Phytohemagglutinin improves the development and ultrastructure of in vitro-cultured goat (Capra hircus) preantral follicles

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.

The ovary of Lagostomus maximus (Mammalia, Rodentia): an analysis by confocal microscopy

Lagostomus maximus is a notable mammalian model for reproductive studies. Females have an extremely high ovulation rate, which is due to down-regulation of the follicular apoptosis pathway, which ensures a large pool of developing follicles. This large pool is supported by the convoluted anatomy of the mature ovary, whose germinal tissue is found in irregularly curved ridges throughout the cortex. Medullary tissue is restricted to a minimum. Lyso Tracker Red reconstruction under confocal laser scanning microscopy was used to recognize and measure all follicular stages from primordial to antral. Unlike most mammals in which early primordial follicles are just found in fetal life, the adult ovary shows regions packed with early primordial follicles. Follicle size ranged from 24 to 316 µm. We discuss the relationships of L. maximus follicles size with regard to other species of mammals and propose that the physiology of the adult viscacha ovary obeys to a neoteny process in the evolution of this species.

Ovarian folliculogenesis in the smallnose fanskate sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae) / Foliculogénesis ovárica en la raya marmorada, sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae)

Chondrichthyes have become an important economic resource in the last decades, with Argentina as one of the countries that exploits more sharks and skates, even at levels that exceed de limits of many species. However, there is a scarce knowledge of the reproductive biology of this group, particularly from species inhabiting the Southern hemisphere. This work shows the most relevant facts during folliculogenesis in Sympterygia bonapartii. Results show that germinal cells are present in immature and maturing females. The most important facts that vary along de follicular development are the number of types and layers of follicular cells, the establishment of thin projections from the follicular cells and the degree of development of the thecae. Follicular cells are, at least, of two different types and both of them emit projections that break through the zona pellucida. The outer theca shows signs of synthetic activity. Atretic follicles of different sizes are present in exemplars of all the reproductive stages. These results are discussed in a physiological and adaptive context.

Los Condrictios se han convertido en un recurso económico importante en las últimas décadas, siendo Argentina uno de los países que más explota tiburones y rayas, incluso a niveles que exceden los límites de varias especies. A pesar de esto, es poco lo que se conoce sobre la biología reproductiva de este grupo, particularmente en especies del Hemisferio Sur. En este trabajo se estudian los estadios más relevantes de la foliculogénesis en Sympterygia bonapartii. Los resultados muestran que las ovogonias están presentes tanto en ejemplares inmaduros como subadultos. Las características más importantes que varían a lo largo del desarrollo folicular son el número de capas y tipos celulares que constituyen el epitelio folicular, el desarrollo de proyecciones de las células de la granulosa y el grado de desarrollo de las tecas. Las células foliculares son, al menos, de dos tipos y ambos emiten proyecciones que atraviesan la zona pelúcida. La teca externa presenta características compatibles con la actividad sintética. Folículos atrésicos de distintos tamaños están presentes en ejemplares de todos los estadios de madurez sexual. Estos resultados se discuten en un marco fisiológico y adaptativo.

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.

Impact of pituitary FSH purification on in vitro early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, o nly Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.

Different Follicle-Stimulating Hormone (FSH) sources influence caprine preantral follicle viability and development in vitro / Diferentes origens do Hormônio Folículo Estimulante (FSH) influenciam a viabilidade e o desenvolvimento de folículos pré-antrais caprinos

The aim of this study was to evaluate the effects of pituitary (pFSH) or recombinant (rFSH) FSH on the survival and growth of caprine preantral follicles. Caprine ovarian tissues were in vitro cultured for one or seven days in Minimum Essential Medium (MEM) alone or containing 10, 50, 100 and 1000 ng/ml of pFSH or rFSH. Control tissues (non-cultured) and those cultured were processed for histological and ultrastructural studies. In addition, follicular and oocyte diameter were analysed. After seven days of culture, only 50ng/ml of rFSH maintained the percentage of normal follicles similar to control. Moreover, 10 ng/ml of pFSH and all the concentrations of rFSH promoted primordial follicles activation. In addition, the presence of 50 ng/ml of rFSH promoted the highest follicular diameter at day seven of culture. In conclusion, 50 ng/ml of rFSH maintained the ultrastructural integrity of caprine preantral follicles, promoted primordial follicles activation and further growth of cultured follicles.

