Comparison of bioactivities, binding properties and intra-follicular levels of bovine follistatins

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, 41kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin- and heparan sulphate proteoglycan (HSP)-binding properties and bio-potencies in neutralization of activin-A action in vitro revealed that all five isoforms bound activin-A, but with different affinities. Only the 31kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency with 35 and 41kDa isoforms being least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analyzed bFF from dominant (DF) and subordinate (SF) follicles collected at strategic times during a synchronized estrous cycle. Total FST, activin-A and activin-AB were measured by immunoassay while individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with estrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin-A (r=-0.34), activin-AB (r=-0.80) and ‘total’ FST (r=-0.70) levels. Follicle diameter was positively correlated with abundance of the 41 kDa isoform (r=0.59) but negatively correlated with abundance of 33 and 31 kDa isoforms (r=-0.56, -0.41). Both follicle status (DF vs SF) and cycle stage affected total FST, activin-A, activin-B levels while follicle status, but not cycle stage, affected abundance of 41, 37, 33 and 31kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms that differ in their ability to bind and neutralise activins and associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may have an important negative role, either directly or via inhibition of the positive effects of activins, on follicle growth and function during follicular waves.

Dysregulation of granulosal bone morphogenetic protein receptor 1B density is associated with reduced ovarian reserve and the age-related decline in human fertility

Reproductive ageing is linked to the depletion of ovarian primordial follicles, which causes an irreversible change to ovarian cellular function and the capacity to reproduce. The current study aimed to profile the expression of bone morphogenetic protein receptor, (BMPR1B) in 53 IVF patients exhibiting different degrees of primordial follicle depletion. The granulosa cell receptor density was measured in 403 follicles via flow cytometry. A decline in BMPR1B density occurred at the time of dominant follicle selection and during the terminal stage of folliculogenesis in the 23-30 y good ovarian reserve patients. The 40+ y poor ovarian reserve patients experienced a reversal of this pattern. The results demonstrate an association between age-induced depletion of the ovarian reserve and BMPR1B receptor density at the two critical time points of dominant follicle selection and pre-ovulatory follicle maturation. Dysregulation of BMP receptor signalling may inhibit the normal steroidogenic differentiation required for maturation in older patients.

A retrospective model of oocyte competence: global mRNA and housekeeping transcripts are not associated with in vitro developmental outcome

Oocyte developmental competence depends on maternal stores that support development throughout a transcriptionally silent period during early embryogenesis. Previous attempts to investigate transcripts associated with oocyte competence have relied on prospective models, which are mostly based on morphological. criteria. Using a retrospective model, we quantitatively compared mRNA among oocytes with different embryo development competence. A cytoplasm biopsy was removed from in vitro matured oocytes to perform comparative analysis of amounts of global polyadenylated (polyA) mRNA and housekeeping gene transcripts. After parthenogenetic activation of biopsied oocytes, presumptive zygotes were cultured individually in vitro and oocytes were classified according to embryo development: (i) blocked before the 8-cell stage; (ii) blocked between the 8-cell and morulae stages; or (iii) developed to the blastocyst stage. Sham-manipulated controls confirmed that biopsies did not alter development outcome. Total polyA mRNA amounts correlate with oocyte diameter but not with the ability to develop to the 8-cell and blastocyst stages. The last was also confirmed by relative quantification of GAPDH, H2A and Hprt1 transcripts. In conclusion, we describe a novel retrospective model to identify putative markers of development competence in single oocytes and demonstrate that global mRNA amounts at the metaphase II stage do not correlate with embryo development in vitro.

Embryo Mitochondrial DNA Depletion Is Reversed During Early Embryogenesis in Cattle

The extensive replication of mitochondria during oogenesis and the wide variability in mitochondrial DNA ( mtDNA) copy numbers present in fully grown oocytes indicate that mtDNA amount may play an important role during early embryogenesis. Using bovine oocytes derived from follicles of different sizes to study the influence of mtDNA content on development, we showed that oocytes obtained from small follicles, known to be less competent in developing into blastocysts, contain less mtDNA than those originating from larger follicles. However, because of the high variability in copy number, a more accurate approach was examined in which parthenogenetic one-cell embryos were biopsied to measure their mtDNA content and then cultured to assess development capacity. Contrasting with previous findings, mtDNA copy number in biopsies was not different between competent and incompetent embryos, indicating that mtDNA content is not related to early developmental competence. To further examine the importance of mtDNA on development, one-cell embryos were partially depleted of their mtDNA (64% +/- 4.1% less) by centrifugation followed by the removal of the mitochondrial-enriched cytoplasmic fraction. Surprisingly, depleted embryos developed normally into blastocysts, which contained mtDNA copy numbers similar to nonmanipulated controls. Development in depleted embryos was accompanied by an increase in the expression of genes (TFAM and NRF1) controlling mtDNA replication and transcription, indicating an intrinsic ability to restore the content of mtDNA at the blastocyst stage. Therefore, we concluded that competent bovine embryos are able to regulate their mtDNA content at the blastocyst stage regardless of the copy numbers accumulated during oogenesis.

