Characterisation of sperm piRNAs and their correlation with semen quality traits in swine.

Piwi-interacting RNAs (piRNAs) are a class of non-coding RNAs that are essential in the transcriptional silencing of transposable elements and warrant genome stability in the mammalian germline. In this study, we have identified piRNAs in porcine sperm using male germline and zygote datasets from human, mice, cow and pig, and evaluated the relation between their abundances and sperm quality traits. In our analysis, we identified 283 382 piRNAs, 1355 of which correlated with P ≤ 0.01 to at least one semen quality trait. Fifty-seven percent of the correlated piRNAs mapped less than 50 kb apart from any other piRNA in the pig genome. Furthermore, piRNA location was significantly enriched near long interspersed nuclear elements. Moreover, some of the significant piRNAs mapped within or close to genes relevant for fertility or spermatogenesis such as CSNK1G2 and PSMF1.

Chlorogenic acid improves the quality of boar semen subjected to cooled storage at 15°C.

The aim of this study was to investigate the effect of chlorogenic acid (ChA) in boar semen stored at 15°C. Twelve ejaculates were processed into insemination doses at different concentrations of ChA (0.0, 1.5, 3.0, 4.0 and 6.0 mg/ml) or vitamin E (200 µl/ml) as positive control. Semen was analysed after 0, 24 and 72 hr of storage. ChA improved (p < .05) sperm motility, acrosome integrity and mitochondrial activity in all periods of storage. Furthermore, after 24 hr of storage, ChA above 1.5 mg/ml supported the sperm viability until 120 min after reheating (p < .05). Both ChA and vitamin E were similarly efficient in increasing the antioxidant capacity of semen, reducing the malondialdehyde levels before and after 72 hr of storage (p < .05). However, with 72 hr of storage, ChA at 3.0 mg/ml improved the mitochondrial activity over vitamin E (p < .05). In conclusion, results suggest that the concentration of 3.2 mg/ml of ChA is the best for semen stored for up to 24 hr. However, for semen stored for a longer period, 6.0 mg/ml or more should be used.

Aquaporins in the male reproductive tract and sperm: Functional implications and cryobiology.

Aquaporins (AQPs) play a vital role for the transport of water and solutes across cell membranes. Classification of these ubiquitous proteins into three categories (orthodox AQPs, aquaglyceroporins and superaquaporins) is based on their sequence similarity and substrate selectivity. In the male reproductive tract of mammals, most AQPs (except AQP6 and AQP12) are found in different organs (including testis, efferent ducts and epididymis). AQP1 and AQP9 are the most abundant AQPs in the efferent ducts and epididymis and play a crucial role for the secretion/reabsorption dynamics of luminal fluid during sperm transport and maturation. AQP3, AQP7, AQP8 and AQP11 are the most abundant AQPs in sperm and are involved in the regulation of their volume, which is required for the differentiation of spermatids into spermatozoa during spermatogenesis, as well as in sperm transit along environments of different osmolality (male and female reproductive tracts). While different studies conducted in oocytes and embryos have demonstrated that AQPs are important for cryotolerance, data in sperm are scarce. At present, mounting evidence indicates that AQP3, AQP7 and AQP11 are involved in the sperm response to variations of osmolality and to freeze-thawing procedures. All these studies contribute to understand the physiology of both male reproductive tract and sperm, and open up new research ventures on the improvement of sperm cryopreservation protocols.

First evidence for the presence of aquaporins in stallion sperm.

