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.

Human sperm decondensation in vitro is related to cleavage rate and embryo quality in IVF.

PURPOSE: To investigate whether the ability of human spermatozoa to decondense in vitro in the presence of heparin (Hep) and glutathione (GSH) is related to assisted reproduction (ART) success. METHODS: Cross-sectional pilot study involving male partners of 129 infertile couples undergoing ICSI with (45) or without (84) donor oocytes at two infertility clinics in CABA, Argentina, between October 2012 and December 2013. In vitro decondensation kinetics with Hep and GSH and DNA fragmentation (TUNEL) were determined on the same sample used for ICSI. The possible relationship of decondensation parameters (maximum decondensation and decondensation velocity) and TUNEL values with ART success was evaluated. RESULTS: Embryo quality correlated positively with decondensation velocity (D60/D30) (Spearman's correlation, p < 0.05). According to D60/D30 values, patients were classified as slow decondensers (SlowD) (n = 68) or fast decondensers (FastD) (n = 61). Embryo quality was better in FastD (unpaired t test, p < 0.05). FastD and SlowD were subdivided according to use of donor oocytes. Among SlowD, biochemical and clinical pregnancy rates per transfer were significantly higher in donor (n = 19) vs. in non-donor (n = 31) cycles (Fisher's exact test, p < 0.05). TUNEL values were not related to embryo quality, but no clinical pregnancies or live births were achieved in TUNEL+ SlowD (n = 7). CONCLUSION: Decondensation kinetics of human spermatozoa in vitro with Hep and GSH could be related to embryo quality and ART success.

Sperm pretreatment with heparin and l-glutathione, sex-sorting, and double cryopreservation to improve intracytoplasmic sperm injection in bovine.

In bovine, intracytoplasmic sperm injection (ICSI) remains inefficient partially due to low levels of sperm decondensation. The aim of this study was to determine whether the injection of normal size sperm pretreated with heparin (Hep) and l-glutathione (GSH), the use of sex-sorted sperm, the double round of sperm freezing/thawing (re frozen), or the combination of these approaches can improve sperm decondensation and embryo development after ICSI in cattle. Cleavage and blastocyst rates were evaluated on days 2 and 7 post ICSI. Quality of ICSI blastocysts was analyzed by the relative expression of four genes by qPCR and the DNA fragmentation index by TUNEL assay. For all assays, semen samples were co-incubated with pCX-EGFP 50 ng/µl before ICSI. GFP expression, which can be detected by fluorescence microscopy, was used as a tool to estimate the success of sperm decondensation after ICSI. The use of normal size sperm pretreated with 80 µM Hep-15 mM GSH for 20 h (Hep-GSH) increased cleavage, blastocyst and EGFP + blastocysts rates (60.8, 19.4 and 61.9%) compared to control ICSI (35, 4.9 and 20%) (p < 0.05). Moreover, HMGN1, GLUT5, AQP3 and POU5F1 transcription levels did not differ between ICSI Hep-GSH and IVF embryos. The use of sex-sorted sperm (X, Y) improved cleavage rates and EGFP expression at day 4 (83 and 30.2% for ICSI Y and 83.2 and 31.7% for ICSI X) compared to non-sorted group (50.9 and 15.1%), not showing differences at the blastocyst stage. Finally, sex sorting (X) was combined with Hep-GSH and/or re frozen treatments. The use of Hep-GSH diminished cleavage rates from ICSI X re frozen group (80.4% vs. 94.2%) and blastocyst development from ICSI X group (3.3% vs. 10%), compared with their controls (p < 0.05). While Hep-GSH pretreatment induced lower transgene expression at day 4, no differences were found at the blastocyst stage between ICSI groups (from 58.3 to 80%). TUNEL assay showed higher DNA fragmentation indexes for all ICSI treatments (p < 0.05), except for ICSI X Hep-GSH, which did not differ from IVF X control. In conclusion, the use of normal size sperm pretreated with Hep-GSH, combined or not with sex-sorting by flow cytometry could improve ICSI outcomes in cattle.

Heterogeneous distribution of histone methylation in mature human sperm.

PURPOSE: To analyze the presence of various histone modifications in ejaculated human spermatozoa METHODS: In this prospective study, seminal ejaculates from 39 normozoospermic individuals were evaluated for semen analysis and the presence of histone modifications in isolated nuclei. RESULTS: We observed heterogeneous presence of histone methylation in normal mature human sperm. We observed that 12 to 30 % of the nuclei of normal sperm contain a heterogeneous distribution of the marks H3K4Me1, H3K9Me2, H3K4Me3, H3K79Me2, and H3K36Me3. Moreover, the presence of these marks is higher in the poor motile fraction of the ejaculate, which is associated with poor morphology and functional quality. In contrast, we did not observe histone acetylation (H3K4Ac and H4K5Ac) in normal or abnormal mature human sperm CONCLUSIONS: Defects in the process of spermatogenesis may alter the correct epigenetic programming in mature sperm. Further studies are required to evaluate the impact of these findings in human infertility.

