Paternal alcohol consumption has intergenerational consequences in male offspring.

PURPOSE: Alcoholism is a heterogeneous set of disorders caused by ethanol intake. Harmful effects of paternal consumption on the offspring are poorly explored and not fully understood. We analyzed the effect of paternal alcohol consumption on both their own reproductive capacity and that of their male offspring. METHODS: We used a model of ethanol consumption (15% v/v in drinking water) for 12 days in adult CF-1 male mice. DNA integrity and post-translational modifications of histones were assessed in sperm; testicular weight, histology, and DNA fragmentation were analyzed. Treated or untreated male mice were mated with non-treated females to obtain two cell embryos that were cultured for 7 days; morphology and embryonic cell death were evaluated. Males of both groups were mated with non-treated females. Adult male offspring was euthanized, and sperm and testicular parameters determined. RESULTS: Paternal ethanol consumption caused histological and epigenetic changes, as well as damage in DNA integrity in the testicular germline and sperm. These alterations gave rise to deleterious effects on embryonic development and to testicular and spermatic changes in the offspring. CONCLUSION: This study provides critical information on reproductive disturbances brought about by paternal alcohol consumption and the profound impact these could have on the male progeny. The need to explore the effects of paternal alcohol consumption in detail and warn about the importance of controlling alcohol intake for the well-being of future generations should not be underscored.

Murine sperm capacitation, oocyte penetration and decondensation following moderate alcohol intake.

Male chronic alcohol abuse causes testicular failure and infertility. We analyzed the effects of moderate sub-chronic alcohol intake on sperm morphology, capacitation, fertilization and sperm head decondensation. CF-1 male mice were administered 15% ethanol in drinking water for 15 days; control mice received ethanol-free water. Similar patterns of tyrosine phosphorylation were observed in capacitated spermatozoa of control and treated males. Percentage of hyperactivation (H) and spontaneous (SAR) and progesterone-induced (IAR) acrosome reaction significantly decreased at 120 and 150 min of capacitation in treated males compared to controls (H: 14.1 ± 2.5 vs 23.7 ± 2.6, P < 0.05; SAR-T120 min: 17.9 ± 2.5 vs 32.9 ± 4.1, P < 0.01; IAR-150 min: 43.3 ± 3.5 vs 73.1 ± 1.1, P < 0.001, n = 6). During in vitro fertilization (2.5, 3.5 and 4.5 h post-insemination), there was an increased percentage of fertilized oocytes (with a decondensed sperm head and one or two pronuclei) in treated males (P < 0.001, n = 7). After 60 min of in vitro decondensation with glutathione plus heparin, the percentage of decondensed sperm heads was significantly higher in treated males than in controls (mean ± s.d.: 57.1 ± 5.6 vs 48.3 ± 4.5, P < 0.05, n = 5). The percentage of morphologically normal sperm heads was significantly decreased in treated males with respect to controls (P < 0.001, n = 9). These results show that short-term moderate alcohol consumption in outbred mice affect sperm morphology, hyperactivation, acrosomal exocytosis, and the dynamics of in vitro fertilization and in vitro sperm nuclear decondensation.

NF-κB transcriptional activity is modulated by FK506-binding proteins FKBP51 and FKBP52: a role for peptidyl-prolyl isomerase activity.