Aquaporins (AQPs) are transmembrane proteins found in all cells and are responsible for the transport of water and small solutes. While these proteins have been found in the spermatozoa of humans, rodents, pigs and cattle, where not only do they play a role for the regulation of sperm volume but are also related with the sperm resilience to withstand freeze-thawing procedures, their presence in stallion sperm is yet to be reported. Therefore, the objectives of this work were as follows: (i) to determine whether AQP3, AQP7 and AQP11 are present in stallion sperm and (ii) to investigate whether the relative amounts of these three AQPs play any role in the cryopreservation success. With this purpose, a total of five ejaculates from healthy stallions were collected. Evaluation of sperm quality and immunoblotting against these three proteins were performed before and after cryopreservation. Immunoblots confirmed the presence of AQP3, AQP7 and AQP11 in all examined samples. Subsequently, ejaculates were classified as GFE (good) and PFE (poor freezability ejaculates), according to their sperm viability and motility at 0 and 2 hr post-thaw. Relative AQP3 and AQP11 contents in stallion fresh semen were found to be significantly (p < .05) higher in GFE than in PFE. In conclusion, the current study has confirmed, for the first time, the presence of AQP3, AQP7 and AQP11 in stallion sperm. In addition, despite preliminary, our results suggest that AQP3 and AQP11 are involved in the resilience of stallion sperm to withstand cryopreservation. Ongoing research is aimed at increasing the sample size and includes immunolocalization studies.

Voltage-dependent anion channel 2 is involved in in vitro capacitation of boar sperm.

Ion channels play an important role during sperm capacitation allowing the transport through plasma and mitochondrial membranes of specific molecules that are essential for the achievement of this physiologic status. Given that voltage-dependent anion channel 2 (VDAC2) is present in boar spermatozoa and is known to be involved in calcium transport in somatic cells, this study aimed at determining whether it is implicated in sperm capacitation and the acrosome reaction. With this purpose, boar spermatozoa were capacitated in vitro for 4 hr, and acrosome reaction was induced with progesterone for a further hour, with or without the presence of two VDAC2-inhibitors (erastin and olesoxime) at two different concentrations (10 and 100 µM). At different time points (0, 120, 240, 270 and 300 min), an aliquot was taken and sperm motility, membrane integrity and lipid disorder were evaluated using computer-assisted sperm analysis and flow cytometry. The addition of the two inhibitors resulted in opposite effects. While erastin 100 µM reduced the percentage of non-capacitated spermatozoa, the presence of olesoxime at the same concentration prevented the increase in membrane lipid disorder, which is a feature of sperm capacitation. Such prevention was concomitant with a maintaining effect on sperm membrane integrity evaluated through SYBR14/PI. Our results suggest that VDAC2 is involved in the regulation of sperm capacitation, despite the fact that the mechanisms through which erastin and olesoxime act are different.

'In vitro' capacitation and further 'in vitro' progesterone-induced acrosome exocytosis are linked to specific changes in the expression and acrosome location of protein phosphorylation in serine residues of boar spermatozoa.

The aim of this study is to determine changes in the expression and location of protein serine phosphorylation (pSer) during 'in vitro' capacitation (IVC) and 'in vitro' acrosome exocytosis (IVAE) in boar spermatozoa. This was performed in both mono- and bi-dimensional analyses of protein expression through Western blot, as well as through immunocytochemistry. Furthermore, IVC was induced through incubation in an IVC medium, and afterwards, progesterone-induced IVAE was performed. The mono-dimensional Western blot analysis showed the presence of a predominant pSer band of approximately 70-75 kDa, which was accompanied by fainter bands, especially three with molecular weights of approximately 50, 35 and 32 kDa. Neither IVC nor IVAE significantly modified this pattern. Bi-dimensional analyses showed a more complex pattern, with at least five protein clusters. The attainment of IVC caused the disappearance of the proteins with the highest molecular weight concomitantly with the appearance of pSer proteins of 75-kDa/pI 9.5 and 80-kDa/pI 10. The induction of IVAE caused the appearance of new pSer proteins of a 75-kDa/pI 6.5-7.5 and 75-kDa/pI 10. Immunocytochemistry showed that the main pSer expression in boar expression before the attainment of IVC was located at the midpiece. The IVC induced the appearance of acrosomal pSer, which was greatly increased during IVAE. Our results indicate that the changes in serine protein phosphorylation associated with IVC and IVAE comprise not only the appearance of specific phosphorylated proteins, such as the pSer-75 kDa, but also changes in pI and displacements in the sperm location of phosphorylated proteins, like the specific acrosomal pSer signal induced during IVC.

