Biochemical and immunohistochemical examination of the effects of ephedrine in rat ovary tissue

Purpose: It was aimed to investigate the biochemical and immunohistochemical effects of ephedrine (EPH) in bilateral ovariectomized rats. Methods: Twenty-four Sprague Dawley female rats were divided into three groups: control group: The abdomen was opened and closed without any treatment; ischemia-reperfusion (IR) group: 2 h of ischemia followed by 2 h of reperfusion were allowed to cause IR injury; IR+EPH group: oral EPH solution (5 mg/kg) was administered for 28 days. Results: Biochemical parameters were statistically significant in group comparisons. Increased interleukin-6 (IL-6) expression, degenerative preantral and antral follicle cells and inflammatory cells around blood vessels were seen in IR group. Negative IL-6 expression was observed in seminal epithelial cells, preantral and antral follicle cells in IR+EPH group. While caspase-3 activity increased in granulosa cells and stromal cells in IR group, caspase-3 expression was negative in preantral and antral follicle cells in the germinal epithelium and cortex in IR+EPH group. Conclusion: The effect of apoptosis, which occurs with the signaling that starts in the cell nucleus, caused the cessation of the stimulating effect at the nuclear level after EPH administration, and a decrease in the antioxidative effect in IR damage and inflammation in the apoptotic process.

El consumo semicrónico moderado de alcohol altera la foliculogénesis y la calidad núcleo-citoplasmática oocitaria, en el ratón / Moderate semi-chronic alcohol consumption alters folliculogenesis and oocyte nuclear-cytoplasmic quality in mice

Resumen El consumo crónico de alcohol es un problema de salud mundial que afecta particularmente a la población femenina. Sin embargo, los efectos de la ingesta semicrónica en cantidades moderadas a bajas en el ovario y el oocito son poco conocidos. En un modelo murino, se administró etanol al 10% en agua de bebida (hembras tratadas) o agua (hembras control) por 15 días, y luego de la superovulación o no (ovulación espontánea), se analizó el ciclo estral y la calidad ovárico-gamética. En las hembras tratadas, la frecuencia y duración del diestro aumentó, y las frecuencias de folículos y cuerpos lúteos disminuyeron vs hembras controles, valores que se restauraron luego de la superovulación. Sin embargo, en las hembras tratadas, la tasa de proliferación celular folicular y el desbalance de la expresión ovárica de VEGF (factor de crecimiento endotelial) persistieron luego de la superovulación. El número de ovocitos ovulados con metafase II anormal, fragmentados y activados partenogenéticamente fue mayor en las hembras tratadas respecto las controles. En conclusión, el consumo semicrónico moderado de alcohol produce anestro, ciclo estral irregular, foliculogénesis deficiente y anomalías núcleo-citoplasmáticas en los oocitos ovulados. Estas alteraciones podrían constituirse en un factor etiológico de pérdida gestacional temprana y desarrollo embrionario anormal luego del consumo de alcohol.

Abstract Chronic alcohol consumption is a global health problem that particularly affects the female population. However, the ef-fects of semi-chronic ethanol intake in low-moderate amounts on the ovary and oocyte are poorly understood. In a mouse model, 10% ethanol was administered in drinking water (treated females) or water (control females) for 15 days, and after superovulation or not (spontaneous ovulation), the estrous cycle and ovarian-gametic quality were analyzed. In treated females, the frequency and duration of the diestrus increased, and the frequencies of follicles and corpus luteum decreased vs control females, values that restored after superovulation. However, in treated females, the follicular cell proliferation rate and the imbalance in ovarian expression of VEGF (endothelial growth factor) persisted after superovulation. The number of ovulated oocytes with abnormal metaphase II, fragmented and parthenogenetically activated was higher in treated females than in control ones. In conclusion, moderate semi-chronic alcohol consumption produces anestrum, irregular estrous cycle, poor folliculogenesis, and nuclear-cytoplasmic abnormalities in ovulated oocytes. These alterations could constitute an etiological factor of early gestational loss and abnormal embryonic development after alcohol consumption.

