Maturação in vitro de oócitos caninos: influência da suplementação hormonal seriada sobre a ultraestrutura oocitária

Pós-graduação em Medicina Veterinária - FCAV

Indução a espermiação do mandi-pintado, Pimelodus britskii no período reprodutivo (Teleostei: Pimelodidae)

Pós-graduação em Aquicultura - FCAV

Expressão gênica e potencial de desenvolvimento in vitro dos complexos cúmulos-oócitos ovinos tratados com roscovitina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Controle do crescimento e da seleção folicular por fatores locais e sistêmicos na espécie bovina

The regulation of ovarian activity is an integrated process involving extraovarian signals and intrafollicular factors. In such process, gonadotropins have an important role, but not always crucial. The initiation of primordial follicle growth and the early stages of folliculogenesis can occur without gonadotropins, however FSH may affect the rate of preantral follicle growth. On the other hand, the antral follicle development from 1 to 4 mm in diameter is completely gonadotropin-dependent in bovine, and the alternation of dependency from FSH to LH may be part of the mechanism involved in follicular selection. Locally produced growth factors, such as the IGF system, work in association with gonadotropins throughout the follicular growth and can influence follicular selection. Thus, the integration of these extraovarian signals and intrafollicular factors seems to determine whether a follicle will continue to develop or go atretic. This review focuses on the interaction between gonadotropins and intrafollicular factors in follicular development and selection in cattle.

Atividade dos neurônios do núcleo anterovental periventricular e sua participação no envelhecimento reprodutivo de ratas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Aspiração folicular por videolaparoscopia e maturação oocitária in vitro em pacas (Cuniculus paca – linnaeus, 1766) mantidas em cativeiro e submetidas a protocolos de superestimulação ovariana

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diâmetro folicular e taxa de prenhez em protocolo de sincronização com GnRH ou Benzoato de Estradiol no dia 0 em vacas de corte

This work aimed to evaluate the pregnancy rate and follicular diameter using EB or GnRH on the insertion of progesterone implant (D0) in lactating beef cows. Two groups were tested in two experiments. In Exp. 1 were used 61 Nelore cows divided into two groups: G-BE (n = 32) and G-GnRH (n = 29), on D0 was inserted P4 implant (CIDR ) and applied 2 mL of BE (G-BE) or 2.5 mL GnRH (G-GnRH). In D9 was performed ultrasonography (U.S.) to measure the diameter of the dominant follicle (DF) present in the ovary and the implant was removed, with concomitant administration of 2.5 mL of PGF2a and estradiol cypionate (ECP ) followed by calves removal. After 48 hours all the cows were inseminated and the calves returned. In Exp. 2 50 cows were used following the same protocol described above, but the pregnancy was assessed without performing ovarian US. There was no difference (p>0.05) in pregnancy rate between treatments, BE (55%) or GnRH (41%), but the follicular diameter was significantly higher (p<0.05) in pregnant cows treated with EB (10.7 mm vs. 8.5 mm) and in cows treated with GnRH there was no difference (p>0.05) between pregnant and no pregnant cows (11.6 mm vs. 10.2 mm). We concluded the use of GnRH on D0 did not improve the pregnancy rate in lactating beef cows and follicular diameter was greater (p <0.05) in pregnant cows compared to non-pregnant only in G-BE.

Glyphosate impairs male offspring reproductive development by disrupting gonadotropin expression

Sexual differentiation in the brain takes place from late gestation to the early postnatal days. This is dependent on the conversion of circulating testosterone into estradiol by the enzyme aromatase. The glyphosate was shown to alter aromatase activity and decrease serum testosterone concentrations. Thus, the aim of this study was to investigate the effect of gestational maternal glyphosate exposure (50 mg/kg, NOAEL for reproductive toxicity) on the reproductive development of male offspring. Sixty-day-old male rat offspring were evaluated for sexual behavior and partner preference; serum testosterone concentrations, estradiol, FSH and LH; the mRNA and protein content of LH and FSH; sperm production and the morphology of the seminiferous epithelium; and the weight of the testes, epididymis and seminal vesicles. The growth, the weight and age at puberty of the animals were also recorded to evaluate the effect of the treatment. The most important findings were increases in sexual partner preference scores and the latency time to the first mount; testosterone and estradiol serum concentrations; the mRNA expression and protein content in the pituitary gland and the serum concentration of LH; sperm production and reserves; and the height of the germinal epithelium of seminiferous tubules. We also observed an early onset of puberty but no effect on the body growth in these animals. These results suggest that maternal exposure to glyphosate disturbed the masculinization process and promoted behavioral changes and histological and endocrine problems in reproductive parameters. These changes associated with the hypersecretion of androgens increased gonadal activity and sperm production.