The onset of age-related benign prostatic hyperplasia is concomitant with increased serum and prostatic expression of VEGF in rats: Potential role of VEGF as a marker for early prostatic alterations.

The onset of age-related benign prostate hyperplasia (BPH) is linked with changes in the expression of specific prostatic chemokines. The aim of this work was to characterize those most relevant changes through the simultaneous analysis of 34 chemokines in both prostatic tissue and serum in rats at different ages with the aim to identify clinically workable parameters for the detection of early prostatic alterations. The study included 28 healthy Sprague-Dawley male rats that were distributed in four groups, 1 month-old (prepuberal; n = 7), 3 months-old (young; n = 7), 6 months-old (mature; n = 7) and 12 months-old (elder; n = 7). Chemokines were analyzed through a commercial mini-array system specially designed for rat tissues. Serum testosterone levels and prostatic histological status were also evaluated. Histological lesions indicative of BPH were detected in three mature rats and in all elder ones. Mini-arrays from prostatic tissue showed that young animals had an overall decreased expression of most of the analyzed chemokines when compared with prepuberal rats, with the exception of agrin, which showed a significant (P < 0.05) increase (100.0 ± 1.3, arbitrary units in prepuberal rats vs.148.2 ± 4.1, arbitrary units in young ones). Older animals showed further specific changes in 4 out 34 analyzed chemokines, namely agrin, platelet-derived growth factor (PDGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and vascular endothelial growth factor (VEGF). Additionally, elder rats showed the lowest intensity levels of agrin combined with the highest ones for PDGF, TIMP1 and VEGF when compared with all other groups. Finally, a significant increase of serum VEGF was detected in elder, BPH-affected rats when compared with young ones. Results indicated that the onset of both rat puberty and BPH would be related with specific changes in the prostatic expression of chemokines such as VEGF. Otherwise, the observed changes in serum VEGF levels could suggest the future possible utilization of serum VEGF levels to detect early pathological prostatic processes.

Rat age-related benign prostate hyperplasia is concomitant with an increase in the secretion of low ramified α-glycosydic polysaccharides.

This work analysed the expression of prostate polysaccharides in rats with age-related benign prostatic hyperplasia (BPH) for a better understanding of the possible relationship between prostate polysaccharides secretion and BPH onset. For this, prostatic glands from 1 month-old, 3 months-old, 6 months-old and 12 months-old Sprague-Dawley rats were processed in order to identify their overall polysaccharide content. Additionally, serum testosterone was also determined. One-month old rats showed significantly (P < 0.05) lower testosterone levels (0.77 ng/mL±0.12 ng/mL) compared with the other groups, which showed no significant difference among them. PAS staining showed positive polysaccharides markings in both the prostatic lumen and inside of luminal prostatic cells in all groups. Semiquantitative analysis of intraluminal PAS showed that one month-old rats had significantly (P < 0.005) lower PAS intensity when compared with all other groups (100.0 ± 0.5, arbitrary units vs. 107.3 ± 0.6, arbitrary units in 3 months-old ones), whereas 12 months-old ones showed significantly (P < 0.005) higher values when compared with all other groups (133.6 ± 3.5, arbitrary units in 12 months-old rats vs. 108.6 ± 1.4, arbitrary units in 6 months-old ones). The PAS + content practically disappeared when tissues were pre-incubated with either α-amylase or amyloglucosidase, regardless of a previous incubation with proteinase K. Incubation of prostate extracts from 12 months-old rats for 2 h with α-amylase yielded a significantly higher amount of free glucose (1.47 nmol/mg protein±0.23 nmol/mg protein vs. 0.32 nmol/mg protein±0.01 nmol/mg protein in untreated extracts). Similar results were obtained when extracts were pre-incubated with amyloglucosidase. Contrarily, pre-incubation with N-glycosidase induced a significantly (P < 0.05), much lower increase of free glucose. Pre-treatment with proteinase K did not significantly modify these results, which indicate that BPH is related to an increase in the secretion of low ramified ductal α-glycosydic polysaccharides that were not protected against lysis by any type of protein protective core. These changes seem to not be related with concomitant variations in serum testosterone levels.

