalis/genética , Digitalis/metabolismo , Oxirredutases/genética , Proteínas de Plantas/genética , Butanonas/farmacologia , Butionina Sulfoximina/farmacologia , Digitalis/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Glutationa/farmacologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Progesterona/farmacologia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34070207

RESUMO

In domestic ruminants, endometrial receptivity is related to successful pregnancy and economic efficiency. Despite several molecules having been reported in the past regarding endometrial receptivity regulation, much regarding the mechanism of endometrial receptivity regulation remains unknown due to the complex nature of the trait. In this work, we demonstrated that the cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1 (CRIM1) served as a novel regulator in the regulation of goat endometrial receptivity in vitro. Our results showed that hormones and IFN-τ increased the expression of CRIM1 in goat endometrial epithelial cells (EECs). Knockdown of CRIM1 via specific shRNA hindered cell proliferation, cell adhesion and prostaglandins (PGs) secretion and thus derailed normal endometrial receptivity. We further confirmed that receptivity defect phenotypes due to CRIM1 interference were restored by ATG7 overexpression in EECs while a loss of ATG7 further impaired receptivity phenotypes. Moreover, our results showed that changing the expression of ATG7 affected the reactive oxygen species (ROS) production. Moreover, mR-143-5p was shown to be a potential upstream factor of CRIM1-regulated endometrial receptivity in EECs. Overall, these results suggest that CRIM1, as the downstream target of miR-143-5p, has effects on ATG7-dependent autophagy, regulating cell proliferation, cell adhesion and PG secretion, and provides a new target for the diagnosis and treatment of early pregnancy failure and for improving the success rates of artificial reproduction.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/fisiologia , Implantação do Embrião/genética , Endométrio/fisiologia , Cabras/fisiologia , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Autofagia/fisiologia , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Receptores de Proteínas Morfogenéticas Ósseas/genética , Adesão Celular , Proliferação de Células , Células Cultivadas , Implantação do Embrião/fisiologia , Endométrio/citologia , Endométrio/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Estradiol/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Cabras/genética , Interferon Tipo I/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Gravidez , Proteínas da Gravidez/farmacologia , Progesterona/farmacologia , Prostaglandinas/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima
5.
Biochem Biophys Res Commun ; 562: 105-111, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34049203

RESUMO

Sperm head-to-head agglutination is a well-known known phenomenon in mammalian and non-mammalian species. Although several factors have been reported to induce sperm agglutination, information on the trigger and process of sperm detachment from the agglutination is scarce. Since hyperactivated motility is involved in bovine sperm detachment from the oviduct, we focused on caffeine, a well-known hyperactivation inducer, and aimed to determine the role of caffeine in sperm detachment from agglutination. Agglutination rate of bovine sperm was significantly decreased upon incubation with caffeine following pre-incubation without caffeine. Additionally, we observed that bovine sperm were detached from agglutination only when the medium contained caffeine. The detached sperm showed more asymmetrical flagellar beating compared to the undetached motile sperm, regardless of whether before or after the detachment. Intriguingly, some sperm that detached from agglutination re-agglutinated with different sperm agglutination. These findings indicated caffeine as a trigger for sperm detachment from the agglutination in bull. Furthermore, another well-known hyperactivation inducer, thimerosal, also significantly reduced the sperm agglutination rate. Overall, the study demonstrated the complete process of sperm detachment from sperm head-to-head agglutination and proposed that hyperactivated motility facilitates sperm detachment from another sperm. These findings would provide a better understanding of sperm physiology and fertilization process in mammals.


