Josep Rutllant - Academia.edu (original) (raw)
Papers by Josep Rutllant
Gamebird Medicine and Management
Anatomia, Histologia, Embryologia, 2022
Argentine black and white tegus (Salvator merianae) are omnivorous lizards native to southeastern... more Argentine black and white tegus (Salvator merianae) are omnivorous lizards native to southeastern Brazil, Uruguay, eastern Paraguay and northern Argentina, and are invasive species in Florida and Georgia, USA. They are opportunistic feeders, which is what allow them to have such a diverse variety of foods. Tegus raised a particular concern due to their adaptive capability to different environments. Our goal was to provide a micromorphology baseline of oesophagus and stomach and correlate findings with their dietary and invasive capabilities. Four Argentine black and white tegus were used for this study. We collected and processed specimens from oesophagus and stomach using standard histological techniques and stained tissue sections using Haematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS), Alcian Blue (AB) and Verhoef's elastic stains. The oesophagus was lined with ciliated pseudostratified columnar epithelium (PSCE) with goblet cells. Gut-associated lymphoid tissues (GALT) were present occasionally in the oesophagus and more frequently in the stomach. Tunica muscularis (Tm) of the oesophageal-gastric junction had distinct smooth muscle which could function as a sphincter. The mucosa of the stomach was lined with simple columnar epithelium (SC). The glands had neck and dark oxyntico-peptic cells. The pyloric sphincter had more GALT and mucus cells than other regions. The Tm outer layer is thinner than the inner. Presence of large number of goblet cells would support faster transit of the bolus. The short digestive tract and the histological features observed are consistent with the ability of tegus consumption of large amount of food.
Anatomia, Histologia, Embryologia, 2022
Anomalies in the subclavian and common carotid arteries can be of interest in cases of cranial me... more Anomalies in the subclavian and common carotid arteries can be of interest in cases of cranial mediastinal surgeries, as well as to diagnose the cause of oesophageal constrictions leading to clinical signs of dysphagia (dysphagia lusoria). The development and regression of the aortic arches are of key importance in understanding the origin of these type of vascular anomalies. This report describes the congenital anomalous aortic origin of the common carotid and the subclavian arteries in a 14-year-old dog and the plausible developmental pattern failure. Academic dissection revealed a common bicarotid trunk and bisubclavian trunk arising from the most cranial aspect of the aortic arch. Despite the abnormal origin, these vessels displayed a predominantly standard anatomical course. All the anticipated branches were identified and described. Cardiac abnormalities were also noted including right atrial dilation, coronary sinus enlargement, right and left valvular endocardiosis, a patent foramen ovale and marked concentric left ventricular hypertrophy with compensatory left atrial dilation. Additionally, the right recurrent laryngeal nerve demonstrated an aberrant course consistent with a 'non-recurrent laryngeal nerve' (non-RLN). Awareness of the anatomical variations of the aortic arch is important for surgical interventions of the cranial mediastinum as well as radiological interpretation. Although infrequent, the variants similar to the one described here have been reported in different species.
ABSTRACT: Fundamental cryobiological characteristics of rhe-sus spermatozoa must be determined fo... more ABSTRACT: Fundamental cryobiological characteristics of rhe-sus spermatozoa must be determined for successful cryopres-ervation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and os-motic tolerance limits of rhesus macaque spermatozoa. Cell vol-ume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of mem-brane integrity and mitochondrial membrane potential was per-formed using flow cytometry. Rhesus monkey spermatozoa be-have as linear osmometers in the osmotic range tested (75–900 mOsmol kg21), as shown by the Boyle van’t Hoff plot (r 2 5.99). Rhesus spermatozoa have a mean cell volume of 36.8 6 0.5 mm3 at 228C, with 77.2 % of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane integri...