Glucose and fructose as functional modulators of overall dog, but not boar sperm function.

The main aim of the present work was to test the effects of glucose and fructose on the phosphorylation levels of proteins linked to the control of overall sperm function in two species with very different metabolic characteristics, dog and boar. Incubation of dog spermatozoa with 10mM glucose increased serine phosphorylation of proteins related to cell cycle and signal transduction including cyclins B and E, Cdk2, Cdk6, Cdc6, PYK2, c-kit, Raf-1, TRK and several protein phosphatases. Incubation of dog spermatozoa with 10mM fructose decreased serine phosphorylation levels of cyclins B and D3, Cdk1/Cdc2, Cdk2, Cdk6, Akt, PI3 kinase, ERK-1 and protein kinase C. Incubation of boar spermatozoa with glucose or fructose did not modify any of the phosphorylation patterns studied. Given that one important difference between dog and boar spermatozoa is the presence of glucokinase (GK) in dog but not in boar, GK-transfected COS7 cells were incubated with either 10mM glucose or 10mM fructose. Incubation of GK-transfected cells with fructose decreased serine phosphorylation of cyclin A, ERK-2 and Hsp-70. In contrast, incubation of control COS7 cells with fructose increased serine phosphorylation of Cdk6, Cdk1/Cdc2, protein kinase C and Hsp-70. Incubation with glucose did not induce any significant effect. Our results indicate that monosaccharides act as signalling compounds in dog spermatozoa after ejaculation through changes in the phosphorylation levels of specific proteins. One of the factors that may be related to the action of sugars is the equilibrium of the total sperm hexokinase activity, in which the presence or absence of GK appears to be relevant.

Partial foetal retention following aglepristone treatment in a bitch.

This short communication describes the case of partial foetal retention in an 18-month-old female French bulldog following induction of abortion owing to an undesired mating. Abortion was induced with aglepristone administered in two consecutive protocols of a dual injection 1 day apart. After failure of the first treatment to achieve abortion, 15 days later, a second treatment was administered. Delivering of aborted foetus occurred 2 days after the last administration. Five weeks after the abortion, the female showed a weak haemorrhagic vaginal discharge. On ultrasound examination, the presence of uterine wall distension as well as a puppy skull inside the uterus was observed. This clinical case makes clear that although aglepristone is a very reliable drug, follow-up of the female during treatment and in the immediate post-partum is necessary to ensure a good outcome.

'In vitro' capacitation and acrosome reaction are concomitant with specific changes in mitochondrial activity in boar sperm: evidence for a nucleated mitochondrial activation and for the existence of a capacitation-sensitive subpopulational structure.

The main scope of this manuscript is to analyse the dynamics of mitochondrial activity in boar sperm subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome reaction (IVAR). This was determined after analysis of the rhythm of O(2) consumption and concomitant changes in the mitochondria activity-specific JC-1 staining. Results showed that IVC, and especially IVAR, was concomitant with a peak in O(2) consumption (from 1.61 ± 0.08 nmol O(2)/min/10(7) viable sperm at 0 h of incubation to 2.62 ± 0.12 nmol O(2) /min/10(7) viable sperm after 5 min of IVAR induction). These results were accompanied by parallel changes in the mean intensity of JC-1 staining. Based on JC-1, mitochondrial activation followed a nucleated pattern, with specific, activation starting points at the midpiece from which mitochondrial activation was spread. Moreover, four separate sperm subpopulations were detected following the JC-1 orange-red/green ratio, and the observed changes in the mean JC-1 staining during IVC and IVAR were related to concomitant changes in both the orange-red/green JC-1 ratio and the percentage of sperm included in each subpopulation. All of these results indicate that IVC and the first minutes of IVAR are accompanied by a progressive increase in mitochondrial activity, which reached a peak coincidental with the achievement of IVAR. Moreover, results suggest the presence of separate sperm subpopulations, which show a different mitochondrial sensitivity to IVC and IVAR. Finally, mitochondrial activation, at least under JC-1 staining, seems to originate in concrete nucleation points at the midpiece, thus suggesting thus a well-coordinated pattern in boar-sperm mitochondrial activity modulation.

