RESUMO
NEW FINDINGS: What is the central question of this study? Giot1, the gene for gonadotropin inducible ovarian transcription factor 1 (GIOT1), is upregulated in osmotically challenged rats: does Giot1 gene expression in the paraventricular nucleus have a role in controlling fluid intake following dehydration and what is the role of ovarian hormones in the modulation of GIOT1 actions? What is the main finding and its importance? GIOT1 acts to regulate water and salt intake as well as hormone secretion after dehydration. The identification of genes that participate in the hormone and behavioural responses involved with hydromineral homeostasis is essential for future exploration of novel drug targets for the treatment of metabolic disease. ABSTRACT: In order to maintain body fluid balance after dehydration, hypothalamic neurons of the paraventricular nucleus (PVN) are activated to promote secretion of vasopressin (AVP) and oxytocin (OXT) from the neurohypophysis, and to modulate the behavioural allostatic responses of thirst and salt appetite. Gonadotropin inducible transcription factor (GIOT1) is a Krüppel-type zinc finger protein induced by gonadotropins and oestradiol (E2). This transcription factor is expressed in the hypothalamus, specifically in the PVN where expression of Giot1 mRNA increases following hydromineral challenges such as water deprivation or salt loading, although its physiological role is not clear. We hypothesize that GIOT1 has a central role in the integrated homeostatic and allostatic responses to disturbances in hydromineral balance, especially in the presence of female gonadal hormones. Female rats with intact ovaries or ovariectomized rats were subjected to specific microinjection of a lentiviral vector mediating Giot1 knockdown in the PVN. Three weeks after injection, rats were subjected to 48 h water deprivation, and thereafter water and salt intake were evaluated. Giot1 knockdown in PVN reduced water and saline intake as well as AVP and OXT secretion. Furthermore, Giot1 knockdown had profound effects on gene expression in the PVN, reducing the abundance of transcripts encoded by the Avp, Oxt, Nr4a1 and Crh genes. In conclusion, the present study shows for the first time that GIOT1 in the PVN regulates both transcription and fluid intake, although any connection to ovarian hormones remains to be established.
Assuntos
Desidratação , Núcleo Hipotalâmico Paraventricular , Animais , Arginina Vasopressina/metabolismo , Ingestão de Líquidos , Feminino , Gonadotropinas/metabolismo , Gonadotropinas/farmacologia , Ovário/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Fatores de TranscriçãoRESUMO
AIM: To evaluate the overall performance and oocyte quality of follicular phase stimulation (FPS) vs. luteal phase stimulation (LPS) among patients undergoing double ovarian stimulation (DuoStim). MATERIALS AND METHODS: Observational retrospective two-center cohort study including 79 infertile women who underwent a total of 87 DuoStim cycles between January 2017 and May 2019. Besides assessing baseline characteristics in order to determine the patients' clinical profile, we analyzed the FPS and LPS regarding the total dose of gonadotropin received, the duration of stimulation, the number and maturity of oocytes, fertilization and blastocyst formation rates, and the number of blastocysts obtained. RESULTS: The patients' baseline characteristics were compatible with a diminished ovarian reserve and poor reproductive prognosis. While the luteal phase needed longer stimulation (12 days (5-19) vs. 11 (7-16), p < .001) and slightly higher gonadotropin doses (2946 ± 890 IU vs. 2550 ± 970 IU, p < .001), no significant differences were detected in the oocyte maturity, fertilization, and blastocyst formation rates. However, the number of oocytes retrieved (5 (0-16) vs. 4 (0-15), p = .006), mature oocytes (4 (0-15) vs. 3 (0-11), p = .032), and blastocysts obtained (70 vs. 53) were substantially greater after LPS. CONCLUSIONS: The DuoStim strategy in poor prognosis patients increases the number of oocytes and blastocysts available. Moreover, the number of oocytes and blastocysts obtained are higher after LPS when compared to FPS. Thus, it should be considered for selected patients in order to not only improve reproductive outcomes but also shorten the time to pregnancy.
