Tyrosine phosphorylation is not a relevant mechanism to modulate aquaporin 2 activity in gestational queen endometrium and placenta.

Aquaporins have been shown to be regulated by phosphorylation of serine residues, but the possible role of tyrosine residues phosphorylation has not been evaluated. Changes in the localization of aquaporin 2 (AQP2) in the queen endometrium have been related to serum progesterone levels. The aim of this study was to determine whether these AQP2-localization changes are mediated by variations in its tyrosine phosphorylation levels. Twelve queens were included in the study and divided into (a) non-macroscopically pregnant with low levels of progesterone; (b) non-macroscopically pregnant with high levels of progesterone; (c) 30 days of pregnancy; and (d) 60 days of pregnancy. Samples from endometrium and placental transference zone were obtained, immunoprecipitated and analysed by immunoblotting to determine the abundance of AQP2 and its relative levels of tyrosine phosphorylation. No significant differences in the tyrosine phosphorylation levels of immunoprecipated-AQP2 were observed between groups. We can thus conclude that changes in the localization of AQP2 in the queen endometrium are not modulated by tyrosine phosphorylation.

Reduced glutathione and procaine hydrochloride protect the nucleoprotein structure of boar spermatozoa during freeze-thawing by stabilising disulfide bonds.

One important change the head of boar spermatozoa during freeze-thawing is the destabilisation of its nucleoprotein structure due to a disruption of disulfide bonds. With the aim of better understanding these changes in frozen-thawed spermatozoa, two agents, namely reduced glutathione (GSH) and procaine hydrochloride (ProHCl), were added at different concentrations to the freezing media at different concentrations and combinations over the range 1-2mM. Then, 30 and 240 min after thawing, cysteine-free residue levels of boar sperm nucleoproteins, DNA fragmentation and other sperm functional parameters were evaluated. Both GSH and ProHCl, at final concentrations of 2mM, induced a significant (P<0.05) increase in the number of non-disrupted sperm head disulfide bonds 30 and 240 min after thawing compared with the frozen-thawed control. This effect was accompanied by a significant (P<0.05) decrease in DNA fragmentation 240 min after thawing. Concomitantly, 1 and 2mM GSH, but not ProHCl at any of the concentrations tested, partially counteracted the detrimental effects caused by freeze-thawing on sperm peroxide levels, motility patterns and plasma membrane integrity. In conclusion, the results show that both GSH and ProHCl have a stabilising effect on the nucleoprotein structure of frozen-thawed spermatozoa, although only GSH exerts an appreciable effect on sperm viability.

Evaluation of the Probiotic In Vitro Potential of Lactic Acid-Producing Bacteria from Canine Vagina: Possible Role in Vaginal Health.

Lactic acid-producing bacteria (LAB) are being widely studied due to their probiotic potential. The aim of the present study was to determine and identify the presence of LAB from canine vaginal samples, as well as to evaluate their probiotic in vitro potential. Ninety-four bitches were included in the study. Vaginal samples were obtained by means of a sterile swab and streaked on Man Rogosa Sharpe agar plates. A total of 100 LAB strains were obtained and submitted to Gram stains and basic biochemical tests, which included catalase, oxidase and haemolysis tests. Thirteen strains belonging to the genera Lactobacillus (n = 10), Lactococcus (n = 2) and Pediococcus (n = 1) were selected as potential probiotics and further subjected to evaluation of resistance to gastrointestinal conditions (pH, lysozyme, bile salts and hydrogen peroxide) and safety and efficacy in vitro (resistance to antibiotics and antimicrobial capacity). Only three strains, one Lactobacillus lactis and two Lactobacillus plantarum, accomplished the requirements for being considered as potential in vitro probiotics.

Endometrial Status in Queens Evaluated by Histopathology Findings and Two Cytological Techniques: Low-Volume Uterine Lavage and Uterine Swabbing.