O objetivo deste estudo foi avaliar os efeitos do FSH pituitário (pFSH)ou recombinante (rFSH) sobre a sobrevivência e o crescimento de folículos pré-antrais caprinos. O tecido ovariano foi cultivado in vitro por um ou sete dias em Meio Essencial Mínimo (MEM) sozinho, ou contendo 10, 50, 100 e 1000 ng/ml de pFSH ou rFSH. O grupo controle (não cultivado) e aqueles cultivados foram processados para análises histológica e ultra-estrutural. Além disso, os diâmetros folicular e oocitário foram avaliados. Após sete dias de cultivo, apenas 50 ng/ml de rFSH manteve o percentual de folículos normais semelhante ao controle. Além disso, 10 ng/ml de pFSH e todas as concentrações de rFSH promoveram ativação de folículos primordiais. A presença de50 ng/ml de rFSH promoveu o maior diâmetro folicular após sete dias de cultivo. Em conclusão, 50 ng/ml de rFSH manteve a integridadede folículos pré-antrais caprinos e promoveu a ativação e o crescimento dos folículos cultivados.

Característica histológica, ultra-estrutural e produção de nitrito de folículos pré-antrais caprinos cultivados in vitro na ausência ou presença de soro / Histological and ultrastructural feature and nitrite production of caprine preantral follicles in vitro cultured in the presence or absence of serum

Avaliou-se o efeito da adição de diferentes tipos e concentrações de soro sobre o desenvolvimento e a sobrevivência de folículos ovarianos pré-antrais (FOPA) caprinos in vitro. Além disso, verificou-se a relação entre as concentrações de nitrito presentes no meio de cultivo e a viabilidade folicular. Cada par ovariano foi dividido em 29 fragmentos, sendo um destinado ao controle. Os fragmentos foram cultivados por um ou sete dias em meio essencial mínimo suplementado (MEM+) ou MEM+ com diferentes concentrações (10 ou 20 por cento) de soro fetal bovino (SFB), soro de cabra em estro (SCE) ou soro de cabra em diestro (SCD). Na análise morfológica após sete dias, apenas o tratamento com 10 por cento de SFB apresentou percentual de FOPA normais similar ao MEM+ (P>0,05). A análise ultra-estrutural dos folículos cultivados por sete dias com MEM+ ou MEM+ com 10 por cento de SFB mostrou danos oocitários, porém células da granulosa normais. A análise do meio de cultivo revelou correlação positiva entre a viabilidade folicular e a produção de nitrito. A suplementação com soro não melhorou a viabilidade de FOPA e a concentração de nitrito no meio de cultivo funcionou como um indicador da viabilidade das células da granulosa de FOPA caprinos cultivados in vitro.

The effect of the addition of different types and concentrations of sera on the viability and development of caprine preantal follicles (PAF) in vitro cultured was analyzed. In addition, it was evaluated the correlation between nitrite concentrations in culture medium and folicular viability. Each ovarian pair was divided in 29 fragments and one was used as control. The fragments were cultured for one or seven days in minimal essential medium (MEM+) or MEM+ with different concentrations of (10 or 20 percent) bovine fetal serum (BFS), estrous goat serum (EGS), or diestrous goat serum (DGS). After seven days, the morphological analysis showed that only the treatment with 10 percent BFS maintained the percentage of normal PAF similar to MEM+ (P>0.05). The ultrastructural analysis of follicles cultured for seven days in MEM+ or MEM+ with 10 percent BFS showed some oocyte damage, although the granulosa cells were normal. Analysis of culture medium revealed a positive correlation between follicular viability and nitrite production. Supplementation with serum did not improve the viability of PAF and nitrite levels in culture medium served as an indicator of viability of granulose cells from caprine PAF in vitro cultured.

