Production of Bovine Hand-Made Cloned Embryos by Zygote-Oocyte Cytoplasmic Hemi-complementation

The aim of this study was to evaluate the effect of the cytoplast type and activation process on development of cloned embryos. Bovine oocytes (MII) or zygotes at the one-cell stage (IVF) were manually bisected and segregated in MII or IVF hemi-cytoplasts or hemi-karyoplasts. Adult skin cells from a bovine female were used as nucleus donors (SC). Experimental groups were composed of IVF embryos; parthenogenetic embryos; handmade cloned (HMC) embryos; and reconstructed HMC embryos using IVF hemi-cytoplast + MII hemi-cytoplast + SC (G-I); IVF hemi-cytoplast + IVF hemi-cytoplast + SC (G-II); MII hemi-cytoplast + IVF hemi-karyoplast (G-III); and IVF hemi-cytoplast + IVF hemi-karyoplast (G-IV). Embryos from G-I to G-IV were allocated to subgroups as sperm-activated (SA) or were further chemically activated (SA + CA). Embryos from all groups and subgroups were in vitro cultured in the WOW system. Blastocyst development in subgroup G-I SA (28.2%) was similar to IVF (27.0%) and HMC (31.4%) controls, perhaps due to a to a more suitable activation process and/or better complementation of cytoplasmic reprogramming factors, with the other groups and subgroups having lower levels of development. No blastocyst development was observed when using IVF hemi-karyoplasts (G-III and G-IV), possibly due to the manipulation process during a sensitive biological period. In summary, the presence of cytoplasmic factors from MII hemi-oocytes and the sperm activation process from hemi-zygotes appear to be necessary for adequate in vitro development, as only the zygote-oocyte hemi-complementation was as efficient as controls for the generation of bovine cloned blastocysts.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Frontonasal Dysplasia, Severe Neuropsychological Delay, and Midline Central Nervous System Anomalies: Report of 10 Brazilian Male Patients

Here we report on 10 male patients with frontonasal dysplasia, cleft lip/palate, mental retardation, lack of language acquisition, and severe central nervous system involvement. Imaging studies disclosed absence of the corpus callosum, midline cysts, and an abnormally modeled cerebellum. Neuronal heterotopias were present in five patients and parieto-occipital encephalocele in three patients. We suggest that this pattern found exclusively in males, most likely represents a newly recognized syndrome distilled from the group of disorders subsumed under frontonasal dysplasia. (C) 2009 Wiley-Liss, Inc.

Color inheritance and pigment characterization of red (wild-type), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta)

Species of Gracilaria are some of the most useful algae in the world for the production of agar. As a consequence of its economic importance, the genus has been the subject of many studies worldwide. Color variants of Gracilaria birdiae have been found in the natural population on the Brazilian coast, and they have also been isolated from plants cultivated in laboratory. These findings raised new questions regarding intraspecific variation and the prospects of cultivating such variants for their agar production. Therefore, this work aimed to determine the mode of color inheritance for two G. birdiae strains: a greenish-brown strain (gb) found in a natural population and a green strain (gr) which had arisen as a spontaneous mutation in a red plant cultured in the laboratory. The pigment contents of these strains, as well as the red wildtype (rd), were also characterized. Crosses between female and male plants of the same color (rd, gr, or gb) and between different colors were performed. Crosses between plants of the same color showed tetrasporophytic and gametophytic descendents of the parental color. Recessive nuclear inheritance was found in the greenish-brown strain, and cytoplasmic maternal inheritance was found in the green strain; both had lower phycoerythrin and higher concentrations of allophycocyanin and phycocyanin than the wild-type. Chlorophyll a contents were similar among all strains. Taken together, our results contribute to knowledge about the variability of this important red algae. In addition, since greenish-brown and green strains showed stability of color, both could be selected and tested in experimental sea cultivation to evaluate if mutants have advantageous performance when compared with red strain.