Assuntos
Fase Folicular/fisiologia , Infertilidade Feminina/terapia , Indução da Ovulação/métodos , Adulto , Estudos de Coortes , Feminino , Fertilização in vitro/métodos , Fase Folicular/efeitos dos fármacos , Gonadotropinas/farmacologia , Gonadotropinas/uso terapêutico , Humanos , Infertilidade Feminina/diagnóstico , Infertilidade Feminina/epidemiologia , Infertilidade Feminina/patologia , Fase Luteal/efeitos dos fármacos , Fase Luteal/fisiologia , Recuperação de Oócitos/métodos , Recuperação de Oócitos/normas , Oócitos/efeitos dos fármacos , Oócitos/patologia , Gravidez , Taxa de Gravidez , Prognóstico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
This study analyzes the effects of neonatal androgenization on follicular growth and first ovulation in response to gonadotrophins, using a model of exogenous stimulation or the use of subcutaneous ovary grafts in castrated animals to replace the hypothalamus-pituitary signal. Neonatal rats (days 1-5) were treated with testosterone, dihydrotestosterone or vehicle. At juvenile period, rats were stimulated with PMSG, hCG (alone or combined) or used as ovarian donors to be grafted on castrated adult female rats. Ovulation and ovarian histology were analyzed in both groups. Animals treated with vehicle or dihydrotestosterone stimulated with gonadotrophins (pharmacological or by using an ovary graft) ovulated, showing a normal histological morphology whereas rats exposed to testosterone and injected with the same doses of gonadotrophins did not it. In this group, ovulation was reached using a higher dose of hCG. Ovaries in the testosterone group were characterized by the presence of follicles with atretic appearance and a larger size than those observed in control or dihydrotestosterone groups. A similar appearance was observed in testosterone ovary grafts although luteinization and some corpora lutea were also identified. Our findings suggest that neonatal exposure to aromatizable androgens induces a more drastic signalling on the ovarian tissue that those driven by non-aromatizable androgens in response to gonadotrophins.
Assuntos
Androgênios/farmacologia , Gonadotropinas/farmacologia , Ovário/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Gonadotropina Coriônica/farmacologia , Di-Hidrotestosterona/farmacologia , Feminino , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/crescimento & desenvolvimento , Ovulação/efeitos dos fármacos , Ratos , Testosterona/farmacologiaRESUMO
PURPOSE: We hypothesized that the chemokine SDF1/CXCR4 system was present in feline cumulus-oocyte complexes (COCs) and that COCs cultured with SDF1 would directly upregulate gene expression in the ovulatory cascade. METHODS: Ovaries (n = 50) were obtained from adult domestic cats during the breeding season and COCs were recovered from antral follicles. Because IVM media triggers cumulus-oocyte expansion, culture conditions needed to be optimized to study periovulatory genes. After optimization, the effects of 25 ng/ml SDF1 and the CXCR4 inhibitor were examined in a COC culture for 3, 12, and 24 h. RESULTS: MEM-hepes with 1% of charcoal stripped-FBS was the optimized culture medium, assessed by the expansion of COCs at 24 h in the gonadotropin (GNT) group but not in the media with serum alone. The mRNA expression of HAS2, TNFAIP6, PTX3, and AREG peaked at 3 h in GNT group as compared to all other groups (p < 0.05). COCs cultured with SDF1 showed increased HAS2 and TNFAIP6 mRNA expression at 3 h compared to negative controls and to the CXCR4 inhibitor group. CXCR4 and SDF1 immunostaining was present in both cumulus cells and the oocyte. CONCLUSIONS: These results demonstrate that GNT stimulation upregulates key periovulatory genes and expansion in feline COCs from antral follicles, and support the use of this culture system to examine molecular processes within the COC. In addition, SDF1 directly promotes key periovulatory genes in feline COCs, suggesting that the SDF1-CXCR4 pathway may extend its function beyond a chemoattractant, and may play a direct role within the COC.