Structural and ultrastructural aspects of folliculogenesis in Didelphis albiventris, the South-American opossum / Aspectos estructurales y ultraestructurales de la foliculogénesis de Didelphis albiventris, la zarigüeña sudamericana

The ovarian histology, the structural and the ultrastructural characteristics of the folliculogenesis in Didelphis albiventris were described in detail. Recent studies suggest that methatherian mammals have unusual reproductive cycle but there are few informations regarding the marsupials reproductive life. Despite of the opossum folliculogenesis pattern resembles methatherian and eutherian pattern in many aspects, the analysis shows some peculiar features of the oocyte structure and ultrastructure that make available new data on the reproductive biology of marsupials.

Fueron descritas con detalles la histología ovárica, las características estructurales y ultra-estructurales de la foliculogénesis del Didelphys albiventris. Estudios recientes sugieren que mamíferos metaterios tienen un ciclo reproductivo inusual, pero existen pocas informaciones en relación a la vida reproductiva de los marsupiales. A pesar de que el modelo de foliculogénesis de la zarigüeya se parece al modelo metaterio y euterio en muchos aspectos, el análisis muestra algunos rasgos peculiares de las estructura y ultra-estructura del oocito, que colocan a disposición nuevos datos en la biología reproductiva de los marsupiales.

Ovarian follicles development and atresia during the estrous cycle in adult female albino rats. immunohistochemical, light and scanning electron microscopic study

Maturation of ovarian follicles results from a complex interplay between proliferation, development and atresia within tire ovary. It is dependant not only on hormonal control but also on important paracrine and autocrine regulation exerted by local intraovarian factors. Polypeptide factors as insulin like growth factors [IGF] may play role in tire regulation of the development of follicles in animals arid humans. For better understanding the regulatory mechanisms of foilicular development, this work was conducted to characterize pattern of ovarian follicle development and atresia during various stages of the estrous cycle. So, twenty adult female a/intro rats were used and daily vaginal smears were done to determine tire estrous cycle stage. The animals were sacrificed at the appropriate stage and blood samples were collected for measurement of FSH and LH levels then the ovaries were removed and processed for light and scanning electron microscopic examination. Expression of p53 and JGF-l in the follicular cells was done. Marked atretic c/ranges, increased expression of p53 and decreased expression of IGF-I in the follicular cells were main/v detected in the preantral and tire antral follicles proestrous. This was accompanied by 4.7 ng/ml mean level of FSH and 8.5 ng/ml mean level of LH. On the other hand, minimal atretic changes decreased expression of p53 and increased expression of JGF-I were seen mainly in the large antral follicles at estrous. This was accompanied by 6.2 ng/ml mean level of FSH and 12.4 ng/ml mean el of LH. It was concluded that ovarian follicular atresia is an apoptotic mechanism through 53 tired/a/ed pathway Furthermore, sufficient hormonal concentrations of FSH and LH in addition to increased expression of IGF-I in time follicular cells are important for the ovarian follicles to reach the mature stage

Ultrastructure and morphometry of ovarian follicles in the armadillo Chaetophractus villosus (Mammalia, Dasypodidae)

Ultrastructural and morphometric changes in oocyte and surrounding granulosa cells during armadillo follicular growth are described. Primordial, intermediary, early and late primary, secondary, tertiary and preovulatory Graafian follicles were found. From primordial to Graafian follicle, granulosa cells increase in height, become multilayered while fluid-filled spaces arise among them in the tertiary follicle stage. As the follicle expands the oocyte is located eccentrically and grows in size. From primordial to secondary stage organelles develop in number and complexity and many vesicles start arising in the cytoplasm at the secondary stage. From tertiary follicle stage the number of mitochondria decreases. In follicular cells, secreting-related organelles expand, but mitochondrial number decreases according to the follicle expansion. Intercellular junctions between both cell types increase. The morphometrical study shows a biphasic pattern of oocyte and follicle growth. The morphological baseline here provided will be essential for further comprehension of the reproductive biology in armadillos

Ultrastructure of ovarian follicular epithelium of the Amazonian fish Pseudotylosurus microps (Teleostei: Belonidae): morphological and histochemical characterization of the intercellular deposits

The present paper reports the presence of great quantities of electrondense intercellular material in the follicular epithelium of P. microps. The material apparently is uptaken from circulation and enter the follicle through the intercellular spaces accumulating in the epithelial median-apical intercellular spaces and in perioocytic space. The accumulation starts in the early growth of the primary oocyte and proceed until vitellogenesis. The possible chemical nature and function of the deposits are discussed

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