RESUMO
The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro-oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion - ovarian nerve plexus - ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3ß-HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3ß-HSD activity and gene expression. The ovarian 3ß-HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3ß-HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the "fine tuning" of neurosecretory functions as a potent modulator of reproductive processes in female rats.
Assuntos
3-Hidroxiesteroide Desidrogenases/metabolismo , Hipotálamo/efeitos dos fármacos , Neuroesteroides/administração & dosagem , Ovário/efeitos dos fármacos , Pregnanolona/administração & dosagem , Animais , Diestro/efeitos dos fármacos , Diestro/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Hipotálamo/enzimologia , Injeções Intraventriculares , Ovário/metabolismo , Proestro/efeitos dos fármacos , Proestro/metabolismo , Progesterona/sangue , RatosRESUMO
ABSTRACT This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.
Assuntos
Animais , Feminino , Cães , Diestro/fisiologia , Colesterol/sangue , Proliferação de Células/fisiologia , Endométrio/citologia , Glucose/análise , Fatores de Tempo , Diestro/metabolismo , Endométrio/fisiologiaRESUMO
This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.
Assuntos
Proliferação de Células/fisiologia , Colesterol/sangue , Diestro/fisiologia , Endométrio/citologia , Glucose/análise , Animais , Diestro/metabolismo , Cães , Endométrio/fisiologia , Feminino , Fatores de TempoRESUMO
We hypothesised that different endocrine profiles associated with pre-ovulatory follicle (POF) size would impact on uterine prostanoid pathways and thereby modulate the histotroph composition. Beef cows (n=15 per group) were hormonally manipulated to have small (SF-SCL group) or large (LF-LCL group) pre-ovulatory follicles (POF) and corpora lutea (CL). Seven days after induction of ovulation, animals were slaughtered and uterine tissues and flushings were collected for quantification of prostanoids. The POF and CL size and the circulating progesterone concentrations at Day 7 were greater (P<0.05) in the LF-LCL cows than in the SF-SCL group, as expected. The abundance of 5 out of 19 genes involved in prostanoid regulation was different between groups. Transcript abundance of prostaglandin F2α, E2 and I2 synthases was upregulated (P<0.05) and phospholipase A2 was downregulated (P<0.05) in endometrium of the LF-LCL group. No difference (P>0.1) in prostanoid concentrations in the endometrium or in uterine flushings was detected between groups. However, prostaglandin F2α and E2 concentrations in the uterine flushings were positively correlated with the abundance of transcripts for prostaglandin endoperoxide synthase 2 (0.779 and 0.865, respectively; P<0.002). We conclude that endometrial gene expression related to prostanoid synthesis is modulated by the peri-ovulatory endocrine profile associated with POF size, but at early dioestrus differences in transcript abundance were not reflected in changes in prostanoid concentrations in the uterine tissue and fluid.
Assuntos
Diestro/metabolismo , Dinoprosta/metabolismo , Dinoprostona/metabolismo , Útero/metabolismo , Animais , Bovinos , Regulação para Baixo , Endométrio/metabolismo , Feminino , Indução da Ovulação , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
BACKGROUND: Muscarinic receptors (mAChRs) of the preoptic and anterior hypothalamus areas (POA-AHA) regulate ovulation in an asymmetric manner during the estrous cycle. The aims of the present study were to analyze the effects of a temporal blockade of mAChRs on either side of the POA-AHA performed in diestrus-2 rats on ovulation, the levels of estradiol, follicle stimulating hormone (FSH) and luteinizing hormone (LH) and the mechanisms involved in changes in ovulation. METHODS: Cyclic rats on diestrus-2 day were anesthetized and randomly assigned to the following groups: 1) microinjection of 1 µl of saline or atropine solution (62.5 ng) in the left or right POA-AHA; 2) removal (unilateral ovariectomty, ULO) of the left (L-ULO) or right (R-ULO) ovary, and 3) rats microinjected with atropine into the left or right POA-AHA plus L-ULO or R-ULO. The ovulation rate and the number of ova shed were measured during the predicted estrus, as well as the levels of estradiol, FSH and LH during the predicted proestrus and the effects of injecting synthetic LH-releasing hormone (LHRH) or estradiol benzoate (EB). RESULTS: Atropine in the left POA-AHA decreased both the ovulation rate and estradiol and LH levels on the afternoon of proestrus, also LHRH or EB injection restored ovulation. L- or R-ULO resulted in a lower ovulation rate and smaller number of ova shed, and only injection of LHRH restored ovulation. EB injection at diestrus-2 restored ovulation in animals with L-ULO only. The levels of estradiol, FSH and LH in rats with L-ULO were higher than in animals with unilateral laparotomy. In the group microinjected with atropine in the left POA-AHA, ovulation was similar to that in ULO rats. In contrast, atropine in the right POA-AHA of ULO rats blocked ovulation, an action that was restored by either LHRH or EB injection. CONCLUSIONS: These results indicated that the removal of a single ovary at noon on diestrus-2 day perturbed the neuronal pathways regulating LH secretion, which was mediated by the muscarinic system connecting the right POA-AHA and the ovaries.
