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Resumen Se han encontrado asociaciones entre polimorfismos genéticos y parámetros reproductivos, este abordaje adicionalmente permitiría proponer explicaciones a diferentes fenómenos estudiados. FSHr, InhA, AMH y AMHr son genes asociados al desarrollo folicular, que tienen una alta homología y la misma función en bovinos. El objetivo del presente trabajo fue evaluar en bovinos muy relacionados si se encuentran algunos polimorfismos asociados a parámetros reproductivos reportados en otras especies. Durante el 2018, en el Magdalena Medio Colombiano fueron tomadas muestras de sangre anticoagulada de 50 búfalas mestizas (Bubalus bubalis) y 50 vacas cebú comercial (Bos indicus). Se registró la paridad, días abiertos, intervalo parto primer servicio y edad al primer parto de cada animal, sin alteraciones anatómicas, con edad y peso similar, mantenidos en las mismas condiciones de alimentación y manejo. Se extrajo el ADN por el método de Salting out y se evaluaron los polimorfismos de acuerdo con lo reportado en la literatura. Se compararon los datos usando la prueba Mann Whitney, se consideró significativo un valor de p<0,05. Se encontró que los búfalos tienen menores niveles de AMH, edad al primer parto, intervalo ente partos que las vacas (p<0,001), mayor paridad (p=0,005). No se confirmaron los po- limorfismos reportados en Holstein y en humanos en ninguna de las muestras y especies analizadas. No confirmar los hallazgos en bovinos de otras razas y espe- cies, aunque las especies tengan diferentes parámetros reproductivos, muestra la necesidad de replantear el abordaje del estudio de la asociación de los fenómenos reproductivos y genéticos.
Abstract Associations have been found between genetic polymorphisms and reproductive parameters, this approach additionally would allow us to propose explanations to the phenomena studied. FSHr, InhA, AMH and AMHr are genes associated with follicular development, which have a high homology and the same function in bovines. The objective of this work was to evaluate if some polymorphisms described in other species associated with reproductive parameters are found in two closely related bovines. During 2018, in the Colombian Magdalena Medio anticoagulated blood samples from 50 buffaloes (Bubalus bubalis) and 50 commercial zebu cows (Bos indicus) were taken. All animals without anatomical abnormalities and similar weight and age were taken and the. parity, intercalving period and age at first calving were also recorded. DNA were extracted using Salting out method and the reported polymorphisms of the above mentioned genes were evaluated. Data were compared using Mann Whitney test and p<0.05 value were considered significant. Buffaloes have lower AMH levels, age at first calving, calving interval than Bos indicus cows (p<0.001) and higher parity (p=0.005). The polymorphisms reported in Holstein and humans were not confirmed in any of the samples and species analyzed. The results, are in some way paradoxical because there are differences in reproductive parameters but nothing different in the studies genes. It shows the need to rethink the approach of the study of the association of reproductive and genetic phenomena
Resumo Foram encontradas associações entre polimorfismos genéticos e parâmetros repro- dutivos; essa abordagem também permitiria propor explicações para os diferentes fenômenos estudados. FSHr, InhA, AMH e AMHr são genes associados ao desenvol- vimento folicular, que apresentam alta homologia e a mesma função em bovinos. O objetivo do presente trabalho foi avaliar em bovinos intimamente relacionados se alguns polimorfismos associados a parâmetros reprodutivos relatados em outras espécies. Em 2018, 50 vacas de búfalo (Bubalus bubalis) e 50 de zebu comerciais (Bos indicus) foram amostradas na Magdalena Medio colombiana. Foram registra- das paridade, dias abertos, intervalo de nascimento do primeiro serviço e idade no primeiro nascimento de cada animal, sem alterações anatômicas, com idade e peso semelhantes, mantidas nas mesmas condições de alimentação e manuseio. O DNA foi extraído pelo método Salting out e os polimorfismos foram avaliados de acordo com o relatado na literatura. Os dados foram comparados pelo teste de Mann Whitney, sendo considerado significativo um valor de p <0,05. Verificou-se que os búfalos apresentam níveis mais baixos de AMH, idade do primeiro nascimento, intervalo entre os nascimentos que as vacas (p <0,001), maior paridade (p = 0,005). Os polimorfis- mos relatados em Holstein e em humanos não foram confirmados em nenhuma das amostras e espécies analisadas. A não confirmação dos achados em bovinos de outras raças e espécies, embora as espécies possuam diferentes parâmetros repro- dutivos, mostra a necessidade de repensar a abordagem do estudo da associação dos fenômenos reprodutivos e genéticos.