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

Improvement in Ovarian Tissue Quality with Supplementation of Antifreeze Protein during Warming of Vitrified Mouse Ovarian Tissue

Ice easily recrystallizes during warming after vitrification, and antifreeze protein (AFP) can inhibit the re-crystallization. However, no study has evaluated the effect of AFP treatment only thereon during warming. This study sought to compare AFP treatment protocols: a conventional protocol with AFP treatment during vitrification and first-step warming and a new protocol with AFP treatment during the first-step warming only. According to the protocols, 10 mg/mL of LeIBP (a type of AFP) was used. Five-week-old B6D2F1 mouse ovaries were randomly divided into a vitrified-warmed control and two experimental groups, one treated with the conventional AFP treatment protocol (LeIBP-all) and the other with the new AFP treatment protocol (LeIBP-w). For evaluation, ratios of ovarian follicle integrity, apoptosis, and DNA double-strand (DDS) damage/repairing were analyzed. The LeIBP-treated groups showed significantly higher intact follicle ratios than the control, and the results were similar between the LeIBP-treated groups. Apoptotic follicle ratios were significantly lower in both LeIBP-treated groups than the control, and the results were not significantly different between the LeIBP-treated groups. With regard to DDS damage/repairing follicle ratio, significantly lower ratios were recorded in both LeIBP-treated groups, compared to the control, and the results were similar between the LeIBP-treated groups. This study demonstrated that both protocols with LeIBP had a beneficial effect on maintaining follicle integrity and preventing follicle apoptosis and DDS damage. Moreover, the new protocol showed similar results to the conventional protocol. This new protocol could optimize the mouse ovary vitrification-warming procedure using AFP, while minimizing the treatment steps.

Characterization of the ovary fatty acids composition of Rhamdia quelen (Quoy & Gaimard) (Teleostei: Siluriformes), throughout their reproductive cycle

Knowledge about gonad fatty acid composition is important for broodstock diet formulation. This study characterized ovary fatty acid composition of wild female jundiá catfish (Rhamdia quelen) in their different gonadal maturation stages. Female jundiá (n = 36, average weight= 383.8 + 208.8 g) were captured in the rio Uruguay, comprising all seasons. Ovaries were extracted and classified according to their gonadal maturation stage. Gonad-somatic ratio varied significantly among seasons, being higher in spring (3.7), followed by summer (2.2), winter (0.9) and autumn (0.6). Main fatty acids groups detected were: saturated (SFA= 35.5%), monounsaturated (MUFA= 28.1%) and polyunsaturated fatty acids (PUFA= 33.5%). Over the four seasons, palmitic acid was recorded in large quantities, followed by docosahexaenoic acid (DHA) and arachidonic acid (ARA). ARA was present in higher concentrations in immature or maturing ovaries, and its content decreased along the maturation process. Conversely, DHA and eicosapentaenoic acid (EPA) contents increased during maturation. Such variation resulted in an increase in EPA/ARA and DHA/ARA ratios in mature gonads, which can be important for successful breeding. Such findings suggest that jundiá broodstock diets should contain lipids that provide long chain polyunsaturated fatty acids from both the n-3 and n-6 series to ensure gonadal maturation completion.

O conhecimento da composição de ácidos graxos da gônada, como dos fatores que a influenciam são importantes na formulação de dietas para reprodutores de qualquer espécie. O presente estudo caracteriza a composição de ácidos graxos da gônada de fêmeas de jundiá (Rhamdia quelen) selvagens em seus diferentes estágios de maturação gonadal. Fêmeas de jundiá (n = 36) com peso médio de 383,8 + 208,8 g foram capturadas no alto rio Uruguai, ao longo de um ano, abrangendo as quatro estações. As gônadas foram extraídas, classificadas e posteriormente sua composição de ácidos graxos foi determinada. A relação gônado-somática variou significativamente entre as estações, sendo maior na primavera (3,7), seguida de verão (2,2), inverno (0,9) e no outono (0,6). Os principais grupos de ácidos graxos detectados foram: saturados (35,5 + 3,5%), monoinsaturados (28,1 + 4,3%) e ácidos graxos poli-insaturados (33,5 + 3,0%), com aproximadamente 30% para cada grupo. Ao longo das quatro estações, o ácido palmítico foi registrado em grandes quantidades, seguido do ácido docosahexaenóico (DHA) e ácido araquidônico (ARA). O ácido araquidônico estava presente em concentrações mais elevadas nas gônadas imaturas que nas gônadas maturas, e seu conteúdo diminuído ao longo do processo de maturação. Inversamente, o conteúdo de DHA e ácido eicosapentaenoico (EPA) aumentaram durante o processo de maturação. Tal variação resultou num aumento das relações EPA/ARA e DHA/ARA nas gônadas maturas, fato que pode ser importante para o sucesso da reprodução no jundiá. Tais resultados sugerem que dietas para reprodutores da espécie devem conter fontes de lipídios que proporcionem ácidos graxos de cadeia longa de ambas series (n-3 e n-6) para assegurar uma correta maturação final da gônada.