Assuntos
Cafeína/farmacologia , Aglutinação Espermática/efeitos dos fármacos , Cabeça do Espermatozoide/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia , Animais , Bovinos , Masculino , Progesterona/farmacologia , Timerosal/farmacologia
6.
Front Immunol ; 12: 672168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34054852

RESUMO

The changes in progesterone (P4) levels during and after pregnancy coincide with the temporary improvement and worsening of several autoimmune diseases like multiple sclerosis (MS) and rheumatoid arthritis (RA). Most likely immune-endocrine interactions play a major role in these pregnancy-induced effects. In this study, we used next generation sequencing to investigate the direct effects of P4 on CD4+ T cell activation, key event in pregnancy and disease. We report profound dampening effects of P4 on T cell activation, altering the gene and protein expression profile and reversing many of the changes induced during the activation. The transcriptomic changes induced by P4 were significantly enriched for genes associated with diseases known to be modulated during pregnancy such as MS, RA and psoriasis. STAT1 and STAT3 were significantly downregulated by P4 and their downstream targets were significantly enriched among the disease-associated genes. Several of these genes included well-known and disease-relevant cytokines, such as IL-12ß, CXCL10 and OSM, which were further validated also at the protein level using proximity extension assay. Our results extend the previous knowledge of P4 as an immune regulatory hormone and support its importance during pregnancy for regulating potentially detrimental immune responses towards the semi-allogenic fetus. Further, our results also point toward a potential role for P4 in the pregnancy-induced disease immunomodulation and highlight the need for further studies evaluating P4 as a future treatment option.


Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Ativação Linfocitária/imunologia , Complicações na Gravidez/imunologia , Progesterona/farmacologia , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Gravidez
7.
Gene ; 790: 145699, 2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-33964380

RESUMO

Progesterone (P4) is an anti-androgen compound whose role in sperm maturation and functionality remains unclear in sheep. Here, we aimed to investigate the regulation mechanism of P4 on the epididymal secretion of dihydrotestosterone (DHT). To this end, we performed enzyme-linked immunosorbent assays, immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction to detect P4 concentration as well as StAR, P450scc, and 3ß-HSD expression in sheep epididymis. Besides, cauda epithelial cells were cultured at different concentrations of P4 (10-9-10-5 g ml-1) as well as with or without the P4 receptor (PGR) inhibitor RU486 (10-7 M) or the PI3K-AKT inhibitor LY294006 (10-7 M) to explore the effect of P4 on DHT secretion and the underlying regulatory mechanism. The results showed that the caput, corpus, and cauda of sheep epididymis could synthesize P4 but had different synthesis ability. The PGR expression levels were the highest in the cauda, followed by the corpus. In vitro cell culture showed that P4 inhibition of DHT secretion and 5α-reductase 1 and 2 expression in epididymal epithelial cells could be moderately mitigated by RU486 but not by LY294002. Our results indicated that the paracrine and autocrine P4 could affect the secretion of DHT in epididymal cells through PGR. Overall, this study provides new data regarding the involvement of P4 in sperm maturation and functionality in sheep.


Assuntos
Di-Hidrotestosterona/metabolismo , Epididimo/metabolismo , Progesterona/farmacologia , Animais , Células Cultivadas , Epididimo/efeitos dos fármacos , Feminino , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ovinos
8.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805726

RESUMO

An electromagnetic field (EMF) may affect the functions of uterine tissues. This study hypothesized that EMF changes the estrogenic activity of pig myometrium during the peri-implantation period. Tissue was collected on days 15-16 of the gestation and incubated in the presence of EMF (50 and 120 Hz, 2 and 4 h). The cytochrome P450 aromatase type 3 (CYP19A3) and hydroxysteroid 17ß dehydrogenase type 4 (HSD17B4) mRNA transcript abundance, cytochrome P450arom (aromatase), and 17ß hydroxysteroid dehydrogenase 17ßHSD) protein abundance and estrone (E1) and estradiol-17ß (E2) release were examined using Real-Time PCR, Western blot and radioimmunoassay. Selected myometrial slices were treated with progesterone (P4) to determine whether it functions as a protector against EMF. CYP19A3 mRNA transcript abundance in slices treated with EMF was less at 50 Hz (2 h) and greater at 120 Hz (2 and 4 h). HSD17B4 mRNA transcript was greater in slices treated with EMF at 120 Hz (2 h). Progesterone diminished EMF-related effects on CYP19A3 and HSD17B4. When P4 was added, EMF had suppressive (50 and 120 Hz, 2 h) or enhancing (50 Hz, 4 h) effects on aromatase abundance. The E1 release was lower after 4 h of EMF treatment at 50 Hz and P4 did not protect myometrial E1 release. In conclusion, EMF alters the synthesis and release of E1 and did not affect E2 release in the myometrium during the peri-implantation period.