Patterns of oocyte development in natural habitat and captive Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae)

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments-the TietA(a) River (natural) and captivity-and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the TietA(a) River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.

The Autoimmune Regulator (AIRE), Which Is Defective in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients, Is Expressed in Human Epidermal and Follicular Keratinocytes and Associates With the Intermediate Filament Protein Cytokeratin 17

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is caused by mutation of the autoimmune regulator (AIRE) gene, is a highly variable disease characterized by multiple endocrine failure, chronic mucocutaneous candidiasis, and various ectodermal defects. AIRE is a transcriptional regulator classically expressed in medullary thymic epithelial cells, monocytes, macrophages, and dendritic cells. Previous studies have suggested that AIRE can shuttle between the nucleus and cytoplasm of cells, although its cytoplasmic functions are poorly characterized. Through mass spectrometry analysis of proteins co-immunoprecipitating with cytoplasmic AIRE, we identified a novel association of AIRE with the intermediate filament protein cytokeratin 17 (K17) in the THP-1 monocyte cell line. We confirmed AIRE expression in HaCaT epidermal keratinocytes, as well as its interaction with K17. Confocal microscopy of human fetal and adult scalp hair follicles demonstrated a cytoplasmic pattern of AIRE staining that moderately colocalized with K17. The cytoplasmic association of AIRE with the intermediate filament network in human epidermal and follicular keratinocytes may provide a new path to understanding the ectodermal abnormalities associated with the APECED syndrome. (Am J Pathol 2011, 178:983-988; DOI: 10.1016/j.ajpath.2010.12.007)

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. (C) 2011 Elsevier Ltd. All rights reserved.

Infecção por HELICOBACTER PYLORI em crianças com dor abdominal crônica e sua associação com a gastrite endoscópica nodular

A infecção por Helicobacter pylori (Hp) é uma das infecções bacterianas mais comuns em todo o mundo. As maiores prevalências da infecção foram encontradas nos países em desenvolvimento, onde, em geral são altas já na infância. O método diagnóstico considerado mais acurado para a infecção por Hp, em crianças, é o exame endoscópico com biópsias gástricas. Alguns autores referem que o único aspecto macroscópico que pode predizer a infecção é o da presença de nodosidades na mucosa gástrica. Este aspecto é denominado de gastrite endoscópica nodular. A especificidade da gastrite endoscópica nodular para a infecção por Hp, entretanto, recentemente foi questionada por outros autores. Realizamos um estudo transversal em uma amostra de crianças (um a 12 anos) com dor abdominal crônica, que preenchiam os critérios para a realização de endoscopia digestiva alta, no Hospital da Criança Conceição e no Hospital de Clínicas de Porto Alegre, de setembro de 1997 a setembro de 1999. O objetivo principal foi verificar a associação entre a infecção por Hp e a gastrite endoscópica nodular nessas crianças. A amostra foi constituída de 185 crianças de ambos os sexos, com baixa renda familiar, cujos pais apresentavam baixo nível de escolaridade. Foi realizado estudo histológico das lâminas de biópsia gástrica (no mínimo cinco fragmentos, corados com H-E ou Giemsa), conforme o Sistema Sydney modificado. A infecção por Hp foi caracterizada pela presença de Hp na lâminas de biópsias gástricas dos pacientes e a gastrite folicular, pela presença de folículos linfóides bem formados, em mucosa gástrica inflamada. A prevalência da infecção por Hp nas crianças com dor abdominal crônica foi de 27% (IC 95%: 20,8-34,0). Foi demonstrada uma associação muito forte entre a infecção por Hp e a gastrite endoscópica nodular nessas crianças (P<0,001; RP = 29,7). Houve um aumento da prevalência tanto da infecção por Hp como da gastrite endoscópica nodular com a idade dos pacientes. A gastrite endoscópica nodular , embora tenha demostrado uma baixa sensibilidade (44,0%), apresentou um valor preditivo positivo de 91,7% para a infecção por Hp. Tanto o teste de urease, como a gastrite endoscópica nodular mostraram-se muito específicas, 94,5% e 98,5%, respectivamente, para o diagnóstico da infecção. Quando se combinou o teste de urease com o aspecto de gastrite endoscópica nodular, encontrou-se, uma sensibilidade muito baixa (34,7%), mas uma especificidade de 100% para a infecção por Hp. A sensibilidade do teste de urease, isolado, para a infecção foi de 60,4% e o seu valor preditivo positivo de 80,5%. O aspecto endoscópico (gastrite endoscópica nodular) teve associação com o microscópico (gastrite folicular) (P<0,001). Houve uma forte e significativa associação entre a infecção por Hp e a gastrite crônica ativa ( P<0,001; RP = 10,8). O mesmo foi demonstrado entre a gastrite nodular e a gastrite crônica ativa (P<0,001; RP = 8,6). Também foi verificado um nítido aumento das razões de prevalência da gastrite crônica ativa e da gastrite endoscópica nodular, com a acentuação dos graus de densidade de Hp. Finalmente, foi demonstrada a importante correlação entre o grau de intensidade da gastrite, verificado no exame histológico, e a gastrite endoscópica nodular (r = 0,97; P<0,001). A prevalência da infecção por Hp encontrada em Porto Alegre, nas crianças, foi menor do que a de outras cidades brasileiras e similar àquela registrada em algumas cidades do primeiro mundo. A presença de nodosidade na mucosa gástrica foi a alteração, à endoscopia, mais freqüentemente verificada nas crianças com infecção por Hp. Considerando a baixa prevalência da infecção encontrada na nossa amostra, a presença de gastrite endoscópica nodular significa uma elevada probabilidade de infecção por Hp, dado o alto valor preditivo verificado. O achado negativo para a gastrite endoscópica nodular, entretanto, não exclui a possibilidade da presença de infecção por Hp. Uma maior colonização bacteriana da mucosa gástrica estaria associada ao aparecimento da gastrite endoscópica nodular, já que a sua prevalência aumentou com os graus de densidade de Hp, assim como ocorreu com a gastrite crônica ativa. E quando ocorre, nas crianças, há maior probabilidade de se tratar de uma gastrite mais ativa e mais intensa.

