Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp (125I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when 125I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a Kdof 7.1 ± 0.9 x 10-8M and a maximal binding capacity of 430 ± 40 ng 125I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.

Estradiol and testosterone concentrations in follicular fluid as criteria to discriminate between mature and immature oocytes

The objective of the present study was to examine the association between follicular fluid (FF) steroid concentration and oocyte maturity and fertilization rates. Seventeen infertile patients were submitted to ovulation induction with urinary human follicle-stimulating hormone, human menopausal gonadotropin and human chorionic gonadotropin (hCG). A total of 107 follicles were aspirated after hCG administration, the oocytes were analyzed for maturity and 81 of them were incubated and inseminated in vitro. Progesterone, estradiol (E2), estrone, androstenedione, and testosterone were measured in the FF. E2 and testosterone levels were significantly higher in FF containing immature oocytes (median = 618.2 and 16 ng/ml, respectively) than in FF containing mature oocytes (median = 368 and 5.7 ng/ml, respectively; P < 0.05). Progesterone, androstenedione and estrone levels were not significantly different between mature and immature oocytes. The application of the receiver-operating characteristic curve statistical approach to determine the best cut-off point for the discrimination between mature and immature oocytes indicated levels of 505.8 ng/ml for E2 (81.0% sensitivity and 81.8% specificity) and of 10.4 ng/ml for testosterone (90.9% sensitivity and 82.4% specificity). Follicular diameter was associated negatively with E2 and testosterone levels in FF. There was a significant increase in progesterone/testosterone, progesterone/E2 and E2/testosterone ratios in FF containing mature oocytes, suggesting a reduction in conversion of C21 to C19, but not in aromatase activity. The overall fertility rate was 61% but there was no correlation between the steroid levels or their ratios and the fertilization rates. E2 and testosterone levels in FF may be used as a predictive parameter of oocyte maturity, but not for the in vitro fertilization rate.

Maturation of pig oocytes in vitro in a medium with pyruvate

The aim of in vitro maturation oocyte systems is to produce oocytes of comparable quality to those derived in vivo. The present study was designed to examine the surface morphological changes of the cumulus-oocyte complex (COC) and nuclear maturation in a culture system containing pyruvate. Ovaries were obtained from a slaughterhouseand transported to the laboratory within 2 h at 35-39ºC,and rinsed three times in 0.9% NaCl. The COCs were harvested from the ovaries and in vitro maturation was evaluated in San Marcos (SM) medium, a chemically defined culture system containing 22.3 mM sodium pyruvate. Oocytes were cultured in SM, SM + porcine follicular fluid (pFF) and in SM + pFF + gonadotropins (eCG and hCG) for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, the degree of cumulus expansion and frequency of nuclear maturation were determined. Oocytes matured in SM (40.9%) and SM + pFF (42.9%) showed moderate cumulus expansion, whereas oocytes matured in SM + pFF + gonadotropins (54.6%) showed high cumulus expansion. The maturation rate of cultured oocytes, measured in function of the presence of the polar corpuscle, did not differ significantly between SM (40.9 ± 3.6%) and SM + pFF (42.9 ± 3.7%). These results indicate that pig oocytes can be successfully matured in a chemically definedmedium and suggest a possible bifunctional role of pyruvate as an energy substrate and as an antioxidant protecting oocytes against the stress of the in vitro environment.

Purification, partial characterization and role in lipid transport to developing oocytes of a novel lipophorin from the cowpea weevil, Callosobruchus maculatus

Lipid transport in arthropods is achieved by highly specialized lipoproteins, which resemble those described in vertebrate blood. Here we describe purification and characterization of the lipid-apolipoprotein complex, lipophorin (Lp), from adults and larvae of the cowpea weevil Callosobruchus maculatus. We also describe the Lp-mediated lipid transfer to developing oocytes. Lps were isolated from homogenates of C. maculatus larvae and adults by potassio bromide gradient and characterized with respect to physicochemical properties and lipid content. The weevil Lp (465 kDa) and larval Lp (585 kDa), with hydrated densities of 1.22 and 1.14 g/mL, contained 34 and 56% lipids and 9 and 7% carbohydrates, respectively. In both Lps, mannose was the predominant monosaccharide detected by paper chromatography. SDS-PAGE revealed two apolipoproteins in each Lp with molecular masses of 225 kDa (apolipoprotein-I) and 79 kDa (apolipoprotein-II). The lipids were extracted and analyzed by thin-layer chromatography. The major phospholipids found were phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in adult Lp, and phosphatidylcholine, phosphatidylethanolamine and sphingomyelin in larval Lp. Hydrocarbons, fatty acids and triacylglycerol were the major neutral lipids found in both Lps. Lps labeled in the protein moiety with radioactive iodine (125I-iodine) or in the lipid moiety with fluorescent lipids revealed direct evidence of endocytic uptake of Lps in live oocytes of C. maculatus.