Morphological indicators of initial reproductive commitment in Mustelus schmitti (Springer 1939) (Chondrichthyes, Triakidae): folliculogenesis and ovarian structure over the life cycle / Indicadores morfológicos de compromisso reprodutivo inicial em Mustelus schmitti (Springer 1939) (Chondrichthyes, Triakidae): folliculogenesis e estrutura ovariana ao longo do ciclo de vida

This work provides information about the sexual commitment and the folliculogenesis of the gatuzo, Mustelus schmitti. A total of 112 females of all maturity stages were fished in the Bahía Blanca estuary, between 2009 and 2010. The oogonia were present throughout the life cycle of the animals. The folliculogenesis follows a pattern similar to other elasmobranchs. The granulosa layer keeps monolayered throughout the folliculogenesis, but with two cell types in the vitellogenic follicle. The zona pellucida forms in the primordial follicles. The thecal system shows a connective inner layer and a glandular outer sheath. The microscopic beginning of the sexual commitment, indicated by the vitello hoarding, takes place in follicles from 500 micrometres, while the macroscopic evidence appears in follicles of 2500-3000 micrometres. The results presented in this study suggest that the fishery pressure may affect a susceptible range of sizes of the species, not previously considered and provides a biological framework for the development of fisheries policy.

Este trabalho provê informações sobre o compromisso sexual e da foliculogênese do gatuzo, Mustelus schmitti. Um total de 112 fêmeas de todas as fases de maturidade foram pescados no estuário Bahía Blanca, entre 2009 e 2010. O oogônias foi presentes durante todo do ciclo de vida dos animais. A foliculogênese segue um padrão semelhante a outros elasmobrânquios. A capa granulosa mantém-se simples durante toda a foliculogénese, mas com dois tipos de células no folículo vitelogênico. A zona pelúcida forma-se nos folículos primordiais. O sistema mostra uma capa tecal interior de tecido conjuntivo e uma bainha exterior glandular. O início microscópico do compromisso sexual, indicado pela acumulação do vitello, realiza-se em folículos de 500 micrómetros, enquanto que a evidência macroscópica aparece em folículos de 2500-3000 micrómetros. Os resultados apresentados neste estudo sugerem que a pressão da pesca pode afetar um amplo intervalo de tamanho das espécies não considerado anteriormente, e fornece uma base biológica para o desenvolvimento de política comum da pesca.

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens.

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with nonmotile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens. Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin. Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

Dynamics of ovarian maturation during the reproductive cycle of Metynnis maculatus, a reservoir invasive fish species (Teleostei: Characiformes)

In this study, we evaluated the dynamics of ovarian maturation and the spawning processes during the reproductive cycle of Metynnis maculatus. Adult females (n = 36) were collected bimonthly between April 2010 and March 2011. The mean gonadosomatic index (GSI) was determined, ovarian and blood samples were submitted for morphometric evaluation and the steroid plasma concentration was determined by ELISA. This species demonstrated asynchronous ovarian development with multiple spawns. This study revealed that, although defined as a multiple spawning species, the ovaries of M. maculatus have a pattern of development with a predominance of vitellogenesis between April and August and have an intensification in spawning in September; in October, a drop in the mean GSI values occurred, and the highest frequencies of post-ovulatory follicles (POFs) were observed. We observed a positive correlation between the POF and the levels of 17α-hydroxyprogesterone. Metynnis maculatus has the potential to be used as a source of pituitary tissue for the preparation of crude extracts for hormonal induction; the theoretical period for use is from September to December, but specific studies to determine the feasibility of this approach must be conducted.