Hsp90 binding immunophilins FKBP51 and FKBP52 modulate steroid receptor trafficking and hormone-dependent biological responses. With the purpose to expand this model to other nuclear factors that are also subject to nuclear-cytoplasmic shuttling, we analyzed whether these immunophilins modulate NF-κB signaling. It is demonstrated that FKBP51 impairs both the nuclear translocation rate of NF-κB and its transcriptional activity. The inhibitory action of FKBP51 requires neither the peptidylprolyl-isomerase activity of the immunophilin nor its association with Hsp90. The TPR domain of FKBP51 is essential. On the other hand, FKBP52 favors the nuclear retention time of RelA, its association to a DNA consensus binding sequence, and NF-κB transcriptional activity, the latter effect being strongly dependent on the peptidylprolyl-isomerase activity and also on the TPR domain of FKBP52, but its interaction with Hsp90 is not required. In unstimulated cells, FKBP51 forms endogenous complexes with cytoplasmic RelA. Upon cell stimulation with phorbol ester, the NF-κB soluble complex exchanges FKBP51 for FKBP52, and the NF-κB biological effect is triggered. Importantly, FKBP52 is functionally recruited to the promoter region of NF-κB target genes, whereas FKBP51 is released. Competition assays demonstrated that both immunophilins antagonize one another, and binding assays with purified proteins suggest that the association of RelA and immunophilins could be direct. These observations suggest that the biological action of NF-κB in different cell types could be positively regulated by a high FKBP52/FKBP51 expression ratio by favoring NF-κB nuclear retention, recruitment to the promoter regions of target genes, and transcriptional activity.

Vasoactive intestinal peptide deficiency promotes ovarian dysfunction associated to a proinflammatory microenvironment reminiscent of premature aging.

Complex immune regulation during pregnancy is required to ensure a successful pregnancy outcome. Vasoactive intestinal peptide (VIP) has local immunoregulatory effects on the ovary, uterus and maternal-fetal interface that favor a tolerogenic maternal microenvironment. Since the VIP Knockout (KO) mice are subfertile, we investigated the mechanisms underlying the effects of VIP deficiency on ovarian physiology and immune homeostasis. Therefore, we studied VIP KO, deficient (HT) and wild type (WT) female mice in estrus at 3 or 8 months of age. Young KO mice showed abnormal cycle timing and regularity associated with dysfunctional ovaries. Ovaries presented higher number of atretic follicles and reduced number of corpora lutea leading to a lower ovulation rates. Part of the VIP KO mice (25 %) failed to ovulate or ovulated oocytes incompetent to be fertilized (50 %). In particular, ovaries of young KO mice exhibited features of premature aging accompanied by a pro-inflammatory milieu with increased levels of IL-1ß. A unique macrophage subpopulation identified as "foamy macrophages" was found. On the other hand, aged VIP KO females did not gain body weight probably due to the sustained production of E2. Finally, the adoptive transfer of FOXP3+ cells to infertile VIP KO females resulted in their selective recruitment to the ovary. It increased FOXP3/RORγt and TGFß/IL-6 ratio improving ovarian microenvironment and pregnancy rate. The present results suggest that VIP contributes to ovarian homeostatic mechanisms required for a successful pregnancy.

Limited contextual memory and transcriptional dysregulation in the medial prefrontal cortex of mice exposed to early protein malnutrition are intergenerationally transmitted.

Memory contextualization is vital for the subsequent retrieval of relevant memories in specific situations and is a critical dimension of social cognition. The inability to properly contextualize information has been described as characteristic of psychiatric disorders like autism spectrum disorders, schizophrenia, and post-traumatic stress disorder. The exposure to early-life adversities, such as nutritional deficiency, increases the risk to trigger alterations in different domains of cognition related to those observed in mental diseases. In this work, we explored the consequences of exposure to perinatal protein malnutrition on contextual memory in a mouse model and assessed whether these consequences are transmitted to the next generation. Female mice were fed with a normal or hypoproteic diet during pregnancy and lactation. To evaluate contextual memory, the object-context mismatch test was performed in both sexes of F1 offspring and in the subsequent F2 generation. We observed that contextual memory was altered in mice of both sexes that had been subjected to maternal protein malnutrition and that the deficit in contextual memory was transmitted to the next generation. The basis of this alteration seems to be a transcriptional dysregulation of genes involved in the excitatory and inhibitory balance and immediate-early genes within the medial prefrontal cortex (mPFC) of both generations. The expression of genes encoding enzymes that regulate H3K27me3 levels was altered in the mPFC and partially in sperm of F1 malnourished mice. These results support the hypothesis that early nutritional deficiency represents a risk factor for the emergence of symptoms associated with mental disorders.

Interleukin-1 beta regulates metalloproteinase activity and leptin secretion in a cytotrophoblast model.