ABSTRACT
Resumen OBJETIVO Evaluar los resultados en ciclos de FIV-ICSI de dos protocolos de estimulación ovárica en mujeres mayores de 35 años e investigar si agregar hormona luteinizante recombinante a FSH-r en un protocolo de estimulación mejora la respuesta ovárica y, en consecuencia, las tasas de embarazo en este grupo poblacional. MATERIALES Y MÉTODOS Estudio longitudinal, observacional y retrospectivo efectuado en pacientes de la Clínica de Reproducción Hisparep del Hospital Español con diagnóstico de infertilidad, mayores de 35 años, que recibieron un ciclo de hiperestimulación ovárica controlada con FIV-ICSI durante el periodo 2014-2016. El análisis estadístico se efectuó con la prueba de t de Student para muestras independientes. Los estudios se analizaron con el paquete estadístico SPSS IBM, versión 22. RESULTADOS Se analizaron 201 mujeres con infertilidad, mayores de 35 años. El grupo 1 (n = 101) de FIV-ICSI recibió estimulación con hormona folículo estimulante recombinante y hormona luteinizante recombinante 2:1 con menotropinas (Pergoveris® y Merapur®) a partir del segundo día del ciclo. El grupo 2 (n = 100) recibió hormona folículo estimulante recombinante y menotropinas (Gonal F® y Merapur®); en ambos esquemas se utilizó antagonista de GnRH a partir del día 7 del ciclo. La media de ovocitos aspirados fue de 7.5 en el grupo 1 y 9.1 en el grupo 2 (p = 0.058). La media de ovocitos maduros fue 6.2 en el Grupo 1 vs 7.4 en el grupo 2 (p = 0.085). La tasa de fecundación en el grupo 1 fue de 57 vs 67% en el grupo 2 (p = 0.045). En el grupo 1 la tasa de implantación por embrión transferido en fresco fue 24.1 vs 10.3% en el Grupo 2 (p = 0.40), la tasa de recién nacido vivo fue de 30% en el Grupo 1 vs 20.6% en el Grupo 2. La media de embriones vitrificados en el Grupo 1 fue 1.47 vs 1.38 en el Grupo 2. CONCLUSIONES La probable ventaja de la complementación con hormona folículo estimulante recombinante durante la estimulación ovárica en mujeres mayores de 35 años es de interés y se requiere su evaluación en estudios posteriores.
Abstract OBJECTIVE To evaluate the reproductive effects when recombinant luteinizing hormone is added and to compare two stimulation schemes by number of aspirated oocytes, mature oocytes, fertilization and implantation rates, live newborn and number of vitrified embryos. MATERIALS AND METHODS Longitudinal, observational and retrospective study carried out in patients of the Hisparep Reproduction Clinic of the Spanish Hospital with diagnosis of infertility, over 35 years old, who received a controlled ovarian hyperstimulation cycle with IVF-ICSI during the period 2014-2016. The statistical analysis was carried out with the Student t test for independent samples. The studies were analyzed with the IBM SPSS statistical package, version 22. RESULTS We analyzed 201 women with infertility, over 35 years of age. Group 1 (n = 101) of IVF-ICSI received stimulation with recombinant follicle-stimulating hormone and recombinant luteinizing hormone 2: 1 with menotropins (Pergoveris® and Merapur®) from the second day of the cycle. Group 2 (n = 100) received recombinant follicle stimulating hormone and menotropins (Gonal F® and Merapur®); in both schemes, GnRH antagonist was used from day 7 of the cycle. The average number of aspirated oocytes was 7.5 in Group 1 and 9.1 in Group 2 (p = 0.058). Mean mature oocytes were 6.2 in Group 1 vs 7.4 in Group 2 (p = 0.085). The fertilization rate in group 1 was 57 vs. 67% in Group 2 (p = 0.045). In Group 1 the implantation rate per embryo transferred fresh was 24.1 vs 10.3% in Group 2 (p = 0.40), the live newborn rate was 30% in Group 1 vs 20.6% in Group 2. The mean number of vitrified embryos in Group 1 was 1.47 vs 1.38 in Group 2. CONCLUSIONS The probable advantage of supplementation with recombinant follicle-stimulating hormone during ovarian stimulation in women over 35 years of age is of interest and its evaluation is required in subsequent studies.