Neste estudo, avaliamos a dinâmica da maturação ovariana a desova durante o ciclo reprodutivo de Metynnis maculatus. Fêmeas adultas (n = 36) foram coletadas bimestralmente entre abril de 2010 e março de 2011. O índice gonadossomático (IGS) foi calculado e amostras de ovário e de sangue foram submetidas à avaliação morfométrica e das concentrações plasmáticas dos esteroides por ELISA, respectivamente. A espécie apresenta desenvolvimento ovariano assincrônico, com múltiplas desovas. Neste estudo revelamos que mesmo sendo de desova parcelada, os ovários do M. maculatus mostraram um padrão de desenvolvimento com predomínio de atividade vitelogênica entre abril a agosto e intensificação da desova em setembro. Em outubro houve uma diminuição nos valores médios de IGS, bem como registramos as maiores frequências de folículos pós-ovulatórios (FPOs). Observamos uma correlação positiva entre a frequência de FPOs e a concentração plasmática de 17 α-OHP. O M. maculatus tem potencial para ser usado como fonte para uso de hipófise para preparo de extrato bruto para indução hormonal, sendo o período teórico para coleta de hipófises de setembro a outubro, mas estudos específicos para esta finalidade ainda precisam ser desenvolvidos.

Evaluation of apoptosis as a mechanism of follicular cell atresia in the ovaries of cattle (bos indicus) and buffalo (bubalus bubalis) fetuses

The objective of this study was to investigate the occurrence of apoptosis in the ovaries of cattle and buffalo fetuses between 4 and 8 months old by the terminal deoxynucleotidyltransferase - mediated dUTP nick end labeling (TUNEL) assay . Histological analysis of the ovarian t issue showed that apoptosis occurred at all ages evaluated , presenting a similar pattern among different fetal stages in both species . Within species, secondary follicles displayed a higher (P 0.05) of apoptotic follicular cells among the three follicular classes compared . C omparing resul ts between species, secondary follicles had a higher (P < 0.05) mean number of TUN EL positive cells in bovine fetuses; however , this difference was proportional to the larger number of follicular cells present in secondary follicles in this species . In summary, the TUNEL method was effective for the detection of apoptosis in the support ing cells of ovarian follicles from bovine and buffalo fetuses with apoptosis occurring at similar rates in both species between 4 and 8 months of gestational age. Further studies are needed to better understand the dynamics of apoptosis as a regulator of follicular atresia in fetal ovaries from these species, as well as the potential involvement of the oocyte in this process.

Vitellogenin levels in hemolymph, ovary and hepatopancreas of the freshwater crayfish Cherax quadricarinatus (Decapoda: Parastacidae) during the reproductive cycle

The freshwater crayfish Cherax quadricarinatus is a tropical species of great interest for aquaculture. Vitellogenin (Vg), a lipoprotein precursor of the vitellum accumulated in spawned eggs, can be synthesized in the ovary and/or hepatopancreas of most crustaceans, being the hemolymph the way for transporting Vg throughout the reproductive cycle. Concentration of Vg in hemolymph, ovary and hepatopancreas of Cherax quadricarinatus adult females was measured by means of ELISA, specifically developed after purifying the native Vg. Measurements were made at four periods of the reproductive cycle: pre-reproductive, mid-reproductive, late reproductive and post-reproductive. Besides, both hepatosomatic (HSI) and gonadosomatic (GSI) indexes were determined in each period. Significant variations in Vg levels were detected in both hemolymph and hepatopancreas, being the highest values observed during the mid-reproductive period. Besides, such variations were positively correlated to the HSI. A positive correlation between Vg levels in hepatopancreas and ovary was also seen. These results support previous evidences about the central role of the hepatopancreas as a site of Vg synthesis in the studied species, together with the relevancy of hemolymph for transporting Vg from the hepatopancreas to the ovary. For aquaculture purposes, Vg monitoring in hemolymph could be used as a non-injurious method, to check the reproductive activity of C. quadricarinatus females.

