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.

Reproductive biology, morphology, and morphometry of ovaries and oviducts of Trachemys scripta elegans in Brazilian Cerrado / Biologia reprodutiva, morfologia e morfometriade ovários e ovidutos de Trachemys scripta elegans no Cerrado Brasileiro

Although Trachemys scripta elegans is an exotic species popular as a pet in Brazil, studies on reproductive biology and capacity are non-existent in the Brazilian Cerrado. This study analyzed ovarian and oviduct characteristics and the egg production capacity of T. scripta elegans grown in this biome. The findings will associate with the size of the specimens and the sexual maturity, aiming at comparisons with native and exotic populations, as well as interspecific and contributing to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. Thus, 39 females had evaluated the body biometry and the morphology and morphometry of the ovaries and oviducts. G2 (N=20): with Class I (>5-10mm) follicles, with Class I and Class II (>10-fold) follicles, 25mm) and G3 (N=9) with Class I, Class II and Class III (>25mm) follicles. Analysis of variance, Scott-Knott's test, and Pearson's correlation analysis showed that there was no significant difference between the groups in body biometry; in the mean gonadosomatic index and gonadal morphometry, only the width of the oviducts in the right antimer and the mass and width in the left antimer were higher in G3, the only one that presented eggs. There was positive and harmonic development between body mass, carapace, and plastron, and gonadal growth occurred concomitantly with body growth, indicating a higher reproductive potential and a positive relationship between the size of the litter and the female litter. The gonadosomatic index proved to be an excellent reproductive indicator, and the ovarian evaluation was a better indicator of sexual maturity than the maximum carapace length. Ovaries were irregular structures, without delimitation between the cortical and medullary regions and filled with vitelogenic follicles of different diameters, atresic follicles, and corpora lutea, which reflected the ovarian complexity of the species and the presence of follicular hierarchy. In the scarce stroma, two germinative beds were observed per ovary and the presence of gaps very close to the follicles and associated with the blood vessels. Analysis of gonadal tissue revealed three types of oocytes according to cytoplasmic characteristics: homogeneous, vesicular or vesicular in the cortex with apparent granules. Oviducts were functional and separated, joining only in the final portion to form the cloaca and subdivided into infundibulum, tuba, isthmus, uterus, and vagina. The structure of the uterine tube was composed of serosa, muscular and mucous, which was full of glands. The presence of eggs in the oviducts indicated that the specimens can reproduce in the Brazilian Cerrado. This study provides necessary and relevant information on the reproductive biology and capacity of T. scripta elegans in the Brazilian Cerrado and can contribute to the understanding of its impact on the invaded ecosystems and the establishment of eradication programs. The extraction of females with capacity can reduce the annual reproductive yield of the species and decrease its effect on local biodiversity.(AU)

Embora Trachemys scripta elegans seja uma espécie exótica popular como animal de estimação no Brasil, estudos sobre biologia e capacidade reprodutivas são inexistentes no Cerrado brasileiro. Este estudo analisou características ovarianas e do oviduto e a capacidade de produção de ovos em T. scripta elegans criadas neste bioma, correlacionando estes achados ao tamanho dos espécimes e a maturidade sexual, visando comparações com populações nativas e exóticas, bem como interespecíficas e contribuir para a compreensão de seu impacto nos ecossistemas invadidos e com o estabelecimento de programas de erradicação. Assim, 39 fêmeas tiveram avaliadas a biometria corporal e a morfologia e morfometria dos ovários e ovidutos. De acordo com o tamanho dos folículos ovarianos as fêmeas foram separadas em G1 (N= 10): com folículos Classe I (>5-10 mm), G2 (N= 20): com folículos Classe I e Classe II (>10-25 mm) e G3 (N= 9) com folículos Classe I, Classe II e Classe III (>25 mm). À análise de variância, teste de Scott-Knott e à análise de correlação de Pearson verificou-se que não houve diferença significativa entre os grupos na biometria corporal; no índice gonadossomático médio e na morfometria gonadal, apenas a largura dos ovidutos no antímero direito e a massa e a largura no antímero esquerdo foram maiores no G3, o único que apresentou ovos. Houve desenvolvimento positivo e harmônico entre massa corporal, carapaça e plastrão e o crescimento gonadal ocorreu concomitante ao crescimento corporal, indicando maior potencial reprodutivo e relação positiva entre o tamanho da ninhada de ovos e o da fêmea. O índice gonadossomático mostrou-se um bom indicador reprodutivo e a avaliação ovariana um melhor indicador da maturidade sexual que o comprimento máximo da carapaça. Ovários foram estruturas irregulares, sem delimitação entre a região cortical e medular e repletos de folículos vitelogênicos de diferentes diâmetros, folículos atrésicos e corpos lúteos, que refletiram a complexidade ovariana da espécie e a presença de hierarquia folicular. No estroma escasso foram observados dois leitos germinativos por ovário e a presença de lacunas muito próximas aos folículos e associadas aos vasos sanguíneos. A análise do tecido gonadal revelou três tipos de oócitos de acordo com as características do citoplasma: homogêneo, vesicular ou vesicular no córtex com grânulos aparentes. Ovidutos eram funcionais e separados, unindo-se apenas na porção final para formar a cloaca e subdividiam-se em infundíbulo, tuba uterina, istmo, útero e vagina. A estrutura da tuba uterina era constituída de serosa, muscular e mucosa, a qual era repleta de glândulas. A presença de ovos nos ovidutos indicou que os espécimes podem se reproduzir no cerrado brasileiro. Este estudo fornece informações básicas e relevantes da biologia e capacidade reprodutivas de T. scripta elegans no Cerrado brasileiro e pode contribuir com a compreensão de seu impacto nos ecossistemas invadidos e com o estabelecimento de programas de erradicação, uma vez que a extração de fêmeas com capacidade reprodutiva pode contribuir com a diminuição do rendimento reprodutivo anual da espécie e diminuir seu efeito sobre a biodiversidade local.(AU)