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Objective@#To explore the effects of BPA on the expression of N-cadherin, Vimentin and FSHR in rat Sertoli cells.@*Methods@#Primary Sertoli cells collected from prepuberty rats (18-21 d) were cultured for 48 h, and then they were treated with 0, 30, 50, 70 μmol/L BPA respectively for 24 h. The methods of MTT, real-time quantitative PCR and Western blotting were utilized to measure the cell ability of Sertoli cells, the mRNA and protein expression levels of N-cadherin, Vimentin and FSHR respectively.@*Results@#Compared with control, the cell abilities of Sertoli cells in 50 μmol/L BPA group and 70 μmol/L BPA group increased significantly (P<0.05) . The cell abilities of Sertoli cells decreased with the increases of exposure doses of BPA. Compared with control, the expression of N-cadherin mRNA only increased in 30 μmol/L BPA group (P<0.05) , the expression of Vimentin mRNA decreased significantly in all doses group of BPA (P<0.05) , the expression of FSHR mRNA increased in all doses group of BPA (P<0.05) . Compared with the control, the protein levels of N-cadherin increased significantly in 50 μmol/L BPA group (P<0.05) , the protein levels of Vimentin decreased significantly in all doses group of BPA (P<0.05) , the protein levels of FSHR decreased significantly in 50 μmol/L BPA group and 70 μmol/L BPA group (P<0.05) .@*Conclusion@#The mechanism of testicular toxicity from BPA might be the alterations of N-cadherin, Vimentin and FSHR by disturbing normal spermatogenesis.
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The objective this study was to determine the effect of phytohemagglutinin (PHA) on survival, growth and gene expression in caprine secondary follicles cultured in vitro. Secondary follicles (∼0.2 mm) were isolated from the cortex of caprine ovaries and cultured individually for 6 days in α-MEM+ supplemented with PHA (0, 1, 10, 50, 100, or 200 µg/mL). After 6 days of culture, follicle diameter and survival, antrum formation, ultrastructure and expression of mRNA for FSH receptors (FSH-R), proliferating cell nuclear antigen (PCNA), and neuronal nitric oxide synthase were determined. All treatments maintained follicular survival [α-MEM+ (94.59%); 1 µg/mL PHA (96.43%); 10 µg/mL PHA (84.85%); 50 µg/mL PHA (85.29%); 100 µg/mL PHA (88.57%), and 200 µg/mL PHA (87.50)], but the presence of 10 µg/mL PHA in the culture medium increased the antrum formation rate (21.21%) when compared with control (5.41%, P < 0.05) and ensured the maintenance of oocyte and granulosa cell ultrastructures after 6 days of culture. The expression of mRNA for FSH-R (2.7 ± 0.1) and PCNA (4.4 ± 0.2) was also significantly increased in follicles cultured with 10 µg/mL PHA in relation to those cultured in α-MEM+ (1.0 ± 0.1). In conclusion, supplementation of culture medium with 10 µg/mL PHA maintains the follicular viability and ultrastructure, and promotes the formation of antral cavity after 6 days of culture in vitro.