Assuntos
Quimiocina CXCL12/metabolismo , Células do Cúmulo/fisiologia , Regulação da Expressão Gênica , Oócitos/fisiologia , Ovulação/genética , Animais , Gatos , Técnicas de Cultura de Células/métodos , Quimiocina CXCL12/farmacologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Receptores CXCR4/metabolismoRESUMO
A reproductive analysis of a captive group of jaguars (Panthera onca; n = 6) at the Santacruz Zoological Foundation in Cundinamarca, Colombia, was conducted by performing a longitudinal, noninvasive, hormonal analysis of estradiol and progestogens in females and of androgens in males. During four seasons, female jaguars confined in solitary were evaluated for ovarian activity and spontaneous ovulation, male jaguars for testicular activity. A second hormonal follow-up was conducted in the females after administration of gonadotropins. Hormones were extracted from fecal samples of three females (n = 3) and two males (n = 2). Estradiol measurements were obtained by RIA and progestogens by enzyme immunoassay. The linear mixed-effect regression showed that there was a significant effect of seasons in the concentrations of estradiol (chi square = 15.97, degrees of freedom = 3, P < 0.01). Posthoc comparisons of all pairs of seasonal means were conducted according to Tukey's honest significant difference, revealing significant differences between seasons: Dry 1 versus Rains 2 (P < 0.01), Rains 1 versus Rains 2 (P < 0.05), and Dry 2 versus Rains 2 (P < 0.05). Elevations of progestogens compatible with spontaneous ovulation occurred in three jaguars, and the linear mixed-effect regression showed that there was also a significant effect of seasons (chi square = 28.56, degrees of freedom = 3, P < 0.01). Posthoc comparisons showed significant differences only between seasons: Dry 2 versus Rains 2 (P < 0.01). The season with the lowest average concentration was Rains 2 (October, November, and December). During this season, periods of anestrous were registered that lasted between 31 and 83 days. The three females presented estradiol peaks after the administration of eCG. A noninvasive longitudinal analysis for androgens was also made (males 1 and 2) over the course of 1 year, and no significant differences were found between the different seasons. A seminal analysis of three adult male jaguars (Panthera onca; n = 3) was also performed after electroejaculation under general anesthesia (male 1 and 2) and by a postmortem epididymal wash (male 3). The mean concentration of spermatozoids was 5.7 × 106 ± 1.1 × 106 spermatozoa/mL. The progressive motility + standard deviation averaged 80%. The percentage of normal spermatozoids obtained by electroejaculation was 80 ± 2.8%, and the abnormalities found more frequently were head defects (7 ± 1.4%). The seminal fluid obtained by epididymal flush contained 35 ± 1.4% normal spermatozoids, and the most frequent abnormalities found corresponded to distal cytoplasmic droplets (39 ± 11.3%).
Assuntos
Fertilidade , Panthera/fisiologia , Androgênios/metabolismo , Animais , Colômbia , Estradiol/metabolismo , Feminino , Gonadotropinas/farmacologia , Modelos Lineares , Estudos Longitudinais , Masculino , Detecção da Ovulação/veterinária , Indução da Ovulação/veterinária , Progestinas/metabolismo , Estações do Ano , Análise do Sêmen/veterináriaRESUMO
The objective of this study was to evaluate the effect of biostimulation and temporary weaning on the follicular dynamics and pregnancy rates in Nelore cows. We used three groups of 75 cows: a control group without biostimulation and suckling calves (WB), a group that was biostimulated and had suckling calves (BE) and a group that was biostimulated and subjected to temporary weaning for 56 h (BETW). Ovarian dynamics were examined using ultrasonography. All groups showed follicular atresia. The interval between beginning of the treatment and wave emergence was 3.25 ± 0.30 days for BE, 3.40 ± 0.27 days for BETW and 3.37 ± 0.50 days for WB. The time between implant removal and ovulation was 64.50 ± 1.88 h for BE, 66.75 ± 1.35 h for BETW and 60.85 ± 3.10 h for WB. Eight cows were submitted to ultrasound analysis, and the percentages of cows that had ovulatory follicles of the new follicular wave with maximum diameters greater than 0.80 cm were 100 % (8/8) in BE (1.28 ± 0.12 cm), 100 % (8/8) in BETW (1.52 ± 0.07 cm) and 87.5 % (7/8) in WB (1.21 ± 0.10 cm). The pregnancy rate was 52 % (39/75) in BE, 69.3 % (52/75) in BETW and 37.3 % (28/75) in WB. The association of biostimulation and temporary weaning increased follicular development, ovulation synchronisation and, consequently, the pregnancy rate in beef cows.
Assuntos
Folículo Ovariano/fisiologia , Ovulação/fisiologia , Prenhez/fisiologia , Desmame , Animais , Animais Lactentes , Bovinos , Cloprostenol/administração & dosagem , Cloprostenol/farmacologia , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Gonadotropinas/administração & dosagem , Gonadotropinas/farmacologia , Carne , Ovulação/efeitos dos fármacos , Gravidez , Taxa de Gravidez , ReproduçãoRESUMO
Guanosine 3',5'-cyclic monophosphate (cGMP), as a second messenger, plays potential roles in ovarian functions. To elucidate the role of phosphodiesterase (PDE) in cGMP signaling during ovarian follicular development, the present study was conducted to investigate ovarian cGMP level and cGMP-PDE activity by radioimmunoassay (RIA) in postnatal rats, immature rats during gonadotropin-primed follicular development, ovulation and luteinization, adult rats during normal estrous cycle, and aged rats that spontaneously developed persistent estrus (PE). All four rat models were confirmed by histological examination of one ovary, and the other ovary was used for RIA. In postnatal rats, cGMP level was high at birth and decreased dramatically by Day 5, and then, it increased maximally at Day 10 and declined at Day 21. However, cGMP-PDE activity did not significantly change during Days 1 to 10, but increased significantly on Day 21. In immature female rats, cGMP level markedly decreased upon treatment with equine chorionic gonadotropin (eCG), while cGMP-PDE activity did not show any significant changes; however, ovarian cGMP level and cGMP-PDE activity increased after injection of an ovulatory dose of human chorionic gonadotropin (hCG) for induction of ovulation and luteinization. In adult rats during normal estrous cycle, cGMP level was high on proestrus and metestrus days, while cGMP-PDE activity was high on estrus day. In PE rats, ovarian cGMP level was similar to that in adult rats on estrus and diestrus days but lower than that on proestrus and metestrus days; ovarian cGMP-PDE activity was lower than that on estrus days but similar as the other estrous cycle days. In addition, there was a significant negative correlation between ovarian cGMP level and cGMP-PDE activity during normal estrous cycles in the adult rat (r = -0.7715, N = 16, P < 0.05), but not in the postnatal rat (r = -0.1055, N = 20, P > 0.05). Together, the results of our present study indicated that ovarian cGMP levels were not dependent on cGMP-PDE activity during early postnatal development, but highly dependent on cGMP-PDE activity in the adult rat. This implies that mechanisms of cGMP signaling involved in ovarian functions are stage-specific in the rat.
