The SLC7A11: sperm mitochondrial function and non-canonical glutamate metabolism.

Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter for mitochondrial functionality was studied using flow cytometry and UHPLC/MS/MS approaches. Intracellular GSH increased in the presence of cystine, but was reduced in the presence of Buthionine sulphoximine (BSO), a γ-glutamylcysteine synthetase inhibitor (P < 0.001). Inhibition of the SLC7A11 antiporter with sulfasalazine caused a dramatic drop in intracellular GSH (P < 0.001) and in the percentage of spermatozoa showing active mitochondria (P < 0.001). These findings suggest that proper functionality of this antiporter is required for the mitochondrial function of spermatozoa. We also describe that under some conditions, glutamate may be metabolized following non-conventional pathways, also contributing to sperm functionality. We provide evidences, that the stallion spermatozoa have important metabolic plasticity, and also of the relation between redox regulation and metabolic regulation. These findings may have important implications for the understanding of sperm biology and the development of new strategies for sperm conservation and treatment of male factor infertility.

The incorporation of cystine by the soluble carrier family 7 member 11 (SLC7A11) is a component of the redox regulatory mechanism in stallion spermatozoa†.

Oxidative stress is considered a major mechanism causing sperm damage during cryopreservation and storage, and underlies male factor infertility. Currently, oxidative stress is no longer believed to be caused only by the overproduction of reactive oxygen species, but rather by the deregulation of redox signaling and control mechanisms. With this concept in mind, here, we describe for the first time the presence of the soluble carrier family 7 member 11 (SLC7A11) antiporter, which exchanges extracellular cystine (Cyss) for intracellular glutamate, in stallion spermatozoa, as well as its impact on sperm function using the specific inhibitor sulfasalazine. Spermatozoa incubated with Cyss exhibited an increased intracellular GSH content compared with controls (P < 0.01): 50% in fresh extended stallion spermatozoa and 30% in frozen-thawed spermatozoa. This effect was prevented by the addition of sulfasalazine to the media. Cystine supplementation also reduced the oxidation-reduction potential of spermatozoa, with sulfasalazine only preventing this effect on fresh spermatozoa that were incubated for 3 h at 37°C, but not in frozen-thawed spermatozoa. While sulfasalazine reduced the motility of frozen-thawed spermatozoa, it increased motility in fresh samples. The present findings provide new and relevant data on the mechanism regulating the redox status of spermatozoa and suggest that a different redox regulatory mechanism exists in cryopreserved spermatozoa, thus providing new clues to improve current cryopreservation technologies and treat male factor infertility.

Pseudorabies virus infection (Aujeszky's disease) in an Iberian lynx (Lynx pardinus) in Spain: a case report.

BACKGROUND: The only natural hosts of Pseudorabies virus (PRV) are members of the family Suidae (Sus scrofa scrofa). In species other than suids infection is normally fatal. In these mammals, including carnivores, PRV typically causes serious neurologic disease. The endangered Iberian lynx (Lynx pardinus) is a wild feline endemic to south-western Europe (Iberian Peninsula). The Iberian lynx was found to be the world's most endangered felid species in 2002. In wild felines, PRV infection has only been previously reported once in a Florida panther in 1994. No seropositive lynxes have ever been found, nor has PRV been detected in dead Iberian lynxes to date. CASE PRESENTATION: We describe the first reported case of pseudorabies in an Iberian lynx (Lynx pardinus). Pseudorabies was diagnosed in a young wild Iberian lynx from Extremadura (SW Spain) by histopathological examination, immunohistochemistry, polymerase chain reaction (PCR) and sequence analysis. Gross lesions included alopecia of the ventral neck, bloody gastro-intestinal contents and congestion of the brain. Histopathological analysis showed a moderate nonsuppurative meningoencephalitis with diffuse areas of demyelination, necrotizing gastritis and enteritis of the small intestine. Pseudorabies virus (PRV) antigen was found in neuronal and non-neuronal cells of the brain, tonsils, and gastric glandular epithelial cells by immunohistochemical analysis. The presence of the virus in the brain was confirmed by nested PCR. The sequence analysis of the 146 bp fragment (from the viral glycoprotein B gene) showed that the amplified sequence matched (with 100% identity) the PRV genome. Furthermore, specific DNA from glycoprotein D and E encoding-genes was detected by conventional and real-time PCR, respectively, confirming the latter that this infection was produced by a wild-type PRV strain. CONCLUSIONS: This study supports the suspicion that PRV could infect the Iberian lynx. The detection of PRV in a dead Iberian lynx suggests that the virus may have a negative impact on the survival of endangered lynxes in the wild. However, because this is the first verified instance of lynx mortality resulting from pseudorabies, its true impact on the population is unknown.