Conditional male dimorphism and alternative reproductive tactics in a Neotropical arachnid (Opiliones)

In arthropods, most cases of morphological dimorphism within males are the result of a conditional evolutionarily stable strategy (ESS) with status-dependent tactics. In conditionally male-dimorphic species, the status` distributions of male morphs often overlap, and the environmentally cued threshold model (ET) states that the degree of overlap depends on the genetic variation in the distribution of the switchpoints that determine which morph is expressed in each value of status. Here we describe male dimorphism and alternative mating behaviors in the harvestman Serracutisoma proximum. Majors express elongated second legs and use them in territorial fights; minors possess short second legs and do not fight, but rather sneak into majors` territories and copulate with egg-guarding females. The static allometry of second legs reveals that major phenotype expression depends on body size (status), and that the switchpoint underlying the dimorphism presents a large amount of genetic variation in the population, which probably results from weak selective pressure on this trait. With a mark-recapture study, we show that major phenotype expression does not result in survival costs, which is consistent with our hypothesis that there is weak selection on the switchpoint. Finally, we demonstrate that switchpoint is independent of status distribution. In conclusion, our data support the ET model prediction that the genetic correlation between status and switchpoint is low, allowing the status distribution to evolve or to fluctuate seasonally, without any effect on the position of the mean switchpoint.

Efficiency of uniparental male and female care against egg predators in two closely related syntopic harvestmen

Although the benefits of maternal care have been investigated in many species, the caring role of males in species with exclusive paternal care has received less attention. We experimentally quantified the protective role of paternal care in the harvestman Iporangaia pustulosa. Additionally, we compared the effectiveness of paternal care against predation in this species with a syntopic harvestman with maternal care, Acutisoma proximum. We demonstrated that nearly one-third of the unprotected Iporangaia clutches disappeared entirely in 12 days, while the other two-thirds suffered a mean reduction of 55% in egg number. Conversely, 50% of the control clutches did not suffer any reduction, and only one was entirely consumed by predators. We also demonstrated that the mucus coat that covers Iporangaia clutches has an important deterrent role against predation by conspecifics: 58.3% of the clutches without mucus were attacked and three of them were entirely consumed, whereas only three clutches with mucus were attacked, suffering a reduction of up to three eggs. Iporangaia males were as efficient as Acutisoma females in protecting eggs. However, unattended Acutisoma eggs were attacked 20% more frequently than unattended Iporangaia eggs. Unattended Iporangaia eggs are protected by a mucus coat that prevents or decreases predation rate, whereas Acutisoma eggs are more susceptible to predation, probably because they lack this mucus coat. Thus, besides the fact that Iporangaia males efficiently protect the offspring against egg predators, females also contribute to egg protection by providing a mucus coat that deters egg predators. (C) 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

UBE2A Deficiency Syndrome: Mild to Severe Intellectual Disability Accompanied by Seizures, Absent Speech, Urogenital, and Skin Anomalies in Male Patients

We describe three patients with a comparable deletion encompassing SLC25A43, SLC25A5, CXorf56, UBE2A, NKRF, and two non-coding RNA genes, U1 and LOC100303728. Moderate to severe intellectual disability (ID), psychomotor retardation, severely impaired/absent speech, seizures, and urogenital anomalies were present in all three patients. Facial dysmorphisms include ocular hypertelorism, synophrys, and a depressed nasal bridge. These clinical features overlap with those described in two patients from a family with a similar deletion at Xq24 that also includes UBE2A, and in several patients of Brazilian and Polish families with point mutations in UBE2A. Notably, all five patients with an Xq24 deletion have ventricular septal defects that are not present inpatients with a point mutation, which might be attributed to the deletion of SLC25A5. Taken together, the UBE2A deficiency syndrome in male patients with a mutation in or a deletion of UBE2A is characterized by ID, absent speech, seizures, urogenital anomalies, frequently including a small penis, and skin abnormalities, which include generalized hirsutism, low posterior hairline, myxedematous appearance, widely spaced nipples, and hair whorls. Facial dysmorphisms include a wide face, a depressed nasal bridge, a large mouth with downturned corners, thin vermilion, and a short, broad neck. (C) 2010 Wiley-Liss, Inc.

Wolbachia in Anastrepha Fruit Flies (Diptera: Tephritidae)

Endosymbiotic bacteria of the genus Wolbachia are widespread among arthropods and cause a variety of reproductive abnormalities, such as cytoplasmic incompatibility, thelytokous parthenogenesis, male-killing, and host feminization. In this study, we used three sets of Wolbachia-specific primers (16S rDNA, ftsZ, and wsp) in conjunction with the polymerase chain reaction (PCR), cloning and sequencing to study the infection of fruit flies (Anastrepha spp. and Ceratitis capitata) by Wolbachia. The flies were collected at several localities in Brazil and at Guayaquil, Ecuador. All of the fruit flies studied were infected with Wolbachia supergroup A, in agreement with the high prevalence of this group in South America. Phylogenetic analysis showed that the wsp gene was the most sensitive gene for studying the relationships among Wolbachia strains. The Wolbachia sequences detected in these fruit flies were similar to those such as wMel reported for other fruit flies. These results show that the infection of Anastrepha fruit flies by Wolbachia is much more widespread than previously thought.