La langosta de agua dulce Cherax quadricarinatus es una especie tropical de gran interés para la acuicultura. Se midió la concentración de vitelogenina (Vg) en hemolinfa, ovario y hepatopáncreas de hembras adultas de esta especie, por medio de ELISA. Las mediciones fueron hechas en los cuatro períodos del ciclo reproductivo: pre-reproductivo, reproductivo medio, reproductivo tardío y post-reproductivo. Se detectaron variaciones significativas en los niveles de Vg tanto en hemolinfa como en hepatopáncreas, se observó el mayor valor durante el período reproductivo medio. Además, tales variaciones se correlacionaron positivamente con el índice hepatosomático. Se observó además una correlación positiva de los niveles de Vg entre hepatopáncreas y ovario. Estos resultados apoyan evidencias previas sobre el papel central del hepatopáncreas como sitio de síntesis de Vg, en esta especie, y también enfatizan la importancia de la hemolinfa para el transporte de la Vg del hepatopáncreas al ovario. Para propósitos de acuicultura, la medición de Vg en hemolinfa podría ser utilizada como un método no lesivo, con el fin de constatar la actividad reproductiva de hembras de C. quadricarinatus.

Effects of echinomycin on endothelin-2 expression and ovulation in immature rats primed with gonadotropins

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.

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.

Estructura y la ultraestructura del ovario de Cichlasoma urophthalmus (Perciformes: Cichlidae)

Structure and ultrastructure of the ovary of Cichlasoma urophthalmus (Osteichthyes: Cichlidae). The study of the normal development, differentiation, structure and function of various components of developing follicles in the ovaries of numerous fish species have been a consistent focus of comparative reproduction. The structural and ultrastructural features of gonads from Cichlasoma urophthalmus have received scarce attention. In this work, we realized a descriptive study of female gonads of Cichlasoma urophthalmus. A total of 40 samples were collected in the Veracruz Alvarado Lagoon, Mexico in 2007-2008 period including the windy, dry and rainy seasons. Female gonads were extracted and a portion was fixed in 4% formaldehyde for treatment for routine histology hematoxylin and eosin (HE) and another part was processed for transmission electron microscopy (TEM). The gonads were fixed in 3% glutaraldehyde and 2% osmium tetroxide, followed by dehydrated in ethanol 50%, 70%, 80%, 95% and 100% for inclusion in Epon, thin sections were then prepared and were contrasted with lead citrate and uranyl acetate. The process of oocyte development can be divided into five distinct stages (formation of oocytes from oogonia, primary growth, lipid stage, vitellogenesis and maturation). In this work, we found that the primary growth stage is characterized by intense RNA synthesis and the differentiation of the vitelline envelope. Secondary growth starts with the accumulation of lipid droplets in the oocyte cytoplasm (lipid stage), which is then followed by massive uptake and processing of proteins into yolk platelets (vitellogenic stage). During the maturation stage, the lipid inclusions coalesce into a single oil droplet, and hydrolysis of the yolk platelets leads to the formation of a homogeneous mass of fluid yolk in mature eggs. In conclusion, further studies should elucidate structure and ultrastructural changes in the ovarian follicular components, in C. urophthalmus during different stages of oocyte growth. Rev. Biol. Trop. 59 (2): 743-750. Epub 2011 June 01.

Se realizó un estudio descriptivo de las gónadas femeninas de Cichlasoma urophthalmus. Las muestras fueron recolectadas en la Laguna de Alvarado Veracruz, México en el período 2007-2008 que incluyó las temporadas de Nortes, Secas y Lluvias. Se extrajeron las gónadas femeninas y una parte se fijó en formol al 4% para su tratamiento por técnica histológica de rutina hematoxilina y Eosina (H-E) y otra parte se procesó para microscopia electrónica de transmisión. Las gónadas se fijaron en glutaraldehído al 3% y OsO4 al 2%, se deshidrataron en etanol de 50 al 100% para ser incluidas en Epón. Se realizaron cortes finos y semifinos contrastados con citrato de plomo y acetato de uranilo. Los ovarios de C. urophthalmus son pareados presentan un desarrollo asincrónico con ovocitos previtelogénicos en estadio perinuclear tardío, asociados a las lamelas ovígeras y ovocitos vitelogénicos del VII estadio, éstos últimos presentan una zona radiada bien definida, con gránulos de vítelo lipídico y vesículas de vítelo proteico que se distribuyen en capas concéntricas, que rodean al núcleo. El presente estudio, permitió conocer más a fondo los cambios de la estructura y ultraestructura de los componentes de los folículos ováricos, en C. urophthalmus durante las diferentes etapas de crecimiento de los oocitos.

