Study on the relationship between expression patterns of cocaine-and amphetamine regulated transcript and hormones secretion in porcine ovarian follicles

Abstract Background Cocaine-and amphetamine regulated transcript (CART) is an endogenous neuropeptide, which is widespread in animals, plays a key role in regulation of follicular atresia in cattle and sheep. Among animal ovaries, CART mRNA was firstly found in the cattle ovaries. CART was localized in the antral follicles oocytes, granulosa and cumulus cells by immunohistochemistry and in situ hybridization. Further research found that secretion of E2 was inhibited in granulosa cells with a certain dose of CART, the effect depends on the stage of cell differentiation, suggesting that CART could play a crucial role in regulating follicle atresia. The objective of this study was to characterize the CART expression model and hormones secretion in vivo and vitro in pig follicle granulosa cells, preliminarily studied whether CART have an effect on granulosa cells proliferation and hormones secretion in multiparous animals such as pigs. Methods The expression levels of CART mRNA in granulosa cells of different follicles were analyzed using qRT-PCR technology. Immunohistochemistry technology was used to localize CART peptide. Granulosa cells were cultured in medium supplemented with different concentrations of CART and FSH for 168 h using Long-term culture system, and observed using a microscope. The concentration of Estradiol (E2) and progesterone (P) in follicular fluids of different test groups were detected by enzyme linked immunosorbent assay (ELISA). Results Results showed that expression level of CART mRNA was highest in medium follicles, and significantly higher than that in large and small follicles (P < 0.05). Immunohistochemical results showed that CART were expressed both in granulosa cells and theca cells of large follicles, while CART were detected only in theca cells of medium and small follicles. After the granulosa cells were cultured for 168 h, and found that concentrations of E2 increase with concentrations of follicle-stimulating hormone (FSH) increase when the CART concentration was 0 μM. And the concentration of FSH reached 25 ng/mL, the concentration of E2 is greatest. It shows that the production of E2 needs induction of FSH in granulosa cells of pig ovarian follicles. With the increasing of CART concentrations (0.01, 0.1, 1 μM), E2 concentration has a declining trend, when the FSH concentrations were 25 and 50 ng/mL in the medium, respectively. Conclusions These results suggested that CART plays a role to inhibit granulosa cells proliferation and E2 production, which induced by FSH in porcine ovarian follicular granulosa cells in vitro, but the inhibition effect is not significant. So we hypothesis CART maybe not a main local negative regulatory factor during porcine follicular development, which is different from the single fetal animals.

Characterization of OAZ1 and its potential functions in goose follicular development

Background: Ornithine decarboxylase antizyme 1 (OAZ1) is an important regulator of polyamine synthesis and uptake. Our previous studies indicated that high OAZ1 expression in the ovaries of laying geese is responsible for poor egg production. In the present study, the molecular characterization of goose OAZ1 gene was analyzed, as well as the expression profile in various follicular tissues. Results: An 873-bp cDNA sequence of the OAZ1 gene (Accession No. KC845302) with a +1 frameshift site (+175T) was obtained. The sequence consisted of a 652-bp two overlapping open reading frames (a putative protein with 216 amino acids). The OAZ domain, OAZ signature and OAZ super family domain were prominent conserved regions among species. As the follicle size increased, OAZ1 abundance showed an increasing trend during follicular development, while it decreased during follicular regression. The level of OAZ1 mRNA expression was the lowest in the fifth largest preovulatory follicle, and was 0.65-fold compared to the small white follicle (P b 0.05). OAZ1 mRNA expression in the largest preovulatory and postovulatory follicle was 2.11- and 2.49-fold compared to the small white follicle, respectively (P b 0.05). Conclusions: The goose OAZ1 structure confirms that OAZ1 plays an important role in ornithine decarboxylase-mediated regulation of polyamine homeostasis. Our findings provide an evidence for a potential function of OAZ1 in follicular development, ovulation and regression.