LH surge in response to the treatment with GnRH analog or estradiol in ovariectomized buffaloes with or without progesterone pre-exposition

The aim of the present study was to evaluate the LH surge after EB (estradiol benzoate) or GnRH administration with or without P4 (progesterone) pre-exposure in ovariectomized (OVX) buffalo cows. Females were randomly assigned to receive an intravaginal P4 device (D0–D9). They were then given EB 24 h or GnRH 36 h post-P4 device removal (factorial 2×2, n=6 per group). Blood collection for LH measurement began 36 h after the P4 device removal and continued at 3 h intervals. The area under the LH curve (AUC; 30.2 ng2 and 13.41 ng2; P=0.007) and the area of the LH peak (AP; 19.0 ng2 and 8.9 ng2; P=0.009) were greater for EB than GnRH. We did not observe an effect of P4 pre-exposure on the AUC and AP. Furthermore, there was no interaction between P4 pre-exposure and EB or GnRH treatment on the AUC and AP. However, there was an interaction (P<0.01) between P4 pre-exposure and the type of inducer (EB or GnRH) to release a preovulatory-like LH surge at the beginning (BP), final (FP) and time (TP) of the LH peak. The P4 pre-exposure anticipated the BP (2.5 and 7.4 h), TP (6.0 and 12.0 h) and FP (11.5 and 17.1 h) when EB was used to induce a preovulatory-like LH surge (P<0.01). However, there was no effect of P4 pre-exposure on BP (0.4 and 0.4 h), TP (3.0 and 3.0 h) and FP (5.9 and 6.1 h) with GnRH treatment. There was also no effect of the pre-exposure to P4, type of inducer or interaction on the amplitude of the LH peak. We concluded that EB therefore led to greater LH release than GnRH, and pre-exposure to P4 before EB administration anticipated the preovulatory-like LH surge in buffalo cows.

Hipogonadismo : revisão a propósito de 3 casos clínicos

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Efeito dos andrógenos na estabilização da preferência manual em machos adultos de sagui comum (Callithrix jacchus)

The role of steroids hormones on the behavior of vertebrates have been described as organizational and activational effects. These actions occur in different periods of the ontogenetic development as fetal, early post natal and during puberty (organizational effect) or modifying the expression of behavioral patterns during time life (activational effects). Studies on brain lateralization in hand use in human and non-human primates have shown that sexual hormones seems to participate in the process of handedness strength that begins in the puberal period and is stabilized at the adult age. The aim of this study was to investigate in adult male Callithrix jacchus if the strength of use of the hand in common marmoset adult male is stable (organizational effect) or androgens variations could affect its stability (activational effect). The preferential use of one hand in 14 common marmoset (Callithrix jacchus was studied in two contexts: (1) spontaneous holding food and directing the food to mouth (feeding episodes), and (2) forced reaching food tests where the animal have to reach the food through a hole within a cover plate with a central hole that allow the use of one hand only to reach the food. The records were made during 5 sessions/20 bouts each during baseline totalizing 100 episodes before two treatments. Firstly it was used GnRH antagonist: a single subcutaneous injection of 100µg de Cetrotide – acetate of cetrorrelix (Baxter Oncology GmbH, Germany) (n=10). Secondly, a single GnRH injection of 0.2mg of GnRH (Sigma – Aldrich) (n= 8) was used. After injections 20 successful attempts of hand use episodes was recorded in the 1st , 2 nd, 7th, 15th and 30 th days, totalizing in the whole period 100 episodes for each context, after both treatments. Fecal sampling to measure extracted fecal androgens was performed in all days of data collection across the length of the basal and during the experimental periods. Statistical analysis by mixed model, Tukey test to compare mean values after the two treatments, and Levene test to compare mean variance were used, all for p-value < 0.05. In basal phase 6 animals used preferentially the right hand, 5 the left and 3 were ambidextrous. Mean handedness index in basal phase were different from that after both treatment starting at 7th day. Mean variance of handedness index for spontaneous and forced activities does not differs before and after both treatments but the mean values for GnRH index were higher than that observed for its antagonist. These findings suggested that androgens have an activational effect on handedness in adult male C. jacchus

