Effects of GDF-9 and FSH on mRNA expression for FSH-R, GDF-9 and BMPs in in vitro cultured goat preantral follicles

The aim of the present study was to determine the role of GDF-9 and/or FSH on the growth and mRNA expression for FSH-R, GDF-9, and BMPs in goat secondary follicles after culture in vitro. Goat secondary follicles (~200µm) were isolated and cultured for six days in minimum essential medium (MEM) supplemented with GDF-9 (200 ng/mL), FSH (50 ng/mL) or both. At the beginning and end of culture, the follicular diameter was evaluated and compared. The levels of mRNA for GDF-9, FSH-R and BMPs -2, -4, -6, -7 and -15 in cultured follicles were quantified by real time PCR. The results showed that a significant increase of follicle diameter after six days when compared to day 0, but the presence of GDF-9 and FSH did not influence the follicular growth in comparison with those cultured in MEM. Real time PCR showed that GDF-9 down-regulated the levels of mRNA for BMPs -2 and -15, while FSH either alone or in combination with GDF-9 did not affect the expression of GDF-9, FSH-R and BMPs. In conclusion, GDF-9 reduced the expression of BMP-2 and -15 in caprine preantral follicles after their culture, but FSH either alone or in association with GDF-9 did not control the expression of GDF-9, FSH-R and BMPs.

Real time PCR and importance of housekeepings genes for normalization and quantification of mRNA expression in different tissues

The aim of this review was to evaluate the importance of the real-time PCR (qRT-PCR) as a technique for mRNA expression analysis in different tissues. Real-time PCR is widely used for quantification of mRNA levels and is a fundamental tool for basic research, molecular medicine and biotechnology.Genes of references are expressed in a wide variety of tissues and cells with minimal variations in their expression levels, and thus are used to normalize data of mRNA quantification. Software programs, such as geNorm, BestKeeper and NormFinder, have been developed to perform the normalization of data, which help to choose the most stable reference gene. Several genes, such as GAPDH, β-actin, β-tubulin, PGK, UBQ, RPL-19 and 18S rRNA have been suggested as standards in PCR studies, but these genes can have variation in their expression in different tissues, reinforcing the idea that there is no ideal reference gene.

Goat ovarian follicles express different levels of mRNA for inhibin-ßA subunit and activin-A stimulates secondary follicle growth in vitro / Folículos ovarianos caprinos expressam diferentes níveis de RNAm para subunidade ßA da inibina e ativina-A promove o crescimento de folículos secundários in vitro

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.

Alterações na expressão de genes para citocinas, fatores de crescimento e seus receptores estão associadas com as neoplasias epiteliais ovarianas / Changes in gene expression for cytokines, growth factors and the irreceptors are associated with epithelial ovarian cancer

O câncer de ovário é o câncer ginecológico de maior letalidade e mais difícil de ser diagnosticado. Cerca de 3/4 dos tumores malignos de ovário apresentam-se em estágio avançado no momento do diagnóstico inicial. A etiologia do câncer de ovário parece ser multifatorial, incluindo fatores reprodutivos, familiares e pessoais. Diversos fatores envolvidos no desenvolvimento do câncer de ovário vêm sendo estudados, buscando melhor entender seu desenvolvimento. O tumor originado do epitélio superficial do ovário é o tipo mais frequente e tem sido associado com alterações na expressão de kit ligante (KL), do receptor de fator epidermal de crescimento (EGF-R), assim como a super expressão e ativação de genes como o HER, myc, ras e p53. O objetivo desta revisão é descrever as características dos tipos de tumores ovarianos e discutir a influência das gonadotrofinas e as alterações na expressão de genes para citocinas, fatores de crescimento e seus receptores nas neoplasias ovarianas.

Ovarian cancer is the most lethal of gynecologic cancer and more difficult to diagnose. Nearly three fourths of malignant tumors of the ovary presents at an advanced stage during early diagnosis. The etiology of ovarian cancer is multifactorial, including reproductive, family and personal factors. Several factors involved in the development of ovarian cancer has being studied, seeking to better understand its development. The tumor originated from the superficial epithelium of the ovary is the most frequent type and it has been associated with alteration in expression of kit ligand (KL), epidermal growth factor receptor (EGF-R), HER, myc, ras and p53. The aim of this review is to describe the characteristics of ovarian tumors and to discuss the influences of gonadotrophins and the changes in gene expression for cytokines, growth factors and their receptors in ovarian tumors.