Expression stability of putative reference genes in equine endometrial, testicular, and conceptus... more Expression stability of putative reference genes in equine endometrial, testicular, and conceptus tissues
The Anatomical Record, 2013
The process of water movement in the excurrent duct system of the male reproductive tract is pivo... more The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, 24, 25, 28, 210, and 211); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, 23, 27, and 29). Overall, we inferred that fluid transport in the stallion testicle involved a collaboration of AQP subtypes (primarily AQP2, 25, 27, and 28). Based on immunohistochemistry, expression of AQP subtypes analyzed (i.e., AQP0, 22, 25, and 29) was localized to Leydig cells and elongated and round spermatids. Functional significance of AQP expression by Leydig cells remained uncertain. In elongated and round spermatids, AQP s likely contributed to the volume reduction observed during spermatogenesis. Subtypes AQP2 and AQP9 were the predominant forms expressed in epididymal tissue. Regulation of AQP2 expression, especially in the epididymal head, seemed to occur at the post-transcriptional level, as protein expression upon immunohistochemistry was pronounced, despite low transcript abundance. In epididymal tissue, AQPs likely contributed to fluid resorbtion, given their localization on the apical membrane of principal cells.
Reproduction in Domestic Animals, 2012
The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA ... more The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non-pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up-regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine-specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.
BMC Research Notes, 2011
Background: Quantitative RT-PCR data are commonly normalized using a reference gene. A reference ... more Background: Quantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue. Findings: The expression stability of four (uterine tissue) and six (testicular and conceptus tissue) was assessed using descriptive data analysis and the software programs Normfinder and geNorm. In uterine samples, 18S showed the largest degree of variation in expression while GAPDH, B2M, and ACTB were stably expressed. B2M and GAPDH were identified as the most stably expressed genes in testicular samples, while 18S showed some extent of regulation between samples. Conceptus tissue overall was characterized by very low variability of the transcripts analyzed with GAPDH, YWHZ, and 18S being the most stably expressed genes. Conclusions: In equine endometrium, GAPDH, B2M, and ACTB transcript levels are equally stable, while 18S is less stably expressed. In testes and associated structures, B2M and GAPDH are the transcripts showing the least amount of variation, while in conceptus tissue GAPDH, YWHZ, and 18S were identified as the most suitable reference genes. Overall, transcripts analyzed in conceptus tissue were characterized by less variation than transcripts analyzed in uterine and testicular tissue.
Biology of Reproduction, 2003
Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism r... more Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons of tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh and frozen-thawed sperm. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine, as well as methyl -cyclodextrin-which causes cholesterol efflux from the spermatozoa-and inhibited by the protein kinase A (PK-A) inhibitor H-89. Our results indicate that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases and that cryopreserved sperm may be more sensitive to inducers of capacitation, which could explain their limited life span when compared with fresh sperm.
Clínica veterinaria de …, 2002
Biology of Reproduction, 2001
It is generally accepted that spermatozoa become functionally mature during epididymal transit. T... more It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n ؍ 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal antiequine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.
Molecular Reproduction and Development, 2000
The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched wi... more The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched with the presence of a glycogenlike staining in the head and midpiece. Glycogen levels lowered to 0.05 mol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 mol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35 Ϯ 0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25 Ϯ 0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo. Mol.
El presente trabajo ha tenido como objetivo general la descripcion y caracterizacion de propiedad... more El presente trabajo ha tenido como objetivo general la descripcion y caracterizacion de propiedades reologicas y ultraestructurales del fluido vaginal bovino, obtenido durante el periodo concreto del ciclo ovarico en el que se manifiesta la sintomatologia del celo. Para cumplir este objetivo, se estudiaron en primer lugar, en un grupo de 8 animales, las caracteristicas reologicas de comportamiento al flujo y tixotropia del fluido vaginal obtenido cada 8 horas a partir de la primera manifestacion del estro. Ademas, se estudiaron las propiedades reologicas en muestras de fluido vaginal obtenidas en el momento de realizar una inseminacion en 208 vacas. Los resultados demostraron una disminucion del indice de consistencia del fluido vaginal aproximadamente hacia la mitad del periodo estral (8-16 horas tras la deteccion del inicio del estro), observandose ademas que el comportamiento reologico del conjunto de muestras era muy variable cualitativa y cuantitativamente perdiendose, incluso,...
Genetics Research International, 2016
Comparative genomics approaches provide a means of leveraging functional genomics information fro... more Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism’s genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism’s genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation ofPython bivittatusgenes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotatedPython bivittatusgenome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction c...