Descripción histológica del aparato reproductor de avestruz hembra (Strutio camelus var. domesticus) / Histological description of the ostrich female reproductive system (Strutio camelus var. domesticus)

El auge de la producción intensiva del avestruz, comenzó en la década de los noventa impulsada por la calidad de su carne y potencialidad de sus subproductos. La raza empleada para producción por la calidad nutricional y sabor de su carne es el híbrido llamado African black (Struthio camelus var. domesticus). En cuanto a la reproducción, el avestruz hembra alcanza su madurez sexual a partir de los 2,5 años. Es importante considerar el aparato genital en aves de producción, ya que una alteración en él, puede generar deficiencias en la fertilidad que se traducen en un menor número de crías. El estudio histológico del aparato reproductor de la hembra será una herramienta más que permitirá resolver problemas reproductivos. Para este análisis se obtuvo muestras de los diferentes segmentos del aparato reproductor de 6 avestruces hembras en edad reproductiva y se procesaron de acuerdo a las técnicas histológicas de rutina. Los cortes fueron observados, fotografiados y analizados bajo microscopio de luz. Obtenidas las fotografías, se analizó comparativamente su morfología con la descrita en la gallina (Gallus gallus). El aparato reproductor de la hembra tiene la particularidad de tener desarrollado solo el ovario y oviducto izquierdo. El ovario es de gran tamaño y en forma de racimo, el cual varía según la estacionalidad. Presenta folículos primordiales, previtelogénicos, vitelogénicos y atrésicos. Los folículos vitelogénicos presentan células de la granulosa y de la teca interna y externa. El oviducto presenta de cefálico a caudal los siguientes segmentos: infundíbulo, magnum, istmo, útero y vagina, que desemboca en la cloaca a nivel del urodeo. En ellos hay pliegues de variada longitud, grosor y número que comprometen la mucosa y submucosa, con glándulas de secreción mucosa y serosa a excepción de la vagina. El análisis histológico comparativo, permitió establecer que la morfología del aparato reproductor de la hembra es semejante a la observada en la gallina con ciertas diferencias microscópicas (Gallus gallus).

In the 1990's, ostrich production reached a peak in our country, boosted by the special characteristics of its meat and the potential of the derivatives. The breed raised is a hybrid called African Black (Struthio camelus var. domesticus) which has a high quality meat in terms of nutritional factors and flavor. With regard to reproduction, the female ostrich reaches maturity at the age of 2.5 years. Genital organs are very important in fowl's production, because they can generate fertility deficiencies that, in turn can diminish brood number. Histological analysis allows a better understanding of the basic structure of the female's genital organs and is a helpful tool to resolve breeding problems. For this analysis samples were obtained from the different segments of the reproductive system of 6 female ostrich in reproductive age. These samples were processed using standard histological technique. Sections were observed, photographed and analyzed under the light-microscope. Photographs were compared with hen's samples. The ostrich female's reproductive system has the particularity of having just the left ovary and oviduct developed. The ovary has a big size and a cluster shape which varies from season to season. It presents paramount, previtellogenic, vitellogenic and atresic follicles. The vitellogenic follicles have granulosa cells and inner and external theca. The oviduct presents cephalocaudally: infundibulum, magnum, isthmus, uterus and vagina, flowing into the urodeum. It shows long pleats of different length and number, with drusen of mucose and serose secretion, except in the vagina. The comparative histological analysis allowed us to establish that the basic structure of the female reproductive system is similar to that of the hen (Gallus gallus).

Effects of laser-assisted hatching and exposure time to vitrification solution on mouse embryo development / 대한생식의학회지

OBJECTIVE: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. METHODS: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. RESULTS: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p < 0.05). In the control-8 group (22.1±4.6), the cell number of the inner cell mass was higher than in the LAH groups (p < 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group (92.8±8.9) than in the others (p < 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group (19.5±5.1, p < 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group (73.2±12.1) than in the other groups, but without statistical significance. CONCLUSION: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

The female reproductive system of Zearaja chilensis (Quichenot, 1848) (Chondrichthyes, Rajidae): gametogenesis and microscopic validation of maturity criteria / El sistema reproductor femenino de Zearaja chilensis (Quichenot, 1848) (Chondrichthyes, Rajidae): gametogénesis y validación microscópica de los criterios de madurez

Chondrichthyans are extremely susceptible to overfishing due, among other things, to their reproductive adaptations. Basic knowledge of reproductive parameters is essential, both of ecological and for economic points of view. Zearaja chilensis is a valuable economic resource in South America. This work analyzes the microanatomy of female reproductive system, as well as the size of the onset of vitellogenesis. The material was fixed in Bouin and processed using routine histological techniques. Both ovaries are equally functional. Folliculogenesis depicts the same pattern of other Chondrichthyans. Follicles with different degrees of maturation coexist in mature animals, with the exception of oogonia, which were only found in immature individuals. Likewise, atretic follicles were recorded in all stages of maturation. The size of yolk input, microscopically recorded, is lower than the detected at naked eye. Oviductal glands and uterus show similarity with those reported in other lecitotrophic cartilaginous fish. This work reports, for the first time, the morfofunctional microanatomy of the species, and puts to the test the accuracy of the commonly employed criteria for the determination of sexual maturity, a critical data when determining management policies.

