Histological study of capuchin monkey (Cebus apella) ovarian follicles

The present study aimed to obtain quanti-qualitative data about the follicular ovarian population in Cebus apella females. Seven ovaries were obtained from 4 C. apella adult females. The ovaries were subjected to light microscopy. The number of preantral and antral follicles for each ovary was estimated using the Fractionator method. The preantral follicles were classified into primordial, transitional, primary and secondary follicles. Antral follicles were those that presented an antral cavity. All counted follicles were classified as normal or degenerated. The diameter of the follicles, oocytes and their nuclei were determined to accompany the follicular development. All results were represented as mean ± SE. The number of preantral follicles was 56,938 ± 21,888 and 49,133 ± 26,896 for the right and left ovaries, respectively. The percentage of normal follicles was 80 ± 4.95%. The follicular diameter ranged from 22 ± 0.5 µm to 61.2 ± 4.0 µm. Regarding the antral follicles, the number of normal and degenerate follicles per ovary were 60.0 ± 19.0 and 3 ± 1.8 follicles, respectively. The antral follicular diameter was 514.4 + 56.6 µm. In conclusion, the information obtained in this study can be used as a parameter for subsequent in vivo or in vitro studies about folliculogenesis in non-human neotropical primates of the C. apella species.

Analyses of the development and glycoproteins present in the ovarian follicles of Poecilia vivipara (Cyprinodontiformes, Poeciliidae)

The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.

Porcine follicular fluid concentration of free insulin-like growth factor-I collected from different diameter ovarian follicles

The study aimed to quantify the concentrations of free IGF-I in serum and fluid of ovarian follicles in pre-pubertal gilts and describe the ovarian morphology by measuring the size of the ovaries and counting the number of surface follicles. Ovaries (n=1,000) from pre-pubertal gilts were obtained immediately after slaughter. A total of 10 samplings were performed, with ovaries obtained from 50 females for each collection. The follicles situated on the surface of each ovary were classified as small (SFs, 2 to 5mm in diameter) or large (LFs 6 to 10mm in diameter) and the follicular fluid was obtained by follicle aspiration. The collection of serum samples was performed after the gilts exsanguination using sterile tubes. From the pool of serum and follicular fluid obtained from 50 females, the concentration of free IGF-I was determined in each sample using an enzyme immunoassay kit (ELISA). The description of ovarian morphometry was performed in 100 ovaries from randomly selected gilts. The larger and smaller lengths of ovaries were measured, and the total number of SFs and LFs present on the surface of each ovary were also counted. The IGF-I concentration was greater (P<0.05) in LFs (170.92±88.29 ng/mL) compared with SFs (67.39±49.90ng/mL) and serum (73.48±34.63ng/mL). The largest and smallest length of the ovaries was 26.0±3.0 and 19.0mm ±2.0mm, respectively. The number of SFs (70.86±25.76) was greater (P<0.01) than LFs (6.54±5.26). The study concluded that LFs present greater levels of IGF-I when compared with SFs and blood, which is related to increased activity of the LFs and its differentiation to ovulation. In addition, ovaries of pre-pubertal gilts have a higher number of SFs compared to LFs. Therefore, our study demonstrated unique data regarding the physiological concentration of free IGF-I in ovarian follicles, that can be used in future research to evaluate the addition of this hormone in the in vitro production media of porcine embryos with the goal to improve the technique efficiency.

Functional significance of the signal transduction pathways Akt and Erk in ovarian follicles: in vitro and in vivo studies in cattle and sheep

BACKGROUND: The intracellular signalling mechanisms that regulate ovarian follicle development are unclear; however, we have recently shown differences in the Akt and Erk signalling pathways in dominant compared to subordinate follicles. The aim of this study was to investigate the effects of inhibiting Akt and Erk phosphorylation on IGF- and gonadotropin- stimulated granulosa and theca cell function in vitro, and on follicle development in vivo. METHODS: Bovine granulosa and theca cells were cultured for six days and stimulated with FSH and/or IGF, or LH in combination with PD98059 (Erk inhibitor) and/or LY294002 (Akt inhibitor) and their effect on cell number and hormone secretion (estradiol, activin-A, inhibin-A, follistatin, progesterone and androstenedione) determined. In addition, ovarian follicles were treated in vivo with PD98059 and/or LY294002 in ewes on Day 3 of the cycle and follicles were recovered 48 hours later. RESULTS: We have shown that gonadotropin- and IGF-stimulated hormone production by granulosa and theca cells is reduced by treatment with PD98059 and LY294002 in vitro. Furthermore, treatment with PD98059 and LY294002 reduced follicle growth and oestradiol production in vivo. CONCLUSION: These results demonstrate an important functional role for the Akt and Erk signalling pathways in follicle function, growth and development.