Análises histológicas comparativas entre diferentes tamanhos de fragmentos ovarianos bovinos criopreservados

Objetivo: Investigar a influência do tamanho dos fragmentos ovarianos na preservação dos folículos primordiais e primários pós-congelamento. Desenho: Experimento laboratorial controlado. Foram realizadas análises histológicas comparativas em grupos controle e pós-congelamento de fragmentos de ovários bovinos. No grupo dos fragmentos menores, a menor das medidas era de 2mm e no grupo dos fragmentos maiores, todos as medidas eram maiores de 2mm. Local de Realização: Universidade Federal do Rio Grande do Sul (UFRGS), Faculdade de Veterinária da UFRGS e Centro de Reprodução Humana Nilo Frantz, Porto Alegre, RS, Brasil. Animais: Foi utilizado um ovário de vaca raça Hereford. Resultados Principais: Nos fragmentos congelados com medida de 2mm, e com medidas maiores de 2mm, a percentagem de folículos normais foi de 56% e 34%, respectivamente. O risco relativo para a manutenção dos folículos normais póscongelamento nos fragmentos com de 2mm e maiores de 2mm foi de 0,63 e de 0,47, respectivamente. A comparação desse dois riscos relativos mostrou, de forma significativa (qui-quadrado de heterogeneidade p=0,022), que o impacto do congelamento na redução das taxas de sucesso foi maior nos fragmentos com espessuras maiores de 2mm. Conclusões principais: Este estudo mostrou que há um comprometimento maior na preservação folicular em fragmentos ovarianos criopreservados com espessuras maiores de 2mm.

Prevalence of biomicroscopic findings in the anterior segment and ocular adnexa among schoolchildren in Natal/Brazil

Objective: To determine the prevalence of ocular findings of the external structures and anterior segment of the eye, detected by biomicroscopic examination in schoolchildren in Natal (RN) - Brazil. Methods: After previous random selection, 1,024 pupils from elementary and secondary public and private schools in the city of Natal were evaluated from March to June 2001. All were submitted to preestablished standard research norms, consisting of identification, demographic information, ophthalmologic biomicroscopic examination, with slit lamp, performed by ophthalmologists from the “Onofre Lopes” University Hospital. Results: Alterations of the conjunctival and palpebral conditions were the most prevalent (10.4% and 6.2% respectively). Follicles (4.2%) and papillae (3.0%) were the frequent conjunctival lesions, while blepharitis (3.5%) and meibomitis (1.1%) were the most detected abnormalities in the eyelids. Upon examining the cornea, iris, lens and anterior vitreous, the most encountered findings were nubecula (0.5%), papillary membrane reliquiae (0.5%), posterior capsula opacity (0.8%) and hyaloid arteria reliquiae (2.0%). Conclusion: The most prevalent findings affecting the external structures of the eye such as eyelids and conjunctiva, consisted of blepharitis followed by follicular reaction of the conjunctiva. The most prevalent abnormalities in the cornea, iris, lens and anterior vitreous were nubecula, papillary membrane reliquiae, posterior capsular opacity and hyaloid arteria reliquiae, in that order