Biology of Reproduction, 2008
Journal of andrology
Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successfu... more Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successful cryopreservation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and osmotic tolerance limits of rhesus macaque spermatozoa. Cell volume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of membrane integrity and mitochondrial membrane potential was performed using flow cytometry. Rhesus monkey spermatozoa behave as linear osmometers in the osmotic range tested (75-900 mOsmol kg(-1)), as shown by the Boyle van't Hoff plot (r(2) =.99). Rhesus spermatozoa have a mean cell volume of 36.8 +/- 0.5 micro m(3) at 22 degrees C, with 77.2% of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane i...
Theriogenology, Jan 15, 2002
Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. ... more Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150...
Theriogenology, 2002
Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. ... more Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P &amp;lt; 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P &amp;lt; 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P &amp;lt; 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P &amp;lt; 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P &amp;lt; 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.
Molecular Reproduction and Development, 2000
The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched wi... more The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched with the presence of a glycogenlike staining in the head and midpiece. Glycogen levels lowered to 0.05 mol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 mol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35 Ϯ 0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25 Ϯ 0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo. Mol.
Gamebird Medicine and Management
Anatomia, Histologia, Embryologia, 2022
Argentine black and white tegus (Salvator merianae) are omnivorous lizards native to southeastern... more Argentine black and white tegus (Salvator merianae) are omnivorous lizards native to southeastern Brazil, Uruguay, eastern Paraguay and northern Argentina, and are invasive species in Florida and Georgia, USA. They are opportunistic feeders, which is what allow them to have such a diverse variety of foods. Tegus raised a particular concern due to their adaptive capability to different environments. Our goal was to provide a micromorphology baseline of oesophagus and stomach and correlate findings with their dietary and invasive capabilities. Four Argentine black and white tegus were used for this study. We collected and processed specimens from oesophagus and stomach using standard histological techniques and stained tissue sections using Haematoxylin and Eosin (H&E), Periodic Acid Schiff (PAS), Alcian Blue (AB) and Verhoef's elastic stains. The oesophagus was lined with ciliated pseudostratified columnar epithelium (PSCE) with goblet cells. Gut-associated lymphoid tissues (GALT) were present occasionally in the oesophagus and more frequently in the stomach. Tunica muscularis (Tm) of the oesophageal-gastric junction had distinct smooth muscle which could function as a sphincter. The mucosa of the stomach was lined with simple columnar epithelium (SC). The glands had neck and dark oxyntico-peptic cells. The pyloric sphincter had more GALT and mucus cells than other regions. The Tm outer layer is thinner than the inner. Presence of large number of goblet cells would support faster transit of the bolus. The short digestive tract and the histological features observed are consistent with the ability of tegus consumption of large amount of food.
Anatomia, Histologia, Embryologia, 2022
Anomalies in the subclavian and common carotid arteries can be of interest in cases of cranial me... more Anomalies in the subclavian and common carotid arteries can be of interest in cases of cranial mediastinal surgeries, as well as to diagnose the cause of oesophageal constrictions leading to clinical signs of dysphagia (dysphagia lusoria). The development and regression of the aortic arches are of key importance in understanding the origin of these type of vascular anomalies. This report describes the congenital anomalous aortic origin of the common carotid and the subclavian arteries in a 14-year-old dog and the plausible developmental pattern failure. Academic dissection revealed a common bicarotid trunk and bisubclavian trunk arising from the most cranial aspect of the aortic arch. Despite the abnormal origin, these vessels displayed a predominantly standard anatomical course. All the anticipated branches were identified and described. Cardiac abnormalities were also noted including right atrial dilation, coronary sinus enlargement, right and left valvular endocardiosis, a patent foramen ovale and marked concentric left ventricular hypertrophy with compensatory left atrial dilation. Additionally, the right recurrent laryngeal nerve demonstrated an aberrant course consistent with a 'non-recurrent laryngeal nerve' (non-RLN). Awareness of the anatomical variations of the aortic arch is important for surgical interventions of the cranial mediastinum as well as radiological interpretation. Although infrequent, the variants similar to the one described here have been reported in different species.