Cytoskeletal organization of bee ovarian follicles during oogenesis

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

Ultrastructural analysis of bovine oocytes from ovarian follicles with different diameters

In vitro embryo production is an important technique for facilitating the reproduction of animals with high genetic merit. The greatest challenge for the reproducibility of this technique is the quality of the oocyte that is submitted for in vitro maturation. The aim of this work was to evaluate the ultrastructural characteristics of oocytes from follicles of different diameters using transmission electron microscopy. The animals were divided into 2 groups and were given a single i.m. injection of 250 IU FSH (Pluset, Serono, Italy). To synchronize the follicular growth, all follicles > 2 mm were aspirated during the estrous cycle, which was considered day zero (D0). Group 1 (G1; n = 4) received FSH on day 1 (D1), and the 2- to 5-mm follicles were aspirated on day 2 (D2). The animals in group 2 (G2; n = 5) received FSH on day 2 (D2), and their 10- to 15-mm follicles were aspirated on day 5 (D5). After aspiration, the oocytes from both groups were fixed and prepared for ultrastructural analysis. The oocytes analyzed from both groups had similar ultrastructural characteristics. The presence and distribution of organelles in the cytoplasm of the oocytes did not differ between groups, suggesting that, in relation to the ultrastructural characteristics, oocytes from 2 to 5 mm and 10 to 15 mm follicles are similar.

Effects of atrazine on female Wistar rats: Morphological alterations in ovarian follicles and immunocytochemical labeling of 90 kDa heat shock protein

Fertility in female mammals may be affected by a variety of endocrine disrupters present in the environment. Herbicide atrazine is an example of endocrine disrupter employed in agriculture, which disrupts estrous cyclicity in rats. Aiming to characterize morphologically the effect of low and sublethal doses of atrazine on the ovaries of Wistar rats, in an effort to determine the possible intrafollicular target site through which this herbicide acts adult females were submitted to both subacute and subchronic treatments. Additionally, immunocytochemical labeling of 90 kDa heat shock protein (HSP90) was performed in order to evaluate the role played by this protein in the ovary, under stressed conditions induced by herbicide exposure. The results indicated that atrazine induced impaired folliculogenesis, increased follicular atresia and HSP90 depletion in female rats submitted to subacute treatment, while the subchronic treatment with low dose of atrazine could compromise the reproductive capacity reflected by the presence of multioocytic follicle and stress-inducible HSP90. © 2007 Elsevier Ltd. All rights reserved.

Mechanical isolation of capuchin monkey (Cebus apella) preantral ovarian follicles

O presente trabalho objetivou adaptar um procedimento mecânico para o isolamento de folículos pré-antrais a partir de ovários de Cebus apella. Para isso, foi testado o efeito do intervalo de cortes seriados do tissue chopper sobre o número de folículos pré-antrais isolados a partir de ovários (n=6) de três fêmeas de C. apella, duas pré-púberes e uma adulta. Os ovários foram divididos em quatro partes iguais e fragmentados com auxílio de um tissue chopper, ajustado para a realização de secções seriadas a intervalos de 250, 500, 750 e 1000µm, respectivamente. Os folículos isolados foram contados em câmara de Neubauer e classificados em primordiais, primários e secundários. O número (média ± EP) de folículos pré-antrais isolados de 1/4 de ovário variou de 68.330+17.590, no intervalo de corte de 1.000µm, a 300.830+111.460, no intervalo de corte de 500µm, o de melhores resultados. O diâmetro médio dos folículos pré-antrais isolados variou de 11,6µm a 27, 8µm.

Histological study of capuchin monkey (Cebus apella) ovarian follicles

O objetivo do presente trabalho foi obter dados quantitativos e qualitativos da população folicular ovariana de fêmeas de Cebus apella. Foram obtidos 7 ovários de 4 fêmeas adultas de C. apella através de ovariectomia. Os ovários foram submetidos à preparação para histologia ótica de rotina. O número de folículos pré-antrais e antrais por ovário foi estimado utilizando o Método Fracionador. Os folículos pré-antrais foram classificados em primordial, transição, primário e secundário. Foram considerados folículos antrais todos aqueles que apresentavam uma cavidade antral. Todos os folículos contados foram classificados em normais ou degenerados. Com o intuito de acompanhar o desenvolvimento folicular, os diâmetros médios folicular, oocitário e do núcleo do oócito foram determinados. Todos os resultados foram apresentados em Média ± Erro Padrão. A população média de folículos pré-antrais foi de 56.938 ± 21.888 e 49.133 ± 26.896 para os ovários direito e esquerdo, respectivamente. A percentagem de folículos pré-antrais estimados normais foi de 80,00 ± 4,95 %. O diâmetro médio folicular variou de 22,0 ± 0,5 µm a 61,2 ± 4,0 µm. No tocante aos folículos antrais, a população média de folículos normais e degenerados por ovário foi de 60,0 ± 19,0 e 3 ± 1,8 folículos, respectivamente. O diâmetro médio folicular foi de 514,4 ± 56,6 µm. Para concluir, as informações obtidas neste trabalho poderão servir como parâmetro para posteriores estudos in vivo ou in vitro da foliculogênese de primatas não-humanos neotropicais da espécie C. apella.

