The inhibition of spermatic cystine/glutamate antiporter xCT (SLC7A11) influences the ability of cryopreserved stallion sperm to bind to heterologous zonae pellucidae.

Sperm are redox-regulated cells, and deregulation of their redox status is considered to affect male fertility and to reduce their fertilizing ability following biotechnological procedures, such as cryopreservation. Cystine (CysS), after incorporation in sperm via SLC7A11 antiporter, has been demonstrated to increase intracellular GSH content, the most important non enzymatic antioxidant. This study was aimed at investigating the role of SLC7A11 antiporter on frozen-thawed stallion sperm ability to respond to in vitro capacitating environment after post-thaw incubation with CysS and/or Sulfasalazine (SS), a specific inhibitor of SLC7A11 antiporter. Viability, motility, immunolocalization of tyrosine phosphorylated proteins and the ability to bind to heterologous zonae pellucidae were evaluated. Thawed sperm from seven stallions (2 ejaculates/stallion) was washed and resuspended in Tyrodes media; each thawed ejaculate was divided in Control (CTR) and 3 samples supplemented with: 0.5 mM Cystine (CysS), 500 µM Sulfasalazine (SS) and 0.5 mM CysS + 500 µM SS (CysS + SS). After 1 h of incubation at 37 °C, samples were washed twice, resuspended in capacitating BWW medium and incubated at 38 °C under 5% CO2. After 30 and 60 min, sperm motility, viability and tyrosine phosphorylated protein immunolocalization, used as capacitation status index, were evaluated. After 30 min of capacitation, 4 × 105 sperm were co-incubated with denuded pig oocytes in capacitation medium for 30 min for the heterologous binding assay. None of the sperm parameters studied (motility, viability and tyrosine phosphorylation) showed any difference respective to control. The number of sperm bound per oocyte (mean ± SEM) tended to increase in CysS group (44.0 ± 12.3) respect CTR (40.8 ± 10.8) while decreased in SS group (32.4 ± 7.8) (p < 0.01). Moreover, CysS + SS group showed a lower binding rate (32.0 ± 10.0) compared to CysS (p < 0.001). Our results suggest that CysS supplementation of thawed stallion sperm can influence their ability to bind to heterologous zona pellucidae as the inhibition of CysS incorporation by SLC7A11 reduced the number of sperm bound per oocyte. This effect does not seem to be ascribed to a modification of sperm motility, membrane integrity and tyrosine phosphorylation.

Resveratrol and Epigallocatechin-3-gallate addition to thawed boar sperm improves in vitro fertilization.

Thawing is one of the most delicate process after semen cryopreservation as spermatozoa pass from a dormant metabolic stage to a sudden awakening in cellular metabolism. The rapid oxygen utilization leads to an overproduction of reactive oxygen species that can damage sperm cells, thus causing a significant decrease of fertilizing potential of frozen-thawed spermatozoa. Resveratrol (Res) is a natural grape-derived phytoalexin and Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis); both molecules are known to possess high levels of antioxidant activity. The objective of the present study was to assess the effect of different concentrations of Res (0.5, 1 or 2 mM; Experiment 1) or EGCG (25, 50 or 100 µM; Experiment 2) supplementation to thawing boar semen extender on sperm quality parameters (viability and acrosome integrity) and in vitro fertilization (IVF). Semen after thawing and dilution with three volumes of Beltsville Thawing Solution (BTS), was immediately divided in control group without antioxidants addition (CTR) and either Res or EGCG groups. Sperm viability and acrosome integrity were evaluated in CTR, Res or EGCG groups after 1 h of incubation at 37 °C. The addition of different doses of Res or EGCG to thawing extender for 1 h did not induce any effect on boar sperm viability and acrosome integrity. However, both Res and EGCG treated samples exhibited a significantly higher penetration rate compared with CTR when used for IVF. In particular the treatment with all the EGCG concentrations increased the penetration rate (P < 0.01) while only Res 2 mM induced a significant increase of this parameter (P < 0.01). In addition, EGCG 25 and 50 µM supplementation significantly increased total fertilization efficiency as compared to control (EGCG 25 µM: 40.3 ± 8.2 vs 26.8 ± 9.5, P < 0.05; EGCG 50 µM: 40.4 ± 7.8 vs 26.8 ± 9.5, P < 0.01). The same effect was observed with Res 2 mM (51.0 ± 7.6 vs 29.6 ± 11.3, P < 0.01). In conclusion, our results indicate that the addition of different doses of the two antioxidants to thawed spermatozoa for one hour, even if does not exert any effect on sperm viability and acrosome integrity, efficiently improves in vitro penetration rate. Moreover, both molecules (EGCG 25 and 50 µM and Res 2 mM) significantly increases the total efficiency of fertilization.

Epigallocatechin-3-gallate (EGCG) and green tea polyphenols do not improve stallion semen parameters during cooling at 4°C.

Stallion semen storage for artificial insemination is mainly based on liquid cooled storage. In many stallions this technique maintains sperm quality for an extended period of time (24-72 hr) at 7°C. While this technique is commonly used in the horse industry, there can be a decline in fertility in some stallions, due to an inability of their sperm to tolerate the cool storage process. The aim of the present work was to evaluate the effect of two natural antioxidants (epigallocatechin-3-gallate (EGCG) at 20, 60 and 120 µm and green tea polyphenols, and p at .001, .01 and .1 mg/ml) on some sperm parameters (sperm motility, viability/acrosome integrity and DNA quality) in extended semen immediately after its collection (T0) and after 2, 6, 24 and 48 hr of cool storage. Two ejaculates from three trotter stallions were analysed after 48 hr of storage at 4°C. No beneficial effect on the analysed parameters was observed: the two antioxidants were not able to improve sperm quality after 48 hr of storage. These results are in agreement with previous findings on the effect of different antioxidants reported by other researches, who have demonstrated that stallion semen keeps good antioxidant capacity after dilution for 24 hr. In conclusion, the positive effect exerted by antioxidant molecules in other species is not confirmed in the equine one.