ABSTRACT: Fundamental cryobiological characteristics of rhe-sus spermatozoa must be determined fo... more ABSTRACT: Fundamental cryobiological characteristics of rhe-sus spermatozoa must be determined for successful cryopres-ervation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and os-motic tolerance limits of rhesus macaque spermatozoa. Cell vol-ume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of mem-brane integrity and mitochondrial membrane potential was per-formed using flow cytometry. Rhesus monkey spermatozoa be-have as linear osmometers in the osmotic range tested (75–900 mOsmol kg21), as shown by the Boyle van’t Hoff plot (r 2 5.99). Rhesus spermatozoa have a mean cell volume of 36.8 6 0.5 mm3 at 228C, with 77.2 % of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane integri...
Expression stability of putative reference genes in equine endometrial, testicular, and conceptus... more Expression stability of putative reference genes in equine endometrial, testicular, and conceptus tissues
The Anatomical Record, 2013
The process of water movement in the excurrent duct system of the male reproductive tract is pivo... more The process of water movement in the excurrent duct system of the male reproductive tract is pivotal for establishment of male fertility. The objective was to elucidate expression of aquaporin (AQP) water channels in the stallion reproductive tract. Real-time RT-PCR detected expression of AQP0-5 and AQP7-11 in testis, epididymis, and ductus deferens of mature stallions. There were two main expression patterns: (1) higher expression in testis than in epididymis and ductus deferens (AQP0, 24, 25, 28, 210, and 211); and (2) lower expression in testis than in epididymis and ductus deferens (AQP1, 23, 27, and 29). Overall, we inferred that fluid transport in the stallion testicle involved a collaboration of AQP subtypes (primarily AQP2, 25, 27, and 28). Based on immunohistochemistry, expression of AQP subtypes analyzed (i.e., AQP0, 22, 25, and 29) was localized to Leydig cells and elongated and round spermatids. Functional significance of AQP expression by Leydig cells remained uncertain. In elongated and round spermatids, AQP s likely contributed to the volume reduction observed during spermatogenesis. Subtypes AQP2 and AQP9 were the predominant forms expressed in epididymal tissue. Regulation of AQP2 expression, especially in the epididymal head, seemed to occur at the post-transcriptional level, as protein expression upon immunohistochemistry was pronounced, despite low transcript abundance. In epididymal tissue, AQPs likely contributed to fluid resorbtion, given their localization on the apical membrane of principal cells.
Reproduction in Domestic Animals, 2012
The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA ... more The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed highest expression levels. Western blot analysis confirmed protein expression of AQP0, 2 and 5. Immunohistochemistry localized protein expression to luminal and glandular epithelial and stromal cells. AQP0 staining intensity was highest in samples obtained on Day 14 of the oestrous cycle. AQP2 immunoreactivity seemed to be stronger in samples collected 14 days after ovulation from non-pregnant animals, in particular luminal epithelial staining. Samples collected 8 days after ovulation from cyclic animals were characterized by intense AQP5 staining of glandular epithelium, predominantly in the deeper glands. Progesterone treatment of anoestrous mares did not enhance expression of AQPs, indicating that factors other than progesterone are required for the up-regulation of certain AQP subtypes during dioestrus. In conclusion, it seems that an equine-specific collaboration of aquaporin subtypes contributes to changes in endometrial fluid content occurring throughout the oestrous cycle and contributes to endometrial receptivity during early pregnancy in the mare.
BMC Research Notes, 2011
Background: Quantitative RT-PCR data are commonly normalized using a reference gene. A reference ... more Background: Quantitative RT-PCR data are commonly normalized using a reference gene. A reference gene is a transcript which expression does not differ in the tissue of interest independent of the experimental condition. The objective of this study was to evaluate the stability of mRNA expression levels of putative reference genes in three different types of equine tissue, endometrial, testicular, and conceptus tissue. Findings: The expression stability of four (uterine tissue) and six (testicular and conceptus tissue) was assessed using descriptive data analysis and the software programs Normfinder and geNorm. In uterine samples, 18S showed the largest degree of variation in expression while GAPDH, B2M, and ACTB were stably expressed. B2M and GAPDH were identified as the most stably expressed genes in testicular samples, while 18S showed some extent of regulation between samples. Conceptus tissue overall was characterized by very low variability of the transcripts analyzed with GAPDH, YWHZ, and 18S being the most stably expressed genes. Conclusions: In equine endometrium, GAPDH, B2M, and ACTB transcript levels are equally stable, while 18S is less stably expressed. In testes and associated structures, B2M and GAPDH are the transcripts showing the least amount of variation, while in conceptus tissue GAPDH, YWHZ, and 18S were identified as the most suitable reference genes. Overall, transcripts analyzed in conceptus tissue were characterized by less variation than transcripts analyzed in uterine and testicular tissue.