Assuntos
GMP Cíclico/metabolismo , Folículo Ovariano/fisiologia , Ovário/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Ativação Enzimática , Ciclo Estral , Feminino , Gonadotropinas/farmacologia , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Ovário/citologia , Ovário/efeitos dos fármacos , RatosRESUMO
OBJECTIVE: To study the effects of different doses of leptin on the expression of proteins involved in P synthesis, such as steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase (3ßHSD). DESIGN: Experimental studies. SETTING: Research laboratory. ANIMAL(S): Immature rats primed with gonadotropins to induce ovulation. INTERVENTION(S): In vivo studies: rats received either an acute or daily treatment with leptin. In vitro studies: ovarian explants were cultured in the absence or presence of leptin (0.3-500 ng/mL). MAIN OUTCOME MEASURE(S): The expression of both messenger RNA and protein of StAR, P450scc, and 3ßHSD were measured by reverse transcription-polymerase chain reaction (PCR) and Western blot, respectively. RESULT(S): The acute treatment with leptin, which inhibits the ovulatory process, caused a significant reduction in the ovarian expression of P450scc without changes in StAR or 3ßHSD. In contrast, the daily treatment, which induces the ovulatory process, showed an increased expression of the ovarian 3ßHSD protein, without differences in the other proteins measured. We also found that leptin increased the protein of both P450scc and 3ßHSD at physiological levels and inhibited both messenger RNA and protein of 3ßHSD at higher concentrations. CONCLUSION(S): The results indicate that 1) leptin is able to regulate the expression of the 3ßHSD protein in a dose-dependent manner; and 2) leptin seems to exert its dual effects on P synthesis on different targets in a dose-dependent manner.
Assuntos
3-Hidroxiesteroide Desidrogenases/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Leptina/fisiologia , Fosfoproteínas/genética , Progesterona/biossíntese , 3-Hidroxiesteroide Desidrogenases/metabolismo , Fatores Etários , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/fisiologia , Gonadotropinas/farmacologia , Leptina/farmacologia , Ovulação/efeitos dos fármacos , Ovulação/fisiologia , Indução da Ovulação/métodos , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The development of assisted reproductive technologies such as embryo transfer (ET), artificial insemination (AI) and in vitro fertilization (IVF) in South American camelids is considerably behind that of other livestock species. Poor success of the embryo transfer technique has been related to a lack of an effective superstimulatory treatment, low embryo recovery rate, and the recovery of hatched blastocysts that are not conducive to the cryopreservation process. Superstimulation has been attempted using equine chorionic gonadotropin (eCG) and follicle stimulating hormone (FSH) during the luteal, or the sexually receptive phase, sometimes given at follicular wave emergence. The rationale for inducing a luteal phase prior to or during superstimulation in camelids is not clearly understood, but it may simply reflect an empirical bias to conventional methods used in other ruminants. The number of ovulations or CL varies widely among studies, ranging from 2 to more than 15 per animal, with the number of transferable embryos ranging from 0 to 4 per animal. The control of follicular growth combined with superstimulatory protocols has resulted in a more consistent ovarian response and a greater number of follicles available for aspiration and oocyte collection. Recent studies in llamas have demonstrated that the use of ovulation inducing treatments or follicle ablation can synchronize follicular wave emergence allowing the initiation of gonadotropin treatment in the absence of a dominant follicle resulting in a more consistent ovulatory response. Few studies in alpacas have been reported, but it appears from recent field studies that the ovarian response is more variable and that there is a greater number of poor responders than in llamas. A review of superstimulation protocols that have been used in llamas and alpacas in the last 15 years is provided, including a discussion of the potential of protocols designed to initiate treatment at specific stages of follicular growth.