Endometritis is associated with fertility problems in many species, with endometrial biopsy being the main diagnostic tool. In feline queens, the reduced size of the uterus may make it difficult to obtain representative diagnostic samples. Endometrial cytology may represent a valuable diagnostic tool for evaluating the health status of the endometrium in queens. Fifty domestic shorthair queens were included and divided into two cytological diagnostic technique groups, the uterine lavage (UL; n = 28) and uterine swabbing (US; n = 22) groups. Cytological results were compared with histopathological and bacteriological information. Changes in the histopathological patterns were also evaluated and compared with progesterone levels to confirm previous published data. Furthermore, the results from both cytological sampling methods were compared to evaluate the utility of each method. Endometritis was ruled out in all queens by means of histology and microbiology. Leukocyte counts and red blood cell/endometrial cell ratios were significantly higher in US than UL samples. Additionally, UL sampling is less affected by blood contamination and cells are better preserved. The combination of endometrial cytology and uterine culture might be useful for evaluating the endometrial characteristics in queens. The UL evaluation method is more representative of the actual endometrial status than the US technique.

Urine glucose concentration: A useful parameter as a surrogate for glycaemia on the first day of life in canine neonates.

INTRODUCTION: Hypoglycaemia is a well-known risk factor in neonatal puppies and kittens; glycaemia control is crucial during the first days of life. Kidneys immaturity provokes the presence of physiological glycosuria during the first 2-3 weeks of life in small animals. OBJECTIVES: The aim of this study was to evaluate the potential of glycosuria as a predictor of glycaemia in neonatal puppies during the first two weeks of life. METHODS: Prospective study. Thirty-three client-owned healthy neonatal puppies admitted to the Veterinary Teaching Hospital, Autonomous University of Barcelona, were included in the study and divided into four different groups according to the day of sampling (1, 4, 7, and 11 days post-delivery). Glucose levels in blood and urine samples were evaluated and compared between groups. Correlation between glucose levels in blood and urine was also determined. RESULTS: Hypoglycaemia was diagnosed in 17.14% of the puppies and only on day 1 after delivery. A positive and significant correlation between blood and urine glucose concentration on day 1 after delivery was observed. No significant correlation between blood and urine glucose was observed on days 4, 7 and 11 after delivery. CONCLUSIONS: Urine concentration of glucose is a useful parameter to establish glycaemic status on the first day of life in canine puppies.

Preference by Donkeys and Goats among Five Mediterranean Forest Species: Implications for Reducing Fire Hazard.

During the second half of the 20th century, European countries experienced an increase in their forest area due to the global change. Consequently, there has been an increase in large forest fires, mainly in the Mediterranean basin, and this has forced the development of several types of prevention programs. One of them is the control of the understory by livestock. In this sense, browsing with a combination of donkeys and goats could be a good option, as both animals usually feed on forest species. However, little is known about their preferences for the key species of the Mediterranean forest. Using a cafeteria test, the preferences and consumption of both animals have been determined for five typical species of the Mediterranean forest, such as Quercus ilex, Pinus halepensis, Phillyrea latifolia, Rubus ulmifolius, and Brachypodium retusum. Results showed that donkeys and goats could act complementarily in the reduction of the fuel biomass of forests. Donkeys appear to act more on fine fuel, such as B. retusum, and goats on the more pyrophyte species, in this case P. halepensis. In addition, given that donkeys are at severe risk of extinction in Europe, this role of providing ecosystem services could contribute to their conservation. Despite this study only showing that goats and donkeys would consume all five presented plant species and that there are some differences in consumption during a short-term test, it constitutes a useful first step for conservation and fire prevention in the Mediterranean forests.

Uterine and placental specific localization of AQP2 and AQP8 is related with changes of serum progesterone levels in pregnant queens.

Aquaporins play vital roles in reproductive physiology. This study evaluates the expression and localization dynamics of AQP1, AQP2, AQP3 and AQP8 in the endometrium and placental transference zone during pregnancy in queens by means of immunohistochemistry and Western blot. Animals were distributed into six groups: non-pregnant queens with low levels of serum progesterone (P4), non-pregnant animals with high P4 levels, and queens at 30, 40, 50 and 60 days of pregnancy. All AQPs were present in glandular and luminal epithelia and myometrium. AQP1 was also present in the endometrial endothelia. AQP2, AQP3 and AQP8 were found in trophoblast. In endometrial samples with P4 above 2 ng/mL, AQP2 and AQP8 were distributed across plasma membrane and cytoplasm, whereas progesterone levels under 1 ng/mL kept both AQPs confined to the plasma membrane. Western blot showed no significant changes in AQPs expression among the stages. In conclusion, our results indicate that the distribution of AQP2 and AQP8 in the queen reproductive tract is related to P4 levels.

Medium-term effects of the diluted pig semen irradiation with red LED light on the integrity of nucleoprotein structure and resilience to withstand thermal stress.