Effect of supplementation of green tea polyphenols on the developmental competence of bovine oocytes in vitro

The objective of the present study was to examine the effect of green tea polyphenols (GTPs) supplementation during in vitro maturation, in vitro fertilization, and in vitro culture on the developmental competence of bovine oocytes. Cumulus-oocyte complexes aspirated from the ovaries were matured in vitro (38.5ºC for 24 h) and fertilized (38.5ºC for 15-18 h) and embryos were cultured (38.5ºC for 192 h) in a defined conditioned medium with or without GTPs supplementation. The GTPs used in the present study contained 99% catechin derivatives, with the major components being 50% (-)-epigallocatechin gallate, 22% (-)-epicatechin gallate, 18% (-)-epigallocatechin, and 10% (-)-epicatechin. Four replicate trials were done for each type of experiment. GTPs supplementation (15 µM) of the maturation medium led to a significant increase in the rate of blastocyst formation (34.0 vs 21.4%, P < 0.05). However, the rate of blastocyst formation was not improved when higher GTPs concentrations (20 or 25 µM) were added to the in vitro maturation medium. During in vitro fertilization, supplementation with higher GTPs concentrations (20 or 25 µM) significantly reduced the rate of blastocyst formation (P < 0.05). Supplementation of the culture medium with 15 µM GTPs improved the rate of blastocyst formation, while higher GTPs concentrations (25 µM) significantly reduced embryo development (P < 0.05). In conclusion, these results demonstrate that supplementation with GTPs at low concentration (15 µM) during in vitro maturation and in vitro culture improved the developmental competence of bovine oocytes.

Histological study of capuchin monkey (Cebus apella) ovarian follicles

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

Reproductive biology of Ameerega trivittata (Anura: Dendrobatidae) in an area of terra firme forest in eastern Amazonia

The reproductive success of tropical amphibians is influenced by factors such as body size and the characteristics of breeding sites. Data on reproductive biology are important for the understanding of population dynamics and the maintenance of species. The objectives of the present study were to examine the abundance of Ameerega trivittata, analyze the use of microhabitats by calling males and the snout-vent length (SVL) of breeding males and females, the number of tadpoles carried by the males and mature oocytes in the females, as well as the relationship between the SVL of the female and both the number and mean size of the mature oocytes found in the ovaries. Three field trips were conducted between January and September, 2009. A total of 31 plots, with a mean area of 2.3 ha, were surveyed, resulting in records of 235 individuals, with a mean density of 3.26 individuals per hectare. Overall, 66.1% of the individuals sighted were located in the leaf litter, while 17.4% were perched on decaying tree trunks on the forest floor, 15.7% on the aerial roots of Cecropia trees, and 0.8% on lianas. Males were observed transporting a mean of 10.8 tadpoles on their backs. A significant correlation was found between the size of the females and the mean diameter of the oocytes. New data were collected on the size of oocytes and no pattern was found in the type of perches used by calling males of the different Ameerega species.

Biologia reprodutiva de Apidoras fuscoguttatus (Siluriformes, Callichthyidae) em riacho de cabeceira da bacia do alto Rio Paraná

The reproductive biology of Aspidoras fuscoguttatus Nijssen & Isbrücker, 1976 from a stream in São José do Rio Preto, northwestern São Paulo State, Brazil, was monthly investigated in the period of August 1999 to July 2000. Measurements of total length, body weight, gonadal weight and macroscopic assessment of gonadal maturation were performed. Environmental parameters were considered in order to verify associations with the reproductive period. Populational structure showed total length amplitude between 14.2 and 50.8 mm. Pronounced sexual dimorphism was verified. The largest mean values of gonadosomatic relation for females coincided with the rainy season (November to March). Mean length at first sexual maturity was different for males (30.5 mm) and females (37.1 mm). Fecundity varied between 51 and 166 oocytes. Gonadal maturation curve, frequency of maturation stages and size frequency distributions of oocytes in mature ovaries revealed a long reproductive period, suggesting fractional spawning.