In vitro maturation and fertilization of prepubertal and pubertal black Bengal goat oocytes

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle's salt with L-glutamine and sodium bicarbonate) for 27 h at 39degrees C under 5% CO2 in humidified air. The oocytes retrieval and efficiency (mean +/- SD) per prepubertal and pubertal goats were 5.2 +/- 0.6 and 6.8 +/- 0.6, and 77.3 +/- 0.1% and 80.5 +/- 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 +/- 0.8 vs. 2.6 +/- 0.2, p 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 +/- 1.7 vs. 32.2 +/- 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production.

Cytogenetic aberrations in primary cell cultures of the ovarian surface epithelium / Aberraciones citogenéticas en cultivos primarios del epitelio superficial del ovario

Our objective was to determine the presence of chromosomal abnormalities in primary cultures of ovarian surface epithelial cells in women of different ages with no history of cancer. Throughout conventional cytogenetic techniques, we analyzed chromosome spreads of cultured ovarian epithelial cells from 10 donors who were 50 or more years old (B) and 16 controls between 20 and 49 years old (A), belonging to the mestizo population in Bogota DC, Colombia. Of the 26 cultures that were analyzed in passage 1, 61.5% had an abnormal chromosome complement (62.5% in A, and 60% in B). Abnormalities included polyploidies, endoduplications and monosomies. Deletions in chromosomes 3 and 11 were found in just one metaphase. None of the samples showed weaknesses or breakpoints. After transforming and applying the exact student’s t-test for variance heterogeneity, we found significant differences in the frequency of metaphases, that were higher in A than in B (p=0.05), and in the frequency of polyploidies, which were higher in B than in A (p=0.044). Through the application of the Mann-Whitney test, we determined that the frequency of endoduplications was higher in A than in B (p=0.126), without reaching significant differences. There were no significant differences in the frequency of monosomies. The level of significance was set at p £ 0.05. Taking into account that polyploidization is a marker of chromosomal instability and that the risk of cancer arising from the ovarian surface epithelium augments substantially after menopause, the increase in the frequency of age-associated polyploidies could be used as a predictor of ovarian cancer in women from an ethnically homogeneous population as the mestizo one in Bogota DC.

El objetivo del presente trabajo fue determinar la presencia de anormalidades cromosómicas en cultivos primarios de células del epitelio superficial ovárico en mujeres de diferentes edades, sin antecedentes de cáncer. Mediante técnicas de citogenética convencional fueron analizados extendidos de células epiteliales ováricas histológicamente normales, provenientes de cultivos primarios de 10 donantes de 50 o más años (B) y de 16 donantes entre 20 y 49 años que se utilizaron como grupo control (A), pertenecientes a la población mestiza de Bogotá DC, Colombia. De 26 cultivos examinados en pase 1, 61,5% presentó complemento cromosómico anormal, 62,5% en A y 60% en B. Las anomalías numéricas halladas, todas en mosaico, incluyeron poliploidías, endoduplicaciones y monosomías. En una única célula en metafase de un cultivo, se presentaron deleciones en los cromosomas 3 y 11. Ninguna muestra presentó fragilidades o roturas. Previa aplicación de transformaciones, con la prueba exacta t-student para varianzas heterogéneas, se encontraron diferencias significativas en la frecuencia de células con metafase normal, mayor en A que en B (p=0,05) y en la de poliploidías, mayor en B que en A (p=0,044). Con la prueba exacta de Mann-Whitney se determinó que la frecuencia de endoduplicaciones en A fue mayor que en B (p=0,126), sin alcanzar diferencias significativas y que no hubo diferencias significativas en la frecuencia de monosomías. El nivel de significación fue p £ 0,05. Si se tiene en cuenta que la poliploidización es un marcador de inestabilidad cromosómica y, que además, el riesgo de aparición de cáncer derivado del epitelio superficial del ovario aumenta sustancialmente después de la menopausia, el incremento en la frecuencia de poliploidías asociado con la edad podría ser utilizado como predictor de cáncer ovárico en mujeres de una población étnicamente homogénea como la población mestiza de Bogotá DC.