Pyruvate enhances stallion sperm function in high glucose media improving overall metabolic efficiency.

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.1 ± 3.5 and 58.1 ± 1.9 to 73.0 ± 1.1 %; P < 0.01). Spermatozoa incubated in Tyrode's with 67 mM glucose 10 mM pyruvate maintained the viability along the incubation (64.03 ± 15.4 vs 61.3 ± 10.2), while spermatozoa incubated in 67 mM Glucose-Tyrode's showed a decrease in viability (38.01 ± 11.2, P < 0.01). 40 mM oxamate, an inhibitor of the lactate dehydrogenase LDH, reduced sperm viability (P < 0.05, from 76 ± 5 in 67 mM Glucose/10 mM pyruvate to 68.0 ± 4.3 %, P < 0.05). Apoptotic markers increased in the presence of oxamate. (P < 0.01). UHPLC/MS/MS showed that 10 mM pyruvate increased pyruvate, lactate, ATP and NAD+ while phosphoenolpyruvate decreased. The mechanisms that explain the improvement of in presence of 10 mM pyruvate involve the conversion of lactate to pyruvate and increased NAD+ enhancing the efficiency of the glycolysis.

Seminal plasma proteins as potential biomarkers for sperm motility and velocities.

Seminal plasma proteins have important roles in sperm functionality, and different mechanisms including micro-vesicle transport of proteins are involved in the regulation of sperm biology. Due to the role of seminal plasma, we hypothesized that specific proteins present in seminal plasma may be used as discriminant variables with potential to identify stallions producing different quality ejaculates; 10 fertile stallions, with different motility and velocity values (although within normal ranges) were used in this study. Motilities and velocities were studied using computer assisted sperm analysis (CASA), while protein composition of the seminal plasma was studied using UHPLC-MS/MS. Specific proteins were more abundant in samples with poorer percentages of total motility, average path velocity and circular velocity, and were: Secreted phosphoprotein 1, Fructose-bisphosphate aldolase (p = 1,95E-09; q = 0.0005) and Malate dehydrogenase 1 (p = 1,41E-11; q = 0.002), to the contrary samples with better straight-line velocity values were enriched in Glutathione peroxidase (p=0.00013; q=0.04) and Triosephosphate isomerase (p=0.00015; q=0.04).

Prenatal histomorphological development of the reticulum in fallow deer (Dama dama).

The histomorphological changes occurring in the Dama dama reticulum during prenatal development have been investigated. Twenty-five Dama dama embryos were used, from the first stages of prenatal life until birth. Differentiation of the reticulum was observed at 23% gestation. By 25% gestation the reticular wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Primary reticular crests were visible at 38% gestation. Secondary reticular crests were observed at 61% gestation. Neuroendocrine cells were detected by synaptophysin (SYP) at 35% gestation, in the lamina propria-submucosa, tunica muscularis, and serosa. Epithelial Cytokeratin-18 (CK-18) cells were observed at 35% gestation extended throughout the epithelial layers. The glial cells (vimentin -VIM- and glial fibrillary acidic protein-GFAP-markers) were discerned at 25% and 43% gestation, respectively, in myenteric and submucosal plexuses, lamina propria, muscularis mucosae, tunica muscularis, and perivascular connective tissue. The neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) markers were immunodetected at 75% and 80 gestation, respectively, in the lamina propria-submucosa, muscularis mucosae, tunica muscularis, serosa, and myenteric plexuses. The prenatal development of the fallow deer reticular mucosa evidenced a considerable precocity similar to that previously reported in goat and red deer.