The effect of low-level laser irradiation on dog spermatozoa motility is dependent on laser output power.

Biological tissues respond to low-level laser irradiation and so do dog spermatozoa. Among the main parameters to be considered when a biological tissue is irradiated is the output power. We have studied the effects on sperm motility of 655 nm continuous wave diode laser irradiation at different output powers with 3.34 J (5.97 J/cm(2)). The second fraction of fresh dog sperm was divided into five groups: control, and four to be irradiated with an average output power of 6.8 mW, 15.4 mW, 33.1 mW and 49.7 mW, respectively. At 0 min and 45 min after irradiation, pictures were taken and a computer aided sperm analysis (CASA) performed to analyse different motility parameters. The results showed that different output powers affected dog semen motility parameters differently. The highest output power showed the most intense effects. Significant changes in the structure of the motile sperm subpopulation were linked to the different output powers used.

Effects of matrix filtration of low-quality boar semen doses on sperm quality.

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 +/- 0.5 cm), CM Sephadex (length 5 +/- 0.5 cm), glass wool (length 2 +/- 0.5 cm) or glass bead (length 10 +/- 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca((R)) 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l-lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l-lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.

Effects of freezing/thawing on motile sperm subpopulations of boar and donkey ejaculates.

The main aim of this study is to assess the influence of freeze/thawing on motile sperm subpopulations in ejaculates from two phylogenetically different mammalian species, boar and donkey. Our results indicate that, whereas boar and donkey sperm respond very differently in their mean motion characteristics to freezing/thawing, this process did not change the existence of a 4-subpopulations structure in the ejaculates in either species when these subpopulations were defined by taking values of curvilinear velocity (VCL) as reference. Moreover, the freezing/thawing-linked changes in mean sperm-motion characteristics in both boar and donkey semen were especially due to changes in the proportion among each concrete subpopulation. In this way, the freezing/thawing-induced mean increase in motion characteristics observed in boar sperm was a result of the decrease in the percentage of sperm in Subpopulation 1 (from 53.9%+/-4.7% to 31.2%+/-3.9% after thawing) and a concomitant increase of sperm from Subpopulations 3 (from 13.3%+/-2.5% to 32.6%+/-3.9% after thawing) and 4 (from 3.4%+/-0.9% to 8.0%+/-1.1% after thawing). On the contrary, changes in mean motility of frozen/thawed donkey sperm were linked to an increase in the percentage of sperm in Subpopulation 1 (from 31.5%+/-4.3% to 58.8%+/-4.9% after thawing) and a concomitant decrease of sperm from Subpopulations 3 (from 32.4%+/-3.2% to 6.6%+/-1.8% after thawing) and 4 (from 12.2%+/-2.5% to 7.3%+/-1.9% after thawing). In conclusion, our results seem to indicate that motility changes induced by the freezing/thawing protocol are linked to concomitant changes in both the specific parameters and, more importantly, to the specific percentage of each of the motile sperm subpopulations. These changes did not affect the overall proportion of motile sperm present in both boar and donkey, which is conserved despite the detrimental effect caused by freezing/thawing in both species. Finally, the presence of some kind of motile sperm subpopulations structure has been described in mammalian species with a very great phylogenetic distance, thus suggesting that this structure could play some role in the maintenance of the overall function of mammalian ejaculates.

Dynamics of motile-sperm subpopulation structure in boar ejaculates subjected to "in vitro" capacitation and further "in vitro" acrosome reaction.