Assuntos
Campos Eletromagnéticos/efeitos adversos , Implantação do Embrião/efeitos da radiação , Estradiol/metabolismo , Estrona/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Miométrio/efeitos da radiação , Animais , Aromatase/genética , Aromatase/metabolismo , Radiação Eletromagnética , Feminino , Miométrio/metabolismo , Proteína Multifuncional do Peroxissomo-2/genética , Proteína Multifuncional do Peroxissomo-2/metabolismo , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Técnicas de Cultura de Tecidos
9.
FASEB J ; 35(4): e21528, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33742713

RESUMO

We have recently reported two different methodologies that improve sperm functionality. The first method involved transient exposure to the Ca2+ ionophore A23187 , and the second required sperm incubation in the absence of energy nutrients (starvation). Both methods were associated with an initial loss of motility followed by a rescue step involving ionophore removal or addition of energy metabolites, respectively. In this work, we show that starvation is accompanied by an increase in intracellular Ca2+ ([Ca2+ ]i ). Additionally, the starved cells acquire a significantly enhanced capacity to undergo a progesterone-induced acrosome reaction. Electrophysiological measurements show that CatSper channel remains active in starvation conditions. However, the increase in [Ca2+ ]i was also observed in sperm from CatSper null mice. Upon starvation, addition of energy nutrients reversed the effects on [Ca2+ ]i and decreased the effect of progesterone on the acrosome reaction to control levels. These data indicate that both methods have common molecular features.


Assuntos
Cálcio/metabolismo , Progesterona/farmacologia , Capacitação Espermática/efeitos dos fármacos , Inanição/metabolismo , Reação Acrossômica/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Feminino , Masculino , Camundongos , Progesterona/metabolismo , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo
10.
J Steroid Biochem Mol Biol ; 210: 105845, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33652098

RESUMO

Orthologs of human glucocorticoid receptor (GR) and human mineralocorticoid receptor (MR) first appear in cartilaginous fishes. Subsequently, the MR and GR diverged to respond to different steroids: the MR to aldosterone and the GR to cortisol and corticosterone. We report that cortisol, corticosterone and aldosterone activate full-length elephant shark GR, and progesterone, which activates elephant shark MR, does not activate elephant shark GR. However, progesterone inhibits steroid binding to elephant shark GR, but not to human GR. Together, this indicates partial functional divergence of elephant shark GR from the MR. Deletion of the N-terminal domain (NTD) from elephant shark GR (truncated GR) reduced the response to corticosteroids, while truncated and full-length elephant shark MR had similar responses to corticosteroids. Swapping of NTDs of elephant shark GR and MR yielded an elephant shark MR chimera with full-length GR-like increased activation by corticosteroids and progesterone compared to full-length elephant shark MR. Elephant shark MR NTD fused to GR DBD + LBD had similar activation as full-length MR, indicating that the MR NTD lacked GR-like NTD activity. We propose that NTD activation of human GR evolved early in GR divergence from the MR.


Assuntos
Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/química , Receptores de Mineralocorticoides/metabolismo , Regulação Alostérica , Animais , Corticosterona/metabolismo , Corticosterona/farmacologia , Relação Dose-Resposta a Droga , Evolução Molecular , Células HEK293 , Antagonistas de Hormônios/farmacologia , Humanos , Hidrocortisona/metabolismo , Hidrocortisona/farmacologia , Mifepristona/farmacologia , Progesterona/administração & dosagem , Progesterona/metabolismo , Progesterona/farmacologia , Domínios Proteicos , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Receptores de Mineralocorticoides/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tubarões , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia
11.
J Biochem Mol Toxicol ; 35(6): 1-8, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33728745