Rosiglitazone in the thawing medium improves mitochondrial function in stallion spermatozoa through regulating Akt phosphorylation and reduction of caspase 3.

BACKGROUND: The population of stallion spermatozoa that survive thawing experience compromised mitochondrial functionality and accelerated senescence, among other changes. It is known that stallion spermatozoa show very active oxidative phosphorylation that may accelerate sperm senescence through increased production of reactive oxygen species. Rosiglitazone has been proven to enhance the glycolytic capability of stallion spermatozoa maintained at ambient temperature. OBJECTIVES: Thus, we hypothesized that thawed sperm may also benefit from rosiglitazone supplementation. MATERIALS AND METHODS: Thawed sperm were washed and resuspended in Tyrodes media, and the samples were divided and supplemented with 0 or 75 µM rosiglitazone. After one and two hours of incubation, mitochondrial functionality, Akt phosphorylation and caspase 3 activity were evaluated. Additional samples were incubated in the presence of an Akt1/2 inhibitor, compound C (an AMPK inhibitor) or GW9662 (an antagonist of the PPARγ receptor). RESULTS: Rosiglitazone maintained Akt phosphorylation and reduced caspase 3 activation (p<0.01), both of which were prevented by incubation in the presence of the three inhibitors. Rosiglitazone also enhanced mitochondrial functionality (P<0.01). CONCLUSION: We provide the first evidence that the functionality of frozen stallion spermatozoa can be potentially improved after thawing through the activation of pro survival pathways, providing new clues for improving current sperm biotechnology.

Prenatal histomorphological development of the rumen in Dama dama.

This work studies the morphological changes taking place in the Dama dama rumen during prenatal development using histomorphometrics, surface microstructure and immunohistochemistry analysis as well as carrying out a comparative analysis of this species with other wild (red deer) and domestic-type ruminants. A total of 25 fallow deer embryos and fetuses were used, from the first stage of prenatal life until birth. The appearance of the rumen from the primitive gastric tube was observed at 51 days of prenatal life (CRL 3 cm, 21% gestation). By 57 days (CRL 4.3 cm, 24% gestation) the ruminal wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Ruminal pillars were visible at 72 days (CRL 6 cm, 30% gestation), and by 85 days (CRL 7.2 cm, 35% gestation) ruminal papillae were starting to appear. Under scanning electron microscopy, by 80 days (CRL 7 cm, 33% gestation) small ruminal papillae were observed protruding from the surface. Morphometric results showed accelerated growth of the epithelial layer and the tunica muscularis at 180 days (75% gestation). By contrast, the growth-rate of the lamina propria and submucosa declined from the early embryonic stages until birth. The serosa maintained a steady rate of growth until birth. Neuroendocrine cells (synaptophysin) were detected at 85 days (CRL 7.2 cm CRL, 35% gestation), while glial cell markers (glial fibrillary acidic protein and vimentin) were found at 108 days (CRL 31 cm, 45% gestation) and 63 days (CRL 4.4 cm, 26% gestation) respectively. Neuropeptide Y and vasoactive intestinal polypeptide were detected immunohistochemically at 180 days (CRL 33 cm, 75% gestation) and 192 days (CRL 35 cm, 80% gestation) respectively. In comparison to other wild and domestic-type ruminants, histomorphogenesis of the rumen in Dama dama was similar to that reported in red deer and goats, but rather slower than that observed for sheep or cattle.

Comparative analysis of the merino sheep and Iberian red deer abomasum during prenatal development.

The aim of this study is to describe differences in the ontogenesis of the abomasum in sheep (domestic ruminant) and deer (wild ruminant). Histomorphometric and immunohistochemical analysis were carried out on 50 embryos and fetuses of the sheep and 50 red deer from the first prenatal stages until birth. To compare similar periods of gestation in both species, we calculate the percentages of gestation. The appearance of the abomasum was earlier in the red deer (22% gestation) than in the sheep (25% gestation). Throughout development the epithelium happened sequentially, being of the types pseudostratified to simple cylindrical. This important modification was earlier in the red deer than the sheep. At 46% gestation in red deer and 50% in sheep, gastric pits were observed on the surface of abomasal folds. Our studies suggest a close link between the initial formation of these pseudoglandular structures and the clear separation of lamina propria and submucosa separated by de muscularis mucosae. At 54% gestation in red deer and at 60% in sheep, in the bottom of these pits the first outlines of glands were distinguishable. Finally, the presence of neuroendocrine and glial cells were detected in deer at earlier stages than in sheep.