Los condrictios son extremadamente susceptibles a la explotación pesquera debido, entre otras cosas, a sus adaptaciones reproductivas. Los conocimientos básicos de sus parámetros reproductivos son esenciales, ya sea desde el punto de vista ecológico como económico. Zearaja chilensis constituye un importante recurso económico en América del Sur. En este trabajo se analiza la anatomía microscópica del sistema reproductor femenino y la talla de inicio de la vitelogénesis. El material se fijó en Bouin y se procesó mediante técnicas histológicas de rutina. Ambos ovarios son igualmente funcionales. La foliculogénesis presenta el mismo patrón de otros condrictios. Los folículos con diferentes grados de maduración coexisten en animales maduros, con la excepción de las oogonias, que sólo se observaron en los individuos inmaduros. Los folículos atrésicos se registraron en todas las etapas de la maduración. La talla de inicio de la vitelogénesis, microscópicamente registrada, es inferior a la detectada en el ojo desnudo. La glándula oviductal y el útero muestran similitud con lo reportado en otros peces cartilaginosos lecitotróficos. Este trabajo describe por primera vez, la microanatomía morfofuncional de la especie y pone a prueba la precisión de los criterios comúnmente empleados para la determinación de la madurez sexual, una información crítica para la determinación de las políticas de gestión.

The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification / 대한생식의학회지

OBJECTIVE: The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. METHODS: In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. RESULTS: The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). CONCLUSION: The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice.

The effect of various assisted hatching techniques on the mouse early embryo development / 대한생식의학회지

OBJECTIVE: In search of an ideal method of assisted hatching (AH), we compared the effects of conventional micropipette-AH and laser-AH on the blastocyst formation rate (BFR) and blastocyst cell numbers. METHODS: Four- to five-week-old ICR female mice were paired with male mice after superovulation using Pregnant mare's serum gonadotropin (PMSG) and hCG. The two-cell embryos were flushed from the oviducts of female mice. The retrieved two-cell embryos underwent one of five AH procedures: single mechanical assisted hatching (sMAH); cross mechanical assisted hatching (cMAH); single laser assisted hatching (sLAH); quarter laser assisted hatching (qLAH); and quarter laser zona thinning assisted hatching (qLZT-AH). After 72 hours incubation, double immunofluorescence staining was performed. RESULTS: Following a 72 hours incubation, a higher hatching BFR was observed in the control, sMAH, cMAH, and sLAH groups, compared to those in the qLAH and qLZT-AH groups (p<0.05). The hatched BFR was significantly higher in the qLAH and qLZT-AH groups than in the others (p<0.05 for each group). The inner cell mass (ICM) was higher in the control and sMAH group (p<0.05). The trophectoderm cell number was higher in the cMAH and qLAH groups (p<0.05). CONCLUSION: Our results showed that the hatched BFR was higher in groups exposed the the qLAH and qLZT-AH methods compared to groups exposed to other AH methods. In the qLAH group, although the total cell number was significantly higher than in controls, the ICM ratio was significantly lower in than controls.

Oviductal isthmus light microscopic changes induced by clomiphene citrate in albino rats

Clomiphene citrate [CC] therapy for cases of infertility has been linked to increased incidence of tubal ectopic pregnancy. This study aimed to investigate the histological changes produced by CC in the isthmus portion of albino rat oviducts. Twenty adult female albino rats were divided into three groups. The first was the control group. The second group received 1 mg/kg/day CC for 4 days, whereas the third group was allowed a recovery period after receiving CC. The oviductal isthmus sections were stained by H and E, periodic acid-Schiff [PAS], and caspase for light microscopic examinations. An image analyzer was used and the measured data were recorded and statistically analyzed. The CC intake led to increased thickness of the oviductal isthmus lining epithelium, as well as to hyperplasia, bridging, and reduction of the lumen diameter. Furthermore, many degenerative and apoptotic changes as well as significant increase in the PAS optical density and caspase reaction area percentage of the lining epithelium were detected. Also, the isthmic sections of the recovery group revealed increased wall thickness, epithelial degenerative changes, and hyperplasia, in addition to significant reduction in the lumen diameter leading to tubal obstruction. Significant increase in the PAS optical density of the lining epithelium was detected, but its caspase reaction area percentage showed insignificant change compared with the control group sections. This study suggests that CC administration for 4 days at a dose of 1 mg/kg/day induces damage to the albino rat oviductal isthmus that could predispose to occurrence of ectopic pregnancy and that progressively developed despite stoppage of the drug