Bone morphogenetic protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro / A proteína morfogenética óssea-6 (BMP-6) induz a atresia em folículos primordiais caprinos cultivados in vitro

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

O presente estudo investigou os efeitos da proteína morfogenética óssea-6 (BMP-6) no desenvolvimento in vitro de folículos primordiais caprinos. Amostras de córtex ovariano de cabras foram cultivados por 1 ou 7 dias em Meio Essencial Mínimo (meio controle) suplementado com diferentes concentrações de BMP-6. As taxas de sobrevivência, ativação e crescimento foram avaliadas por histologia clássica e microscopia eletrônica de transmissão (MET). Após 7 dias de cultivo, a análise histológica demonstrou que a BMP-6 aumentou o percentual de folículos primordiais degenerados no dia 7 quando comparados ao controle fresco (D0). Além disso, houve um aumento significativo do diâmetro folicular e oocitário em ambos os períodos de cultivo em todos os tratamentos na presença de BMP-6. Com a progressão do cultivo do dia 1 para o dia 7, nos tratamentos com 1 ou 50ng/ml de BMP-6, foi observado um aumento significativo no diâmetro folicular. Entretanto, contrário ao observado no meio controle, a MET revelou que os folículos cultivados nesses tratamentos apresentavam sinais evidentes de atresia. Em conclusão, esse estudo demonstrou que a BMP-6 afeta negativamente a sobrevivência e a ultra-estrutura de folículos primordiais caprinos.

Effect of bone morphogenetic protein-7 (BMP-7) on in vitro survival of caprine preantral follicles / Efeito da proteína morfogenética óssea 7 (BMP-7) para a sobrevivência in vitro de folículos pré-antrais caprinos

This study was conducted in order to verify the effect of different concentrations of BMP-7 in the in vitro survival and development of caprine preantral follicles. Fragments of caprine ovarian cortical tissue were cultured for 1 or 7 days in Minimum Essential Medium (MEM+) supplemented with different concentrations of BMP-7 (1, 10, 50 or 100ng/ml). Non-cultured fragments or those cultured for 1 or 7 days were processed for classical histology and transmission electron microscopy (TEM). Parameters such as follicular survival, activation and growth were evaluated. The results showed that, after 1 or 7 days of culture, the percentage of morphologically normal follicles was significantly reduced in all treatments when compared with fresh control, except at 1ng/ml of BMP-7 for 1 day. In addition, the concentration of 10ng/ml of BMP-7 significantly increases follicular diameter from day 1 to 7 of culture. There was no influence of the other concentrations of BMP-7 regarding to the follicular and oocyte diameter. Ultrastructure studies confirmed follicular integrity after 7 days of culture in 1ng/ml BMP-7. In conclusion, small concentrations of BMP-7 can improve the survival and growth of caprine preantral follicles during in vitro culture.

O presente trabalho foi conduzido de modo a se verificar o efeito de diferentes concentrações da BMP-7 no desenvolvimento in vitro de folículos pré-antrais caprinos. Fragmentos de tecido cortical ovariano caprino foram cultivados por 1 ou 7 dias em Minimum Essential Medium (MEM+) suplementado com diferentes concentrações de BMP-7 (1, 10, 50 ou 100ng/ml). Os fragmentos não cultivados ou aqueles cultivados por 1 ou 7 dias foram processados para histologia clássica e microscopia eletrônica de transmissão (TEM), sendo avaliados parâmetros morfológicos indicativos de viabilidade, ativação e crescimento. Os resultados mostraram que o percentual de folículos morfologicamente normais diminuiu significativamente em todos os tratamentos quando comparados ao controle, exceto na concentração de 1ng/ml por 1 dia de cultivo. Já no D7 todos os tratamentos reduziram significativamente os percentuais de folículos morfologicamente normais. Utilizando 10ng/ml de BMP-7 foi observado um aumento significativo no diâmetro folicular quando comparados os diferentes períodos de cultivo. Não houve influência das demais concentrações de BMP-7 quando avaliados além do diâmetro folicular o diâmetro oocitário. A análise por TEM confirmou a integridade ultra-estrutural nos folículos após 7 dias de cultivo com 1ng/ml de BMP-7 . Em conclusão, o BMP-7 em baixas concentrações pode melhorar a sobrevivência e o crescimento durante o cultivo in vitro de folículos pré-antrais caprinos.

Teste de toxicidade e criopreservação de folículos pré-antrais ovinos isolados utilizando Glicerol, Etilenoglicol, Dimetilsulfóxido e Propanodiol / Toxicity test and cryopreservation of sheep isolated preantral follicles using glycerol, ethylen glycol, dimethil sulfoxyde and propanediol