Dinâmica reprodutiva de Oligosarcus jenynsii e O. robustus (Characiformes, Characidae) na lagoa Fortaleza, Rio Grande do Sul, Brasil

Oligosarcus jenynsii (Günther, 1864) and O. robustus Menezes, 1969 are fish species distributed in Rio Grande do Sul, Brazil, Uruguay, and northern Argentina. The reproductive period and recruitment, sex ratio, absolute and relative fecundity, and body length at first gonadal maturation of the two carnivorous species from Fortaleza Lagoon were analized. The specimens were sampled monthly, from May 2000 to April 2001, with fishing effort of 24 hours/month, using stationary gillnets of different mesh sizes and seine net (three samples per edge). The records of each individual included total length, total weight, gonad weight, sex and gonadal maturity stage. The reproductive period of both O. jenynsii and O. robustus ranges from May/June to November/December, according to the bimonthly variation of the mean values of gonosomatic index, and the relative frequencies of the gonadal maturation stages. Recruitment of new individuals to the population occurs from November/December to March/April. The sex ratio is different from 1:1 for O. jenynsii and similar to 1:1 for O. robustus. The mean absolute fecundity, calculated by counting sub-sampled oocytes from mature females, was 14,483 oocytes for O. jenynsii, and 16,308 oocytes for O. robustus. The first maturation curve shows that O. jenynsii begins to reproduce between 84 mm and 104 mm (total length), and O. robustus between 126 mm and 146 mm, probably at similar ages.

Gonad development in females of fiddler crab Uca rapax (Crustacea, Brachyura, Ocypodidae) using macro and microscopic techniques

The morphology of the ovaries in Uca rapax (Smith, 1870) was described based on macroscopic and microscopic analysis. Females were collected in Itamambuca mangrove, Ubatuba, state of São Paulo, Brazil. In the laboratory, 18 females had their ovaries removed and prepared for histology. Each gonad developmental stage was previously determined based on external and macroscopic morphology and afterwards each stage was microscopically described. The ovaries of U. rapax showed a pronounced macroscopic differentiation in size and coloration with the maturation of the gonad, with six ovarian developmental stages: immature, rudimentary, developing, developed, advanced and spent. During the vitellogenesis, the amount of oocytes in secondary stage increases in the ovary, resulting in a change in coloration of the gonad. Oogonias, primary oocytes, secondary oocytes and follicular cells were histologically described and measured. In female’s ovaries of U. rapax the modifications observed in the oocytes during the process of gonad maturation are similar to descriptions of gonads of other females of brachyuran crustaceans. The similarities are specially found in the morphological changes in the reproductive cells, and also in the presence and arrange of follicle cells during the process of ovary maturation. When external morphological characteristics of the gonads were compared to histological descriptions, it was possible to observe modifications that characterize the process in different developmental stages throughout the ovarian cycle and, consequently, the macroscopic classification of gonad stages agree with the modifications of the reproductive cells.

Advertisement call and female sexual cycle in Uruguayan populations of Physalaemus henselii (Anura, Leiuperidae)

Physalaemus henselii (Peters, 1872) is a little known leiuperid frog that has not been studied since the 1960’s. Herein, we redescribe its advertisement call, and assess the female sexual cycle and the reproductive period on the basis of the macroscopic analysis of the ovaries and field observations. The Ovarian Size Factor (OSF) was calculated. The study was made in Departamento de Rivera, northern Uruguay. The advertisement call consists of short (177 ± 21ms), multipulsed (20 ± 3 pulses/note) notes, with a note repetition rate of 1.57 ± 0.13 notes/s. Physalaemus henselii has a female sexual cycle with unimodal distribution of gravid females, which are present from February to September. The OSF and the ratio "females with mature oocytes / females without mature oocytes" reached the highest values from April to June. The number and size of oocytes were positively correlated with female size. The smallest female (SVL =18.94mm, weight =0.78g) with mature oocytes was found in July. The observed sexual cycle with a single annual reproductive period during the cold season (autumn and early winter), is an uncommon fact for anuran species in the region.

Reproductive biology of Leptodactylus fuscus (Anura, Leptodactylidae) in the subtropical climate, Rio Grande do Sul, Brazil

The aim of this study was to characterize, for the central region of the State of Rio Grande do Sul, Brazil, the reproductive biology of Leptodactylus fuscus (Schneider, 1799), based on the analysis of gonadal development of males and females, reproductive effort, size-fecundity relationships, and occurrence of sexual dimorphism in body size. Mature individuals were found from October 1996 to February 1997 and from October 1997 to December 1997. The highest input of juveniles in the population was recorded in March 1997. There was a positive and significant correlation between the number of mature individuals and the mean monthly temperature. The population did not present sexual dimorphism in size. Males presented significant correlation only between snout-vent length and testes length. All females had oocytes at four different maturation stages and there were no significant correlations regarding size-fecundity variables. The correlation between ovarian size factor and females snout-vent length was not significant either. The main difference between this population and those that inhabit tropical climate was that temperature was responsible for stimulating the reproduction activity, instead of rainfall.