Male pseudohermaphroditism in a Maltese/poodle mixed dog / 동물의과학연구지

Male pseudohermaphroditism is not commonly reported in veterinary medicine. Here, a 3-year-old Maltese/poodle mixed dog presented with malformed external genitalia and episodic hematuria. Inspection and palpation of the external genitals showed a malformed penis, shortened prepuce, external urethral orifice, and cryptorchidism. There was no urethral meatus at the tip of the penis. The urethral opening was situated between the prepuce and the penis. The anterior half of the prepuce was absent, and the penis was free and exposed to both trauma and licking. Plain radiographic examination showed absence of an os penis in the penis. A double-contrast cystograph showed the suspected uterus as well as the cystic calculi. A hypoechoic space was seen at the dorsal portion of the urinary bladder. The space was suspected to be the uterus. A sagital ultrasonograph showed cystic calculi in the urinary bladder. During surgery to remove cystic calculi, hypoplastic testes as well as the uterus were observed. Histological examination of the testes showed the seminiferous tubules and interstitial cells. The sertoli cells and spermatogonia were adjacent to the basement membrane. No evidence of spermatogenesis was found. Striated squamous epithelial cells and smooth muscle cells were found in the uterus. This dog had vestigial oviducts as well as a uterus with male-appearing external genitals.

Purification of heparin binding oviduct specific proteins and its effect on in vitro embryo development in cattle.

The objective of this study was to see the effect of purified heparin binding oviduct specific proteins (OSP) as media supplement on in vitro embryo developmental competence in cattle. The oviduct specific proteins were isolated from abattoir cattle oviducts and precipitated, dialyzed and at the end purified by high performance liquid chromatography system. The SDS-PAGE profile of eluted heparin binding protein (HBP) fraction showed bands between ~66 - ~97 kDa, while heparin unbinding protein (HUBP) fraction showed two bands at ~66 kDa and in total protein (TP) bands were ~60 - ~95 kDa. Collected all three OSP fractions were used as a media supplement in three different concentrations (0, 5 and 20 µg/mL) for in vitro maturation of immature oocytes, in vitro fertilization and culture of presumptive embryos at 38.5 ºC in 5% CO2 incubator with maximum humidity. The highest cleavage rate (73.40±2.36%) was observed at 5 µg/mL concentration level and lowest cleavage rate (27.63±1.89%) was obtained in 20 μg/mL total protein (TP) fraction. The highest blastocyst formation (26.47±1.47%) also occurred in 5 µg/mL concentration of total protein (TP) fraction and the lowest blastocyst rate (3.60±1.80%) was achieved at 20 µg/mL HBP fraction. The highest cleavage rate in the control group was 60.45±2.66% in TP fraction and blastocyst formation was 11.66±2.54% in HUBP fraction which was not significantly differ from HBP fraction. These results indicate that at 5 µg/mL of total OSP fraction (TP) and HBP used as media supplement increased the cleavage rate significantly as compared to HUBP fraction, and total OSP fraction (TP) increased blastocyst formation significantly (P<0.05) as compared to HBP & HUBP fraction.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices

BACKGROUND AND OBJECTIVES: Use of somatic cells as a feeder layer to maintain the embryonic stem cells (ESCs) in undifferentiated state limits the stem cell research design, since experimental data may result from a combined ESCs and feeder cell response to various stimuli. Therefore, present study was designed to evaluate the developmental competence of the buffalo ESCs over different homogenous feeders and compare with various extracellular matrices using different concentrations of LIF. METHODS AND RESULTS: Inner cell masses (ICMs) of in vitro hatched blastocysts were cultured onto homologous feeders viz. fetal fibroblast, granulosa and oviductal cell feeder layers and synthetic matrices viz. fibronectin, collagen type I and matrigel in culture medium. Developmental efficiency was found higher for ESCs cultured on fetal fibroblast and granulosa layers (83.33%) followed by fibronectin (77.78%) at 30 ng LIF. Oviductal feeder was found to be the least efficient feeder showing only 11.11% undifferentiated primary ESC colonies at 30 ng LIF. However, neither feeder layer nor synthetic matrix could support the development of primary colonies at 10 ng LIF. Expression of SSEA-4, TRA-1-60 and Oct-4 were found positive in ESC colonies from all the feeders and synthetic matrices with 20 ng and 30 ng LIF. CONCLUSIONS: Fetal fibroblast and granulosa cell while, amongst synthetic matrices, fibronectin were found to be equally efficient to support the growth and maintenance of ESCs pluripotency with 30 ng LIF. This well-defined culture conditions may provide an animal model for culturing human embryonic stem cells in the xeno-free or feeder-free conditions for future clinical applications.

Morphological and morphometrical assessment of spermathecae of Aedes aegypti females

The vectorial capacity of Aedes aegypti is directly influenced by its high reproductive output. Nevertheless, females are restricted to a single mating event, sufficient to acquire enough sperm to fertilize a lifetime supply of eggs. How Ae. aegypti is able to maintain viable spermatozoa remains a mystery. Male spermatozoa are stored within either of two spermathecae that in Ae. aegypti consist of one large and two smaller organs each. In addition, each organ is divided into reservoir, duct and glandular portions. Many aspects of the morphology of the spermatheca in virgin and inseminated Ae. aegypti were investigated here using a combination of light, confocal, electron and scanning microscopes, as well as histochemistry. The abundance of mitochondria and microvilli in spermathecal gland cells is suggestive of a secretory role and results obtained from periodic acid Schiff assays of cell apexes and lumens indicate that gland cells produce and secrete neutral polysaccharides probably related to maintenance of spermatozoa. These new data contribute to our understanding of gamete maintenance in the spermathecae of Ae. aegypti and to an improved general understanding of mosquito reproductive biology.