Expression of cocaine- and amphetamine-regulated transcript (CART) in hen ovary

BACKGROUND: Cocaine- and amphetamine-regulated transcript (CART), discovered initially by via differential display RT-PCR analysis of brains of rats administered cocaine, is expressed mainly in central nervous system or neuronal origin cells, and is involved in a wide range of behaviors, such as regulation of food intake, energy homeostasis, and reproduction. The hens egg-laying rate mainly depends on the developmental status of follicles, expression of CART have not been identified from hen follicles, the regulatory mechanisms of CART biological activities are still unknown. The objective of this study was to characterize the mRNA expression of CART in hen follicular granulosa cells and determine CART peptide localization and regulatory role during follicular development. METHODS: Small white follicles (1-2 mm in diameter) were treated for RNA isolation; Small white follicles (1-2 mm in diameter) and large white follicles (4-6 mm in diameter) were treated for immunohistochemical localization and large white follicles (4-6 mm in diameter), small yellow follicles (6-8 mm in diameter), large yellow follicles (9-12 mm in diameter), mature follicles (F5, F4, F3, F2, F1, >12 mm in diameter) were treated for RNA isolation and Real time PCR. RESULTS: The results showed that full length of the CDS of hen CART was 336 bp encoding a 111 amino acid polypeptide. In the hen ovary, CART peptide was primarily localized to the theca layer, but not all, the oocyte and granulosa layer, with diffused, weaker staining than relative to the theca cell layer. Further, amount of CART mRNA was more (P < 0.05) in granulosa cells of 6-8 mm follicles compared with that in granulosa cells of other follicles. However, CART mRNA amount was greater in theca cells of 4-6 mm follicles relative to follicles of other sizes (P < 0.05). CONCLUSIONS: Results suggest that CART could play a potential role in developmental regulation of chicken follicles.

Influência da interação entre o oócito e as células da granulosa nos resultados dos procedimentos de reprodução assistida / The influence of interaction between oocyte and granulosa cells on the results of procedures in assisted reproduction

A interação oócito-células da granulosa in vivo e sua influência na qualidade oocitária e embrionária tem sido alvo de inúmeros estudos, mas muitas questões ainda necessitam ser esclarecidas. O objetivo deste trabalho foi revisar a importância dessa comunicação, estabelecendo uma relação com a questão da maturação in vitro de oócitos imaturos humanos aplicando esses conhecimentos para definir possíveis marcadores moleculares que poderiam melhorar a seleção de oócitos e, consequentemente, selecionar embriões de boa qualidade para posterior transferência e sucesso de gravidez de pacientes submetidas ao tratamento da infertilidade conjugal. As células da granulosa têm um importante papel na maturação oocitária in vitro e os benefícios da presença dessas células durante essa etapa podem ser atribuídos à formação de um microambiente favorável (bioquímico e metabólico) ao redor do oócito. Foram identificados nesta revisão vários marcadores em potencial nas células do cumulus de oócitos competentes, incluindo vários genes que poderiam ser usados como preditores da competência oocitária, o que pode contribuir para a formulação de critérios mais objetivos e confiáveis para a seleção de oócitos e embriões, e consequente aprimoramento e otimização das técnicas em reprodução humana assistida que são aplicadas nos procedimentos clínicos atuais de fertilização in vitro.

The interaction of oocyte-granulosa cells in vivo and in vitro and its influence on oocyte and embryo quality has been the subject of numerous studies, but many issues still need to be clarified. The objective of this study was to promote a review about the importance of this communication establishing a connection with the issue of in vitro maturation of immature human oocytes by applying this knowledge to define potential molecular markers that could improve the selection of oocytes and consequently select good quality embryos for later transfer and success of pregnancy in patients undergoing treatment of infertility. The granulosa cells also have an important role in oocyte maturation in vitro and the venefits from the presence of these cells during this process can be atributed to the formation of a favorable micro-environment (biochemical and metabolic) around the oocyte. In this review, we identified several potential markers in the cumulus cells of competent oocytes, including several genes that could be used as predictors of oocyte competence, which contributes for more objective and reliable criteria for the selection of oocytes and embryos, thus improving and optimizing techniques in assisted human reproduction that are applied in current clinical in vitro fertilization.

Sazonalidade na dinâmica folicular ovariana e produção embrionária em novilhas da raça Guzerá / Ovarian follicular dynamic and embryo production seasonality in guzera heifers breed