Implantation is one of the most regulated processes in human reproduction, by endocrine and immunological systems. Cytokines are involved in embryo-maternal communication and an impaired balance could result in pregnancy loss. Here we investigated the effect of interleukin 1-beta on the activity of two important metalloproteinases (MMP-2 and MMP-9) that are involved in extracellular matrix remodeling as well as the secretion of leptin, one of the reproductive hormones actively regulating their activity and secretion. We found that IL-1 beta activates matrix metalloproteinase activity as well as increases leptin secretion. We propose that this interleukin, through the regulation of leptin, in turn activates matrix metalloproteinases which results in an increased cytotrophoblast invasion.

Dermatan sulfate synergizes with heparin in murine sperm chromatin decondensation.

The mammalian sperm nucleus contains an unusually condensed chromatin, due to replacement of the majority of histones by protamines. However, soon after the spermatozoon penetrates the ooplasm at fertilization, decondensation of this densely packed chromatin must occur to allow formation of the male pronucleus and syngamy. Decondensation is accomplished by protamine disulfide bond reduction by oocyte glutathione and replacement of protamines by oocyte histones with the aid of an acceptor molecule. Previous results from our laboratory have demonstrated that heparan sulfate (HS) present in the ooplasm functions as protamine acceptor during human sperm decondensation in vivo. In the present paper, we analyze the role of heparin, structural analogue of HS, and dermatan sulfate (DS) in murine sperm chromatin decondensation in vitro, including the possibility of a synergistic effect between both glycosaminoglycans. Decondensation was assessed under phase contrast microscopy following incubation of murine spermatozoa with glutathione and either heparin, DS, or a combination of both. Ultrastructural changes taking place during decondensation were analyzed by transmission electron microscopy. Both glycosaminoglycans were able to promote the decondensation of murine spermatozoa in vitro but the decondensing ability of heparin was significantly higher. Use of both glycosaminoglycans together revealed the existence of a synergistic effect. Transmission electron microscopy analysis of decondensing spermatozoa supported these findings. Synergism between heparin and DS was observed both in capacitated and non-capacitated spermatozoa but decondensation kinetics was faster in the former. The results obtained indicate a new potential role for dermatan sulfate in murine sperm decondensation at fertilization and provide evidence of differences in the degree of chromatin condensation throughout the murine sperm nucleus.

Matrix metalloproteinase expression and activity in trophoblast-decidual tissues at organogenesis in CF-1 mouse.

During early placentation, matrix metalloproteinases (MMPs) play important roles in decidualization, trophoblast migration, invasion, angiogenesis, vascularization and extracellular matrix (ECM) remodeling of the endometrium. The aim of our study was to analyze the localization, distribution and differential expression of MMP-2 and -9 in the organogenic implantation site and to evaluate in vivo and in vitro decidual MMP-2 and -9 activities on day 10 of gestation in CF-1 mouse. Whole extracts for Western blotting of organogenic E10-decidua expressed MMP-2 and -9 isoforms. MMP-2 immunoreactivity was found in a granular and discrete pattern in ECM of mesometrial decidua (MD) near maternal blood vessels and slightly in non-decidualized endometrium (NDE). Immunoexpression of MMP-9 was also detected in NDE, in cytoplasm of decidual cells and ECM of vascular MD, in trophoblastic area and in growing antimesometrial deciduum. Gelatin zymography showed that MMP-9 activity was significantly lower in CM compared to the active form of direct (not cultured) and cultured decidua. The decidual active MMP-9 was significantly higher than the active MMP-2. These results show differential localization, protein expression and enzymatic activation of MMPs, suggesting specific roles for MMP-2 and MMP-9 in decidual and trophoblast tissues related to organogenic ECM remodeling and vascularization during early establishment of mouse placentation.

Expression of erythroblastic leukemia viral oncogene homolog (erbBS) mRNAs and possible splice variants in 3T3-L1 preadipocytes.