Reproductive strategies of Brazilian lizards of the genus Tropidurus Rodrigues, 1987 (Squamata, Tropiduridae) in the temporal and spatial / Estrategias reproductivas de las lagartijas brasileñas del género Tropidurus Rodrigues, 1987 (Squamata, Tropiduridae) en lo temporal y espacial

Reproductive strategy is the set of adaptations that promote the most efficient way that the species will survive under the particular conditions of a determined environment. Understanding these adaptations is important and can help pinpoint populations indicator of environmental changes. Spermatogenesis is a measurable biological process of these adaptations in spatial and temporal scales. We analyzed the morphology of the testes and oviducts of the lizard species that comprise the genus Tropidurus, taking into account the geographical distribution and sympatric relations. For the analysis and the testes were removed from the middle part of the oviducts from Tropidurus etheridge, T. oreadicus, T. itambere, T. spinulosus and T. Guarani species, collected in different places in the Mato Grosso state, Brazil. The reproductive period is synchronous for males and females and occurs in September, October and November. Reproductive males were characterized. In the testes are seminiferous tubules with germ cells at different stages of spermatogenesis, with a high epithelium, at present light, free spermatozoa in the lumen and reduction of interstitial tissue. For females, the reproduction peak occurs when the oviduct epithelium is high with secretions and basal nucleus. These months are characterized in the sampled areas over a period of heavy rain and high temperatures. The decline of reproductive period was observed in both sexes, between April and August. Low reproduction in males is characterized by ample light, absence of sperm, only germ cells in the early stages of spermatogenesis are observed (a few spermatogonia and spermatocytes) and interstitial tissue wide. In females, the period of reproductive decline is marked by the absence of unicellular glands in the oviduct epithelium, with higher affinity with the dye. This period corresponds to low rainfall periods and lower temperatures. We propose an analysis of zoological samples; this is a proposal ...

Estrategia reproductiva es el conjunto de adaptaciones que promueven la manera más eficiente para una población a sobrevivir en las condiciones específicas de un entorno particular. Comprender esta adaptación es importante y puede identificarse en algunas poblaciones como indicador de cambios ambientales. La espermatogénesis puede ser un proceso biológico medible de estas adaptaciones en escalas espaciales y temporales. Se analizó la morfología de los testículos y oviductos de las especies de lagartijas que componen el género Tropidurus, teniendo en cuenta la amplia distribución geográfica y las relaciones simpátricas. Para el análisis hemos eliminado los testículos de los machos y la parte media de los oviductos de hembras de las especiesTropidurus Etheridge, T. oreadicus, T. itambere, T. spinulosus y T. Guarani recolectadas en diferentes lugares en el estado de Mato Grosso, Brasil. El período reproductivo es sincrónico para machos y hembras, y se produce durante septiembre, octubre y noviembre. Se caracterizaron como machos reproductores por testículos con túbulos seminíferos con células germinales en distintas etapas de la espermatogénesis, un epitelio muy alto, un lumen reducido, numerosos espermatozoides libres en el lumen y tejido intersticial redicudo. En hembras el pico reproductivo se produce cuando los canales de conducción de gametos poseen un epitelio alto, secretor y con núcleos basales. Estos meses son caracterizados en las zonas muestreadas por fuertes lluvias y altas temperaturas. La disminución del período reproductivo se observó en ambos sexos, entre abril y agosto. La baja reproducción en machos se caracteriza por un lumen ancho, ausencia de espermatozoides, presencia de células germinales sólo en las primeras etapas de la espermatogénesis (espermatogonias y pocos espermatocitos) y tejido intersticial amplio. En las mujeres, el período de baja reproducción está marcado por la ausencia de glándulas unicelulares en el epitelio de...

Ultrastructural and functional aspects of the spermatheca in the american harlequin bug, Murgantia histrionica (Hemiptera: Pentatomidae)

The spermatheca of Murgantia histrionica (Hahn) was investigated using fluorescence, scanning and transmission electron microscopy. The aim of the study was to elucidate the structure of this organ, pointing out differences between mated and unmated females. Results have shown an elaborated cuticular structure associated with muscular and glandular tissues. The spermatheca is joined with the common oviduct by the spermathecal duct, forming a thin saccular dilation through two consecutive invaginations. The distal part of the organ is formed by a series of two communicating cuticular chambers. The first cylindrical-shaped chamber, corresponding to the coiled region, is wrapped by longitudinal muscular fibers suspended between two cuticular flanges. The contractions of these fibers compress a deformable zone of the cylinder, pumping the sperm toward the spermathecal duct. Without contractions the cylinder results to be isolated from the proximal part of the spermatheca by means of a valve. The second chamber, corresponding to the spermatheca, is made of two parts: a truncated-conical sub chamber, with a constant cuticular thickness, bearing on itself the distal flange, where muscular fibers are attached. The second part is a bulb-like structure wrapped in a glandular epithelium. The secretory units are composed by two cells: a secretory cell and an associated duct cell. Every evacuating duct shows a little reservoir just after the terminal apparatus, and converge inside the distal bulb after a tortuous path. The functional implications of this structure in the reproductive biology of M. histrionica are discussed.