Objetivou-se avaliar os efeitos da sazonalidade sobre a dinâmicafolicular ovariana e analisar a influência de temperaturas elevadas no desenvolvimento embrionário inicial em novilhas da raça Guzerá.Seis animais foram sincronizados e o dia do estro foi considerado D0. A dinâmica folicular foi acompanhada por dois ciclos estrais consecutivos, na época 1 (verão) e 2 (inverno), utilizando-se um ultrasom Scanner 200 Vet (Pie Medical). Após nove dias do término dosegundo ciclo estral, todos os animais iniciaram o tratamento superovulatório, com duração de quatro dias. Os animais foram inseminados artificialmente, 12 e 24 horas após a detecção do estro. A coleta dos embriões foi realizada sete dias após a primeira inseminação. Houve efeito sazonal no número de ondas foliculares, com maior prevalência de ciclos com uma onda no verão. O intervalo estral e ovulatório apresentaram maior duração no verão. Foi encontrado efeito significativo de época sobre a duração do crescimento do folículo ovulatório, ocorrendo maior persistência no verão. A taxa de crescimento folicular foi menor no verão. A temperatura retal oscilou entre as épocas, evidenciando a influência (P< 0,05) da estação do ano sobre a temperatura corporal interna. O THI (índice de temperatura e umidade) observado no verão foi 94 e no inverno 86, sugerindo a condição de estresse dos animais. O número de estruturas viáveis foi maior na época 2, sugerindo os efeitos de época sobre a fertilização dos oócitos. As concentrações de progesterona não apresentaram efeito de época. As alterações na dinâmica folicular em decorrência do estresse térmico, tais como, taxa de crescimento folicular diminuída e aumento na duração do crescimento folicular, podem comprometer a qualidade do oócito e afetar a subseqüente funcionalidade do corpo lúteo.

The objective was to verify the effects of seasonality on ovarian follicular dynamic and evaluate the influence of heat stress on early embrionic development in Guzerá heifers. Six animals were sincronized and the estrus day was considered D0. Follicular dynamic was followed by two consecutive estrous cycles, at times 1 (summer) and 2 (winter),using an ultrasonographic Scanner 200 Vet (Pie Medical). After nine days of the end of second estrous cycle, all the animals were submitted to superovulatory treatment, with duration of four days. The animals were artificially inseminated, 12 to 24 hours after estrus detection. Embryo collection was realized seven days after the first insemination.There was seasonal effect on the number of follicular waves, with agreater prevalence of cycles with one wave in the summer. The intervals between estrus and ovulations showed a greater duration in the summer. There was a significant effect of time on the duration of the ovulatory follicle growth that was longer in the summer. The follicular growth rate was smaller in the summer. Rectum temperature oscillated between times, become evident the influence (P < 0.05) of season on the internal body temperature. The THI observed on summer was94 and winter was 86, these values suggest the animal stress condition.The number of viable structures was higher in winter, suggesting the effects on time 2 on fertilization of the oocytes. Progesterone concentrations did not have seasonal effect. Thermal stress alterations on follicular dynamic, such do reduced follicular growth rate and increase on duration of follicular growth. Might prejudice the quality of oocyte and affect the subsequent corpus luteum functionality.

Autologous somatic cell nuclear transfer in pigs using recipient oocytes and donor cells from the same animal

The objective of the present study was to examine the feasibility of the production of autologous porcine somatic cell nuclear transfer (SCNT) blastocysts using oocytes and donor cells from slaughtered ovaries. Therefore, we attempted to optimize autologous SCNT by examining the effects of electrical fusion conditions and donor cell type on cell fusion and the development of SCNT embryos. Four types of donor cells were used: 1) denuded cumulus cells (DCCs) collected from in vitro-matured (IVM) oocytes; 2) cumulus cells collected from oocytes after 22 h of IVM and cultured for 18 h (CCCs); 3) follicular cells obtained from follicular contents and cultured for 40 h (CFCs); and 4) adult skin fibroblasts. The DCCs showed a significantly (p > 0.01) lower rate of fusion than the CCCs when two pulses of 170 V/mm DC were applied for 50 microsec (19 +/- 2% vs. 77 +/- 3%). The rate of DCC fusion with oocytes was increased by the application of two DC pulses of 190 V/mm for 30 microsec, although this was still lower than the rate of fusion in the CCCs (33 +/- 1% vs. 80 +/- 2%). The rates of cleavage (57 +/- 5%) and blastocyst formation (1 +/- 1%) in the DCC-derived embryos did not differ from those (55 +/- 6% and 3 +/- 1%, respectively) in the CCC-derived SCNT embryos. Autologous SCNT embryos derived from CFCs (5 +/- 2%) showed higher levels of blastocyst formation (p > 0.01) than CCC-derived autologous SCNT embryos (1 +/- 0%). In conclusion, the results of the present study show that culturing cumulus and follicular cells before SCNT enhances cell fusion with oocytes and that CFCs are superior to CCCs in the production of higher numbers of autologous SCNT blastocysts.

Local regulatory factors in regulation of ovarian function: role of prorenin-renin-angiotensin-system.