Effects of antioxidants on boar spermatozoa during sorting and storage.

Sorting procedures submit sperm cells to a set of stressful steps that can trigger an increase of ROS production and consequently reduce sorted semen quality. This study evaluated the effect of supplementation with different antioxidants (EGCG, Na pyruvate+catalase, SOD) on acrosome and plasma membrane integrity, activation of caspases (as assayed by FITC-VAD/PI staining) and immunolocalization of Hsp70 of boar spermatozoa after sperm preparation (Hoechst 33342 staining) and sorting procedure. The effect of antioxidants, with or without seminal plasma, on sorted spermatozoa stored for 24h at 15°C was also evaluated. Antioxidants did not exert any preventive action on sperm modification induced by staining and sorting. After 24h of storage at 15°C, sorted samples supplemented with either EGCG or SOD plus seminal plasma showed a significant (p<0.05) increase of the percentage of viable spermatozoa, while no positive effect on the other tested parameters was observed; EGCG seems to exert an inhibition on caspase activation in that a decrease of the number of dead cells FITC-VAD+/PI+ was recorded. In conclusion, our results indicate that EGCG and SOD in association with seminal plasma are effective in exerting some compensatory protection against the detrimental effects of storage of sorted semen while their action is not evident during steps of the sorting procedure.

Effects of epigallocatechin-3-gallate (EGCG) on in vitro maturation and fertilization of porcine oocytes.

The beneficial properties of green tea and especially of its principal active polyphenol, epigallocatechin-3-gallate (EGCG), have led to an increased demand for dietary supplements with highly enriched EGCG concentrations. In order to investigate the possible reproductive-related consequence of EGCG supplementation, the effects of this catechin on in vitro maturation (IVM) and fertilization (IVF) of oocyte, using the pig as experimental model, were examined. In the first series of experiments EGCG, at concentrations ranging from 0 to 25 microg/ml, was added during in vitro maturation of pig oocytes. EGCG had no effect on nuclear maturation of pig oocytes and on fertilization traits considered after IVF at any of the doses tested. By contrast, a significant (p<0.05) decrease in the number of embryos that developed to blastocysts following parthenogenetic activation was recorded when 25 microg/ml EGCG was added to IVM medium; in addition this catechin concentration significantly (p<0.05) inhibited progesterone production by cumulus cells after 48 h of culture. When induction of sperm capacitation was performed in presence of EGCG, a significantly lower percentage of spermatozoa showing a Hsp70-capacitated pattern and a significant reduction of sperm H(2)O(2) production were evident at a concentration of 25 microg/ml EGCG (p<0.05). During gamete coincubation EGCG reduced, in a dose response manner, the number of reacted spermatozoa suspended in fertilization medium and increased the number of sperm bound to ZP. Supplementation of 10 microg/ml EGCG during IVF significantly increased the fertilization rate while higher EGCG concentrations (25 microg/ml) decreased the percentage of fertilized oocytes (p<0.05). In conclusion, our data suggest that high EGCG concentrations could affect in vitro maturation and fertilization in pig; it cannot be totally excluded that excessive EGCG concentrations could induce reproductive-related consequences also in vivo.

Selenium stimulates estradiol production in bovine granulosa cells: possible involvement of nitric oxide.

Reduction in fertility is well known to be possibly related to selenium deficiencies, even if target organ for selenium action is, at present, unclear. The present study was aimed to examine whether selenium directly influences granulosa cells. Bovine granulosa cells from different size follicles were used to investigate the effect of selenium (5 ng/ml), with or without bovine follicle-stimulating hormone (bFSH) (100 ng/ml), on proliferation and steroidogenesis. In addition, we sought to determine if selenium modulates the production of nitric oxide, which is known to play an important role in ovarian activity. Our data demonstrate that selenium significantly (P < 0.001) stimulates the proliferation of the cells from small follicles; moreover, it further potentiates the stimulatory effect of the gonadotropin in the same cells. Furthermore, selenium significantly (P < 0.01) augments E2 output by cells from both kinds of follicles. bFSH increases E2 production (P < 0.01) by cells from large follicles, whereas it exerts a stimulatory (P < 0.01) effect only in the presence of selenium in the cells from the small ones. The production of nitric oxide is significantly increased (P < 0.001) by bFSH, but only in cells from small follicles. Selenium inhibits (P < 0.001) nitric oxide production in cells from both kinds of follicles and significantly decreases (P < 0.001) bFSH-induced nitric oxide production in cells from the small ones. We conclude that selenium acts on granulosa cells by modulating their proliferation and E2 synthesis; moreover, its effect could be mediated, at least in part, through an inhibition of nitric oxide.

Plasma cortisol variations induced in the stallion by mating.

Plasma cortisol variations have been determined by radioimmunoassay in 5 stallions during mating and in 2 teasers during oestrous female exposure. In all the animals, cortisol plasma levels consistently increase (71.1 ng/ml vs 44.0 and 63.0 ng/ml vs 35.1, in the stallions and in the teasers, respectively) 7-30 min after female exposure; 120 min after exposure, cortisol concentrations are again low.