Subject(s)
Animals , Female , Follicle Stimulating Hormone/metabolism , Mitogens/pharmacology , Ovarian Follicle/drug effects , Phytohemagglutinins/pharmacology , Follicle Stimulating Hormone/genetics , Goats , In Vitro Techniques , Ovarian Follicle/growth & development , Ovarian Follicle/ultrastructure , Proliferating Cell Nuclear Antigen/metabolism , RNA, Messenger/metabolismABSTRACT
This study investigated the levels of messenger ribonucleic acids (mRNA) for inhibin-ßA subunit in goat primordial, primary and secondary follicles, as well as in cumulus-oocyte complexes (COCs) and mural granulosa / theca cells of antral follicles. The effects of activin-A (100ng mL-1) and/or follicle stimulating hormone (FSH, 50ng mL-1) on growth and expression of mRNA for activin-A and FSH receptor (FSH-R) in secondary follicles cultured for six days were evaluated. The data showed that the expression of inhibin-ßA is lower in secondary follicles than in primary follicles and is higher in large antral follicles than in small antral follicles. After culture, activin-A and/or FSH promoted growth of secondary follicles, while FSH increased the levels of mRNA for inhibin-ßA, and activin-A increased the levels of FSH-R mRNA. In conclusion, mRNA for inhibin-ßA is expressed at different levels in pre-antral and antral follicles and activin-A acts as a stimulator of the FSH-R expression in goat follicles. On its turn, the expression of inhibin-ßA is stimulated by FSH, which together with activin-A promotes secondary follicle growth in-vitro.
Este estudo investigou os níveis de ácidos ribonucleicos (RNAm) para a subunidade ßA da inibina em folículos primordiais, primários e secundários caprinos, bem como em complexos cumulus-oócitos (CCOs) e células da granulosa mural/teca de folículos antrais. Além disso, avaliaram-se os efeitos da ativina-A (100ng mL-1) e/ou hormônio folículo estimulante (FSH, 50ng mL-1) sobre o crescimento e a expressão do RNAm para inibina-ßA e receptores de FSH (FSH-R) em folículos secundários cultivados por seis dias. Os dados mostraram que a expressão de inibina-ßA é menor em folículos secundários do que em folículos primários e é maior em grandes folículos antrais que nos pequenos folículos antrais. Após o cultivo, ativina-A e/ou FSH promoveram o crescimento de folículos secundários. Enquanto o FSH aumentou os níveis de RNAm para inibina-ßA, a ativina-A aumentou os níveis de RNAm para FSH-R. Em conclusão, a inibina-ßA é expressa em diferentes níveis em folículos pré-antrais e antrais e a ativina-A atua como um estimulador da expressão de FSH-R em folículos caprinos. Por sua vez, a expressão de inibina-ßA é estimulada pelo FSH, que, juntamente com ativina, promove o crescimento de folículos secundários in vitro.
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Exogenous gonadotropins are widely used in controlled ovarian stimulation (COH) for patients undergoing in-vitro fertilization (IVF),because of the effect of follicle maturation and ovulation.Except for environmental factors such as age and ovarian reserve,genetic variability seems also to be a key factor in determining the ovarian response to the exogenous gonadotropins.In this review, the reason for the interindividual difference in ovarian resoponse as well as the influence of the genetic polymorphisms of FSHR and ESR on COH and IVF outcome has been summarized.
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Objective To sequence follicle stimulating hormone receptro (FSHR) promoter of the ovarian granulocyte and initially research the molecular mechanism of the poor ovarian response. Methods To study the relationship between FSHR promoter mutation of ovarian granulocyte and ovarian respone. The 263 bp DNA fragments before FSHR 5'initiation site in 70 cases of patients with poor ovarian respone and 88 cases of patients with ovarian normal respone who were in the cycle of IVF-ET were sequenced, Results There were 63 cases which occurred 29th site G → A point mutation in 158 women and the mutation rate was 40. 0%. Mutation rate [ 60. 0% ( 42/70 ) ] of 29th site G → A in group of poor ovarian respone was significantly higher(χ2 = 21. 450,P < 0. 01 ) than normal response group [ 23.9% ( 21/88 ) ]. There was no obviously variability ( t = 0. 457, P 0. 05 ) of basic FSH values between two groups [ G/G group was (7.2 ± 2. 3) U/L, G/A & A/A group was (7. 1±2. 0) U/L];there was obviously variability (t = 35. 81 ,P < 0. 05 ) in the number of follicles sinus between two groups ( G/G group was 14. 2±1.3, G/A & A/A group was 4. 5±0. 8 ) ;there was obviously variability ( t = 40. 35, P < 0. 05 ) in the number of ovum pick-up between two groups ( G/G group was 14. 0±1.2, G/A & A/A group was 4. 5±1.1 ) ;there was obviously variability (t =25. 80,P <0.05) of FE2-peak value between two groups [G/G group was (2 865±557) pmol/L, G/A & A/A group was (880±211 ) pmol/L] ;there was obviously variability (t =40. 22 ,P <0. 05) in the number of mature eggs ( G/G group was 13.6±1.2, G/A&A/Agroupwas4.3±0. 9).Conclusion The 29th site of FSHR promoter significantly affect the activity of FSHR promoter. Mutation of G→A can weaken promoter activity, so that ovarian granulocyte poor respone to FSH.