Annual profile of fecal androgen and glucocorticoid levels in free-living male American kestrels from southern mid-latitude areas

Fecal samples and behavioral data were collected at a fortnightly basis during 11 months period from free-living male American kestrels living in southeast Brazil (22 degrees S latitude). The aim was to investigate the seasonal changes in testicular and adrenal steroidogenic activity and their correlation to reproductive behaviors and environmental factors. The results revealed that monthly mean of fecal glucocorticoid metabolites in May and June were higher than those estimated in November. in parallel, monthly mean of androgen metabolites in September was higher than those from January to April and from October to November. Molt took place from January to March, whereas copulation was observed from June to October but peaked in September. Nest activity and food transfer to females occurred predominantly in October, and parental behavior was noticed only in November. Territorial aggressions were rare and scattered throughout the year. Multiple regression analysis revealed that fecal androgen levels are predicted by photoperiod and copulation, while fecal glucocorticoid levels are only predicted by photoperiod. Bivariate correlations showed that fecal androgen metabolites were positively correlated with fecal glucocorticoid metabolites and copulation, but negatively correlated with molt. Additionally, copulation was positively correlated with food transfer to females and nest activity, but negatively correlated with molt. These findings suggest that male American kestrels living in southeast Brazil exhibit significant seasonal changes in fecal androgen and glucocorticoid concentrations, which seem to be stimulated by decreasing daylength but not by rainfall or temperature. (C) 2009 Elsevier Inc. All rights reserved.

Do individuals in better condition survive for longer? Field survival estimates according to male alternative reproductive tactics and sex

There is a gap in terms of the supposed survival differences recorded in the field according to individual condition. This is partly due to our inability to assess survival in the wild. Here we applied modern statistical techniques to field-gathered data in two damselfly species whose males practice alternative reproductive tactics (ARTs) and whose indicators of condition in both sexes are known. In Paraphlebia zoe, there are two ART: a larger black-winged (BW) male which defends mating territories and a smaller hyaline-winged (HW) male that usually acts as a satellite. In this species, condition in both morphs is correlated with body size. In Calopteryx haemorrhoidalis, males follow tactics according to their condition with males in better condition practicing a territorial ART. In addition, in this species, condition correlates positively with wing pigmentation in both sexes. Our prediction for both species was that males practicing the territorial tactic will survive less longer than males using a nonterritorial tactic, and larger or more pigmented animals will survive for longer. In P. zoe, BW males survived less than females but did not differ from HW males, and not necessarily larger individuals survived for longer. In fact, size affected survival but only when group identity was analysed, showing a positive relationship in females and a slightly negative relationship in both male morphs. For C. haemorrhoidalis, survival was larger for more pigmented males and females, but size was not a good survival predictor. Our results partially confirm assumptions based on the maintenance of ARTs. Our results also indicate that female pigmentation, correlates with a fitness component - survival - as proposed by recent sexual selection ideas applied to females.

The gynoecium of male Anchietea pyrifolia (Violaceae): Preserved structure with a new function

Structure of inflorescences and flowers and flowering behaviour are reported for the woody liana Anchietea pyrifolia (Violaceae) from Brazil. The specimen studied is grown for some decades now in the greenhouses of Halle Botanical Garden and turned out unisexually male, which adds a further example of dioecism to the family Violaceae, in which this type of sex distribution is rarely encountered. The flowers are exceptional also for the strongly asymmetric anterior petal, which represents a rare case of a species with enantiomorphic flowers pollinated by Lepidoptera. They have a fully developed gynoecium with a complicated architecture comparable to the pistil of bisexual Violaceae flowers, though without ovules. The style head is capable to release viscose liquid on tactile stimulation or pressure, which is known to act as pollen-gathering mechanism in bisexual Violaceae species with usually dry pollen and buzz-pollination. This function has switched in male A. pyrifolia to a mechanism for efficient pollen release mediated by insect pollinators from its short-lived flowers. (C) 2009 Elsevier GmbH. All rights reserved.