Biology of Reproduction, 2003
Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism r... more Phosphorylation of tyrosine residues on sperm proteins is one important intracellular mechanism regulating sperm function that may be a meaningful indicator of capacitation. There is substantial evidence that cryopreservation promotes the capacitation of sperm and this cryocapacitation is frequently cited as one factor associated with the reduced longevity of cryopreserved sperm in the female reproductive tract. This study was designed to determine whether stallion sperm express different levels of tyrosine phosphorylation after in vitro capacitation and whether thawed sperm display similar phosphorylation characteristics in comparison with freshly ejaculated sperm. Experiments were performed to facilitate comparisons of tyrosine phosphorylation, motility, and viability of sperm prior to and following in vitro capacitation in fresh and frozen-thawed sperm. We hypothesized that equine spermatozoa undergo tyrosine phosphorylation during capacitation and that this phosphorylation is modified when sperm have been cryopreserved. We also hypothesized that tyrosine phosphorylation could be enhanced by the use of the activators dibutyryl cAMP (db cAMP) and caffeine, as well as methyl -cyclodextrin-which causes cholesterol efflux from the spermatozoa-and inhibited by the protein kinase A (PK-A) inhibitor H-89. Our results indicate that equine sperm capacitation is mediated by a signaling pathway that involves cAMP-dependent PK-A and tyrosine kinases and that cryopreserved sperm may be more sensitive to inducers of capacitation, which could explain their limited life span when compared with fresh sperm.
Clínica veterinaria de …, 2002
Biology of Reproduction, 2001
It is generally accepted that spermatozoa become functionally mature during epididymal transit. T... more It is generally accepted that spermatozoa become functionally mature during epididymal transit. The objective of this study was to determine whether the cellular location of equine PH-20 is modified during epididymal transit and, if so, the mechanism for such modification. Sperm were isolated from caput and cauda epididymal regions from stallions undergoing castration (n ؍ 7) and used as whole sperm cell or subjected to nitrogen cavitation for isolation of plasma membrane proteins. Both caput and cauda sperm and sperm protein extracts were subjected to N-deglycosylation, O-deglycosylation, or trypsinization. The SDS-PAGE and Western blot analysis using a polyclonal antiequine PH-20 IgG were performed in sperm extracts, and indirect immunofluorescence on whole sperm was also performed to determine the cellular distribution of plasma membrane PH-20 following similar treatments (deglycosylation or trypsinization). Hyaluronan substrate gel electrophoresis was performed to detect hyaluronidase activity in SDS-PAGE proteins. Western blots revealed significant differences in electrophoretic migration of PH-20 proteins from caput and cauda epididymal sperm. No effect was seen from deglycosylation treatments on the Western blot pattern; caput protein extracts exposed to trypsin showed the same band pattern as extracts from the cauda epididymis. N-deglycosylation resulted in the loss of hyaluronidase activity of sperm from both epididymal regions, whereas O-deglycosylation or trypsinization did not affect hyaluronidase activity. In caput epididymal sperm, the PH-20 protein is distributed over the entire sperm head; in cauda epididymal sperm, it is restricted to the postacrosomal region. No effect from deglycosylation on the cellular distribution of PH-20 was observed; however, treatment with trypsin changed the cellular distribution of PH-20 in caput sperm similar to that of the distribution of cauda sperm. These results suggest that PH-20 distribution during epididymal maturation is dependent on proteolytic trypsin-like mechanisms and, possibly, on complementary membrane-associated factors.
Molecular Reproduction and Development, 2000
The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched wi... more The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched with the presence of a glycogenlike staining in the head and midpiece. Glycogen levels lowered to 0.05 mol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 mol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35 Ϯ 0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25 Ϯ 0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo. Mol.