During reproductive life in the female, there is a continuous flow of growth, maturation and demise of ovarian follicles, unless pregnancy occurs. Although ovarian function is primarily controlled by the hypothalamus-pituitary-axis, there is no doubt that a hormonal microenvironment specific for each individual follicle is established, that finally determines whether a follicle ovulates and becomes a corpus luteum or undergoes atresia. In this respect, autocrine and paracrine factors that act alone or modulate gonadotropins action are of paramount importance. In this article, we want to introduce the ovarian prorenin-renin-angiotensin-system (PRAS) and summarize what is actually known about its involvement in ovarian physiology and pathology.

Typological series for ovarian follicles of sexually mature Myocastor coypus (coypu)

We are herein putting forward the results derived from the careful examination of the ovary of a sexually mature Myocastor coypus which was carried out to establish a follicular typological series. Sexually mature virgin females from breeding farms were used and their ovaries were processed by routinely histological techniques. The following analysis criteria were considered for the follicular classification: size of the oocyte in follicles at different stages of development, size of the follicle regarding the number of follicular cells, and follicular morphology. Complementary characteristics were also analyzed: mean follicular diameter, presence and thickness of the pellucid zone, mean size of follicular cells, their shape in all follicular types, presence and extent of the antrum, and presence of thecas. By combining these different criteria, a follicular typological series was obtained according to Pedersen and Peters (1968) nomenclature together with a qualitative and quantitative characterization of the follicular types.

Inhibin in individual buffalo ovarian follicles in relation to size.

A sensitive and specific radioimmunoassay was validated and applied for measurement of inhibin in follicular fluid (bFF) obtained from individual buffalo ovarian follicles. Follicular size was measured with an ultrasound machine and follicles were categorized as small, medium and large. Presence of inhibin was detected in all the antral follicles above 3 mm diameter. Inhibin concentration showed a positive relationship (R = 0.27, P < 0.01) with follicular diameter and was significantly higher (P < 0.05) in bFF from medium and large follicles (8.44 +/- 0.54 and 7.70 +/- 0.45 micrograms/ml, respectively) in comparison to that from small follicles (5.74 +/- 0.80 micrograms/ml). Total inhibin content was highly correlated (R = 0.92, P < 0.001) with follicular diameter and the inhibin content was higher (P < 0.001) in large > medium > small follicles.

RNA metabolism in the follicle cells of Bufo arenarum oocytes: I. Biochemical studies

Se determinó la captación de 3H adenosina en células foliculares y ovocitos ováricos totalmente crecidos de Bufo arenarum, y la incorporación del trazador radioactivo en ARN total y en la fracción reistente a ribonucleasas. Se incubaron 3H adenosina en folículos y envolturas foliculares (I-FE). En el primer caso, luego de la incubación las envolturas foliculares fueron manualmente separadas (O-FE). En el primer caso, luego de la incubación las envolturas foliculares fueron manualmente separadas (O-FE) y procesadas independiente de los ovocitos . I-FE incorporó 2,9 veces más 3H adenosina que O-FE. Los medios de incubación de I-FE y O-FE fueron analizados; en el primer caso se encontró 10.2 veces más radioactividad en macromoléculas que en el segundo caso. Se realizó una caracterización parcial de la ARN recientemente sintetizada, mediante electroforesis, encontrándose diferentes patrones para ARN de I-FE y O-FE. Una posible explicación para los resultados obtenidos es que las células foliculares transfieren ARN recientemente sintetizado al ovocito y, en ausencia de esto, al medio de incubación

RNA metabolism in the follicle cells of Bufo arenarum oocytes: II. Autoradiographic studies

Se incubaron folículos ováricos, conteniendo ovocitos totalmente crecidos de Bufo arenarum en 3H adenosina por períodos de hasta 16 horas. Autorradiografías de cortes de los folículos mostraron que los núcleos de las células foliculares y los nucleolos de las vesículas germinales de los ovocitos se presentaron fuertemente marcados. El nucleoplasma y la region cortical del ovocito están más radioactivos que las zonas subcortical y la periferia de la vesícula germinal. La región cortical es la única que muestra incrementos significativos en la concentración de granos de plata luego de un período de "chase". Esto sugiere que los granos de plata de la región cortical provienen de las células foliculares y no de la vesícula germinal. El agregado de Actinomicina D a los medios de incubación reduce notablemente los depósitos de plata, indicando que las moléculas marcadas son ARN