CONSIDERATIONS ON THE REINTRODUCTION AND RECOVERY OF THE ALAGOAS CURASSOW MITU MITU (LINNAEUS, 1766) FROM EXTINCTION USING A POTENTIALLY HYBRID CAPTIVE STOCK

The Alagoas Curassow Mitu mitu is considered extinct in the wild. Since 1979, two females and a male caught in the wild have bred successfully in captivity, and, in 1990, hybridizations between M. mitu and Razor-billed Mitu M. tuberosum were performed. By June 2008, there were around 130 living birds in two different aviaries. We sequenced two regions of the mitochondrial DNA of both captive stocks of Alagoas Curassows. We unequivocally identified hybrids that have haplotype typical of M. tuberosum. However, unless the original studbook can be recovered there is no confident way to discriminate ""pure"" M. mitu birds for breeding and reintroduction purposes. Allied with morphological data gathered in an independent study, we suggest that conservation actions need to focus on specimens with diagnostic phenotypic characters of M. mitu, and avoid birds with mitochondria, genetic contribution of M. tuberosum. Although we have detected low levels of genetic variability among captive birds, the steady increase of the captive population suggests that inbreeding depression and hybridization are not a reproductive hindrance. Reintroduction of some of these potential hybrid birds in the original area of occurrence of the Alagoas Curassow may be the only hope to fill in the ecological niche left vacant. An educational program involving local communities to conserve future reintroduction of curassows and their restored habitat is highly recommended. Accepted 12 November 2009.

Influence of Radiotransmitters on Fecal Glucocorticoid Levels of Free-Ranging Male American Kestrels

Although radiotelemetry is considered a valuable technique for ornithological field studies, several assumptions have been made about the impact that transmitters may have on the estimation of behavioral, ecological, and reproductive parameters. To assess the potential effects of backpack radiotransmitters, we captured and assigned 8 male American kestrels (Falco sparverius) into 2 groups: radiotagged (n = 6) and control individuals (leg-banded, n = 2). Thereafter, we collected feces approximately 2 hours after capture (day -1), and subsequently during days 0 (releasing day), 4, 7, 15, 30, 40, and 55. Prior to fecal analysis, we validated the corticosterone enzyme immunoassay using standard procedures (e. g., parallelism, dose-response curve), and we confirmed physiological significance of fecal glucocorticoid metabolites through adrenocorticotropin challenge, which induced an increase of 4-fold (446.10 +/- 60.73 ng/g) above baseline (114.27 +/- 15.23 ng/g) within 4 hours (P < 0.001). Both groups exhibited a significant increase in fecal glucocorticoids during day 0 (P < 0.001), but concentrations returned to preattachment values within 4 days. Fecal glucocorticoid concentrations did not differ between samples of radiotagged and leg-banded kestrels (P > 0.05). In spite of the small number of monitored subjects, these findings suggested that radiotransmitters did not affect adrenocortical activity in these male American kestrels. (JOURNAL OF WILDLIFE MANAGEMENT 73(5): 772-778; 2009)

Male and female odors induce Fos expression in chemically defined neuronal population

Olfactory information modulates innate and social behaviors in rodents and other species. Studies have shown that the medial nucleus of the amygdala (MEA) and the ventral premammillary, nucleus (PMV) are recruited by conspecific odor stimulation. However, the chemical identity of these neurons is not determined. We exposed sexually inexperienced male rats to female or male odors and assessed Fos immunoreactivity (Fos-ir) in neurons expressing NADPH diaphorase activity (NADPHd, a nitric oxide synthase), neuropeptide Urocortin 3, or glutamic acid decarboxylase rnRNA (GAD-67, a GABA-synthesizing enzyme) in the MEA and PMV. Male and female odors elicited Fos-ir in the MEA and PMV neurons, but the number of Fos-immunoreactive neurons was higher following female odor exposure, in both nuclei. We found no difference in odor induced Fos-ir ill the MEA and PMV comparing fed and fasted animals. Ill the MEA, NADPHd neurons colocalized Fos-ir only in response to female odors. In addition, Urocortin 3 neurons comprise a distinct population and they do not express Fos-ir after conspecific odor stimulation. We found that 80% of neurons activated by male odors coexpressed GAD-67 mRNA. Following female odor, 50% of Fos neurons coexpressed GAD-67 rnRNA. The PMV expresses very little GAD-67, and virtually no colocalization with Fos was observed. We found intense NADPHd activity in PMV neurons, some of which coexpressed Fos-ir after exposure to both odors. The majority of the PMV neurons expressing NADPHd colocalized cocaine-and amphetamine-regulated transcript (CART). Our findings suggest that female and male odors engage distinct neuronal populations in the MEA, thereby inducing contextualized behavioral responses according to olfactory cues. In the PMV, NADPHd/CART neurons respond to male and female odors, suggesting a role in neuroendocrine regulation in response to olfactory cues. (C) 2009 Elsevier Inc. All rights reserved.