Incubation of diluted boar sperm from fresh ejaculates in a previously established "in vitro" capacitation medium induced a significant, time-dependent increase in several mean parameters of sperm motility, such as curvilinear velocity (VCL), linear velocity (VSL), mean velocity (VAP), linearity coefficient (LIN), straightness coefficient (STR) and wobble coefficient (WOB). Furthermore, motile boar-sperm semen samples were structured in four definite subpopulations. Subpopulation 1 showed the lowest values of VCL, VSL and VAP and also low values of linearity. Subpopulation 2 showed the second lowest values of VCL and VAP and higher values of LIN and STR. Subpopulation 3 was characterized by high values of velocity and low values of linearity. Finally, Subpopulation 4 was characterized by high values of velocity and linearity. "In vitro" capacitation and further acrosome reaction induced changes in the motility characteristics of each subpopulation as well as in their percentage distribution, Subpopulations 3 and 4 being those that showed the most significant changes. However, despite these changes, the observed, overall four-subpopulation structure was firmly maintained during the entire "in vitro" capacitation and acrosome-reaction process. Our results suggest that capacitation-induced motility changes are related to specific changes in the percentage of each motile-sperm subpopulation in the ejaculate without losing the overall, specific four-subpopulation structure. In this way, the maintenance of a four-subpopulation structure seems to be important in the control of the whole ejaculate physiology.

Freeze-thawing induces alterations in the protamine-1/DNA overall structure in boar sperm.

The main aim of this work was to test the effects that freeze-thawing could have on the overall nuclear structure of boar sperm. This was done by analyzing both the DNA fragmentation and the protamine-1-DNA interaction of the boar-sperm nucleus. Our results indicate that freezing-thawing did not induce a significant degree of DNA fragmentation, as manifested through both the Sperm-Sus-Halomax stain and a random primed analysis prior to partial DNA digestion with enzymes BamHI-HinDIII. On the other hand, freeze-thawing induced significant changes in the protamine-1-DNA interaction, as revealed through both Western blot analysis and immunocytochemistry for protamine-1. These alterations caused, in turn, significant changes in the overall nuclear structure of boar sperm after thawing. Protamine-1-DNA alterations started to be apparent during the cooling phase of the freeze-thawing protocol. These results imply that one of the alterations that may be responsible for the loss of fertilizing ability of boar sperm after freeze-thawing may be an alteration in the correct formation of the overall nuclear structure, which, in turn, would induce alterations in the correct formation of the first nuclear structure after oocyte penetration.

The achievement of boar sperm in vitro capacitation is related to an increase of disrupted disulphide bonds and intracellular reactive oxygen species levels.

The aim of this work was to determine the relationship of intracellular reactive oxygen species (ROS) and the disulphide bonds established between sperm proteins with the achievement of capacitation in boar spermatozoa. With this purpose, spermatozoa were incubated in a specifically designed in vitro capacitation medium (CM) in the presence or absence of reduced glutathione (GSH). Incubation of boar spermatozoa in CM for 4 h significantly (p < 0.05) increased free cysteine residues, which is a marker of disrupted disulphide bonds, and also intracellular ROS levels. The addition of GSH to the medium prevented most capacitation-like changes in sperm motility, membrane lipid disorder, mitochondrial membrane potential, intracellular calcium levels and localization of tyrosine-phosphorylated proteins (pTyr), but not in tyrosine phosphorylation of P32. These effects were accompanied by the inhibition of the ability of sperm cells to trigger the acrosome exocytosis in response to progesterone. When GSH was added together with progesterone after 4 h of incubation, acrosome exocytosis was not altered, but the subsequent decrease in intracellular calcium observed in controls cells was inhibited. Furthermore, co-incubation of oocytes with spermatozoa previously incubated in CM in the presence of GSH for 4 h significantly (p < 0.05) increased the number of spermatozoa attached to the oocyte surface but decreased normal fertilization rates. Our results suggest that boar sperm capacitation is related to an increase in disrupted disulphide bonds and intracellular ROS levels and that both events are related to the regulation of hyperactivated motility, intracellular calcium dynamics, sperm binding ability to the oocyte and achievement of proper nuclear decondensation upon oocyte penetration.

Vanadate treatment restores the expression of genes for key enzymes in the glucose and ketone bodies metabolism in the liver of diabetic rats.