Effects of melatonin on ovarian follicles

Objective: To evaluate the histomorphometry and expression of Ki-67 and c-kit in ovarian follicles of pinealectomized or melatonin-treated pinealectomized rats. Study design: Forty adult rats were randomly divided into four groups of 10 animals: Group I – control; Group II – sham-pinealectomized; Group III – pinealectomized (Px), and Group IV – Px treated with melatonin (10 mg/night, per animal). After two months’ treatment, on the night of proestrous, the animals were placed in metabolic cages for night urine collection and subsequent measurement of 6-sulfatoxymelatonin (6-SMT). The rats were anesthetized, blood samples were taken for estrogen and progesterone determinations, and they were then euthanized. The ovaries were dissected out for further histological and immunohistochemical analyses. Data were first submitted to analysis of variance (ANOVA) complemented with the Tukey–Kramer test for multiple comparisons (P < 0.05). Results: The urinary levels of 6-SMT and serum progesterone were lower in the Px group (GIII). Exogenous melatonin treatment restored both blood melatonin and 6-SMT urinary levels. The histomorphometric data in Group III revealed a significant increase of degenerating antral and nonantral follicles with regard to the other groups. In addition no corpora lutea were observed in this group. No significant differences were noticed regarding the number of corpora lutea among the other groups (I, II and IV), but the number of cells and the thickness of the theca interna of Px animals (Group III) were higher than in the other groups. Conversely, the density of progesterone receptors (fmol/g) in the ovaries of Group III was significantly lower than in the other groups. Conclusion: Our data indicate that melatonin exerts a role on the maintenance of a proper follicular function, and is thus important for ovulation and progesterone production.

Effects of aqueous extract from Asparagus officinalis L. roots on hypothalamic-pituitary-gonadal axis hormone levels and the number of ovarian follicles in adult rats

Background: Asparagus is a plant with high nutritional, pharmaceutical, and industrial values. Objective: The present study aimed to evaluate the effect of aqueous extract of asparagus roots on the hypothalamic-pituitary-gonadal axis hormones and oogenesis in female rats. Materials and Methods: In this experimental study, 40 adult female Wistar rats were divided into five groups, which consist 8 rats. Groups included control, sham and three experimental groups receiving different doses (100, 200, 400 mg/kg/bw) of aqueous extract of asparagus roots. All dosages were administered orally for 28 days. Blood samples were taken from rats to evaluate serum levels of Gonadotropin releasing hormone (GnRH), follicular stimulating hormone (FSH), Luteinal hormone (LH), estrogen, and progesterone hormones. The ovaries were removed, weighted, sectioned, and studied by light microscope. Results: Dose-dependent aqueous extract of asparagus roots significantly increased serum levels of GnRH, FSH, LH, estrogen, and progestin hormones compared to control and sham groups. Increase in number of ovarian follicles and corpus luteum in groups treated with asparagus root extract was also observed (p<0.05). Conclusion: Asparagus roots extract stimulates secretion of hypothalamic- pituitary- gonadal axis hormones. This also positively affects oogenesis in female rats.

Ovarian and uterine periovulatory Doppler ultrasonography in bitches

This paper aims to describe the uterine and ovarian ultrasonographic characteristics and Doppler velocimetric features of their arteries in bitches during the periovulatory period. Fifteen estrous cycles in 10 animals were evaluated. The ultrasonographic characteristics, resistance indices (RI) and pulsatility indices (PI) of the uterus and ovaries in each animal were recorded 5 days before and after ovulation (D0). The data were statistically analyzed, and the results were expressed as the mean ± standard error of mean (P<0.05). In results the ultrasonographic features of the uterus were the same on all of the cycles and evaluated days. The uterus had an average diameter of 0.85±0.02cm. An increase in the volume of the ovaries and the diameter of the ovarian follicles were measured. Ovaries had a volume of 0.64±0.06cm³, and the follicles cavities had a diameter of 0.46 ± 0.01 cm on the day of ovulation. After ovulation, it was observed that some follicles not collapse in some cycles. Two days prior to ovulation, the uterine blood perfusion decreased. This decrease remained unchanged until ovulation. Following ovulation, we measured a gradual increase in the uterine perfusion and in the ovarian artery. This artery directed blood flow to the ovaries and increased the intra-ovarian perfusion on the day after ovulation. In conclusion, specific features are observed in the uterus and ovarian ultrasound image and Doppler values of their arteries presented on the periovulatory days and when associated allow to estimate more accurately the date of ovulation.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.