Histomorphometric study of the goat stomach during prenatal development.

The aim of this study was to perform a morphometric analysis of the different structural tissue layers of the goat stomach to study their prenatal growth from mathematical models fitted to these morphometric data. A total of 90 embryos and fetuses were used, from the early stages of prenatal life until birth. The growth rate of the gastric wall was slower than that of body length; rumen was the stomach compartment displaying slowest growth. In the three non-glandular compartments, the epithelial layer grew faster than the gastric wall itself, while the growth rate of the abomasal epithelium declined in the early stages of development. A decline in growth rate was also observed for the lamina propria and submucosa in rumen and reticulum from the early embryonic stages, whereas in omasum and abomasum these layers continued to grow as gestation progressed. The tunica muscularis displayed consistent growth in all compartments, growing faster than the gastric wall. Serosa thickness increased as gestation progressed, displaying a decline in growth-rate only in the omasum. In conclusion, the dynamics of gastric wall growth were governed by the growth rate of each of the component tissue layers.

Immunohistochemical evaluation of the goat forestomach during prenatal development.

Here we report the detection and distribution of synaptophysin (SPY), non-neuronal enolase (NNE), glial fibrillary acidic protein (GFAP), vimentin (VIM), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP) expression in the goat forestomach during prenatal development. A total of 140 embryos and fetuses were examined to evaluate protein expression from the first stage of prenatal life until birth. In all cases, SPY immunoreactivity was detected at 53 days gestation in the lamina propria-submucosa, tunica muscularis, serosa, and myenteric plexuses. Immunoreactivity to NNE was observed at 64 days gestation in the same locations as well as the epithelial layer. Glial cells were found at 64 days as indicated by signals corresponding to GFAP and VIM at 39 days. Positive staining for NPY and VIP was observed at 113, 75, and 95 days in the rumen, reticulum, and omasum, respectively, in the lamina propria-submucosa, tunica muscularis, and myenteric plexuses of each of these gastric compartments. These findings indicate possible preparation of the fetal goat forestomach for postnatal function. Compared to other ruminant species, neuroendocrine cells, glial cells and peptidergic innervations markers were detected earlier compared to sheep but at around the same stage as in deer.

Histomorphometric and immunohistochemical study of the goat omasum during prenatal development.

This work studies the morphological changes taking place in the goat omasum during prenatal development, using scanning electron microscope, light microscopy and immunohistochemical analysis. A total of 140 goat embryos and fetuses were used, from the first stages of prenatal life until birth. Differentiation of the omasum as a separate compartment of the primitive gastric tube was observed at 35 days of prenatal life ([crown-rump length (CRL)] 3 cm, 23% gestation). By 38 days (CRL 4.3 cm, 25% gestation) the omasal wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Omasal laminae appeared in the following order: primary at 38 days (CRL 4.3 cm, 25% gestation), secondary at 50 days (CRL 7.7 cm, 33% gestation), tertiary at 59 days (CRL 12 cm, 39% gestation) and quaternary at 64 days (CRL 13.5 cm, 43% gestation). Neuroendocrine cells were detected by synaptophysin (SYP) at 52 days (CRL 8 cm, 35% gestation), while glial cell markers (glial fibrillary acidic protein - GFAP, and vimentin-VIM) were observed at 64 days (CRL 13.5 cm, 43% gestation) and 38 days (CRL 4.3 cm, 25% gestation), respectively. Sympathetic and parasympathetic nerve fibers and nerve bodies were detected via neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) at 95 days (CRL 20 cm, 63% gestation). In conclusion, prenatal development of the omasum - like that of the rumen - appears to take place somewhat earlier in goats than in sheep or cattle, but at a similar stage to that reported in deer.

Histomorphometric and immunohistochemical study of the goat rumen during prenatal development.