RESUMO

Endocrine disruptors are a major concern due to their possible association with hormone-dependent carcinogenesis. Some examples of compounds with such properties are organochlorine pesticides (OCPs). OCPs are persistent pollutants with high lipophilicity, long half-life, and bioaccumulation potential. In the past, some of the most commonly used OCPs were dichlorodiphenyltrichloroethane (DDT) and endosulfan. Here, we investigated the effects of o,p'-DDT, p,p'-DDT, and endosulfan and of hormones estradiol, testosterone, and progesterone on the expression of estrogen, progesterone, and androgen receptors (ER, PR, and AR) and of their target genes (KLF4, VEGFA, CCND1, PRLR, CDKN1A, and BCL6) in MCF-7 and MDA-MB-231 cells. The results confirmed that under the action of the insecticides, there are dose- and time-dependent changes in the expression of these receptors and target genes. As corroborated by an experiment with ER, PR, and AR negative MDA-MB-231 cells, the change in the expression of KLF4, VEGFA, CCND1, and PRLR in MCF-7 cells treated with o,p'-DDT and the change in CDKN1A and PRLR expression in MCF-7 cells treated with p,p'-DDT are likely mediated by ER, PR, and AR pathways. In conclusion, we have identified some targets of DDT and endosulfan and confirmed that the effects of insecticides on the expression of these target genes differ for breast cancer cell lines with different receptor statuses.


Assuntos
Androgênios/farmacologia , Neoplasias da Mama/metabolismo , DDT/farmacologia , Endossulfano/farmacologia , Estrogênios/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/biossíntese , Progesterona/farmacologia , Neoplasias da Mama/patologia , Disruptores Endócrinos , Feminino , Humanos , Células MCF-7
12.
Fertil Steril ; 116(1): 255-265, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33676751

RESUMO

OBJECTIVE: To test whether mechanical substrate stiffness would influence progesterone receptor B (PRB) signaling in fibroid cells. Uterine fibroids feature an excessive extracellular matrix, increased stiffness, and altered mechanical signaling. Fibroid growth is stimulated by progestins and opposed by anti-progestins, but a functional interaction between progesterone action and mechanical signaling has not been evaluated. DESIGN: Laboratory studies. SETTING: Translational science laboratory. PATIENT(S)/ANIMAL(S): Human fibroid cell lines and patient-matched fibroid and myometrial cell lines. INTERVENTION(S): Progesterone receptor B-dependent reporter assays and messenger RNA quantitation in cells cultured on stiff polystyrene plates (3GPa) or soft silicone plates (930KPa). Pharmacologic inhibitors of extracellular signal-related protein kinase (ERK) kinase 1/2 (MEK 1/2; PD98059), p38 mitogen-activated protein kinase (SB202190), receptor tyrosine kinases (RTKs; nintedanib), RhoA (A13), and Rho-associated coiled-coil kinase (ROCK; Y27632). MAIN OUTCOME MEASURE(S): Progesterone-responsive reporter activation. RESULT(S): Fibroid cells exhibited higher PRB-dependent reporter activity with progesterone (P4) in cells cultured on stiff vs. soft plates. Mechanically induced PRB activation with P4 was decreased 62% by PD98059, 78% by nintedanib, 38% by A13, and 50% by Y27632. Overexpression of the Rho-guanine nucleotide exchange factor (Rho-GEF), AKAP13, significantly increased PRB-dependent reporter activity. Collagen 1 messenger RNA levels were higher in fibroid cells grown on stiff vs. soft plates with P4. CONCLUSION(S): Cells cultured on mechanically stiff substrates had enhanced PRB activation via a mechanism that required MEK 1/2 and AKAP13/RhoA/ROCK signaling pathways. These studies provide a framework to explore the mechanisms by which mechanical stiffness affects progesterone receptor activation.