El presente trabajo ha tenido como objetivo general la descripcion y caracterizacion de propiedad... more El presente trabajo ha tenido como objetivo general la descripcion y caracterizacion de propiedades reologicas y ultraestructurales del fluido vaginal bovino, obtenido durante el periodo concreto del ciclo ovarico en el que se manifiesta la sintomatologia del celo. Para cumplir este objetivo, se estudiaron en primer lugar, en un grupo de 8 animales, las caracteristicas reologicas de comportamiento al flujo y tixotropia del fluido vaginal obtenido cada 8 horas a partir de la primera manifestacion del estro. Ademas, se estudiaron las propiedades reologicas en muestras de fluido vaginal obtenidas en el momento de realizar una inseminacion en 208 vacas. Los resultados demostraron una disminucion del indice de consistencia del fluido vaginal aproximadamente hacia la mitad del periodo estral (8-16 horas tras la deteccion del inicio del estro), observandose ademas que el comportamiento reologico del conjunto de muestras era muy variable cualitativa y cuantitativamente perdiendose, incluso,...
Genetics Research International, 2016
Comparative genomics approaches provide a means of leveraging functional genomics information fro... more Comparative genomics approaches provide a means of leveraging functional genomics information from a highly annotated model organism’s genome (such as the mouse genome) in order to make physiological inferences about the role of genes and proteins in a less characterized organism’s genome (such as the Burmese python). We employed a comparative genomics approach to produce the functional annotation ofPython bivittatusgenes encoding proteins associated with sperm phenotypes. We identify 129 gene-phenotype relationships in the python which are implicated in 10 specific sperm phenotypes. Results obtained through our systematic analysis identified subsets of python genes exhibiting associations with gene ontology annotation terms. Functional annotation data was represented in a semantic scatter plot. Together, these newly annotatedPython bivittatusgenome resources provide a high resolution framework from which the biology relating to reptile spermatogenesis, fertility, and reproduction c...
Biology of Reproduction, 2008
Journal of andrology
Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successfu... more Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successful cryopreservation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and osmotic tolerance limits of rhesus macaque spermatozoa. Cell volume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of membrane integrity and mitochondrial membrane potential was performed using flow cytometry. Rhesus monkey spermatozoa behave as linear osmometers in the osmotic range tested (75-900 mOsmol kg(-1)), as shown by the Boyle van't Hoff plot (r(2) =.99). Rhesus spermatozoa have a mean cell volume of 36.8 +/- 0.5 micro m(3) at 22 degrees C, with 77.2% of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane i...
Theriogenology, Jan 15, 2002
Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. ... more Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P < 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P < 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150...
Theriogenology, 2002
Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. ... more Cryopreservation requires exposure of sperm to extreme variations in temperature and osmolality. The goal of this experiment was to determine the osmotic tolerance levels of equine sperm by analyzing motility, viability, mitochondrial membrane potential (MMP), and mean cell volume (MCV). Spermatozoa were incubated at 22 degrees C for 10 min in isosmolal TALP (300 mOsm/kg), or a range of anisosmolal TALP solutions (75-900 mOsm/kg), for initial analysis, and then returned to isosmolal conditions for 10 min for further analysis. Total sperm motility was lower (P &amp;lt; 0.05) in anisosmolal conditions compared to sperm motility in control medium. When cells were returned to isosmolal conditions, only sperm previously incubated in 450 mOsm/kg TALP were able to recover to control levels of motility. Sperm viability and MMP were lower (P &amp;lt; 0.05) when exposed to hypotonic solutions in comparison to control solutions. Sperm suspensions that were returned to isosmolal conditions from 75, 150, and 900 mOsm/kg had lower (P &amp;lt; 0.05) percentages of viable sperm than control suspensions (300 mOsm/kg). MMP was lower (P &amp;lt; 0.05) in cells previously incubated in 75 and 900 mOsm/kg when returned to isosmolal, as compared to control cells. MCV differed (P &amp;lt; 0.05) from control cell volume in all anisosmolal solutions. Cells in all treatments were able to recover initial volume when returned to isosmolal medium. Although most spermatozoa are able to recover initial volume after osmotic stress, irreversible damage to cell membranes may render some sperm incapable of fertilizing an oocyte following cryopreservation.
Molecular Reproduction and Development, 2000
The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched wi... more The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched with the presence of a glycogenlike staining in the head and midpiece. Glycogen levels lowered to 0.05 mol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 mol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35 Ϯ 0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25 Ϯ 0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo. Mol.