This study sought to chart the ontogenesis of the goat rumen by histomorphometric examination, scanning electron microscopy and immunohistochemical analysis. A total of 140 goat embryos and fetuses were used, from the first stage of prenatal life until birth. The appearance of the rumen from the primitive gastric tube was observed at 35 days of prenatal life (CRL 3 cm, 23% gestation). By 38 days (CRL 4.3 cm CRL, 25% gestation) the ruminal wall comprised three layers: an internal epithelial layer, a middle layer of pluripotential blastemic tissue and an external layer or serosa. Ruminal pillars were visible at 46 days (CRL 6 cm, 30% gestation), and by 76 days (CRL 18 cm, 50% gestation) ruminal papillae were starting to appear. Under scanning electron microscopy, by 50 days (CRL 7.7 cm, 33% gestation) small ruminal papillae were observed protruding from the surface. Finally, neuroendocrine cells (synaptophysin, SYP) were detected at 53 days (CRL 9 cm CRL, 35% gestation), while glial cell markers (glial fibrillary acidic protein--GFAP, and vimentin-VIM) were found at 108 days (CRL 31 cm, 72% gestation) and 39 days (CRL 4.4 cm, 26% gestation), respectively. Neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were detected immunohistochemically at 113 days (CRL 33 cm, 75% gestation) and 120 days (CRL 35 cm, 80% gestation), respectively. In conclusion, histomorphogenesis of the rumen in goats was similar to that reported in deer, but rather slower than observed for sheep or cattle.

Ontogenesis of the rumen: a comparative analysis of the Merino sheep and Iberian red deer.

The aim of this study is to describe differences in the ontogenesis of the rumen in the sheep (domestic ruminant) and deer (wild ruminant). A total of 50 embryos and fetuses of Merino sheep and 50 of Iberian deer were used, from the first stages of prenatal life until birth. For the study, the animals were divided into five experimental groups according to the most relevant histological characteristics. The appearance of the rumen from the primitive gastric tube was earlier in the sheep (22% gestation, 33 days) than in the deer (25% gestation, 66 days). In both cases it displayed a primitive epithelium of a stratified, cylindrical, non-ciliary type. At around 28% gestation in the sheep (42 days) and 26% (67 days) in the deer, the rumen was configured of three clearly-differentiated layers: internal or mucosal, middle or muscular and external or serosal. In both species the stratification of the epithelial layer was accompanied by modifications in its structure with the appearance of the ruminal pillars and papillae. The pillars appeared before the papillae and the appearance of both structures was always earlier in the deer (pillars: 70 days, 27% gestation; papillae: 97 days, 36% gestation) than in the sheep (pillars: 42 days, 28% gestation; papillae: 57 days, 38% gestation). The outlines of the ruminal papillae appeared as evaginations of the basal zone toward the ruminal lumen, dragging in their formation the basal membrane, the lamina propria and the submucosa. The tegumentary mucosa of the rumen was without secretion capability in the first embryonic phases. From 67 days (26% gestation) the neutral mucopolysaccharides appeared in the deer and at 46 days (30% gestation) in the sheep. In both cases they continued to decrease until birth, this diminution being more pronounced in the deer. Finally, the presence of neuroendocrine and glial cells was detected in the deer at earlier stages than in the sheep.

Arginase I induction during Leishmania major infection mediates the development of disease.

In a previous work, we demonstrated that the induction of arginase I favored the replication of Leishmania inside macrophages. Now we have analyzed the differential expression of this enzyme in the mouse model of L. major infection. Ours results show that arginase I is induced in both susceptible and resistant mice during the development of the disease. However, in BALB/c-infected tissues, the induction of this protein parallels the time of infection, while in C57BL/6 mice, the enzyme is upregulated only during footpad swelling. The induction of the host arginase in both strains is mediated by the balance between interleukin-4 (IL-4) and IL-12 and opposite to nitric oxide synthase II expression. Moreover, inhibition of arginase reduces the number of parasites and delays disease outcome in BALB/c mice, while treatment with l-ornithine increases the susceptibility of C57BL/6 mice. Therefore, arginase I induction could be considered a marker of disease in leishmaniasis.