Assuntos
Camelídeos Americanos/fisiologia , Gonadotropinas/farmacologia , Superovulação/efeitos dos fármacos , Animais , Transferência Embrionária/veterinária , Feminino , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Superovulação/fisiologiaRESUMO
BACKGROUND: It is estimated that 15% of couples living in industrialized countries are infertile, ie have failed to conceive, reproductive age, after 12 months ormore of regular intercourse without contraception. During the past decade has increased the demand for fertility treatments because they believe are moreeffective now. OBJECTIVE: To unify the therapeutic approach and service to patients and set a precedent for a Mexican Official Standard respect and support for the legislation of these procedures. METHOD: Consensus by technical experts group panel with the participation of 34 national centers accredited for use in assisted reproduction. He organized seven workshops with the following themes: 1) selection of patients for assisted reproduction treatment, 2) schemes controlled ovarian stimulation for assisted reproduction techniques of high complexity, 3) preparation and egg retrieval technique, 4) transferembryo; 5) luteal phase supplementation; 6) indications and techniques of cryopreservation and 7) informed consent. Each table had a coordinator who wrote and presented the findings to the full, it made a number of observations until they reached unanimity of criteria, which are reflected in this document. RESULTS: Patient selection for assisted reproduction techniques is the first step of the process. Proper selection lead to success, in the same way that a bad pick up for failure. In the case of egg donation the most important recommendation is that only one to two embryos transferred in order to reduce multiple pregnancy rates and maintaining high pregnancy rates.
Assuntos
Técnicas de Reprodução Assistida/normas , Blastocisto , Manutenção do Corpo Lúteo , Criopreservação/métodos , Destinação do Embrião , Transferência Embrionária/normas , Feminino , Gonadotropinas/administração & dosagem , Gonadotropinas/isolamento & purificação , Gonadotropinas/farmacologia , Humanos , Infertilidade Feminina/etiologia , Infertilidade Feminina/terapia , Infertilidade Masculina/etiologia , Infertilidade Masculina/terapia , Consentimento Livre e Esclarecido , Inseminação Artificial/normas , Masculino , Doação de Oócitos/normas , Recuperação de Oócitos/métodos , Recuperação de Oócitos/normas , Ovário , Indução da Ovulação/métodos , Indução da Ovulação/normas , Seleção de Pacientes , Gravidez , Taxa de Gravidez , Progesterona/administração & dosagem , Progesterona/farmacologia , Preservação do Sêmen/métodos , Preservação do Sêmen/normas , Testículo , Preservação de Tecido/métodos , Preservação de Tecido/normasRESUMO
The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A(1) (with a dense cumulus), A(2) (with a translucent cumulus), B(1) (with the corona radiata), B(2) (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A(1) COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.
Assuntos
Células do Cúmulo/fisiologia , Gonadotropinas/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Suínos , Amônia/metabolismo , Animais , Contagem de Células , Células Cultivadas , Células do Cúmulo/citologia , Feminino , Fertilização in vitro/veterinária , Hormônio Foliculoestimulante/farmacologia , Glucose/metabolismo , Glicólise , Ácido Láctico/metabolismo , Lipídeos/análise , Lipólise , Hormônio Luteinizante/farmacologia , Metaboloma , Oócitos/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/análiseRESUMO
Cadherins are adhesion molecules that play a crucial role in tissue morphogenesis. Studies on N-cadherin and E-cadherin in the ovary of fetal hamster suggest that these adhesion molecules are involved in primordial follicle formation. In chicken embryo, present results demonstrate that N-cadherin is located on the surface epithelium and in the cortical cords of the ovary. Moreover, N-cadherin is identified in germ cells on day 14 of chicken embryo development. Quantification of mRNA of N-cadherin and E-cadherin demonstrates that treatments with follicle-stimulating hormone (FSH) or human chorionic gonadotropin (hCG), increase N-cadherin expression. Whereas, E-cadherin expression is decreased by gonadotropin treatments. The negative correlation between both cadherins expression is demonstrated after 18 h of hormonal treatment. Regulation of cadherin expression by gonadotropins and the presence of N-cadherin in the ovarian cortex suggest that these adhesion molecules are involved in ovarian morphogenesis in the chicken embryo.