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.

α(2)-Adrenoceptors enhance cell proliferation and mammary tumor growth acting through both the stroma and the tumor cells.

We have previously described enhanced human breast cancer cell proliferation and mouse mammary tumor growth induced by α(2)-adrenoceptor (α(2)-AR) expression in epithelial cells. The aim of the present work was to assess if stromal fibroblasts can contribute to this effect. α(2)-AR expression was assessed by immunocytochemistry and immunohistochemistry, cell proliferation by [(3)H]-Thymidine incorporation and tumor growth by measuring with caliper. All tested mouse and human fibroblasts expressed at least two α(2)-AR subtypes and α(2)-adrenergic agonists enhanced fibroblast proliferation. In vivo, the α(2)-adrenergic agonist clonidine significantly enhanced tumor growth. The α(2)-adrenergic antagonist rauwolscine reversed this effect, but when administered alone, significantly inhibited tumor growth. Clonidine significantly stimulated cell proliferation in the epithelial-enriched fraction, the cancer associated fibroblast-enriched fraction and the co-culture of both fractions in primary cultures from both tumors (IBH-4 and IBH-6). Rauwolscine reversed clonidine stimulation in every fraction. However, when incubated alone, the inhibitory effect was observed in fractions from IBH-4 tumors but not from IBH-6 tumors. These experiments show that fibroblasts from tumor stroma are also influenced by α(2)-adrenergic compounds through the α(2)-ARs expressed in these cells. Moreover, the α(2)-adrenergic antagonist rauwolscine could eventually block in both epithelial and stromal cells, the mitogenic effect of catecholamines released during stress, providing a potential additional treatment for breast cancer patients. Chemists synthesizing adrenergic compounds should consider their action in breast cancer patients.

Further evidence on the role of heparan sulfate as protamine acceptor during the decondensation of human spermatozoa.

BACKGROUND: Human spermatozoa decondense in vitro upon exposure to heparin and glutathione. Glutathione is also the disulfide bond reducer in vivo, and heparan sulfate, a functional analogue of heparin, has been proposed as the protamine acceptor. The aim of this study was to evaluate the decondensing ability of chemically modified heparins and different glycosaminoglycans (GAGs) on isolated sperm nuclei in vitro, and to analyse the possible role of different GAGs as protamine acceptors. METHODS: Capacitated spermatozoa and isolated sperm nuclei from normospermic semen samples were decondensed in the presence of heparin (or its equivalent) and glutathione. After fixation with glutaraldehyde, the percentage of decondensed spermatozoa and nuclei was determined under phase-contrast. Proteins were extracted from sperm nuclei previously incubated in the presence of gluhathione and different GAGs by incubation with urea-beta-meracptoethanol-NaCl, and analysed by acid polyacrylamide gel electrophoresis. RESULTS: The ability of desulfated heparins and other GAGs to decondense isolated nuclei mirrored exactly the decondensation of capacitated spermatozoa, the only difference being the level of maximum decondensation achieved. Heparan sulfate and heparin, but not other GAGs, were able to release protamines from sperm chromatin. CONCLUSIONS: Heparan sulfate could be functioning as protamine acceptor in vivo during human sperm nuclear decondensation.

Heparan sulphate: a putative decondensing agent for human spermatozoa in vivo.

BACKGROUND: Human sperm decondense in vitro upon exposure to heparin and glutathione. The aim of this study was to evaluate whether this decondensing ability of heparin in vitro is related to structural characteristics of the molecule and to test the in-vitro decondensing ability of other glycosaminoglycans. METHODS: Capacitated sperm obtained from normospermic semen samples were decondensed in the presence of heparin (or its equivalent) and glutathione. After fixation with glutaraldehyde, the percentage of decondensed sperm was determined under phase contrast. RESULTS: The decondensing ability of heparin was related to sulphation characteristics of the molecule: heparin, O-desulphated heparin and N-desulphated-N-acetylated heparin had similar decondensing abilities; N-desulphated was less active and O/N-desulphated-N-acetylated heparin was completely inactive. On the other hand, the decondensing ability of heparin was not affected by molecular weight, within the range 3000-18,000 kDa. When the decondensing ability of different glycosaminoglycans was tested, heparin and heparin sulphate were equally active, while chondroitin sulphate and hyaluronic acid were completely inactive and dermatan sulphate was slightly active. CONCLUSIONS: Our results indicate that heparin's decondensing ability in vitro is related to sulphation characteristics of the molecule and suggest that heparan sulphate, a structural analogue of heparin, could be a sperm-decondensing agent in vivo.