Construction and expression characterization of transgenic chicken bioreactor vector / 生物工程学报

We constructed transgenic chicken bioreactor vector, driven by chicken ovalbumin promoter, lentiviral vector and cytomegalovirus (CMV) promoter control vector encoding green fluorescent protein (GFP) and luciferase (Luc) as reporter genes. The three vectors were used to transfect or infect chicken primary oviduct epithelial cells, embryo fibroblasts cells, mouse 3T3-L1 preadipocytes cells and bovine mammary epithelial cells. High efficient and specific expression vector for transgenic chicken bioreactor was determined by detecting fluorescence and luciferase activity. Reporter gene analysis showed that chicken ovalbumin promoter expression vector was not cell type-specific in these four different cells. Additionally, luciferase reporter analysis illustrated that the chicken ovalbumin promoter activity was over 100 times lower than that of the CMV promoter in four different cells. Both of these two reporter genes were expressed in those four different cells infected by lentiviral expression vectors. Similarly, the GFP reached the similar expression level in cells infected by lentivirus and cells transfected with CMV promoter plasmid vectors when the multiplicity of infection was 20. In conclusion, the transgenic chicken bioreactor vector under the control of chicken ovalbumin promoter was not highly efficient and cell type-specific. However, the efficient expression and extensiveness oflentiviral vector could be used for studying chicken oviduct bioreactor.

Impact of coenzyme Q10 on the histological structure and immunohistochemical localization of leptin in the ampulla of rat oviduct after monosodium glutamate administration

The female reproductive system is very sensitive to different environmental chemicals and food additives such as monosodium glutamate. Coenzyme Q10 [CoQ10] is a naturally occurring compound and a potent antioxidant. To investigate the structural changes and the immunohistochemical distribution of leptin in the ampulla of oviduct in adult female albino rats after administration of monosodium glutamate, and to study the effects of CoQ10 supplementation. Fifty adult female albino rats were divided into four equal groups: group I control rats, group II receiving monosodium glutamate, group III receiving CoQ10, and group IV receiving monosodium glutamate and CoQ10. After 2 months, rats were weighed and killed during the diestrous phase. Blood samples were collected for assessment of serum cholesterol. Oviducts were prepared for histological study and immunohistochemical localization of leptin. Control group showed positive immune reaction for leptin. Group II showed a significant increase in body weight and serum cholesterol associated with structural and ultrastructural changes, in addition to negative immune reaction for leptin. Group III showed similar structure to the control group. The increase in body weight and serum cholesterol in group IV was not significant. There were no changes in the histological structure of the oviduct. A positive immune reaction for leptin was detected. Administration of monosodium glutamate alters the histological structure and expression of leptin in the oviduct. The coadministration of CoQ10 with monosodium glutamate partially prevented these changes, suggesting a protective effect of CoQ10

Porcine oviduct sperm binding glycoprotein and its deleterious effect on sperm: a mechanism for negative selection of sperm?

In their journey through the oviduct some subpopulations of sperm are preserved in a reservoir, while others are negatively selected. Sperm binding glycoprotein (SBG) is a pig oviductal epithelial cell glycoprotein that produces, under capacitating conditions, acrosome alteration, p97 tyrosine-phosphorylation and reduction of the motility of sperm. In this paper, we show that SBG is accessible at the extracellular surface of the oviductal epithelial cells, supporting a sperm interaction biological role in situ. We analyze the possible dependence of the tyrosine-phosphorylation of p97 on the PKA mechanism, finding that apparently it is not PKA dependent. Also, after SBG treatment the phosphorylated proteins locate mainly at the detached periacrosomal region and at the tail of sperm; the latter may be related to SBG's motility reduction effect. The study of the time course effect of SBG on sperm as detected by chlortetracycline (CTC) staining and of its binding to sperm by immunodetection in conjunction with CTC, shows results in agreement with the hypothesis that this glycoprotein is involved in the alteration of acrosomes in a specific sperm subpopulation. The results suggest that SBG may be part of a mechanism for negative selection of sperm.

Efectos en el tracto reproductivo, digestivo y pérdida de pesocorporal del ave semipesada sometida a descanso ovárico / Efeitos no trato reprodutivo, digestivo e perda do peso de aves semi-pesadossubmetidas a descanso ovárico / The effects on the reproductive and digestive tract and loss of body weight of the Brown egg layers submitted to ovarian rest