Previously, we studied the erythroblastic leukemia viral oncogene homolog (erbB) family of tyrosine kinase growth factor receptors in terms of protein expression, modulation and activation in the 3T3-L1 cell line. In the present study, the presence of full-length erbB mRNAs, and splice or proteolytic erbB variants, was evaluated using RT-PCR. Epidermal growth factor receptor (EGFR)/erbB1 expression was confirmed. Wild-type (wt) ErbB2, human erbB2 (HER2)-extracellular domain (ECD) and herstatin mRNA expression were analyzed. Restriction analysis confirmed wt expression. 3T3-L1 cells exhibited HER2-ECD and herstatin mRNA expression, although at extremely low levels (compared to the control cell lines). ErbB3 cDNA was amplified using mouse mammary tumors and rat acines as positive controls. ErbB4 was not positively identified in these cells. In conclusion, this study demonstrates that 3T3-L1 cells express EGFR/erbB1, erbB2 and erbB3, and possibly, certain erbB2 splice or proteolytic variants, which are worth pursuing.

Interferon-gamma inhibits metalloproteinase activity and cytotrophoblast cell migration.

PROBLEM: Establishment of a successful pregnancy relies on a complex fetal-mother communication that starts with the embryo adhering and invading the endometrium. This requires remodeling of extracellular matrix, performed by metalloproteinases. Cytokines, such as interferon-gamma (IFN-gamma), play a role in implantation and could affect the success of pregnancy. METHOD OF STUDY: Using JEG-3 cell line as model, we cultured the cells in the presence or absence of IFN-gamma and determined the activities of MMP-2 and MMP-9 using zymography and the secretion of leptin using Western blot. RESULTS: Interferon-gamma inhibits gelatinase activity from MMP-2 and MMP-9 in a dose-dependent manner, reducing the secretion of leptin (not because of a general inhibition on protein synthesis) and impairs cell migration on Matrigel. CONCLUSION: Our results correlate with previous reports from our laboratory indicating that IFN- gamma is deleterious for mouse embryo outgrowth, having an effect on metalloproteinases activity as well as leptin secretion.

Interleukin-1 beta regulates metalloproteinase activity and leptin secretion in a cytotrophoblast model

Implantation is one of the most regulated processes in human reproduction, by endocrine and immunological systems. Cytokines are involved in embryo-maternal communication and an impaired balance could result in pregnancy loss. Here we investigated the effect of interleukin 1-beta on the activity of two important metalloproteinases (MMP-2 and MMP-9) that are involved in extracellular matrix remodeling as well as the secretion of leptin, one of the reproductive hormones actively regulating their activity and secretion. We found that IL-1 beta activates matrix metalloproteinase activity as well as increases leptin secretion. We propose that this interleukin, through the regulation of leptin, in turn activates matrix metalloproteinases which results in an increased cytotrophoblast invasion.

Exogenous interferon-gamma alters murine inner cell mass and trophoblast development. Effect on the expression of ErbB1, ErbB4 and heparan sulfate proteoglycan (perlecan).

Implantation is a crucial event in human pregnancy. The participation of cytokines in the implantation process has been widely documented, although the role of many of these molecules is still a matter of controversy. In a previous report from our laboratory, we demonstrated that addition of interferon-gamma to the culture medium produces deleterious effects on mouse embryo development. In this study we investigated the effect of this cytokine on outgrowing embryo morphology and on the expression of epidermal growth factor receptors (ErbBs) and heparan sulfate proteoglycan (perlecan) in mouse embryos cultured in vitro. Morphological assessment of inner cell mass and trophoblast development was carried on in-situ fixed and stained outgrowths. Localization of ErbB1, ErbB4 and perlecan on pre- and peri-implantation embryos was investigated by immunocytochemistry. Addition of interferon-gamma produced a deleterious effect on both inner cell mass and trophoblast morphology. Immunostaining demonstrated that ErbB1, ErbB4 and perlecan are present on pre-implantation embryos and blastocysts; interferon-gamma altered the expression of ErbB4 and Perlecan at the blastocyst stage. We propose that the effects produced by this cytokine could be related to the altered acquisition of adhesion competence and low implantation rates observed in certain reproductive immunological disorders.