Heat SHOCK proteins in equine spermatozoa: Expression and correlation to kinetic and environmental parameters.

Heat Shock Proteins are chaperones primary involved in the repair of cellular damages induced by temperature. The harmful effect of temperature on the male gonad is well known, on the contrary knowledge on the effects of the environment on semen quality are still insufficient. The aim of this paper was to learn more about the role of HSPs and the environment in modulating the physiology of equine male gonads. We showed a detailed analysis of equine semen characteristic and the expression level of three HSPs (60-70-90) over a one-year period analyzing the effects of temperature and humidity and the correlation among the different variables. We showed also that the interpretation of results depends strongly on the way in which data are assembled and analyzed, therefore we compared results obtained from three different ways of grouping: according to single months, to weather seasons and to mare reproductive periods. Results showed that the expression of the three HSPs is correlated to the environment through temperature and humidity and that it reaches the highest level in the breeding season and in summer. We found also that HSPs expression is correlated to some variables describing the quality of equine semen (concentration) and the kinetic of spermatozoa (total motility-MOT, %, average path velocity -VAP, µm/s- and lateral head displacement -ALH, µm). No correlation was found between HSPs expression and the mitochondrial membrane potential; while viability and HSP90 expression resulted positively correlated. The month-by-month analysis evidenced that in February equine semen has the highest kinetic characteristics (increased linearity -LIN, %-, straightness -STR, % -and average path velocity -VAP, µm/s) with the highest number of motile, progressive motile and rapid cells. These results may have a great impact in the comprehension of functional aspects of the physiology of equine semen and may have potential implications for breeders who want to understand the period (and/or month) of the year in which equine semen reaches the best characteristics with increased chances for better results in reproductive practice.

Localization and functional modification of L-type voltage-gated calcium channels in equine spermatozoa from fresh and frozen semen.

It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level focusing on the function of L-type calcium channels. We report that in cryopreserved spermatozoa the mean basal value of [Ca(2+)]i is higher than that of spermatozoa from fresh semen (447.130 vs. 288.3 nM; P < 0.001) and L-type channels function differently in response to their agonist and antagonist in relation to semen condition (fresh or frozen-thawed). We found that on addition of agonist to the culture medium, the increase in intracellular calcium concentrations ([Ca(2+)]i) was greater in frozen semen than in fresh semen (Δ[Ca(2+)]i = 124.59 vs. 16.04 nM; P < 0.001), whereas after the addition of antagonist the decrease in [Ca(2+)]i was lower in frozen semen than in fresh semen (Δ[Ca(2+)]i = 32.5 vs. 82.5 nM; P < 0.001). In this article, we also discuss the impact of cryopreservation on sperm physiology.

Lectin-binding sites on ejaculated stallion sperm during breeding and non-breeding periods.

Stallion sperm from semen collected in Southern Italy during the breeding (June-July) and non-breeding (December-January) periods were analyzed by means of twelve lectins to evaluate the glycoconjugate pattern and to verify whether there are any seasonal differences in the glycosylation pattern of the sperm glycocalyx. The acrosomal cap showed reactivity for Maackia amurensis (MAL II), Sambucus nigra (SNA), Arachis hypogaea (PNA), Glycine max (SBA), Helix pomatia (HPA), Canavalia ensiformis (Con A) Triticum vulgaris (WGA), and Griffonia simplicifolia isolectin II (GSA II) in breeding and non-breeding ejaculated sperm, suggesting the presence of oligosaccharides terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc, Neu5Ac alpha 2,6Gal/GalNAc, with Gal beta 1,3GalNAc, alpha/beta GalNAc and glycans with terminal/internal alpha Man and GlcNAc. During the non-breeding period, the acrosomal cap expressed oligosaccharides terminating with Gal beta 1,4GlcNAc (Ricinus communis(120) affinity) (RCA(120)) and L-Fuc alpha 1,2Gal beta 1,4GlcNAc beta (Ulex europaeus affinity) (UEA I). The equatorial segment placed between the acrosomal cap and post-acrosomal region did not display glycans terminating with GalNAc, GlcNAc, and alpha L-Fuc. The post-acrosomal region of sperm collected in the breeding and non-breeding periods bound Con A, MAL II, SNA, and SBA, thus showing the presence of N-linked oligosaccharides from high-Man content, terminating with Neu5Ac alpha 2,3Gal beta 1,4GlcNAc, Neu5Ac alpha 2,6Gal/GalNAc and GalNAc. In winter, the post-acrosomal region also expressed oligosaccharides terminating with alpha GalNAc, GlcNAc, and L-Fuc alpha 1,2Gal beta 1,4GlcNAc beta (HPA, GSA II, and UEA I staining). The tail of sperm from semen collected during the breeding and non-breeding periods showed a lectin binding pattern similar to the post-acrosomal region, except for the absence of HPA staining in sperm collected during the winter season. These results indicate that the surface of stallion sperm contains different glycocalyx domains and that the glycosylation pattern undergoes changes during the breeding and non-breeding periods.

Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa.

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean (X -DFI), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, alpha-zearalenol, and beta-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.

In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI.

The interleukin-1 (IL-1) system is thought to be involved in periovulatory events in the mare. Previous in vivo studies have demonstrated that IL-1beta induces oocyte maturation, but depresses the pregnancy rate 14 days after ovulation. To better understand the role of IL-1 in oocyte maturation and fertilization, the effects of IL-1 on the in vitro maturation rate of equine oocytes in pure follicular fluid were evaluated and fertilization rate assessed following intracytoplasmic sperm injection (ICSI). Oocytes collected from slaughterhouse ovaries were cultured in four different media for 30 h prior to fertilization. Two experiments were performed, each using three maturation media as the experimental treatments. Medium 1 was pure follicular fluid from subordinate follicles. Medium 2 was medium 1 plus 50 ng/ml recombinant human IL-1beta. Medium 3 was pure follicular fluid collected from mares administered crude equine gonadotropin (CEG). Medium 4 was medium 2 plus 50 ng/ml of recombinant human IL-1 receptor antagonist. Media 1, 2 and 3 were compared in experiment 1. In experiment 2, media 1, 2 and 4 were compared. After maturation, metaphase II oocytes were submitted to microinjection and assessed for signs of fertilization. In experiment 1, 101 oocytes were evaluated. The rate of polar body extrusion was 66, 51 and 68% and the proportions of normally fertilized oocytes after ICSI were 40, 18 and 38% for media 1, 2 and 3, respectively. In experiment 2, 122 oocytes were evaluated. The rate of polar body extrusion was 55, 48 and 42% and the proportions showing normal fertilization after ICSI were 14, 25 and 29% for media 1, 2 and 4, respectively. There was no positive effect of IL-1beta on maturation in both experiments, but the fertilization rate and percentage of embryos reaching four-cell were low in the presence of IL-1beta, indicating that this cytokine may interfere with fertilization and early embryo development.