Assuntos
Leiomioma/enzimologia , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Mecanotransdução Celular , Receptores de Progesterona/metabolismo , Neoplasias Uterinas/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Leiomioma/genética , Leiomioma/patologia , Ligantes , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Mecanotransdução Celular/efeitos dos fármacos , Poliestirenos/química , Progesterona/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Receptores de Progesterona/agonistas , Silicones/química , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores
13.
Int J Mol Sci ; 22(4)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671517

RESUMO

Recently, it has been suggested that progesterone affects the contractile activity of pregnant myometrium via nongenomic pathways; therefore, we aimed to clarify whether progesterone causes and/or inhibits pregnant myometrial contractions via nongenomic pathways. Our in vitro experiments using myometrial strips obtained from rats at 20 days of gestation revealed that progesterone caused myometrial contractions in a concentration- and time-dependent manner at concentrations up to 5 × 10-7 M; however, this effect decreased at concentrations higher than 5 × 10-5 M. Similarly, progesterone enhanced oxytocin-induced contractions up to 5 × 10-7 M and inhibited contractions at concentrations higher than 5 × 10-5 M. Conversely, progesterone did not enhance high-KCl-induced contractions but inhibited contractions in a concentration- and time-dependent manner at concentrations higher than 5 × 10-7 M. We also found that RU486 did not affect progesterone-induced contractions or the progesterone-induced inhibition of high-KCl-induced contractions; however, progesterone-induced contractions were blocked by calcium-free phosphate saline solution, verapamil, and nifedipine. In addition, FPL64176, an activator of L-type voltage-dependent calcium channels, enhanced high-KCl-induced contractions and rescued the decrease in high-KCl-induced contractions caused by progesterone. Together, these results suggest that progesterone exerts conflicting nongenomic effects on the contractions of pregnant myometrium via putative L-type voltage-dependent calcium channels.


Assuntos
Miométrio/fisiologia , Progesterona/fisiologia , Contração Uterina/fisiologia , Animais , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Feminino , Antagonistas de Hormônios/farmacologia , Mifepristona/farmacologia , Miométrio/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Cultura de Órgãos , Ocitocina/farmacologia , Cloreto de Potássio/farmacologia , Gravidez , Progesterona/farmacologia , Pirróis/farmacologia , Ratos Wistar , Contração Uterina/efeitos dos fármacos , Verapamil/farmacologia
14.
J Int Med Res ; 49(3): 300060521999527, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33752482

RESUMO

OBJECTIVE: This was a prospective study to investigate whether progesterone affects sperm activity by regulating the cyclic AMP-protein kinase A (cAMP-PKA) signalling pathway via α/ß hydrolase domain-containing protein 2 (ABHD2). METHODS: Spermatozoa were collected from healthy and infertile men (with oligoasthenospermia or abnormal acrosome; n = 30/group). The expression of and mutations in ABHD2 were detected by quantitative PCR, western blot, and gene sequencing. The expression of ABHD2 in the presence of progesterone was detected in all groups, and cAMP and PKA levels were detected by ELISA in fertile men after treatment with ABHD2 antibody and PKA inhibitor H-89, respectively. RESULTS: Expression of ABHD2 mRNA and protein were reduced in spermatozoa from infertile compared with fertile men. Four gene mutation sites were detected in spermatozoa from the infertile groups. Progesterone increased mRNA and protein levels of ABHD2 in healthy spermatozoa but not in spermatozoa from infertile men. The levels of cAMP and PKA were increased by progesterone in healthy spermatozoa, and the progesterone-increased cAMP and PKA were decreased by ABHD2 antibody and H-89, respectively. CONCLUSION: Progesterone regulates the ABHD2-mediated cAMP-PKA signalling pathway in healthy spermatozoa, which provides a new target for clinical diagnosis and treatment of infertility.


Assuntos
AMP Cíclico , Progesterona , Reação Acrossômica , Proteínas Quinases Dependentes de AMP Cíclico/genética , Humanos , Hidrolases , Masculino , Progesterona/farmacologia , Estudos Prospectivos , Espermatozoides
15.
J Vis Exp ; (168)2021 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-33645583

RESUMO

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.


Assuntos
Membrana Celular/fisiologia , Fenômenos Eletrofisiológicos , Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Tamanho Celular , Dissecação , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Flagelos/efeitos dos fármacos , Flagelos/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/efeitos dos fármacos , Macaca mulatta , Masculino , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Perfusão , Progesterona/farmacologia , Soluções , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos
16.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33671466

RESUMO

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.