Assuntos
Núcleo Hipotalâmico Anterior/metabolismo , Diestro/metabolismo , Estradiol/metabolismo , Hormônio Foliculoestimulante/metabolismo , Hormônio Luteinizante/metabolismo , Ovulação/metabolismo , Área Pré-Óptica/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Núcleo Hipotalâmico Anterior/efeitos dos fármacos , Atropina/farmacologia , Anticoncepcionais/farmacologia , Diestro/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Luteinizante/efeitos dos fármacos , Antagonistas Muscarínicos/farmacologia , Ovariectomia , Ovário/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Área Pré-Óptica/efeitos dos fármacos , Proestro/efeitos dos fármacos , Proestro/metabolismo , Ratos , Receptores Muscarínicos/efeitos dos fármacosRESUMO
Phospholipid metabolism and signaling influences on early pregnancy events in cattle are unknown. This study aimed to characterize global phospholipid composition of oviduct and uterus during early diestrus in a model of contrasting embryo receptivity. Beef cows were treated to ovulate a larger (LF-LCL group, associated with greater receptivity) or smaller (SF-SCL group) follicle and, consequently, to present greater or smaller plasma concentrations of estradiol during proestrus-estrus, as well as progesterone during early diestrus. Oviduct and uterus (4 days after gonadotropin-releasing hormone-induced ovulation; D4) as well as the uterus (D7) were collected, and lipid profiles were monitored by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This technique allowed the identification and tissue localization of sphingomyelins (SM), phosphatidylcholines (PC), ceramides (Cer), and phosphatidylethanolamines (PE). Multivariate statistics were used to separate samples into groups with distinctly different phospholipid profiles in the uterus at D4 and D7. Different abundance of ions corresponding to specific lipids were detected on D4 (Cer [42:1], PC [31:0], PC [32:1], PC [34:4], and PC [36:4] greater for LF-LCL group; and PC [38:7], PC [38:5], PC [38:4], PC [40:7], and PC [40:6] greater for SF-SCL group) and D7 (SM [34:2], SM [34:1], PC [32:1], and PC [35:2] greater for LF-LCL group). The MALDI-MS imaging showed the spatial distributions of major phospholipids. In conclusion, distinct phospholipid profiles were associated with animals treated to show contrasting receptivity to the embryo. Functional roles of the identified phospholipids on uterine function and preimplantation embryo development deserve further studies.