Assuntos
Caderinas/genética , Embrião de Galinha/metabolismo , Gonadotropinas/fisiologia , Ovário/metabolismo , Animais , Caderinas/metabolismo , Células Cultivadas , Gonadotropina Coriônica/farmacologia , Feminino , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Gonadotropinas/farmacologia , Humanos , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Morfogênese/fisiologia , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/embriologia , Cultura Primária de Células , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Aiming to improve the developmental competence of bovine oocytes during meiotic block, this study evaluated the effects of a serum replacer (Knockout SR®) and hormones (gonadotropins and estradiol) supplementation of prematuration medium (TCM119 with 0.5 mM IBMX [IBMX group] or 25 µM roscovitine [ROSC group]) on the kinetics of oocyte nuclear maturation and embryo development. Most IBMX and ROSC oocytes prematured for 8 h culture remained in the GV stage (70.3% and 73.1%, respectively; p > 0.05) similar to Control 8 h (63.5%) and to control immature oocytes (Control 0 h, 92.5%). After prematuration for 16 h, no oocytes remained in the GV stage at similar rates to those recently aspirated (p < 0.05); GV rates in ROSC (32.4%) were higher (p < 0.05) than in the Control 16 h group (8.6%), but similar (p > 0.05) to IBMX (9.7%). After in vitro maturation (IMV) for 24 h, metaphase II (MII) rates for oocytes prematured during 8 h were similar (p > 0.05) between control and treatments (65.0-71.7%). Similarly, MII rates oocytes prematured during 16 h were similar (p > 0.05) between all groups (45.9-60.4%). Cleavage rates (67.8-78.2%), embryonic development in day-7 (25.0-35.6%) and hatching rates in day-8 (2.5-11.3%) oocytes blocked during 8 h were similar for all groups (p > 0.05). Results indicate that addition of Knockout SR® and hormones to meiotic block culture with IBMX and roscovitine negatively affected meiotic arrest, but did not impair oocyte nuclear maturation and acquisition of developmental competence.
Assuntos
Meiose/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Bovinos , Núcleo Celular , Meios de Cultura/química , Desenvolvimento Embrionário/efeitos dos fármacos , Estradiol/farmacologia , Feminino , Fertilização in vitro , Gonadotropinas/farmacologia , Purinas/farmacologia , RoscovitinaRESUMO
During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17beta and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 microg ml(-1)) of estradiol-17beta and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17beta), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17beta supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.
Assuntos
Estradiol/farmacologia , Gonadotropinas/farmacologia , Oócitos/efeitos dos fármacos , Progesterona/farmacologia , Animais , Técnicas de Cultura de Células , Divisão do Núcleo Celular/efeitos dos fármacos , Meios de Cultura , Cães , Oócitos/citologiaRESUMO
Os índices de prenhez após inseminação artificial em felídeos selvagens não são satisfatórios devido ao variável ambiente endócrino após a estimulação com gonadotropinas. O objetivo deste estudo consistiuem aumentar a taxa de sucesso em programas de inseminação artificial em gatas domésticas (animal modelo). As fêmeas (n=9) foram divididas em três grupos, cada um com três animais, sendo: 1) controle(C), somente 200 UI eCG/ 100 UI hCG ; 2) levonorgestrel oral (L)(0,075 mg) durante 37 dias + eCG/hCG; 3) etonogestrel (E), implante subdérmico durante 37 dias + eCG/hCG. Foram submetidas ao exame laparoscópico 29-39 horas após a administração de hCG para verificação da resposta ovariana e realização de esfregaço vaginal para monitoração da fase do ciclo estral. Foram coletadas amostras de fezes 60 dias antes e 60 dias após o tratamento com gonadotropinas para dosagem hormonal de estrógenos. Os resultados foram avaliados através do Teste ANOVA. Os níveis de significância mostraram que o Grupo E, em contraste com o Grupo C e o Grupo L, apresentou inibição satisfatória das concentrações de estrógenos durante a sua utilização. O grupo L não apresentou inibição ovariana durante o tratamento e diferença significativa em relação ao Grupo C. No exame laparoscópico todas as fêmeas dos grupos C, L e E apresentaram folículos e 77% das fêmeas apresentaram corpo lúteo. Também apresentaram células epiteliais superficiais anucleadas e nucleadas características de estro. Concluiu-se que a utilização de implantes de etonogestrel em gatas domésticas mostrou-se eficaz, possibilitando asua utilização prévia aos programas de inseminação artificial, aspiração folicular e também para a contracepção.