Assuntos
Acrossomo/metabolismo , Exocitose/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Progesterona/farmacologia , Capacitação Espermática/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Paxilina/farmacologia , Quinina/farmacologia , Motilidade Espermática/efeitos dos fármacos , Suínos
17.
Nat Commun ; 12(1): 1837, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758202

RESUMO

Oocytes are held in meiotic prophase for prolonged periods until hormonal signals trigger meiotic divisions. Key players of M-phase entry are the opposing Cdk1 kinase and PP2A-B55δ phosphatase. In Xenopus, the protein Arpp19, phosphorylated at serine 67 by Greatwall, plays an essential role in inhibiting PP2A-B55δ, promoting Cdk1 activation. Furthermore, Arpp19 has an earlier role in maintaining the prophase arrest through a second serine (S109) phosphorylated by PKA. Prophase release, induced by progesterone, relies on Arpp19 dephosphorylation at S109, owing to an unknown phosphatase. Here, we identified this phosphatase as PP2A-B55δ. In prophase, PKA and PP2A-B55δ are simultaneously active, suggesting the presence of other important targets for both enzymes. The drop in PKA activity induced by progesterone enables PP2A-B55δ to dephosphorylate S109, unlocking the prophase block. Hence, PP2A-B55δ acts critically on Arpp19 on two distinct sites, opposing PKA and Greatwall to orchestrate the prophase release and M-phase entry.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Meiose , Oócitos/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Cromatografia Líquida , Feminino , Meiose/efeitos dos fármacos , Meiose/genética , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Ácido Okadáico/toxicidade , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Progesterona/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/isolamento & purificação , Proteínas Recombinantes , Espectrometria de Massas em Tandem , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Proteínas de Xenopus/isolamento & purificação , Xenopus laevis
18.
Am J Physiol Regul Integr Comp Physiol ; 320(5): R719-R727, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33533305

RESUMO

Preeclampsia (PE) is characterized by new-onset hypertension in association with elevated natural killer (NK) cells and inflammatory cytokines, which are likely culprits for decreased fetal weight during PE pregnancies. As progesterone increases during normal pregnancy, it stimulates progesterone-induced blocking factor (PIBF). PIBF has been shown to decrease inflammation and cytolytic NK cells, both of which are increased during PE. We hypothesized that PIBF reduces inflammation as a mechanism to improve hypertension in the preclinical reduced uterine perfusion pressure (RUPP) rat model of PE. PIBF (2.0 µg/mL) was administered intraperitoneally on gestational day 15 to either RUPP or normal pregnant (NP) rats. On day 18, carotid catheters were inserted. Mean arterial blood pressure (MAP) and samples were collected on day 19. MAP in NP rats (n = 11) was 100 ± 2 mmHg and 105 ± 3 mmHg in NP + PIBF rats (n = 8) and 122 ± 1 mmHg in RUPP rats (n = 10), which improved to 110 ± 2 mmHg in RUPP + PIBF rats (n = 11), P < 0.05. Pup weight was 2.4 ± 0.1 g in NP, 2.5 ± 0.1 g in NP + PIBF, 1.9 ± 0.1 g in RUPP, and improved to 2.1 ± 0.1 g in RUPP + PIBF rats. Circulating and placental cytolytic NK cells, IL-17, and IL-6 were significantly reduced while IL-4 and T helper (TH) 2 cells were significantly increased in RUPP rats after PIBF administration. Importantly, vasoactive pathways preproendothelin-1, nitric oxide, and soluble fms-Like tyrosine Kinase-1 (sFlt-1) were normalized in RUPP + PIBF rats compared with RUPP rats, P < 0.05. Our findings suggest that PIBF normalized IL-4/TH2 cells, which was associated with improved inflammation, fetal growth restriction, and blood pressure in the RUPP rat model of PE.


