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.

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.

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.

Addition of chlorogenic acid and caffeine during the processing of cooled boar semen / Adição de ácido clorogênico e cafeína durante o processamento do sêmen suíno resfriado

A study was conducted to evaluate the effect of chlorogenic acid (ChA) added pre-cooling and its combination with caffeine added during warming on cooled-stored boar semen parameters. Ten ejaculates were diluted in commercial extender with or without 4.5mg/ml ChA and stored at 15°C. After 0, 24 and 72 hours of storage, aliquots of these doses were taken and incubated at 37°C in the presence or absence of 8.0mM caffeine. Semen quality was evaluated after 10 and 120 minutes of incubation. The ChA increased (P <0.01) the sperm motility, viability, acrosomal integrity and the percentage of spermatozoa with high mitochondrial activity (PMHA), however, decreased (P <0.01) the malondialdehyde (MDA) concentration. Caffeine increased (P<0.05) the sperm motility, viability, PMHA and the MDA concentration and reduced (P <0.05) the acrosome integrity. When associated (ChA+caffeine), there was an increase (P <0.05) in sperm motility and viability, PMHA and acrosome integrity. The addition of ChA to the dilution medium improves the quality of the swine inseminating doses. The addition of caffeine during re-warming is only recommended when the semen is stored for prolonged periods (72h), and the inseminating dose should be used immediately after its addition.(AU)

O objetivo deste estudo foi avaliar os efeitos da adição de ácido clorogênico (ChA) antes do resfriamento e sua combinação com cafeína adicionada durante o reaquecimento sobre a qualidade do sêmen suíno resfriado. Dez ejaculados foram diluídos em diluidor comercial com adição ou não de 4,5mg/mL de ChA e armazenados a 15°C. Após zero, 24 e 72 horas de armazenamento, 10mL foram retirados e incubados a 37°C na presença ou ausência de 8,0mM de cafeína. A qualidade seminal foi avaliada após 10 e 120 minutos de incubação. O ChA aumentou (P<0,01) a motilidade, a viabilidade, a integridade acrosomal e a porcentagem de espermatozoides com alta atividade mitocondrial (PMHA), entretanto diminuiu (P<0,01) a concentração de malondialdeído (MDA). A cafeína aumentou (P<0,05) a motilidade, a viabilidade, a PMHA e a concentração de MDA e reduziu a integridade acrossomal. Quando associados (ChA+cafeína), houve aumento (P<0,05) na motilidade, na PMHA, na viabilidade e na integridade acrossomal. Conclui-se que a adição de ChA ao meio de diluição melhora a qualidade das doses inseminantes de suínos. A adição de cafeína durante o reaquecimento só é recomendada ao sêmen adicionado de ChA quando esse for armazenado por períodos prolongados (72h), devendo a dose inseminante ser utilizada imediatamente após sua adição.(AU)

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.

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.

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.

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.

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.

Effect of different light sources on reproductive anatomy and physiology of Japanese quail (Coturnix coturnix japonica).

Artificial lights are essential for controlling the reproductive tract development of birds during puberty and therefore influence reproductive quality. The aim of this study was to evaluate the effect of different light sources on reproductive anatomic and physiological characteristics of female Japanese quail (Coturnix coturnix japonica). A total of 270 birds from one day of age were housed in a masonry shed divided into six rooms with light isolation. Each room was equipped with a different type of light bulb and contained seven cages with five birds in each. The light bulbs tested were: incandescent; compact fluorescent; and light-emitting diode (LED) in the colors white, blue, red and green. The experimental design was completely randomized with six treatments and seven replications of individual birds each. The anatomic and physiological condition of the birds was evaluated at four, eight and 12 weeks of age. The white LED bulb advanced (P<0.05) the sexual maturity by one week, resulted (P<0.05) in higher live weights and greater weight and relative percentage of ovarian stroma, oviduct and ovarian tissue at eight weeks of age. Higher plasma concentrations of estradiol and lipids were also observed (P<0.05) at eight weeks under the white LED bulb. At 12 weeks of age, the magnum and isthmus folding characteristics were better (P<0.05) with the red LED bulb. In conclusion, the photostimulation with the white LED bulb was more efficient at activating the reproductive cycle, hastening the onset of sexual maturity and increasing the development of reproductive organs after puberty.

