José Pertusa

Long-Term Effects of Delayed Fatherhood in Mice on Postnatal Development and Behavioral Traits of Offspring

Abstract This study aims to analyze, in mice, the long-term effects of delayed fatherhood on postnatal development, spontaneous motor activity, and learning capacity of offspring. Hybrid parental-generation (F0) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The resulting first-generation (F1) offspring were tested for several developmental and behavioral variables. Cumulative percentage of F1 pups that attained immediate righting in the 120-wk group was lower than that found in the 12-, 70-, and 100-wk groups. Furthermore, the postnatal day of attaining immediate righting was higher in pups from the 120-wk group when compared to pups from the other age-groups. At the age of 20 wk, F1 offspring from the 120-wk group displayed lower counts of motor activity than offspring from the 12-, 70-, and 100-wk groups. One week later, a higher percentage of offspring from the 100- and 120-wk groups entered the dark compartment during the retention trial of the passive-avoidance test when compared to offspring from the 12-wk group. Offspring from the 120-wk group exhibited also lower step-through latency in the retention trial than offspring from the 12-, 70-, and 100-wk groups. These results show that advanced paternal age at conception has long-term effects on preweaning development, spontaneous motor activity, and reduced passive-avoidance learning capacity of mouse offspring.

Carboplatin treatment induces dose-dependent increases in the frequency of micronuclei in Ehrlich ascites tumor cells

The effect of various doses (75-300 mg/kg b.w.) of carboplatin on the induction of micronuclei in Ehrlich ascites tumor (EAT) cells was studied. Carboplatin treatment resulted in a dose-dependent increase in the frequency of micronucleated EAT cells. Carboplatin treatment also decreased the mitotic index. The dose-effect curves were linear-quadratic for all the parameters studied. The maximal effect of the drug was obtained in all the treatments after 48 h.

The Phosphorylated Pathway of Serine Biosynthesis Is Essential Both for Male Gametophyte and Embryo Development and for Root Growth in Arabidopsis

This works characterizes the effects of loss- or gain-of-function of phosphoserine phosphatase (PSP1) of the phosphorylated pathway of Ser biosynthesis (PPSB). It provides evidence for a crucial role of the PPSB in embryo, pollen, and root development and suggests that the pathway is an important link between primary metabolism and development. This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.

Oral administration of pharmacological doses of Vitamins C and E reduces reproductive fitness and impairs the ovarian and uterine functions of female mice

This study aims to ascertain whether oral administration of pharmacological doses of Vitamins C and E has any detrimental effect on reproductive fitness of female mice. We fed hybrid female mice from the first day of weaning a standard diet supplemented or not supplemented with pharmacological doses of Vitamins C and E. At the age of 28 weeks, we individually caged females with a male for the rest of their reproductive life. We performed a series of mating experiments to ascertain the number of oocytes ovulated and the potential for embryo development in vitro to the blastocyst stage and in vivo to Day 12 of gestation. The antioxidant diet decreased the frequency of litters, litter size, total number of offspring born and survival of male pups to weaning. This effect was associated with lower number of corpora lutea in the left ovary, decreased percentage of viable fetuses, and higher number of fetal resorptions in the left uterine horn when compared to the control group. The strategy of supplementing the diet with antioxidant vitamins to prevent the age associated decrease in reproductive potential should not be implemented in human beings until a safe and efficient diet is designed.

Delayed Fatherhood in Mice Decreases Reproductive Fitness and Longevity of Offspring

Abstract This study aims to analyze, in mice, the long-term effects of delayed fatherhood on reproductive fitness and longevity of offspring. Hybrid parental-generation (F0) males, at the age of 12, 70, 100, and 120 wk, were individually housed with a randomly selected 12-wk-old hybrid female. The reproductive fitness of first-generation (F1) females was tested from the age of 25 wk until the end of their reproductive life. In F1 males, the testing period ranged from the age of 52 wk until death. Breeding F1 females from the 120-wk group displayed interbirth intervals longer than females from the 12-, 70-, and 100-wk groups. Furthermore, F2 pups begotten by F1 studs exhibited weaning weights lower than pups from the 12- and 70-wk groups. Offspring from the 120-wk group exhibited shorter survival times associated with lower incidence of tumorigenesis and higher loss of body weight when approaching death when compared to F1 offspring from younger age-groups. The results indicate that advanced paternal age at conception has negative long-term effects on reproductive fitness and longevity of offspring in the mouse model.