Assuntos
Antígenos de Neoplasias/farmacologia , Pressão Sanguínea/fisiologia , Inflamação/tratamento farmacológico , Progesterona/farmacologia , Útero/efeitos dos fármacos , Animais , Citocinas/metabolismo , Feminino , Retardo do Crescimento Fetal/fisiopatologia , Feto/efeitos dos fármacos , Feto/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Isquemia/fisiopatologia , Células Matadoras Naturais/metabolismo , Placenta/metabolismo , Gravidez , Ratos , Artéria Uterina/efeitos dos fármacos , Artéria Uterina/fisiopatologia , Útero/fisiopatologia
19.
Am J Physiol Endocrinol Metab ; 320(4): E747-E759, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33554778

RESUMO

Prostaglandin G/H synthase 2 (PTGS2) is a rate-limiting enzyme in prostaglandin synthesis. The present study assessed the role of the uterine circadian clock on Ptgs2 transcription in response to steroid hormones during early pregnancy. We demonstrated that the core clock genes (Bmal1, Per2, Nr1d1, and Dbp), Vegf, and Ptgs2, and their encoded proteins, have rhythmic expression in the mouse uterus from days 3.5 to 4.5 (D3.5-4.5) of pregnancy. Progesterone (P4) treatment of cultured uterus endometrial stromal cells (UESCs) isolated from mPer2Luciferase reporter gene knock-in mice on D4 induced a phase shift in PER2::LUCIFERASE oscillations. This P4-induced phase shift of PER2::LUCIFERASE oscillations was significantly attenuated by the P4 antagonist RU486. Additionally, the amplitude of PER2::LUCIFERASE oscillations was increased by estradiol (E2) treatment in the presence of P4. Consistently, the mRNA levels of clock genes (Bmal1 and Per2), Vegf, and Ptgs2 were markedly increased by E2 treatment of UESCs in the presence of P4. Treatment with E2 also promoted prostaglandin E2 (PGE2) synthesis by UESCs. Depletion of Bmal1 in UESCs by small-interfering RNA (siRNA) decreased the transcript levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2 compared with nonsilencing siRNA treatment. Bmal1 knockdown also inhibited PGE2 synthesis. Moreover, the mRNA expression levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2, and their respective proteins were significantly decreased in the uterus of Bmal1-/- mice. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.NEW & NOTEWORTHY Rhythmic expression of Bmal1 and Ptgs2 was observed in the uterus isolated from D3.5-4.5 of pregnant mice. E2 increased the expression of Bmal1 and Ptg2 in UESCs isolated from mice on D4. The expression of Ptgs2 was significantly decreased in Bmal1-siRNA treated UESCs. Bmal1 knockdown also inhibited PGE2 synthesis. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.


Assuntos
Fatores de Transcrição ARNTL/fisiologia , Ciclo-Oxigenase 2/genética , Dinoprostona/biossíntese , Estradiol/sangue , Fatores de Transcrição ARNTL/genética , Animais , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Estradiol/farmacologia , Feminino , Idade Gestacional , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Progesterona/farmacologia , Ativação Transcricional/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/metabolismo
20.
Theranostics ; 11(7): 3512-3526, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33537101

RESUMO

Menstruation occurs in few species and involves a cyclic process of proliferation, breakdown and regeneration under the control of ovarian hormones. Knowledge of normal endometrial physiology, as it pertains to the regulation of menstruation, is essential to understand disorders of menstruation. Accumulating evidence indicates that autophagy in the endometrium, under the regulation of ovarian hormones, can result in the infiltration of immune cells, which plays an indispensable role in the endometrium shedding, tissue repair and prevention of infections during menstruation. In addition, abnormal autophagy levels, together with resulting dysregulated immune system function, are associated with the pathogenesis and progression of endometriosis. Considering its potential value of autophagy as a target for the treatment of menstrual-related and endometrium-related disorders, we review the activity and function of autophagy during menstrual cycles. The role of the estrogen/progesterone-autophagy-immunity axis in endometriosis are also discussed.


Assuntos
Autofagia/imunologia , Endometriose/imunologia , Endométrio/imunologia , Estrogênios/farmacologia , Menstruação/imunologia , Progesterona/farmacologia , Adulto , Autofagossomos/genética , Autofagossomos/imunologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Endometriose/etiologia , Endometriose/genética , Endometriose/patologia , Endométrio/citologia , Endométrio/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Estrogênios/imunologia , Estrogênios/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Progesterona/imunologia , Progesterona/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia
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