Oral administration of vanadate to diabetic streptozotocin-treated rats decreased the high blood glucose and D-3-hydroxybutyrate levels related to diabetes. The increase in the expression of the P-enolpyruvate carboxykinase (PEPCK) gene, the main regulatory enzyme of gluconeogenesis, was counteracted in the liver and the kidney after vanadate administration to diabetic rats. Vanadate also counteracted the induction in tyrosine aminotransferase gene expression due to diabetes and was able to increase the expression of the glucokinase gene to levels even higher than those found in healthy animals. Similarly, an induction in pyruvate kinase mRNA transcripts was observed in diabetic vanadate-treated rats. These effects were correlated with changes on glucokinase and pyruvate kinase activities. Vanadate treatment caused a decrease in the expression of the liver-specific glucose transporter, GLUT-2. Thus, vanadate was able to restore liver glucose utilization and block glucose production in diabetic rats. The increase in the expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCoAS) gene, the key regulatory enzyme in the ketone bodies production pathway, observed in diabetic rats was also blocked by vanadate. Furthermore, a similar pattern in the expression of PEPCK, GLUT-2, HMGCoAS, and the transcription factor CCAAT/enhancer-binding protein alpha genes has been observed. All of these results suggest that the regulation of the expression of genes involved in the glucose and ketone bodies metabolism could be a key step in the normalization process induced by vanadate administration to diabetic rats.

Expression of the GM-CSF receptor in ovine spermatozoa: GM-CSF effect on sperm viability and motility of sperm subpopulations after the freezing-thawing process.

The granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine capable of stimulating proliferation, maturation and function of haematopoietic cells. Receptors for this cytokine are composed of two subunits, alpha and beta, and are expressed in myeloid progenitors and mature mononuclear phagocytes, monocytes, eosinophils and neutrophils, as well as in other non-haematopoietic cells. We have previously demonstrated that bull spermatozoa express functional GM-CSF receptors that signal for increased glucose and vitamin-C uptake and enhance several parameters of sperm motility in the presence of glucose or fructose substrates. In this study, we have analyzed the expression of GM-CSF receptors in ovine spermatozoa and studied the effect of GM-CSF on sperm viability and motility after the freezing-thawing process. Immunolocalization and immunoblotting analyses demonstrated that ovine spermatozoa (Xisqueta race) expressed GM-CSF receptors. In addition, GM-CSF partially counteracted the impairing action of freezing/thawing on the percentage of total motility, as well as on the specific motility patterns of each of the separate, motile sperm subpopulations of ram ejaculates subjected to this protocol. These results suggest that GM-CSF can play a role in the resistance of ram spermatozoa to environmental thermal stress.

Relationship of aquaporins 3 (AQP3), 7 (AQP7), and 11 (AQP11) with boar sperm resilience to withstand freeze-thawing procedures.

Cryopreservation is the most suitable method to preserve boar spermatozoa over long-term storage. However, freeze-thawing protocols inflict extensive damage to sperm cells, reducing their viability and compromising their fertilizing ability. In addition, high individual variability is known to exist between boar ejaculates, which may be classified as of good (GFE) or poor (PFE) freezability. While conventional spermiogram parameters fail to predict sperm cryotolerance in fresh spermatozoa, high levels of certain proteins, also known as freezability markers, have been found to be related to the sperm resilience to withstand freeze-thawing procedures. In this context, the hypothesis of this study was that aquaporins AQP3, AQP7, and AQP11 could be linked to boar sperm cryotolerance. Twenty-nine ejaculates were evaluated and subsequently classified as GFE or PFE based upon their sperm viability and motility at post-thawing. Fourteen ejaculates resulted to be GFE, whereas the other fifteen were found to be PFE. Relative abundances of AQP3, AQP7, and AQP11 and their localization patterns were evaluated in all fresh and frozen-thawed ejaculates through immunoblotting and immunocytochemistry. Prior to cryopreservation, relative amounts of AQP3 and AQP7 were found to be significantly (p < 0.05) higher in GFE than in PFE. In contrast, no significant differences (p > 0.05) between freezability groups were found for AQP11, despite GFE tending to present higher levels of this protein. The localization of AQP7, but not that of AQP3 or AQP11, was observed to be affected by cryopreservation procedures. In conclusion, these results suggest that AQP3 and AQP7 are related to boar sperm cryotolerance and may be used as freezability markers.