Avaliação de diferentes formas de preservação e desenvolvimento in situ de folículos ovarianos pré-antrais caprinos

This study evaluated the effect of indole-3-acetic acid (IAA) in the activation of goats preantral follicles (FOPA). Were used four pairs of ovaries of adult mixed breed goats. Each ovarian pair was divided into 23 fragments. One fragment was fixed for histology and other fragment was using to follicular isolation procedure. The remaining fragments were cultured in 1.0 ml of Minimum Essential Medium (MEM) or MEM supplemented with IAA at concentrations of 10, 40, 100, 500 or 1000 ng/mL. The in vitro culture was performed at 39°C in incubator with 5% CO2 for 1, 3 and 5 days. After in vitro culture was evaluated histological integrity and viability of FOPA. The addition of 100 ng/ml of IAA to MEM showed a significant increase in follicles transition in the third day of in vitro culture, characterizing follicular activation. Moreover, this concentration was obtained maintaining the histological integrity of PAF by the fifth day of in vitro culture. The viability test confirmed the results of histology. Thus, we conclude that IAA can promote the activation of goats FOPA

The administration of exogenous prostaglandin may improve ovulation in pacu (Piaractus mesopotamicus)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Testosterona e gonadotrofina coriônica humana estimulam a esteroidogênese em células da granulosa de folículo pré-ovulatório de égua?

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

Efficacy of linear and convex transducers for ultrasound-guided transvaginal follicle aspiration

A new device was developed to hold linear transducers for transvaginal follicle aspiration. Efficacy of follicle aspiration was compared using a linear 6 MHz and a convex 5 MHz transducer. Fifty-five cows were submitted to follicle aspiration at random days of the estrous cycle. Aspirations were conducted with linear (n = 28) and convex (n = 38) transducers with 18 G needles at a negative pressure corresponding to 13 ml H2O/min. A greater number of follicles were aspirated using convex than to linear probe (12.4 versus 7.8, respectively, P < 0.05). Mean number of oocytes and recovery rates were similar for convex (5.4 and 48.6%) and linear (4.6 and 59.3%) transducers. Limited space between the linear transducer and needle guide restricted access to some portions of the ovary, reducing the number of follicles aspirated using a linear transducer. The newly developed adaptor allowed greater stability, holding the ovaries firmly against the linear transducer. This diminished mobility permitted a similar number of oocytes to be recovered with both transducers. In conclusion, this new adaptor provided a low cost alternative for routine follicle aspiration and oocyte recovery in cattle. (C) 2002 Elsevier B.V. All rights reserved.

Relationship between follicle size and ultrasound-guided transvaginal oocyte recovery

We evaluated the relationship between follicle size and oocyte recovery (OR) using ultrasound-guided follicle aspiration. Thirty Holstein cows were subjected to OR without gonadotrophic therapy. Oocytes were recovered two to four times from each cow in a total of 67 aspiration sessions, Ovarian follicles with diameters less than or equal to4 mm and >4 mm were aspirated in separated groups. Recovered oocytes from each group were kept separate and submitted to in vitro maturation, fertilization, and culture to the blastocyst stage. A total of 430 follicles were aspirated, of which 154 (35.8%) were from follicles >4 mm and 276 (64.2%) were from follicles less than or equal to4 mm. Seventy-seven oocytes (50%) were recovered from follicles >4 mm and 200 (72.2%) were from follicles less than or equal to4 mm. Nineteen blastocysts were obtained from follicles >4 mm, whereas 45 blastocysts were obtained from follicles less than or equal to4 mm. Recovery rate was greater (P < 0.01) in follicles less than or equal to4 mm, Oocyte quality, cleavage rate and blastocyst development did not differ between different follicle sizes. Routine aspiration of small follicles (less than or equal to4 mm) could increase the number of oocytes available for in vitro development. (C) 2001 Elsevier B.V. B.V. All rights reserved.