Search

In the present study, acetylation status of histone H4 and methylation status of the lysine 9 residue of histone H3 (H3K9) were assessed by immunofluorescence in order to determine the effect of vitrification on epigenetic status of pig MII oocytes. Hyperacetylation of H4 and dimethylation of H3K9 were assessed in control oocytes, after cryoprotectant treatment and after vitrification at two time points, immediately after warming and after a post-warming incubation for 2 h. While no changes in the immunopositivity for both the epitopes were recorded after cryoprotectants, the percentage of negative oocytes for dimethyl H3K9 was observed to increase immediately after devitrification. The influence of vitrification was more evident after 2 h post-thaw incubation when acetylation status of H4 significantly increased and a rise in the percentages of both oocytes exhibiting strong positivity and negative oocytes for dimethyl H3K9 was observed. In conclusion, acetylation of H4 and methylation of H3K9 are altered by vitrification procedure that may lead to an aberrant epigenetic presentation of female chromatin to the fertilizing event and may be, at least in part, responsible for the reduction of developmental competence of vitrified pig oocytes.

Taste signalling molecules are found in the gastrointestinal (GI) tract suggesting that they participate to chemosensing. We tested whether fasting and refeeding affect the expression of the taste signalling molecule, α-transducin (Gαtran ), throughout the pig GI tract and the peptide content of Gαtran cells. The highest density of Gαtran -immunoreactive (IR) cells was in the pylorus, followed by the cardiac mucosa, duodenum, rectum, descending colon, jejunum, caecum, ascending colon and ileum. Most Gαtran -IR cells contained chromogranin A. In the stomach, many Gαtran -IR cells contained ghrelin, whereas in the upper small intestine many were gastrin/cholecystokinin-IR and a few somatostatin-IR. Gαtran -IR and Gαgust -IR colocalized in some cells. Fasting (24 h) resulted in a significant decrease in Gαtran -IR cells in the cardiac mucosa (29.3 ± 0.8 versus 64.8 ± 1.3, P 0.05), pylorus (98.8 ± 1.7 versus 190.8 ± 1.9, P 0.0 l), caecum (8 ± 0.01 versus 15.5 ± 0.5, P 0.01), descending colon (17.8 ± 0.3 versus 23 ± 0.6, P 0.05) and rectum (15.3 ± 0.3 versus 27.5 ± 0.7, P 0.05). Refeeding restored the control level of Gαtran -IR cells in the cardiac mucosa. In contrast, in the duodenum and jejunum, Gαtran -IR cells were significantly reduced after refeeding, whereas Gαtran -IR cells density in the ileum was not changed by fasting/refeeding. These findings provide further support to the concept that taste receptors contribute to luminal chemosensing in the GI tract and suggest they are involved in modulation of food intake and GI function induced by feeding and fasting.

Oocyte and embryo cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig oocytes, because of their greater susceptibility to cryoinjuries, have shown a reduced ability to be fertilized in vitro and a lower developmental competence. The aim of this study was to evaluate the apoptotic status of porcine oocytes vitrified by Cryotop method. We assessed three parameters linked to the activation of the apoptotic cascade: the exteriorization of phosphatidylserine using Annexin V, the caspase activation using FITC-VAD-FMK and the alteration of plasma membrane permeability employing YO-PRO-1. These assays were performed on control oocytes, oocytes exposed to vitrification solutions (toxicity control) and vitrified oocytes either immediately after warming or after incubation for 2 h into maturation medium. The exposition to vitrification solutions triggered an increase of the percentage of oocytes both faintly (VAD+ PI−) and strongly (VAD++ PI−) labeled by FITC-VAD-FMK but not a significant modification of the number of oocytes Annexin V (A+ PI−, early apoptotic) and YO-PRO-1(YP+ PI−, apoptotic) positive in comparison with control oocytes. Oocytes submitted to the whole vitrification procedure showed a rise of the percentage of early apoptotic oocytes (A+ PI−) and FITC-VAD-FMK positive oocytes (VAD+/VAD++ PI−) and a contemporaneous increase of the number of dead oocytes (PI+). On the contrary, vitrified oocytes analyzed immediately after warming did not show a significant increase in the percentage of apoptotic oocytes (YO-PRO-1+/PI−) as compared with the control. Post warming incubation for 2 h into maturation medium, in comparison with oocytes analyzed immediately after warming, did not induce any increase in the percentage of early apoptotic (A+ P−) oocytes while a decrease of the percentage of VAD+/PI− oocytes and a contemporaneous increase of VAD−/PI− oocytes were observed. Moreover, the post-warming incubation induced a rise of the percentage of apoptotic oocytes (YO-PRO-1+/PI−). All these data confirm the involvement of apoptotic mechanisms on the injuries induced by vitrification procedure in pig oocytes; explanation of this phenomenon could be useful to improve oocytes’ cryopreservation protocols.

Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates. These proteins are also present in mature sperm cells, which, in fact, need carriers for uptake energetic sources that are important for maintaining cell basic activity as well as specific functions, such as motility and fertilization ability. Likewise, several GLUTs have been studied in various mammalian species (man, bull, rat, mouse, boar, dog, stallion, and donkey) to point out both their actual presence or absence and their localization on plasma membrane. The aim of this work is to give an overall picture of the studies available on GLUTs in mammalian spermatozoa at this moment, pointing out the species peculiarity, the possible role of these proteins, and the potential future research on this item.

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 M ) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development.

GLUTs are a family of proteins that facilitate the transport of glucose and other hexoses through the plasma membrane of the cells. GLUTs are present in mammalian spermatozoon's membrane in different isoforms and they supply metabolic substrates for all the cell's activities such as motility, homoeostasis and fertilization. As studies about donkey spermatozoa and their metabolism are lacking, this study was aimed at detecting GLUTs 1, 2, 3 and 5 presence by western blotting technique and at determining their localization on the plasma membrane by indirect immunofluorescence. Each protein showed a typical localization on the sperm cells' plasma membrane, differencing the one to the other on the basis of the hexose they transport. We also highlighted some differences between GLUTs distribution and molecular weight in donkey spermatozoa and its nearest relative, the horse.

The expression and distribution patterns of the taste signaling molecules, α-gustducin (Gαgust) and α-transducin (Gαtran) G-protein subunits, were studied in the gastrointestinal tract of the chicken (Gallus domesticus) using the immunohistochemical method. Gαgust and Gαtran immunoreactive (-IR) cells were observed in the mucosal layer of all examined segments, except the esophagus, crop, and the saccus cranialis of the gizzard. The highest numbers of Gαgust and Gαtran-IR cells were found in the proventriculus glands and along the villi of the pyloric, duodenum, and rectal mucosa. Gαgust and Gαtran-IR cells located in the villi of the jejunum, ileum, and cloaca were much less numerous, while only a few Gαgust and Gαtran-IR cells were detected in the mucosa of the proventriculus and cecum. In the crypts, IR cells were observed in the small and large intestine as well as in the cloaca. Gαgust and Gαtran-IR cells displayed elongated (“bottle-” or “pear-like”) or rounded shape. The demonstration of Gαgust and Gαtran expression provides evidence for taste receptor mediated mucosal chemosensitivity in the chicken gastrointestinal tract.