Reproductive success in endangered captive small felids species is veryl ow. Due to great variability in endocrine environment post gonadotropin treatment, after artificial insemination pregnancy rates are very low. Nowadays, ovarian activity controll improves the AI success in many species. In this study, new protocols were compared to improve the fertilization rates in artificial insemination programs in domestic cat. Female domestic cats were divided in three treatments: 1) control (eCG/hCG); 2) levonorgestrel (0.075 mg) orally during 37days + eCG/hCG; 3) etonogestrel subdermal implant during 37days + eCG/hCG: Laparoscopies were done 29-39 hours post hCG treatment to verify ovarian activity. Vaginal swabs were collected at laparoscopic procedures. Fecal samples were colected 60 days before, during and 60 days after the gonadotropin treatment for estradiol assay. Means comparisons were done by ANOVA test. Results demonstrated that etonogestrel (implant) and not oral levonorgestrel successfully suppressed ovarian activity. The levonorgestrel group didnot show ovarian inactivity during the administration, presenting oestradiol peaks and without significative diference comparing to control group. All females presented anuclear and nuclear superficial vaginal epithelial cells at laparoscopies. In conclusion, the etonogestrel implant used in the domestic cat was efficient and can be used previous to gonadotropin protocol in artificial insemination programs, follicular aspiration and contraception
Assuntos
Animais , Feminino , Anticoncepcionais Orais Sintéticos/farmacologia , Fertilização/fisiologia , Gatos/fisiologia , Indução da Ovulação/veterinária , Inseminação Artificial/veterinária , Ovário/fisiologia , Análise de Variância , Anticoncepção/métodos , Desogestrel/farmacologia , Fertilização , Gonadotropinas/farmacologia , Inseminação Artificial/normas , Levanogestrel/farmacologiaRESUMO
Despite the probable inhibitory effects of GnRH analogues on ovarian steroidogenesis in vitro, their association with assisted reproduction protocols shows favorable results. This suggests that there are important differences in the behaviors of these drugs when administered in vivo versus in vitro. To clarify these differences, this study was designed to analyze the effect of leuprolide acetate (LA) on ovarian steroidogenesis in women undergoing In Vitro Fertilization (IVF). A prospective, randomized open label study was conducted on 14 women (26-35 years): seven receiving only gonadotrophins (Group 1) and seven receiving gonadotrophin plus LA at 1mg/day (Group 2). The LA in vivo effect was determined with serum and follicular fluid (FF) samples and via luteinized granulosa cell cultivation (GCC), where cells were obtained during oocyte retrieval after ovarian hyperstimulation. In vitro analysis was performed via addition of LA to GCC only for Group 1 (without LA) at progressively higher concentrations (0, 10(-12), 10(-9) and 10(-6)M). In vivo, the main observation was a reduction in androgen production in Group 2, represented by lower androstenedione production in FF (G1=6479+/-3458; G2=3021+/-1119 ng/ml; p=0.04) and a lower testosterone peak in GC at 96h (G1=0.64+/-0.12 ng/ml; G2=0.50+/-0.19 ng/ml; P=0.02), but a higher fertilization rate (G1=67%; G2=83%; p=0.009). In vitro, testosterone, estradiol and progesterone were also reduced by LA, even though this reduction occurred for progesterone only at the highest LA dosage (10(-6)M; 606.0+/-114.3 ng/ml versus 1524.0+/-246.5 ng/ml; p=0.02). Results show that LA reduces ovarian steroidogenesis in vivo by essentially inhibiting androgen synthesis; whereas, in vitro, ovarian steroidogenesis is reduced overall.
Assuntos
Fármacos para a Fertilidade Feminina/uso terapêutico , Fertilização in vitro/efeitos dos fármacos , Infertilidade/tratamento farmacológico , Leuprolida/uso terapêutico , Ovário/efeitos dos fármacos , Esteroides/biossíntese , Adulto , Células Cultivadas , Combinação de Medicamentos , Feminino , Líquido Folicular/citologia , Líquido Folicular/efeitos dos fármacos , Líquido Folicular/metabolismo , Hormônio Liberador de Gonadotropina , Gonadotropinas/farmacologia , Células da Granulosa/citologia , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Humanos , Técnicas In Vitro , Infertilidade/metabolismo , Ovário/metabolismo , Estudos Prospectivos , Esteroides/sangueRESUMO
The angiopoietin (ANGPT) receptor (TEK) system plays a crucial role in blood vessel development and regression. To date, no reports have addressed the actions of the anti-ANGPT1 antibody on gonadotropin-stimulated follicular development and atresia in the ovary. Therefore, in this study we specifically investigated whether ANGPT1 plays a critical intraovarian survival role for gonadotropin-dependent folliculogenesis. In particular, we examined the effect of local administration of anti-ANGPT1 antibody on follicular development, apoptosis, and expression of BCL2 protein family members (BAX, BCL2, and BCL2L1), TNFRSF6, and FASLG in ovarian follicles from prepubertal eCG-treated rats. The inhibition of ANGPT1 caused an increase in the number of atretic follicles and a decrease in the number of both antral follicles (AFs) and preovulatory follicles in gonadotropin-treated rat ovaries. Taking into account that follicular atresia is mediated by apoptosis, we analyzed the effect of the antibody against ANGPT1 on programmed cell death. The inhibition of the action of ANGPT1 caused an increase both in the number of apoptotic granulosa cells in AFs and in the spontaneous DNA fragmentation of AFs cultured in serum-free medium. Besides, AFs obtained from rats treated with intraovarian antibodies against ANGPT1 showed both a decrease in BCL2 and an increase in BAX protein levels. Moreover, a reduction in the BCL2L1(L)/BCL2L1(S) ratio was observed in this group, with a reduction of BCL2L1(L) greater than that of BCL2L1(S), thus showing that the expression of these antiapoptotic proteins is lower in follicles from treated rats than in those from untreated ones. Our findings suggest that the inhibition of ANGPT1 activity causes an increase in the number of atretic follicles mediated by ovarian apoptosis through an imbalance in the ratio of antiapoptotic to proapoptotic proteins. This could take place through a paracrine effect on granulosa cells mediated by the TEK receptor in theca cells. Therefore, these data clearly indicate that ANGPT1 is necessary for follicular development induced by gonadotropins.