O objetivo deste estudo foi avaliar foliculos pré-antrais (FOPA) ovinos isolados após sua exposição e criopreservação utilizando glicerol (GLI), etilenoglicol (EG), propanodiol (PROH) ou dimetilsulfóxido (DMSO) a 1,5 e 3,0 M. Cada par ovariano de 5 ovelhas sem raça definida foi coletado em abatedouro local e submetido ao isolamento folicular. Da suspensão obtida, uma aliquota foi imediatamente destinada à análise da viabilidade folicular com o auxílio do corante vital azul de trypan. O restante da suspensão foi dividida em 16 aliquotas de 0,9 mL, suspensas (v/v) em MEM+ com EG, DMSO, GLI ou PROH a 1,5 ou 3,0 M, para teste de toxicidade e criopreservação. Após o término de cada tratamento, a viabilidade folicular foi analisada e os FOPA considerados viáveis se não corados ou não viáveis, quando corados. A análise dos dados mostrou que após o teste de toxicidade e criopreservação, em todos os crioprotetores e em ambas as concentrações, a percentagem de FOPA viáveis foi significativamente reduzida quando comparada ao controle. No teste de toxicidade, quando os crioprotetores foram comparados entre si nas mesmas concentrações, foram observadas percentagens signifIcativamente menores de FOPA viáveis no PROH 3,0 M (38,9%), apresentando-se, portanto, mais tóxico quando comparado aos demais crioprotetores. Após criopreservação, obteve-se percentagens significativamente maiores de foliculos pré-antrais viáveis quando o EG e o DMSO foram utilizados. Em conclusão, FOPA ovinos isolados podem ser criopreservados com sucesso utilizando-se D MSO e EG a 1,5 e 3,0 M.

The aim of this study was to evaluate isolated sheep preantral follicles (PF) after exposure and cryopreservation using glycerol (GLI), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO) at 1.5 and 3.0 M. Each ovarian pair from 5 mixed breed adult sheeps was obtained at a local slaughterhouse and submited to follicular isolation. From the obtained suspension, one aliquot was immediately analysed with trypan blue. The remaining suspension was divided in 16 aliquots of 0.9 mL, suspended in (v /v) in MEM+with EG, DMSO, GLI or PROH at 1.5 or 3.0 M to the toxicity test and cryopreservation. After the end of each treatment, the follicular viability was analysed and the PF were classified as viable if not dyed or not viable if dyed with trypan blue. The analysis of the results showed that after the toxicity test and cryopreservation, using all cryoprotectants and at both concentrations, the percentage of viable PF was significandy reduced when compared to the control. At the toxicity test, when the cryoprotectants were compared at the same concentrations, the lowest percentage of viable preantral follicles was obtained when 3.0 M PRO H (38,9%) was used, being, more toxic when compared to the others cryoprotectants. After cryopreservation, significantly higher percentual of viable PF was observed when the EG and DMSO were used. In conclusion, sheep PF can be cryopreserved successfully using DMSO and EG at 1.5 and 3.0 M.

Degeneration rate of goat primordial follicles maintained in TCM 199 or PBS at different temperatures and incubation times

The present work has investigated the degeneration rate of goat primordial follicles in situ after preservation in PBS or TCM 199 at different temperatures and incubation times. For each animal the ovarian pair was divided into 19 fragments. One ovarian fragment was taken randomly and immediately fixed (control). The other 18 ovarian fragments were randomly distributed in tubes containing PBS or TCM 199 and stored at 4º, 20º or 39ºC for 4, 12 or 24h. The storage of ovarian fragments in PBS or TCM 199 at 20ºC for 12h and 24h or at 39ºC, in all incubation times tested, increased significantly the percentage of degenerated primordial follicles (P<0.05). In contrast, for both media tested the degeneration rate of primordial follicles preserved at 4ºC for up to 24h and at 20ºC for 4h was similar to control values (P>0.05). In conclusion, this study shows that PBS was as efficient as TCM 199 in the preservation of goat primordial follicles in situ, being the best results observed at 4ºC

Effect of 0.9 percent saline solution and phosphate buffer saline at different temperatures and incubation times on the morphology of goat preantral follicles

The present work investigated the efficiency of 0.9 percent saline solution and Phosphate Buffered Saline (PBS) in the preservation of goat preantral follicles in situ at different temperatures and incubation times. The ovarian pair of each animal was divided into 19 fragments. One ovarian fragment was taken randomly and fixed (control). The other 18 fragments were randomly distributed in tubes containing 0.9 percent saline solution or PBS at 4, 20 or 39 ºC for 4, 12 or 24 h. A total of 5,921 preantral follicles were examined. The quality of preantral follicles was evaluated by classical histology. The storage of ovarian fragments in 0.9 percent saline solution or PBS at 4 ºC did not reduce significantly the percentage of morphologically normal follicles when compared with the control, except after preservation in 0.9 percent saline solution for 24 h. The storage of ovarian fragments at 20 or 39°C reduced the percentage of normal preantral follicles when compared to the control, except after preservation in PBS at 20°C for 4 h. In conclusion, this study showed for the first time that goat preantral follicles can be stored in situ successfully at 4 ºC in 0.9 percent saline solution for 12 h and in PBS for 24 h, and at 20 ºC in PBS for 4 h