Assuntos
Ceramidas/metabolismo , Diestro/metabolismo , Oviductos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Esfingomielinas/metabolismo , Útero/metabolismo , Animais , Bovinos , Estradiol/sangue , Feminino , Progesterona/sangue , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Distribuição TecidualRESUMO
BACKGROUND: In cattle, recent studies have shown positive associations between pre-ovulatory concentrations of estradiol (E2), progesterone (P4) at early diestrus and fertility. However, information on cellular and molecular mechanisms through which sex steroids regulate uterine function to support early pregnancy is lacking. Based on endometrial transcriptome data, objective was to compare function of the redox system in the bovine uterus in response to different periovulatory endocrine milieus. METHODS: We employed an animal model to control growth of the pre-ovulatory follicle and subsequent corpus luteum (CL). The large follicle-large CL group (LF-LCL, N=42) presented greater levels of E2 on the day of GnRH treatment (D0; 2.94 vs. 1.27 pg/mL; P=0.0007) and P4 at slaughter on D7 (3.71 vs. 2.62 ng/mL, P=0.01), compared with the small follicle-small CL group (SF-SCL, N=41). Endometrium and uterine washings (N=9, per group) were collected for analyses of variables associated with the uterine redox system. RESULTS: The SF-SCL group had lower endometrial catalase (0.5 vs. 0.79 U/mg protein, P<0.001) and glutathione peroxidase (GPx; 2.0 vs. 2.43 nmol ß-nicotinamide adenine dinucleotide phosphate reduced/min/mg protein, P=0.04) activity, as well as higher lipid peroxidation (28.5 vs. 17.43 nmol malondialdehyde/mg of protein, P<0.001) and superoxide dismutase (SOD) activity (44.77 vs. 37.76 U; P=0.04). There were no differences in the endometrial reactive species (RS) or glutathione (GSH) concentrations between the groups. The uterine washing samples showed no differences in the concentrations of RS or GSH or in total SOD activity (P>0.1). Additionally, catalase, GPx4, SOD1 and SOD2 gene expression was lower in the SF-SCL group than in the LF-LCL group. CONCLUSIONS: We concluded that the intrauterine environment of cows from the LF-LCL group exhibited higher antioxidant activity than that of the cows from the SF-SCL group. We speculate that uterine receptivity and fertility are associated with an optimal redox environment, such as that present in the animals in the LF-LCL group.
Assuntos
Diestro/metabolismo , Ciclo Estral/metabolismo , Fertilidade , Oxirredução , Animais , Antioxidantes/metabolismo , Bovinos , Estradiol/sangue , Feminino , Peroxidação de Lipídeos , Estresse Oxidativo , Progesterona/sangueRESUMO
The objective of this study was to compare expression of estrogen receptor alpha (ER-α), ß (ER-ß), progesterone receptor (PR), as well as prostaglandin E2 type 2 (EP2) and 4 (EP4) receptors in the equine myometrium and endometrium during estrus, diestrus and early pregnancy. Tissues were collected during estrus, diestrus, and early pregnancy. Transcripts for ER-α (ESR1), ER-ß (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-α, ER-ß, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the ΔΔCT method. Expression for ESR1 (P<0.05) tended to be higher during estrus than diestrus in the endometrium (P=0.1) and myometrium (P=0.06). In addition, ESR1 expression was greater during estrus than pregnancy (P<0.05) in the endometrium and tended to be higher in estrus compared to pregnancy in the myometrium (P=0.1). Expression for PGR was greater (P<0.05) in the endometrium during estrus and diestrus than during pregnancy. In the myometrium, PGR expression was greater in estrus than pregnancy (P=0.05) and tended to be higher during diestrus in relation to pregnancy (P=0.07). There were no differences among reproductive stages in ESR2, PTGER2 and PTGER4 mRNA expression (P>0.05). Immunolabeling in the endometrium appeared to be more intense for ER-α during estrus than diestrus and pregnancy. In addition, immunostaining for PR during pregnancy appeared to be more intense in the stroma and less intense in glands and epithelium compared to estrus and diestrus. EP2 immunoreactivity appeared to be more intense during early pregnancy in both endometrium and myometrium, whereas weak immunolabeling for EP4 was noted across reproductive stages. This study demonstrates differential regulation of estrogen receptor (ER) and PR in the myometrium and endometrium during the reproductive cycle and pregnancy as well as abundant protein expression of EP2 in the endometrium and myometrium during early pregnancy in mares.