Esta investigación evaluó la aplicación del descanso ovárico (DO) en aves semipesadas y el efecto quetienen la edad y la duración del ayuno sobre el tracto reproductivo y gastrointestinal. Se utilizaron 840ponedoras de la línea Hy-Line Brown® con 64 semanas de edad, se evaluaron tres edades de inducciónal DO (65, 70 y 75 semanas) y tres períodos de ayuno (cinco, diez y quince días), en un modelo estadísticocompletamente aleatorizado, anidado, efecto fijo y balanceado. Se sacrificaron aves para evaluar el pesocorporal, peso del tracto digestivo, peso del tracto reproductivo (ovario y oviducto), porcentaje de folículoscon diámetro igual o superior a diez mm (LYF) y el porcentaje de pérdida de peso del ave. El modelo fuesignificativo (p<0.05) para todas las variables evaluadas; la edad presentó efecto significativo (p<0.05) enel peso del ovario y el porcentaje de folículos (LYF), mientras que la duración del ayuno anidado en laedad presentó efecto significativo (p<0.05) en todas las variables, lo que indica un mayor efecto del períodode ayuno en contraste con la edad de aplicación del DO sobre las variables evaluadas. El peso del ovario(edad 65:15.87 ±15.11; edad 70: 7.92 ± 5.46; y edad 75: 7.54 ± 4.96) y porcentaje de folículos (LYF) (edad65: 43.75 ± 21.54; la edad 70 18.05 ± 8.72 y edad 75: 8.33 ± 6.85), mostraron tendencia en la disminucióna medida que aumenta la edad, comportamiento que está asociado con la disminución gradual del númerode folículos y la ampliación del tiempo entre la maduración de una onda folicular y la siguiente normaldel ave a medida que envejece (Kim y Donalson, 2000; North y Bell, 1990). Los porcentajes de pérdida de peso (edad 65: 24.24 ± 8.53%; edad 70: 20.91 ± 5.50% y edad 75: 20.88 ± 7.15%), fueron menores encomparación con los reportes en líneas livianas, posiblemente debido al contenido de reservas lipídicas,lo que ofrece al ave semipesada la capacidad de tolerar el ayuno.

The effects on the reproductive and digestive tract and loss of body weight of the Brown egg layerssubmitted to ovarian rest. This research evaluated the application of the ovarian rest (OR) on brown egglayers and the effect the age and duration of feed withdrawal have over the reproductive and gastrointestinaltract. 840 Hy-Line Brown ® layers were used at 64 weeks of age in which three ages of induction to theOR (65.70 and 75 weeks) and three feed withdrawal periods (five, ten and fifteen days) in a completelyrandomized statistical model, nested, of balanced and fixed effect. Sacrifices were carried out in order toevaluate body weight, weight of the digestive and reproductive tract (ovary and oviduct), follicle percentagewith a diameter equal to or higher than ten mm (LYF) and the percentage of the bird’s weight loss. Themodel was significant (p<0.05) for all the variables evaluated; the age presented a significant effect(p<0.05) over the ovary weight and the follicle percentage (LYF), while the duration of the feed withdrawalrelated to the age had a significant effect (p<0.05) on all the variables, which indicates a greater effect ofthe feed withdrawal period contrasted to the age of the application of the OR upon the variables evaluated.The ovary’s weight (age 65:15.87 to ± 15.11; age 70: 7.92b ± 5.46; and age 75: 7.54b ± 4.96) and thefollicle percentage (65: 43.75 to ± 21.54; age 70 18.05b ± 8.72 and age 75: 8.33b ± 6.85) showed a loweringtendency as the age increased. This type of behavior is associated with the gradual decrease in the numberof follicles and the extended time between the maturation of one follicular wave and the next as the layerages (Kim, Donalson, 2006; North, Bell, 1990). The weight loss percentages (age 65: 24.24 ± 8.53%; age70: 20.91 ± 5.50% and age 75: 20.88 ± 7.15%) were low compared to the reports on white egg layers,possibly due to the content of lipidic reserves which offers the brown egg...

Foi avaliado o descanso ovárico (DO) em galinhas semipesadas e o efeito da idade e a duração do jejumsobre o trato reprodutivo e gastrointestinal. Foram utilizadas 840 galinhas poedoras da linha Hy-LineBrown® com 64 semanas de idade, foram avaliadas três idades de indução ao DO (65, 70 e 75 semanas) etrês períodos de jejum (cinco, dez e quinze dias), utilizando um modelo desenho completamente aleatório,aninhado , efeito fixo e balanceado. As aves foram sacrificadas para avaliar o peso corporal, peso do tratodigestivo, peso do trato reprodutivo (ovário e oviduto), porcentagem de folículos com diâmetro igual ousuperior a dez mm (LYF) e a porcentagem da perdida de peso da ave. O modelo foi significativo (p<0.05)para todas as variáveis avaliadas; a idade foi significativa (p<0.05) no peso do ovário e a LYF e a duraçãodo jejum aninhado na idade apresentou efeito significativo (p<0.05) em todas as variáveis, o que indicouum maior efeito do período de jejum do que a idade sobre as variáveis avaliadas. O peso do ovário (idade65:15.87 ±15.11; idade 70: 7.92 ± 5.46; e idade 75: 7.54 ± 4.96) e percentagem de folículos (idade 65: 43.75± 21.54; idade: 70: 18.05 ± 8.72 e idade 75: 8.33 ± 6.85), mostraram tendência na diminuição à medidaque aumenta a idade. As percentagens de perda de peso (idade 65: 24.24 ± 8.53%; idade 70: 20.91 ± 5.50%e idade 75: 20.88 ± 7.15%) e as três idades apresentaram uma relação direta entre a duração dos períodosde jejum e o efeito na atrofia reprodutiva e perda de peso, permitindo determinar que não é necessáriosubmeter a jejuns superiores a dez dias.