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To explore the effects of long-term treatment of different dietary energy levels on ovarian expression of mRNAs for LHR and FSHR,the present study was performed in nine growth-matched littermate crossbred (Land-race×Large White X Duroc) prepubertal gilts. At approximately 30 kg of body weight and 50 day of age,gilts were housed with individual feeding stalls and placed on a normal level of feeding for 90 days (dl-90) with free ac-cess to water and food throughout the whole research. From d91 ,littermates were divided and randomly assigned to one of the following three treatment lines for 3 weeks till d112:Group H,Group M, and Group L, fed the high energy level diet (n = 3, digestible energy 14.87 MJ/kg), moderate energy level diet (n = 3, digestible energy 12.39 MJ/ kg), and low energy level diet (n = 3, digestible energy 9.98 MJ/kg), respectively. When gilts were slaughtered on d112 after 3 weeks energy treatment, both ovaries of every gilts were collected,snap frozen in liquid nitrogen and re-tained at -80℃ for use to determine and analysis the relative amount of ovarian LHR and FSHR mRNAs using semi-quantitative RT-PCR. Results showed that the effect of dietary energy treatment was notable: the ovarian ex-pression of mRNAs for LHR and FSHR was significantly higher (P<0.05) in gilts treated with high energy diet compared to gilts treated with moderate and low energy diets, while gilts treated with low energy diet had a signifi-cantly lower (P<0.05) ovarian LHR and FSHR expression compared with gilts treated with moderate and high en-ergy diets. These results revealed that ovarian expression of I.HR and FSHR in prepubertal gilts increased as the lev-el of dietary energy intake elevated,i, e. , high energy diet can markedly enhance ovarian expression of mRNAs for LHR and FSHR,whereas energy deficit markedly suppress the expression.
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Women of reproductive ages are varies in their responses to exogenous FSH stimulations. The difference of FSHR genotype due to the polymorphisms in exon 10 is one of its significant factors. To know further whether the core promoter of FSHR is also polymorphic and to know whether those polymorphisms influence the promoter activity, we did polymorphism screening of FSHR promoter to 262 women undergoing IVF/ICSI, followed by functional study to know the impact of polymorphisms to the promoter activity. This study indicated that the core promoter of human FSHR is polymorphic. We found five SNPs at positions –29, –37, –114, –123 and –138 in addition to the variety number of adenines. Polymorphism at position –123 significantly decreased the promoter activity, in contrast, polymorphism at position –37 and –138 significantly increased the promoter activity, whereas polymorphism at position –29, –114 and short adenines stretch did not significantly influence the promoter activity. The differences of the promoter activities due to polymorphisms might change the ovarian sensitivity to FSH.
Subject(s)
Receptors, FSH , Polymorphism, Single NucleotideABSTRACT
Objective To observe the localization and distribution of follicle stimulating hormone receptor(FSHR) in the rat submandibular gland and stomach. Methods Immunohistochemical SABC method was used in the experiment. Results The serous glandular cells,granular convoluted epithelial cells and all other duct epithelial cells in the submandibular gland and the parietal cells of gastric gland showed FSHR positive immunoreactivity.The positive substance was distributed in the cytoplasm with negative nuclei.Conclusion The function of rat submandibular gland and gastric gland might be regulated by follicle stimulating hormone(FSH) through FSHR.