Induction of micronucleated and binucleate cells in Chinese hamster ovary (CHO) cells by cis-diamminedichloroplatinum (II): a morphological and morphometric study

The mutagenicity and cytotoxicity of cis-diamminedichloroplatinum (II) (cisplatin) at doses of 5, 10 and 20 micrograms/ml in Chinese hamster ovary (CHO) cells have been examined. A morphological characterization of several cell types induced by cisplatin was carried out. The frequencies of both cells with micronuclei and binucleate cells as a time-dependent parameter have also been studied. Whilst the number of cells with micronuclei was found to decrease with time, the number of binucleate cells increased. The possible kinetic mechanism for the production of binucleate cells and cells with micronuclei is discussed. A morphometric analysis was also performed. The nuclear area in both treated and control nuclei was measured with the IBAS image analysis system. The results of this analysis show that a continuous reduction in the nuclear size in the control cells is produced. However the size of the treated cells increased after treatment.

Effect of ivermectin on the liver of gilthead sea bream Sparus aurata: A proteomic approach

Gilthead sea bream Sparus aurata is the most commercialized Mediterranean aquacultured fish species. Ivermectin has recently (experimentally) started to be used to control ectoparasitic infestations in Mediterranean cultured marine fish. The potential hepatotoxicity of ivermectin was investigated in gilthead sea bream juveniles (35g) following oral administration at the recommended dose of 0.2 mgkg(-1) fish for 10d. Difference Gel Electrophoresis Technology (DIGE) was used to study the effect of this treatment in gilthead sea bream liver protein profile under routine culture conditions. The 2D-DIGE protein maps obtained were analyzed using the DeCyder 6.5 software. The results obtained showed significant changes in the expression of 36 proteins respect to the control group. Among these proteins, six increased in abundance, and 30 decreased. Spot showing differential expression respect to the control were analyzed by mass spectrometry and database search, which resulted in three positive identifications corresponding to hepatic proteins involved in lipid metabolism (apoA-I), oxidative stress responses and energy generation (beta-globin, ATP synthase subunit beta). These proteins have not been previously associated to invermectin effect.

Possible relationship between micronucleated and binucleated cells induced by cisplatin in cultured CHO cells

Chinese hamster ovary (CHO) cells were treated with a single dose (10 micrograms/ml) of cis-diamminodichloroplatinum (II) (cisplatin) for 1 h and the effect of the drug on the kinetics of proliferation of the cultures was studied. It was found that the drug produces a delay in the proliferation rates of the treated cultures. The induction of micronuclei and binucleated cells (BC) at different times after treatment have also been studied, and the ability of these cells to undergo DNA synthesis (measured as the ability to incorporate [3H]thymidine) is shown. It was also found that cisplatin induced a particular type of BC that contains one or more micronuclei rather than a pure population of BC. The results obtained show a possible relationship between micronuclei and BC. The possibility that some of the micronucleated cells evolve in subsequent cell divisions to BC with micronuclei is suggested.

A method for resolving occlusions when multitracking individuals in a shoal

Studying the collective behavior of fishes often requires tracking a great number of individuals. When many fishes move together, it is common for individuals to move so close to each other that some fishes superimpose themselves on others during one or several units of time, which impacts on tracking accuracy (i.e., loss of fish trajectories, interchange of fish identities). Type 1 occlusions arise when two fishes swim so near each other that they look like one long fish, whereas type 2 occlusions occur when the fishes’ trajectories cross to create a T- or X-shaped individual. We propose an image processing method for resolving these types of occlusions when multitracking shoals in two dimensions. We assessed processing effectiveness after videorecording shoals of 20 and 40 individuals of two species that exhibit different shoal styles: zebrafish (Danio rerio) and black neon tetras (Hyphessobrycon herbertaxelrodi). Results show that, although the number of occlusions depended on both the number of individuals and the species, the method is able to effectively resolve a great deal of occlusions, irrespective of the species and the number of individuals. It also produces images that can be used in a multitracking system to detect individual fish trajectories. Compared to other methods, our approach makes it possible to study shoals with water depths similar to those seen in the natural conditions of the two species studied.

A minimally invasive methodology based on morphometric parameters for day 2 embryo quality assessment

The risk of multiple pregnancy to maternal-fetal health can be minimized by reducing the number of embryos transferred. New tools for selecting embryos with the highest implantation potential should be developed. The aim of this study was to evaluate the ability of morphological and morphometric variables to predict implantation by analysing images of embryos. This was a retrospective study of 135 embryo photographs from 112 IVF-ICSI cycles carried out between January and March 2011. The embryos were photographed immediately before transfer using Cronus 3 software. Their images were analysed using the public program ImageJ. Significant effects (P < 0.05), and higher discriminant power to predict implantation were observed for the morphometric embryo variables compared with morphological ones. The features for successfully implanted embryos were as follows: four cells on day 2 of development; all blastomeres with circular shape (roundness factor greater than 0.9), an average zona pellucida thickness of 13 µm and an average of 17695.1 µm² for the embryo area. Embryo size, which is described by its area and the average roundness factor for each cell, provides two objective variables to consider when predicting implantation. This approach should be further investigated for its potential ability to improve embryo scoring.