Kisspeptina: efeito na maturação in vitro de ovócitos bovinos

This study aimed to evaluate different concentrations of kisspeptin, as well as the interaction of kisspeptin and FSH/LH in vitro maturation and oocyte competence in cattle. In Experiment 1 was determined the minimum concentration of Kisspeptin (Kp) to be used, and in Experiment 2 was evaluated its interection with FSH and LH. The oocytes were collected in a commercial slaughterhouse and only Grade I oocytes were utilized. The oocytes were cultured in TCM-199 medium with bicarbonate plus 10% FBS, sodium pyruvate (22μg/mL), amikacin (83mg/mL), FSH (0.5μg/mL), with different concentrations of Kp, the treatments were: FSH + 0M Kp-10; FSH + 10-7M Kp-10, FSH + 10-6M Kp-10; FSH + 10-5M Kp-10. In Experiment 2, was used better concentration of Kp found in Experiment 1, the following treatments: no hormones; FSH; FSH + Kp-10; FSH + LH; FSH, LH + Kp-10; Kp-10. The oocyte competence was determined by nuclear maturation, mitochondrial distribution, MitoTracker® Orange CMTMRos fluorescence intensity and DCF. The evaluation of nuclear maturation was made after 24 hours incubation and the oocytes were stained with DAPI to determine the nuclear stage (Germinal Vesicle-GV, Metaphase I-MI and Metaphase II-MII).The mitochondrial distribution was classified as peripheral/semiperipheral and diffuse in clusters/granules, evaluated after stained with the MitoTracker® Orange CMTMRos, and was also identified the intensity of it. To determine the intensity of ROS oocytes were stained with DCF. The statistical analysis was performed by SAS GLIMMIX PROC. In Experiment 1 oocytes matured only with the FSH reached a smaller nuclear maturation when compared to those who were matured with Kisspeptin at different concentrations (FSH:13/33; FSH + 10-7M Kp-10: 28/35; FSH + 10-6M Kp-10:30/34; FSH + 10-5M Kp-10:28/32; P=0,0001). There was no statistical difference in mitochondrial distribution between treatments (P>0.05). The fluorescence intensity of MitoTracker did not differ among treatments (P>0.05). The DCF fluorescence intensity was lower when the concentration of Kp was increased in the medium (FSH:12177726,1; FSH + 10-7M Kp-10:10945982,83; FSH + 10-6M Kp-10:9820536,53; FSH + 10-5M Kp-10:9147016,38; P<0,0001). Based in the Experiment 1 results, the concentration of Kp was determined in 10-7M. In Experiment 2 the mitochondrial distribution was different between treatments, because oocytes matured only with Kp or FSH+LH, reached a oocyte competence greater than those maturated with FSH only or without hormone addition (no hormones:66,66%; FSH:66,66%; FSH + Kp-10:75,86%; FSH + LH:91,17%; FSH, LH + Kp-10:82,85%; Kp-10:91,17%; P<0,05). The no hormones resulted in a lower nuclear maturation than the other treatments (no hormones: 5/18; FSH:18/32; FSH + Kp-10:22/29; FSH + LH:26/33; FSH, LH + Kp-10:26/34; Kp-10:25/34; P=0,0094). The fluorescence intensity of probes MitoTracker and DCF was lower when Kp was added to the maturation medium (no hormones:1228363/540069; FSH:2307984/1395751; FSH + Kp-10:1941890/1114948; FSH + LH:2502145/1722376; FSH, LH + Kp-10:2286173/1467782; Kp-10:1859411/979325 P<0,0001). So this is the first study that shows that Kisspeptin stimulates oocyte maturation without the presence of gonadotropins in the maturation medium.

Influência do polimorfismo no gene do receptor da FSH na forma como as pacientes respondem ao tratamento de estimulação ovárica

A estimulação da foliculogénese através da FSH é essencial para o êxito das técnicas de reprodução assistida. Variantes genéticas do receptor da FSH podem influenciar a resposta à estimulação ovárica em pacientes submetidas a estas técnicas. O polimorfismo Asn680Ser do exão 10 do gene do receptor da FSH tem sido implicado na variabilidade da resposta a esta hormona, podendo desempenhar um papel importante na estimulação ovárica controlada. Verificou-se que no grupo das mulheres classificadas como más respondedoras, 44% apresentam o genótipo Ser/Ser, 38% Asn/Ser e 18% Asn/Asn. No grupo das pacientes com resposta normal e resposta elevada as frequências genotípicas foram, respectivamente, 10,3% e 14% para Ser/Ser, 51.5% e 50% para Asn/Ser, 38,2% e 36% para Asn/Asn. Entre as pacientes com genótipo Ser/Ser, 32,4% são más respondedoras, sendo esta frequência de 5,4% para o genótipo Asn/Asn e 8,2% para o genótipo Asn/Ser. No entanto, não se verificaram diferenças entre os três genótipos relativamente aos níveis de FSH basal, número de foliculos recrutados, número de ovócitos obtidos após punção folicular e taxa de gravidez. O genótipo parece ter influência na forma como as mulheres respondem ao processo de estimulação ovárica. As mulheres portadoras do genótipo Asn/Asn ou Asn/Ser apresentam baixa probabilidade de vir a desenvolver uma má resposta ovárica contrariamente às pacientes portadoras do genótipo Ser/Ser. Assim, o estudo do polimorfismo na posição 680 do exão 10 do gene do receptor da FSH poderá ser importante na previsão da resposta ovárica à estimulação com gonadotrofinas.

Infertilidade na síndrome do ovário poliquístico : evidências sobre o tratamento

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Caracterização clínica, metabólica e hormonal de portadores de lipodistrofia parcial familiar

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências da Saúde, Programa de Pós-Graduação em Ciências da Saúde, 2015.