Reproductive characteristics of characid fish species (Teleostei, Characiformes) and their relationship with body size and phylogeny

In this study, I investigated the reproductive biology of fish species from the family Characidae of the order Characiformes. I also investigated the relationship between reproductive biology and body weight and interpreted this relationship in a phylogenetic context. The results of the present study contribute to the understanding of the evolution of the reproductive strategies present in the species of this family. Most larger characid species and other characiforms exhibit a reproductive pattern that is generally characterized by a short seasonal reproductive period that lasts one to three months, between September and April. This is accompanied by total spawning, an extremely high fecundity, and, in many species, a reproductive migration. Many species with lower fecundity exhibit some form of parental care. Although reduction in body size may represent an adaptive advantage, it may also require evolutionary responses to new biological problems that arise. In terms of reproduction, smaller species have a tendency to reduce the number of oocytes that they produce. Many small characids have a reproductive pattern similar to that of larger characiforms. On the other hand they may also exhibit a range of modifications that possibly relate to the decrease in body size and the consequent reduction in fecundity. Examples of changes in the general reproductive pattern include the following: reduction in the size of mature oocytes; increase in fecundity; production of several batches of oocytes; an extended reproductive period or even continuous reproduction that allows individuals to reproduce more than once a year; high growth rates; rapid recruitment of juveniles; presence of more than one reproductive cohort that increases the sexually active population; and multiple independent development of insemination as a reproductive strategy. These changes are possibly associated with adaptive pressures that are related to the reduction in body size. In addition, such reproductive characteristics or novelties may reflect the phylogenetic history of a given species.

Reproductive traits of Gymnogeophagus labiatus (Teleostei, Cichlidae), in an upper stretch of Sinos river, Caraá, Brazil

This work describes the reproduction of Gymnogeophagus labiatus (Hensel, 1870) from an upper stretch of Sinos river, southern Brazil, based on the analysis of 174 males and 132 females captured in monthly samples taken from January to December 2007. Results showed that reproductive activity occur in spring and summer although ripe males were found along the year. The standard length of the smallest ripe male was 104.74 mm (Lt) and the smallest ripe female was 55.00 mm (Lt). There was a significant difference in total sex ratio, with 1.32 males to each female (χ2 = 5.76). Males were much more abundant in March (1.75 males: 1 female) and December (5 males: 1 female). Females were more abundant in the 62├77 mm interval (1 male: 2.36 female) while males were more abundant in the 77├92 mm size interval (2.57 males: 1 female). The largest length intervals were composed of only males. Mean absolute fecundity was 113.4 (± 31.24 sd) and mean relative fecundity was 0.0125 (± 0.0026 sd) oocytes/mg. In ripe ovaries, small-diameter oocytes were observed at high frequencies while larger ones occurred at lower frequencies. This pattern is common in fishes with asynchronous oocyte development. Characteristics of G. labiatus, such as low fecundity, asynchrony in oocyte development, multiple spawning, and its well-known parental care behavior, are consistent with an equilibrium strategy, as proposed for other cichlids.

Reproductive aspects of Dendropsophus sanborni (Anura, Hylidae) in northeastern Argentina

Reproductive aspects of Dendropsophus sanborni (Anura, Hylidae) in northeastern Argentina. The aim of this investigation was to characterize reproductive aspects of Dendropsophus sanborni (Schmidt, 1944) (Anura, Hylidae) in the northwest of Corrientes province, in northeastern Argentina. The reproductive cycles of males and females, the existence of sexual dimorphism and the male’s vocalization period were analyzed. Samplings were made from September 2011 to August 2012. Dendropsophus sanborni showed a prolonged reproductive pattern because reproductive activity was observed during most part of the year. Calling males were recorded all months except in July. The spermatogenic cycle can be characterized as potentially continuous. In males, snout-vent length was significantly correlated with mean testicular volume. The species showed sexual dimorphism in body mass, with females larger than males. Post-ovulatory females were found from March to June and most females had oocytes at different stages of maturity, with abundant post-vitellogenic oocytes from August to December.