Assuntos
Endométrio/metabolismo , Ciclo Estral , Cavalos , Miométrio/metabolismo , Prenhez , Receptores de Prostaglandina E/genética , Receptores de Esteroides/genética , Animais , Diestro/genética , Diestro/metabolismo , Ciclo Estral/genética , Ciclo Estral/metabolismo , Estro/genética , Estro/metabolismo , Feminino , Idade Gestacional , Hormônios Esteroides Gonadais/metabolismo , Cavalos/genética , Cavalos/metabolismo , Ovário/metabolismo , Gravidez , Prenhez/genética , Prenhez/metabolismo , Receptores de Prostaglandina E/metabolismo , Receptores de Esteroides/metabolismoRESUMO
The timing and magnitude of exposure to preovulatory estradiol followed by post-ovulatory progesterone (periovulatory endocrine milieu) in cattle modulate endometrial gene expression, histotroph composition, and conceptus development, but the mechanisms underlying this regulation remain unknown. Using an experimental model based on the modulation of follicle growth, this work aimed to evaluate if the polyamine metabolic pathway is regulated by the periovulatory endocrine milieu. Nelore cows were manipulated to ovulate small (n = 15) or large (n = 15) follicles, then the profiles of polyamines and their synthetic enzymes were compared between groups. Transcripts for the enzymes of this pathway, ornithine decarboxylase 1 (ODC1; the rate-limiting enzyme in polyamine biosynthesis) protein quantification, adenosylmethionine decarboxylase 1 (AMD1) protein immunolocalization, and concentrations of the different polyamines (putrescine, spermidine, and spermine) were respectively quantified by quantitative reverse-transcriptase PCR, immunoblotting, immunohistochemistry, and gas chromatography-mass spectrometry in both the endometrium and uterine flushing. No differences in gene and protein expression or concentration of polyamines were observed between groups. There were significant correlations between the relative abundance of ODC1 and spermidine/spermine N1-acetyltransferase 1 (SAT1) transcripts as well as between antizyme inhibitor 1 (AZIN1) and adenosylmethionine decarboxylase 1 (AMD1) transcripts. In conclusion, our results show that the polyamine metabolic pathway is present and functional, but not regulated by the periovulatory endocrine milieu in the bovine endometrium.
Assuntos
Diestro/metabolismo , Endométrio/metabolismo , Redes e Vias Metabólicas/fisiologia , Poliaminas/metabolismo , Adenosilmetionina Descarboxilase/metabolismo , Animais , Bovinos , Endométrio/química , Endométrio/enzimologia , Feminino , Ornitina Descarboxilase/metabolismo , Poliaminas/análise , Progesterona/análise , Progesterona/metabolismoRESUMO
OBJECTIVE: To investigate whether cholinergic ganglionic stimulus modifies the release of gonadotropin-releasing hormone (GnRH), catecholamines, and progesterone at the ovarian level. DESIGN: Animal study. SETTING: University animal laboratory. ANIMAL(S): Six to eight virgin adult Holtzman rats. INTERVENTION(S): Superior mesenteric ganglion-ovarian nerve plexus-ovary system removed and placed in one cuvette with two compartments, with acetylcholine added to the ganglion in the experimental group. MAIN OUTCOME MEASURE(S): Measurement of ovarian liquid obtained from catecholamines by high-performance liquid chromatography; measurement of progesterone (P(4)), GnRH, and luteinizing hormone (LH) by radioimmunoassay; and measurement of gene expression of 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and 20α-hydroxysteroid dehydrogenase (20α-HSD) by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULT(S): The study focused on the estrus and diestrus II (DII) stages. On the estrus days, the release of GnRH, NA, and 20α-HSD increased, while P(4) and 3ß-HSD decreased. On the DII days, GnRH, P(4), and 3ß-HSD increased, while 20α-HSD and NA decreased. The ovarian liquid with GnRH showed biologic activity, namely, an increase in LH release during the DII stage and a decrease during the estrus stage. CONCLUSION(S): Neural stimulus from the superior mesenteric ganglion influences the release of NA, adrenaline, and GnRH. We also have demonstrated that these neurotransmitters participate in the atretogenic processes of the ovary, thus providing evidence of the necessity of the sympathetic neural pathway.