Actividad de la ß-galactosidasa como marcador de senescencia en cultivos primarios del epitelio superficial del ovario / ß-Galactosidase activity as a marker of senescence in primary cultures of the ovarian surface epithelium

La actividad de la ß-galactosidasa refleja la tasa de envejecimiento celular in vitro. Mediante quimioluminiscencia se cuantificó dicha actividad a pH 6 en células epiteliales ováricas provenientes de 28 donantes sin antecedentes de cáncer. Las células fueron cultivadas en forma seriada hasta alcanzar el estado de detención permanente de crecimiento. Durante la fase de crecimiento exponencial, todos los cultivos mostraron un patrón semejante de crecimiento y una baja actividad ß-galactosidasa. Sin embargo, el inicio de la disminución de la capacidad replicativa que caracteriza el final de dicha fase, así como el inicio de la fase estacionaria o senescente presentaron un aumento significativo en la actividad enzimática. Nuestros resultados mostraron que la actividad ß-galactosidasa puede ser considerada como marcador de senescencia replicativa del epitelio superficial del ovario a pH 6.

ß-galactosidase activity reflects the rate of cellular aging in vitro. Such activity was quantified at pH 6 in ovarian epithelial cells from 28 donors without a history of cancer, by the chemoluminiscent method. The cells were serially cultured until they achieved the state of permanent growth arrest. During the exponential growth phase, all cultures showed a similar pattern of growth and low ß-galactosidase activity. However, both in the onset of decrease replicative activity, as well as in the onset of the stationary phase, there was a significant rise in the enzyme activity. Our results showed that ß-galactosidase activity can be considered as a replicative senescence marker of the ovarian surface epithelium at pH 6.

Culex quinquefasciatus vitellogenesis: morphological and biochemical aspects

The vitellogenic process in Culex quinquefasciatus, which is triggered by a blood meal, involves the synthesis, distribution and storage of the nutrients necessary for embryo development. The fat body of an adult female Cx. quinquefasciatus revealed two cell types: large trophocytes and small, eosinophilic, "oenocyte-like" cells, which show no morphological changes throughout the gonotrophic cycle. Trophocytes, which only begin to synthesise vitellogenin (Vg) 12 h post-blood meal (PBM), undergo a series of morphological changes following engorgement. These changes include the expansion of the rough endoplasmic reticulum (RER) and Golgi complex, which are later destroyed by autophagosomes. At 84 h PBM, trophocytes return to their pre-engorgement morphology. The ovarian follicles of non-blood-fed Cx. quinquefasciatus contain a cluster of eight undifferentiated cells surrounded by follicular epithelium. After engorgement, the oocyte membrane facing the perioocytic space increases its absorptive surface by microvilli development; large amounts of Vg and lipids are stored between 24 and 48 h PBM. Along with yolk storage in the oocyte, follicular cells exhibit the development of RER cisternae and electron-dense granules begin to fill the perioocytic space, possibly giving rise to endochorion. Later in the gonotrophic cycle, electron-dense vesicles, which are possible exochorion precursors, fuse at the apical membrane of follicular cells. This fusion is followed by follicular cell degeneration.

Study on the vaginal smear of rats submitted to autologous ovarian transplant: impact of remote ischemic preconditioning / Estudo do esfregaço vaginal de ratas submetidas a transplante autólogo de ovário: impacto do precondicionamento isquêmico remoto

PURPOSE: To evaluate the remote ischemic preconditioning (R-IPC) impact on the quality of the ovarian graft by means of vaginal smear of transplanted rats. METHODS: Sixty rats were used divided in six groups: Control; Fresh transplant (TxF); Cryopreserved transplant (TxC); R-IPC; R-IPC + fresh transplant (TxF+R-IPC); R-IPC + cryopreserved transplant (TxC+R-IPC). R-IPC was performed in the common iliac artery. Autologous ovarian tissue was implanted integrally in the retro peritoneum. On the first PO day, vaginal smear collection was daily initiated. After 30 days, the PO day when the estrous cycle was re-initiated was considered for analysis as well as the estrous days and the number of estrous cycles. RESULTS: R-IPC showed a tendency to an early estrus re-initiation (p>0.05) as well as increase the number of cycles in the fresh transplanted group while in the cryopreserved transplant the number of cycles was similar, regardless of the stimulus R-IPC (p>0.05). The animals which had undergone fresh grafts had a longer estrous period than the ones which had undergone cryopreserved grafts, with or without R-IPC (p<0.05). CONCLUSION: R-IPC promoted earlier re-initiation of ovarian activity in the PO and greater estrous frequency, with more consistent results in the fresh grafts than in the cryopreserved ones.

OBJETIVO: Avaliar o impacto do precondicionamento isquêmico remoto (PCI-R) na qualidade do enxerto ovariano através dos esfregaços vaginais dos animais transplantados. MÉTODOS: Foram utilizadas 60 ratas, distribuídas em seis grupos de estudo: Controle; Transplante fresco; Transplante criopreservado; PCI-R; PCI-R + Transplante fresco; PCI-R + Transplante criopreservado. O PCI-R foi realizado na artéria ilíaca comum. O tecido ovariano foi implantado íntegro no retroperiônio. No 1º dia de pós-operatório (PO) foram coletados esfregaços vaginais diariamente. Após 30 dias foram considerados para análise o dia de PO de retorno do estro, assim como o número de dias em estro e de ciclos estrais. RESULTADOS: PCI-R mostrou tendência em reinício mais precoce do cilo estral (p>0,05), assim como aumento no número de cilcos estrais nos grupos com transplante fresco enquanto que no criopreservado o número de ciclos foi semelhante, independente do PCI-R (p>0,05). Os animais que receberam enxertos frescos apresentaram mais dias na fase estro do que os criopreservados, com ou sem PCI-R. CONCLUSÃO: O PCI-R promoveu retorno mais precoce da atividade ovariana no PO e maior freqüência de estro, sendo os resultados mais consistentes nos enxertos frescos do que nos criopreservados.

The ovary of the bignose fanskate Sympterygia acuta Garman, 1877 (Chondrichthyes, Rajidae) in the Bahía Blanca estuary, Argentina: morphology and reproductive features / O ovário da bignose fanskate Sympterygia acuta Garman, 1877 (Chondrichthyes, Rajidae) no estuário de Bahía Blanca, Argentina: morfologia e características reprodutivas

Chondrichthyes have become an important economic resource in recent years. In spite of this importance, there is little knowledge about their reproductive biology, especially of species from the south-western Atlantic. In this work, we study the morphology and histology of the ovary of Sympterygia acuta. The results show that oogonia were present only in the immature females (stage I of maturity scale), whereas specimens in stages II and III of maturity displayed oocytes in all developmental steps. The most important histological features that vary throughout oocyte development are the presence or absence of yolk, the number of types and layers of follicular cells and the degree of development of the thecae. Follicular cells are, at least, of two different types. Finally, a new point of view for the determination of maturity stages in the field based on the size of follicles is discussed.

Os Chondrichthyes tornaram-se num importante recurso econômico nos últimos anos. Apesar da sua importância, é muito pouco o que se conhece da sua biologia reprodutiva, especialmente das espécies do Atlântico sul-ocidental. Neste trabalho, foi estudada a morfologia e histologia do ovário de Sympterygia acuta. Os resultados mostraram que as oogonias somente encontraram-se presentes nas fêmeas imaturas (fase I de escala de maturidade) e que os animais em estágio II e III de maturidade exibiram oocitos em todas as etapas de desenvolvimento. As características histológicas mais importantes que variam ao longo de desenvolvimento dos oocitos são a presença da suarda, o tipo e quantidade de celas foliculares e o grau do desenvolvimento do thecae. Existem pelo menos dois tipos de células foliculares. Finalmente se discute um novo ponto de vista para a determinação dos estágios de maturidade no campo, baseado no tamanho dos folículos.