Morphological study of the oviductal gland in the smallnose fanskate Sympterygia bonapartii (Müller and Henle, 1841) (Chondrichthyes, Rajidae) / Estudo morfológico da glândula oviductal no smallnose fanskate Sympterygia bonapartii (Müller and henle, 1841) (Chondrichthyes, Rajidae)

The oviductal gland is an exclusive structure of cartilaginous fishes that produces the egg jelly, forms the tertiary egg envelopes and stores sperm. The biological importance of this structure is related to the special features of the reproductive strategy of the group and to its phylogeny, considering that egg-laying is the ancestral condition in this fish (Dulvy and Reynolds, 1997). This gland of the smallnose fanskate shows four morphofunctional zones. The lining epithelium along the gland is columnar with secretory and ciliated cells. Secretions are mucous and/or proteic according to the zone, and to their specific functions. This is the first report about the microanatomy of the female reproductive tract of S. bonapartii with evidence of sperm storage in the genus.

A glândula oviductal é uma estrutura exclusiva das elasmobrânquias que produz a geléia do ovo, o envoltório terciário do ovo e armazena espermatozóides. Sua importância está relacionada com as características especiais da estratégia reprodutiva do grupo e com a filogenia destes peixes em que oviparidade é a condição ancestral (Dulvy and Reynolds, 1997). A glândula oviductal do smallnose fanskate mostra quatro zonas morfofuncionais. O revestimento em toda a glândula é epitélio colunar ciliado com células glandulares e ciliadas. As secreções são diferentes entre as zonas e relacionada com as suas funções específicas. A informação aqui exposta constitui o primeiro relato sobre a micro-anatomia do trato reprodutivo feminino de S. bonapartii com evidencias de armazenamento do esperma neste gênero.

Ultrasonographic and radiographic determination of egg development of jurarás (Kinosternon scorpioides) in captivity / Determinação ultrassonográfica e radiográfica do desenvolvimento de ovos de jurarás em cativeiro

Kinosternon scorpioides (Linnaeus, 1766), with its common name of jurará, is a quite variable species of turtles, and many different names have been applied to populations throughout its range. Currently, however, four subspecies are considered valid as K. scorpioides arises from southern Panama over most of northern South America and is found in Ecuador, northern Peru, southern Bolivia, northern Argentina, eastern Guyana and Brazil. Thus, an ultrasonographic and radiographic study was performed in order to describe the morphology and development of eggs of 20 female jurará mud turtles K. scorpioides, from September 2005 to August 2006. In the first month, the ovarian cycle was characterized by absence of vitellogenic follicles, atresic follicles or oviduct eggs. From October 2005 to March 2006 on, ultrasonographic scanning allowed to establish the growing vitellogenic follicles. Vitellogenic follicles were observed with spherical to ovoid shapes, with a surrounding echogenic yolk, a nonechogenic albumin layer, and a high echogenic shell. The oviduct eggs were identified by radiography just 180 days after beginning the experiment, when the shell became enough mineralized to impress the radiographic film. This experiment allowed to obtain by means of the 7.5 MHz linear probe images with adequate resolution and penetration for visualization of follicles. Successive ultrasonographic examinations of 20 K. scorpioides females allowed to access initial stages of vitellogenic follicles and oviduct eggs, and radiographic examination revealed to be an easy technique to assess oviduct eggs and to allow evaluation of egg development in jurarás, from 6 months on.

A tartaruga Kinosternon scorpioides (Linnaeus, 1766), com nome popular de jurará, é uma espécie bastante variável, e muitos nomes diferentes são atribuídos às suas populações, de acordo com sua localização geográfica. Atualmente, quatro subespécies foram validadas como K. scorpioides surgindo desde o extremo Sul do Panamá e norte da América do Sul, podendo ser encontradas ainda no Equador, no norte do Peru, sul da Bolívia, norte da Argentina, leste da Guiana e no Brasil. Desde modo, foi realizado um estudo ultrassonográfico e radiológico, a fim de descrever a morfologia e o desenvolvimento dos ovos de K. scorpioides em cativeiro, utilizando-se 20 fêmeas, no período de setembro de 2005 a agosto de 2006. No primeiro mês, o ciclo ovariano foi caracterizado pela ausência de folículos vitelogênicos, atrésicos ou presença de ovos em ovidutos. De outubro de 2005 a março de 2006 a varredura ultrassonográfica permitiu determinar o crescimento vitelogênico dos folículos, os quais foram observados com um formato que variou de ovóide a esférico, envolvidos por um conteúdo ecogênico, uma camada não-ecogência de albumina e uma casca de elevada ecogenicidade. Ovos presentes em ovidutos foram identificados por radiografias apenas 180 dias após início do experimento, quando a casca tornou-se suficientemente mineralizada para impressionar o filme radiográfico. Um transdutor linear de 7,5 MHz promoveu imagens com resolução e penetração adequada para visibilização de folículos. Exames ultrassonográficos sucessivos em fêmeas K. scorpioides permitiram determinar os estágios iniciais de desenvolvimento de folículos vitelogênicos e o exame radiográfico mostrou-se como técnica simples para avaliar a presença de ovos em ovidutos, permitindo fazer inferências sobre desenvolvimento de ovos em jurarás, a partir dos 6 meses de pós-observação por ultrassonografia.