Intracellular calcium movements of boar spermatozoa during 'in vitro' capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular calcium-dependent model.

This work analysed intracellular calcium stores of boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced acrosome exocytosis (IVAE). Intracellular calcium was analysed through two calcium markers with different physico-chemical properties, Fluo-3 and Rhod-5N. Indicative parameters of IVC and IVAE were also evaluated. Fluo-3 was located at both the midpiece and the whole head. Rhod-5N was present at the sperm head. This distribution did not change in any of the assayed conditions. Induction of IVC was concomitant with an increase in both head and midpiece Ca(2+) signals. Additionally, while IVC induction was concurrent with a significant (p < 0.05) increase in sperm membrane permeability, no significant changes were observed in O2 consumption and ATP levels. Incubation of boar spermatozoa in the absence of calcium showed a loss of both Ca(2+) labellings concomitantly with the sperm's inability to achieve IVC. The absence of extracellular calcium also induced a severe decrease in the percentage of spermatozoa exhibiting high mitochondrial membrane potential (hMMP). The IVAE was accompanied by a fast increase in both Ca(2+) signalling in control spermatozoa. These peaks were either not detected or much lessened in the absence of calcium. Remarkably, Fluo-3 marking at the midpiece increased after progesterone addition to sperm cells incubated in a medium without Ca(2+) . The simultaneous addition of progesterone with the calcium chelant EGTA inhibited IVAE, and this was accompanied by a significant (p < 0.05) decrease in the intensity of progesterone Ca(2+) -induced peak, O2 consumption and ATP levels. Our results suggest that boar spermatozoa present different calcium deposits with a dynamic equilibrium among them and with the extracellular environment. Additionally, the modulation role of the intracellular calcium in spermatozoa function seems to rely on its precise localization in boar spermatozoa.

The Wnt1 ligand/Frizzled 3 receptor system plays a regulatory role in the achievement of the 'in vitro' capacitation and subsequent 'in vitro' acrosome exocytosis of porcine spermatozoa.

The aim of this work was to determine the existence of a functional Wnt/ß-catenin signaling pathway in boar spermatozoa, which would be linked with the already well-known GSK-3 signaling pathway. This was first confirmed by detecting the presence of the specific Frizzled 3 receptor in these cells. Furthermore, this signaling pathway was activated in boar spermatozoa subjected to 'in vitro' capacitation (IVC) and subsequent progesterone-induced 'in vitro' acrosome exocytosis (IVAE) by incubating cells with separate concentrations of Wnt1 ligand, the Wnt/ß-catenin signaling pathway-specific effector. Incubation with the Wnt1 ligand increased the rhythm of the time-dependent reduction in sperm viability during the achievement of both IVC and IVAE. This finding was concomitant with an increase in the percentage of spermatozoa with altered membrane fluidity and permeability determined through both merocyanine-540 and YO-PRO-1 stains. While the Wnt1 ligand did not affect total sperm motility during the achievement of the IVC, it induced a fast and transient increase in the overall motility patterns in spermatozoa subjected to IVAE. This IVAE-linked action was related to a decrease in the percentage of cells with high mitochondrial membrane potential and an increase in the percentage of cells with high intracellular Fluo-3-marked Ca(2+) content. In conclusion, our results suggest that the Wnt1 ligand-modulated Wnt/ß-catenin signaling pathway plays a relevant role in the modulation of both IVC and subsequent, progesterone-induced IVAE. Furthermore, our data indicate that the transduction pathways by which the Wnt1 ligand acts on IVC and IVAE are different, and that the Wnt/ß-catenin pathway is independent from GSK-3 activity in the achievement of IVC.

Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus.

Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) and poor (poor freezability ejaculates: PFE) freezability stallion ejaculates in other sperm parameters, including peroxide and superoxide levels, potential of mitochondrial membrane and nuclear integrity. With this purpose, a total of 24 stallion ejaculates were cryopreserved and classified into two groups (GFE vs. PFE), depending on their sperm membrane integrity and motility after freeze-thawing. From the total of 24 ejaculates, 13 were classified as GFE and the other 11 were classified as PFE. Apart from differences in sperm membrane permeability and lipid disorder after freeze-thawing, GFE presented significantly (p < 0.05) higher percentages of viable spermatozoa with high content of peroxides and of superoxides than PFE. In contrast, and despite cryopreservation of stallion spermatozoa increasing DNA fragmentation and disrupting disulphide bonds in sperm head proteins, no significant differences between GFE and PFE were seen. We can thus conclude that good and poor freezability stallion ejaculates differ in their reactive oxygen species levels after cryopreservation, but not in the damage extent on sperm nucleus.