Assuntos
Angiopoietina-1/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Angiopoietina-1/antagonistas & inibidores , Angiopoietina-1/fisiologia , Animais , Anticorpos/administração & dosagem , Apoptose/genética , Contagem de Células , Fragmentação do DNA/efeitos dos fármacos , Endotélio/citologia , Endotélio/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Gonadotropinas/farmacologia , Injeções Intraperitoneais , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/fisiologia , Cavidade Peritoneal , Ratos , Ratos Sprague-Dawley , Maturidade Sexual/efeitos dos fármacosRESUMO
The present study analyses the participation of ovarian innervation during reproductive senescence. We use the model of acute peripheral pharmacological sympathetic denervation with guanethidine in young (3 months old), middle-aged (12 months old) or old (18 months old) rats with spontaneous or induced ovulation. Ovarian levels of norepinephrine (NE) were measured by HPLC and the oestrous cycle, the number of ovulating animals and the percentage of atretic follicles were also assessed. Aged animals showed a progressive reduction in ovulatory capacity and an increase in ovarian NE content. Acute denervation increased the percentage of healthy follicles in 12- and 18-month-old rats compared with control adult animals. Combined treatment of denervation plus stimulation with gonadotrophins doubled the number of ova shed in young adult rats and restablished a partial ovulation in 12-month-old rats. The results suggest that ovarian noradrenergic innervation plays a modulator role in ovarian physiology during the ageing ovary process. The action of ovarian noradrenergic innervation seems to be associated with folliculogenesis and the ovarian response to gonadotrophins.
Assuntos
Envelhecimento/fisiologia , Ciclo Estral/fisiologia , Ovário/inervação , Ovulação/fisiologia , Ratos/fisiologia , Simpatectomia Química/métodos , Animais , Feminino , Gonadotropinas/farmacologia , Guanetidina , Análise Multivariada , Norepinefrina/metabolismo , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/fisiologia , Ovário/fisiologia , Ovulação/efeitos dos fármacos , Ratos Wistar , SimpatolíticosRESUMO
OBJECTIVE: To compare embryo implantation in Wistar rats submitted to ovarian stimulation using recombinant FSH (rFSH) with cetrorelix acetate or leuprolide acetate. DESIGN: Experimental study. SETTING: Faculty of medicine animal facility. PATIENT(S): Fifty-six female Wistar rats with normal estrus cycles and 30 male. INTERVENTION(S): Ovarian stimulation and laparotomy (by the day 13 of gestation). MAIN OUTCOME MEASURE(S): Embryo implantation. RESULT(S): The female rats were subdivided into four groups: group 1, medicated with rFSH, hCG, and cetrorelix acetate; group 2, medicated with rFSH, hCG, and leuprolide acetate; group 3, medicated with rFSH and hCG; and group 4, in which only saline solution was administered. The female rats were mated with fertile male rats on the day of hCG administration with copulation confirmed through cytologic vaginal analysis. The females were killed on the 13th day of gestation. After laparotomy, comparison and identification was done regarding the numbers of corpora lutea and embryo implantations and gestation rates. Group 1 presented lower numbers of corpora lutea and embryo implantations in comparison to the other groups (P<.05). A difference was not found in the gestation rates between the groups. CONCLUSION(S): The number of embryo implantations in Wistar rats medicated with rFSH and cetrorelix acetate is lower than that of rats medicated with rFSH and leuprolide acetate.