This study sought to evaluate the effects of irradiating pig seminal doses with red LED light irradiation on their quality and longevity over liquid-storage at 17 °C. For this purpose, boar ejaculates were diluted in a commercial extender at a final concentration of 3 × 107 sperm/mL and stored at 17 °C for 96 h. Upon arrival to our laboratory (5-6 h within collection), 1.5 mL-aliquots were subjected to irradiation with a temperature-controlled red light-emitting diode (LED) for 1 min, 5 min or 10 min. Controls consisted of non-irradiated spermatozoa. Aliquots were then stored at 17 °C for 96 h, and plasma membrane and acrosome integrity, motility and free cysteine radicals of sperm head proteins were evaluated every 24 h. In addition, the sperm resilience to withstand thermal stress following irradiation was evaluated at 24 h, 48 h, 72 h and 96 h by incubating stored seminal doses at 37 °C for 120 min. In our experimental conditions, light-stimulation for 5 min and 10 min counteracted the decrease in thermal stress observed in non-irradiated samples during the first 48 h of storage. Moreover, all irradiation protocols counteracted the decrease in percentages of spermatozoa with altered acrosomes observed in non-irradiated samples after 72 h of storage. The effects of light-stimulation upon sperm motility parameters were less consistent. While liquid-storage also led to an increase in the free cysteine levels of sperm head proteins, this increment was partially mitigated through light-stimulation for 5 min and 10 min. Our results suggest that effects linked with red LED light irradiation would be consistently maintained in our experimental conditions for the first 48 h. Finally, the maintenance of light effect appears to depend upon the specific experimental design, the analyzed sperm parameters and the utilized irradiation patterns.

Placental and uterine expression of GLUT3, but not GLUT1, is related with serum progesterone levels during the first stages of pregnancy in queens.

The present study investigated the expression of GLUT1 and GLUT3 in the uterus and placental transference zone of non-pregnant and pregnant queens throughout different pregnancy ages, using immunohistochemistry and immunoblotting techniques. Both GLUT1 and GLUT3 were expressed in both uterine glandular and luminal epithelia and myometrium in pregnant and non-pregnant queens. While endometrial endothelia showed expression of GLUT1 in both pregnant and non-pregnant queens, GLUT3 was only expressed in the pregnant counterparts. Regarding placental structures, GLUT3 was present in cytotrophoblasts, syncytiotrophoblasts and chorionic endothelia and GLUT1 showed a similar location but was absent in cytotrophoblasts. The presence of GLUT1 (55 kDa) and GLUT3 (60 kDa) was confirmed in both uterine and placental tissues through immunoblotting. When the expression of both GLUT1 and GLUT3 were analysed as a whole in the total of the pregnancy period, no significant differences in the relative content of both GLUTs were observed between pregnant and non-pregnant queens. However, when GLUTs expression was analysed in a time-period basis and related with progesterone levels, results were different. Thus, whereas the relative content of GLUT1 showed no correlation with serum progesterone levels, a significant (P < 0.05) and negative correlation was found between the relative GLUT3-content in the uterus on days 30 and 40 of pregnancy as well as in the placental transference zone on day 30 and serum progesterone levels. In summary, our results indicate that whereas GLUT1 could be considered as a basal, constant sugar intake system for the whole of pregnancy in queens, GLUT3 is specially required for optimizing glucose uptake during the first half of pregnancy in this species through a progesterone-related mechanism.

Melatonin affects the motility and adhesiveness of in vitro capacitated boar spermatozoa via a mechanism that does not depend on intracellular ROS levels.

This work sought to address the effects of melatonin during in vitro capacitation (IVC) and progesterone-induced acrosome exocytosis (IVAE) in boar spermatozoa. With this purpose, two different experiments were set. In the first one, IVC and IVAE were induced in the absence or presence of melatonin, which was added either at the start of IVC or upon triggering the IVAE with progesterone. Different parameters were evaluated, including intracellular levels of peroxides and superoxides, free cysteine radicals and distribution of specific lectins. While melatonin neither affected most capacitation-associated parameters nor IVAE, it dramatically decreased sperm motility, with a maximal effect at 5 µm. This effect was accompanied by a significant increase in the percentage of agglutinated spermatozoa, which was independent from noticeable changes in the distribution of lectins. Levels of free cysteine radicals were significantly lower in melatonin treatments than in the control after 4 h of incubation in capacitating medium. The second experiment evaluated the effects of melatonin on in vitro fertilising ability of boar spermatozoa. Spermatozoa previously subjected to IVC in the presence of 1 µm melatonin and used for in vitro fertilisation exhibited less ability to bind the zona pellucida (ZP) and higher percentages of monospermy. In conclusion, melatonin affects sperm motility and the stability of nucleoprotein structure and also modulates the ability of in vitro capacitated boar spermatozoa to bind the oocyte ZP. However, such effects do not seem to be related to either its antioxidant properties or changes in the sperm glycocalix.

Pro-inflammatory cytokines: Useful markers for the diagnosis of canine mammary tumours?

The aim of the present study was to analyse the expression of 60 pro-inflammatory cytokines as possible markers of malignancy in canine mammary tumours using a human cytokine antibody array. The cytokines were grouped into two different categories: (1) cytokines in which expression indicated the presence of a mammary tumour and (2) cytokines in which expression differentiated between simple mammary adenoma, tubulopapillary carcinoma or complex carcinoma. These data suggest that specific pro-inflammatory cytokines could be useful as tools for the diagnosis of canine mammary tumours.

Melatonin receptors MT1 and MT2 are expressed in spermatozoa from several seasonal and nonseasonal breeder species.

Melatonin is a ubiquitous and multipurpose molecule, and one of its roles is to regulate reproduction in some seasonal mammals. Our group has previously reported the variation in the melatonin levels in ram seminal plasma along the year and identified MT1 and MT2 receptors in ram spermatozoa. The objective of this study was to elucidate whether the presence of melatonin receptors (MT1 and MT2) in the sperm plasma membrane, and melatonin in the seminal plasma is related to seasonal breeding. For this purpose, the presence of melatonin receptors and the levels of melatonin in seminal plasma have been examined in several species: donkey and stallion as long-day breeders; red deer as a wild, short-day, highly seasonal breeder (epididymal spermatozoa); bull as a conventional nonseasonal breeder; boar as a seasonal breeder under management techniques; and dog as possible a seasonal breeder not regulated by melatonin. We have detected measurable levels of melatonin in the seminal plasma of all ejaculated semen samples (from donkey, stallion, boar, bull, and dog). Also, and for the first time, we have demonstrated the presence of MT1 and MT2 melatonin receptors in the spermatozoa of all these species, regardless their type of reproduction or sperm source (ejaculated or epididymal), using indirect immunofluorescence techniques and Western blotting. Our findings suggest that melatonin and melatonin receptors may be universally distributed in the reproductive system of mammals and that the sperm melatonin receptors cells may not be necessarily related with seasonal reproduction. Furthermore, the presence of MT1 at the cytoplasmic droplet in immature ejaculated stallion spermatozoa found in one sample and epididymal red deer spermatozoa suggests that melatonin may be involved in specific functions during spermatogenesis and sperm maturation, like protecting spermatozoa from oxidative damage, this activity being mediated through these receptors.

Tungstate treatment improves Leydig cell function in streptozotocin-diabetic rats.

Oral administration of sodium tungstate to adult male streptozotocin-diabetic rats for 3 months normalized serum levels of glucose, insulin, luteinizing hormone, and follicle-stimulating hormone. These effects were accompanied by an increase in reproductive performance, which was related to a strong improvement in Leydig cell function markers, such as the recovery of the number of Leydig cells and serum testosterone levels. Moreover, this in vivo recovery was related to a concomitant increase in the cell expression of insulin receptors. Tungstate treatment did not modify Leydig cell function in healthy rats. Furthermore, the addition of tungstate or insulin to the mTLC-1 cell line from Leydig cell origin increased the phosphorylation states of MAP-kinase and glycogen synthase kinase-3. Our results indicate that tungstate treatment in diabetic rats leads to a recovery of reproductive performance by increasing the number of Leydig cells. This increase contributes to the recovery of their functionality, thereby improving the overall function of these cells. We propose that this improvement is caused by the combined effect of the tungstate-induced normalization of insulin glucose and luteinizing hormone serum levels and a direct action of the effector on Leydig cells through modulation of at least MAP-kinase and glycogen synthase kinase-3 activities.

Natural Mediterranean photoperiod does not affect the main parameters of boar-semen quality analysis.

Photoperiod is an important factor in the modulation of male reproduction in mammals. In boars, however, it is a controversial factor. The main aim of this work is to determine the precise effect of the natural, Mediterranean photoperiod on boar-semen quality. To do this, boars were housekept in strictly controlled temperature and humidity conditions, whereas light periods were also strictly adjusted to obtain a light-cycle in the farm. The work was performed over a period of one year, thus allowing for the determination of the putative yearly oscillations of boar-semen quality. Variations of the natural Mediterranean photoperiod do not induce substantial changes in overall semen-quality parameters like the percentages of viability, morphological abnormalities and total motility, the response to the osmotic resistance test and sperm motion characteristics. Only the motile-sperm subpopulation structure was significantly (P<0.05) changed depending on the variations of the natural photoperiod. Furthermore, the boar-semen ability for storage at 15-17 degrees C in a commercial extender was not modified by photoperiod changes. Our results indicate that the natural Mediterranean photoperiod does not induce strong changes in boar-semen characteristics, probably due to boar sperm having a strong capability of adaptation to the light variations of this photoperiod.

The presence of a high-Km hexokinase activity in dog, but not in boar, sperm.

The presence of a high-Km hexokinase activity was tested in both dog and boar spermatozoa. Hexokinase kinetics from dog extracts showed the presence of a specific activity (dog-sperm glucokinase-like protein, DSGLP), in the range of glucose concentrations of 4-10 mM, whereas boar sperm did not show any DSGLP activity. Furthermore, dog-sperm cells, but not those of boar, showed the presence of a protein which specifically reacted against a rat-liver anti-glucokinase antibody. This protein also had a molecular weight equal to that observed in rat-liver extracts, suggesting a close similarity between both the proteins. This glucokinase-like protein was distributed in the peri- and post-acrosomal zones of the head, and the midpiece and principal piece of tail of dog spermatozoa. These results indicate that dog spermatozoa have functional high-Km hexokinase activity, which could contribute to a very fine regulation of their hexose metabolism. This strict regulation could ultimately be very important in optimizing dog-sperm function along its life-time.

Insulin-dependent diabetes affects testicular function by FSH- and LH-linked mechanisms.

A study was conducted to form a unified hypothesis regarding the gonadotropin-related mechanisms that underlie alterations in the male reproductive system in individuals with diabetes. Streptozotocin-induced diabetes resulted in reduced fertility, prolificacy, and libido. Testes showed a marked decrease in the number and function of Leydig cells, the latter manifested as changes in the expression of biochemical markers, including the GLUT-3 hexose transporter, c-kit, insulin-like growth factor I (IGF-I), androgen receptors, and overall tyrosine phosphorylation, as assessed by Western blot and immunocytochemical analyses. The expression of c-kit, IGF-I, insulin, and follicle-stimulating hormone (FSH) receptors in the seminiferous tubules was also affected. Serum levels of luteinizing hormone (LH), FSH, and testosterone significantly decreased. There was a significant (P <.05) correlation between the serum levels of insulin and FSH. No significant correlation was found between the serum levels of insulin or glucose and LH. On the basis of our results, we conclude that, in insulin-dependent diabetes, 1) Leydig cell function and testosterone production decrease because of the absence of the stimulatory effect of insulin on these cells and an insulin-dependent decrease in FSH, which, in turn, reduces LH levels; and 2) sperm output and fertility are reduced because of a decrease in FSH caused by a reduction in insulin.

Regression analyses and motile sperm subpopulation structure study as improving tools in boar semen quality analysis.

A precise estimation of the fertilizing ability of a boar ejaculate would be very useful to improve pig assisted reproduction results. For this purpose, we tested the mathematical combination of several parameters of the boar semen quality analysis, including the computer-assisted semen motility analysis (CASA), as a predictive fertility tool. The utilized mathematical relations among parameters were logistic and linear regressions. Two mathematical models obtained by logistic regression involving Osmotic Resistance Test (ORT Test), Hyperosmotic Resistance Test (HRT Test) and viability of fresh samples, showed a significant (P<0.05) correlation between semen characteristics and conception rate. However, none of the obtained models produced a significant correlation model between semen characteristics and prolificacy. The CASA analyses show that three separate subpopulations of spermatozoa with different motility characteristics coexist in boar ejaculates. There were significant (P<0.001) differences in the distribution of these subpopulations among boars, but no clear relationship between motile subpopulation structure and fertility was obtained. Our results support the belief that the predictive use of the results obtained in a standard boar semen quality analysis can reasonably be achieved by applying logistic correlation analyses among several function parameters of boar semen quality analysis and in vivo conception rates obtained after artificial insemination (AI).

The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17°C is concomitant with a higher cryotolerance of boar spermatozoa.

Boar-sperm cryopreservation is not usually performed immediately after semen collection, but rather a holding time (HT) of 4 h-30 h at 17°C is spent before starting this procedure. Taking this into account, the aim of this study was to go further in-depth into the mechanisms underlying the improving effects of HT at 17°C on boar-sperm cryotolerance by evaluating the effects of two different HTs (3 h and 24 h) on overall boar-sperm function and survival before and after cryopreservation. Given that phospho/dephosphorylation mechanisms are of utmost importance in the overall regulation of sperm function, the phosphorylation levels of serine residues (pSer) in 30 different sperm proteins after a 3 h- or 24 h-HT period were also assessed. We found that a HT of 24 h contributed to a higher sperm resistance to freeze-thawing procedures, whereas mini-array protein analyses showed that a HT of 24 h induced a significant (P<0.05) increase in pSer (from 100.0±1.8 arbitrary units in HT 3 h to 150.2±5.1 arbitrary units in HT 24 h) of HSP70 and, to a lesser extent, in protein kinases GSK3 and total TRK and in the cell-cycle regulatory protein CDC2/CDK1. In the case of HSP70, this increase was confirmed through immunoprecipation analyses. Principal component and multiple regression analyses indicated that a component explaining a percentage of variance higher than 50% in sperm cryotolerance was significantly correlated with pSer levels in HSP70. In addition, from all the parameters evaluated before freeze-thawing, only pSer levels in HSP70 resulted to be able to predict sperm cryotolerance. In conclusion, our results suggest that boar spermatozoa modulate its function during HT, at least partially, by changes in pSer levels of proteins like HSP70, and this is related to a higher cryotolerance.

Coagulation parameters do not change during luteal phase and pregnancy in cats.

Changes in coagulation parameters depending on reproductive status and pregnancy have been previously reported in both human and other veterinary species. The objective of this study was to determine if different reproductive status affects coagulation parameters in queens. Blood samples from 66 queens submitted to spay surgery were obtained. A hemostatic panel including platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen concentration, and D-dimer and also progesterone concentrations were measured before surgery. According to progesterone results and embryo vesicles diameter, four groups were established: (1) nonpregnant queens with low (≤1 ng/mL) progesterone concentration (LP) (n = 33); (2) nonpregnant queens with high (≥2 ng/mL) progesterone concentration (n = 8) (HP); (3) first half of pregnancy (n = 12); and (4) second half of pregnancy (n = 13). None of the evaluated parameters showed statistically significant differences among the different groups. There was no significant linear correlation between progesterone values and coagulation parameters. In conclusion, neither the presence of the embryo nor the higher values of progesterone concentration induced statistically significant changes in the coagulation profile studied.

The presence and function of dopamine type 2 receptors in boar sperm: a possible role for dopamine in viability, capacitation, and modulation of sperm motility.

Several studies have shown that dopamine and other catecholamines are present in oviduct luminal fluid. We recently reported that dopamine type 2 receptors (DRD2) are present in a wide range of mammalian sperm, suggesting a role for dopaminergic signaling in events such as fertilization, capacitation, and sperm motility. In the present study, we used Western blot analysis to show that boar sperm express DRD2 and that their activation with dopamine (100 nM) has a positive effect on cell viability that can be correlated with AKT/PKB phosphorylation. Bromocriptine (100 nM) and dopamine (100 nM and 10 muM) increased tyrosine phosphorylation during the capacitation period. Immunofluorescence analysis indicated that DRD2 localization is dynamic and depends on the capacitation stage, colocalizing with tyrosine phosphorylated proteins in the acrosome and midpiece region of capacitated boar sperm. This association was confirmed by coimmunoprecipitation analysis. We also showed that bromocriptine (100 nM) and low-concentration dopamine (100 nM and 10 muM) increased total and progressive motility of sperm. However, high concentrations of dopamine (1 mM) decreased tyrosine phosphorylation and motility in in vitro sperm capacitation assays. This can be explained by the presence of the dopamine transporters (DAT, official symbol SLC6A3) in sperm, as demonstrated by Western blot analysis and immunocytochemistry. Taken together, our results support the idea that dopamine may have a fundamental role during sperm capacitation and motility in situ in the female upper reproductive tract.