Supplementing cryopreservation media with reduced glutathione increases fertility and prolificacy of sows inseminated with frozen-thawed boar semen.

The main aim of this work was to evaluate how supplementing freezing media with reduced glutathione (GSH) affected the 'in vivo' fertilizing ability of boar semen subjected to cryopreservation procedures. With this purpose, 12 ejaculates coming from 12 boars were cryopreserved in the presence or absence of 2 mm GSH, whereas the same number of extended ejaculates coming from the same boars was used as negative/farm controls. Eight different sperm parameters (levels of free-cysteine residues in sperm nucleoproteins, DNA fragmentation, sperm viability, acrosome-membrane integrity, intracellular peroxide and superoxide levels, and total and progressive sperm motility) were evaluated before freezing and after 30 and 240 min of thawing. In addition, a total of 180 multiparous sows were used in the field fertility trials, the females being randomly divided into three groups and inseminated with extended, frozen-thawed control or frozen-thawed semen supplemented with 2 mm GSH. The presence of GSH in the freezing media significantly (p < 0.05) increased farrowing rates and the number of total born piglets and alive born piglets, and partially counteracted the cryopreservation-induced damages inflicted on frozen-thawed spermatozoa. We can thus conclude that supplementing freezing media with 2 mm GSH greatly improves boar sperm cryopreservation technology, as it significantly improves the fertilizing ability of frozen-thawed spermatozoa.

Modulation of the biochemical composition of amniotic and allantoic fluids as a control mechanism of feline foetal development.

Amniotic fluid exerts a protective function and is an essential component for foetal development and maturation during pregnancy. However, little is known about the exact physiological functions of foetal fluids in this process as well as their biochemical composition in cats. In the present study, the biochemical composition of amniotic and allantoic fluids and maternal serum in pregnant queens was compared after performing an ovariohysterectomy. Fifteen queens were included in the study and distributed in four different groups, D(30), D(40), D(50) and D(60), according to their gestational age. Foetal fluids showed thoroughly greater concentrations of dissociate and total bilirubin, bile acids and gamma-glutamyl transferase than those of maternal serum, whereas albumin, total protein, alanine-transferase, creatine-kinase, amylase, lipase, triglycerides, cholesterol, HDL-cholesterol, and LDL-cholesterol were significantly lower, as compared to maternal serum. Other parameters like alkaline phosphatase, uric acid, creatinine, and electrolytes showed significant differences at specific stages of pregnancy, when compared to maternal serum. Lactate and cortisol significantly increased at the end of the pregnancy in foetal fluids, when compared with maternal serum. No significant differences between foetal fluids and maternal serum were observed for aspartate aminotransferase, lactate dehydrogenase, urea, phosphorus and glucose. According to our results, foetal fluids composition is not a result of simple filtration from maternal blood, the fetus being an active element involved in the production of the same and reflecting organ development and maturation.

'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.

"In vitro" capacitation and subsequent acrosome reaction are related to changes in the expression and location of midpiece actin and mitofusin-2 in boar spermatozoa.

The induction of "in vitro" capacitation (IVC) and subsequent, progesterone-induced "in vitro" acrosome reaction (IVAR) was concomitant with an increase in actin polymerization, also showing an increase in actin presence at the apical area of the midpiece. The presence of mitofusin-2, a protein involved in the regulation of the coordinated mitochondrial function, expanded from midpiece to the principal piece after IVC and IVAR. All of these results indicate that the increase of boar sperm mitochondrial activity during IVC and the first minutes of IVAR is concomitant with changes in the expression and location of both actin and mitofusin-2. Our results suggest that both actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondrial function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure itself.

Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa.

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after "in vitro" capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm.

Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm.

The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P < 0.05) increase in the free cysteine radical content (from 3.1 ± 0.5 nmol/µg protein in fresh samples to 6.7 ± 0.8 nmol/µg protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone.