Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

BackgroundThe in vivo concentration of bicarbonate (HCO3-), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3- concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased.ResultsOnce exposed to the capacitation medium, the intracellular pH (pH(i)) of spermatozoa increased immediately even at low concentrations of HCO3-, but only extracellular concentrations of and above 15mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15mmol/L of HCO3- stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3- concentration to 15mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%).ConclusionsOptimising HCO3- concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15mmol/L of HCO3- in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), the European Regional Development Fund (FEDER), Grants AGL2012-40180-C03-01-02 and AGL2015-66341-R), Fundacion Seneca (20040/GERM/16) and by a grant R01-HD-038082 (to P. E. V.) from the National Institutes of Health (NIH), USA

Regulation of boar sperm functionality by the nitric oxidesynthase/nitric oxide system

Purpose Nitric oxide (NO) is a free radical synthesized mainly by nitric oxide synthases (NOSs). NO regulates many aspects in sperm physiology in different species. However, in vitro studies investigating NOS distribution, and how NO influences sperm capacitation and fertilization (IVF) in porcine, have been lacking. Therefore, our study aimed to clarify these aspects. Methods Two main experiments were conducted: (i) boar spermatozoa were capacitated in the presence/absence of S-nitrosoglutathione (GSNO), a NO donor, and two NOS inhibitors, N-G-nitro-L-arginine methyl ester hydrochloride (L-NAME) and aminoguanidine hemisulfate salt (AG), and (ii) IVF was performed in the presence or not of these supplements, but neither the oocytes nor the sperm were previously incubated in the supplemented media. Results Our results suggest that NOS distribution could be connected to pathways which lead to capacitation. Treatments showed significant differences after 30 min of incubation, compared to time zero in almost all motility parameters (P < 0.05). When NOSs were inhibited, three protein kinase A (PKA) substrates (similar to 75, similar to 55, and similar to 50 kDa) showed lower phosphorylation levels between treatments (P < 0.05). No differences were observed in total tyrosine phosphorylation levels evaluated by Western blotting nor in situ. The percentage of acrosome-reacted sperm and phosphatidylserine translocation was significantly lower with L-NAME. Both inhibitors reduced sperm intracellular calcium concentration and IVF parameters, but L-NAME impaired sperm ability to penetrate denuded oocytes. Conclusions These findings point out to the importance of both sperm and cumulus-oocyte-derived NO in the IVF outcome in porcine.This study was supported by H2020 MSC-ITN-EJD 675526 REP-BIOTECH, the Spanish Ministry of Economy and Competitiveness (MINECO), and the European Regional Development Fund (FEDER), Grant AGL2015-66341-R, and by a grant ESPDOC17/33 (to Jon Romero-Aguirregomezcorta) from the University of the Basque Country (UPV/EHU, Spain)

Manipulation of bicarbonate concentration in sperm capacitation media improves in vitro fertilisation output in porcine species

[EN] Background: The in vivo concentration of bicarbonate (HCO3), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3 concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased. Results: Once exposed to the capacitation medium, the intracellular pH (pHi) of spermatozoa increased immediately even at low concentrations of HCO3, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3 stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3 concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). Conclusions: Optimising HCO3 concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3 in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcineSIThis work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO), the European Regional Development Fund (FEDER), Grants AGL2012–40180-C03–01-02 and AGL2015–66341-R), Fundación Séneca (20040/GERM/16) and by a grant R01-HD-038082 (to P.E.V.) from the National In- stitutes of Health (NIH), US

Sperm selection by rheotaxis improves sperm quality and early embryo development

peer-reviewedThe objective of this work was to elucidate whether a sperm selection method that combines rheotaxis and microfluidics can improve the selection of spermatozoa over density gradient and swim-up. For this purpose human sperm selected by rheotaxis were compared against density gradient, swim-up and a control group of non-selected spermatozoa in split frozen-thawed (FT) and fresh (F) semen samples. Sperm quality was assessed in terms of motility, morphology, DNA fragmentation index (DFI), viability, acrosome integrity and membrane fluidity. Using a mouse model, we compared fertilisation and embryo development rates after performing ICSI with spermatozoa, sorted using rheotaxis or swim-up. Selection by rheotaxis yielded a sperm population with reduced DFI than the control (P < 0.05), improved normal morphology (P < 0.001) and higher total motility (TM; P < 0.001) than the other techniques studied in F and FT samples. Swim-up increased TM compared to density gradient and control in FT or F samples (P < 0.001), and yielded lower DFI than the control with F samples (P < 0.05). In FT samples, selection by rheotaxis yielded sperm with higher viability than control, density gradient and swim-up (P < 0.01) while acrosomal integrity and membrane fluidity were maintained. When mouse spermatozoa were selected for ICSI using rheotaxis compared to swim-up, there was an increase in fertilisation (P < 0.01), implantation (P < 0.001) and foetal development rates (P < 0.05). These results suggest that, in the absence of non-destructive DNA testing, the positive rheotaxis can be used to select a population of low DNA fragmentation spermatozoa with high motility, morphology and viability, leading to improved embryo developmental rates

Efecto del fluido oviductal, α-L-fucosidasa y óxido nítrico sobre la interacción de gametos en la especie porcina = Effect of oviductal fluid,α-L-fucosidase and nitric oxide on porcine gamete interaction

La especie porcina es considerada el modelo animal idóneo para el estudio y desarrollo de terapias contra enfermedades humanas debido a sus similitudes genéticas, anatómicas y fisiológicas con el hombre. Sin embargo, la eficiencia de la producción in vitro (PIV) de embriones porcinos para utilizarlos con este fin sigue siendo muy baja, pues aun persisten los problemas de la fecundación polispérmica y las condiciones de cultivo subóptimas. Por estos motivos, se pretende profundizar en el conocimiento de los diferentes factores relacionados con el oviducto porcino que pudieran ser utilizados para modular el proceso de la fecundación in vitro (FIV) y contribuir a la mejora de los sistemas de PIV de embriones. La presente tesis aborda tres posibles vías de mejora diferentes en cuanto a complejidad e implicaciones fisiológicas consistentes en la adición al medio de cultivo de tres tipos de compuestos: fluido oviductal (FO), α-L-fucosidasa y óxido nítrico (NO). El objetivo del primer estudio fue determinar las condiciones óptimas de inclusión del FO en los protocolos de FIV. Para ello, se preincubaron ovocitos madurados in vitro con FO pre o postovulatorio y se realizó FIV. Se midieron las concentraciones de E2 y P4 en el FO. Finalmente se comparó la eficiencia de la FIV al utilizar semen fresco o congelado con ovocitos preincubados en FO preovulatorio. La eficiencia de la FIV porcina mejoró con la preincubación en FO preovulatorio. Cuando se utilizó semen congelado su capacidad de penetración aumentó comparando con semen fresco. La concentración de E2 en el FO preovulatorio fue mayor que la determinada en sangre para la misma fase del ciclo estral y la adición de E2 al medio de FIV a la concentración detectada en el oviducto produjo un efecto similar al de la adición de FO. Concluimos que la preincubación de los ovocitos en FO preovulatorio o la adición de E2 pueden considerarse protocolos adecuados para aumentar la eficiencia de la FIV porcina con semen fresco. En el segundo estudio se investigó el papel de la α-L-fucosidasa durante la FIV porcina. Para ello se añadió la enzima al medio de FIV y se observó un incremento en la penetración y en la unión de espermatozoides a la zona pelúcida (ZP). Se realizó inmunofluorescencia indirecta para detectar la α-L-fucosidasa espermática y finalmente se realizaron estudios de capacitación espermática, demostrándose que la α-L-fucosidasa aumenta los niveles de [Ca2+] intracelular y la fosforilación de tirosina. Por lo que concluímos que la α-L-fucosidasa estimula eventos relacionados con la capacitación en espermatozoides porcinos, dando lugar a una mayor capacidad de unión a la ZP y de penetración al ovocito. En el tercer estudio se investigó el papel del NO durante la maduración in vitro (MIV) porcina. Para ello se suplementó el medio de MIV con tres inhibidores distintos de la óxido nítrico sintasa (NOS) y con un donante de NO. Se estudió la expansión de las células del cúmulus y la reanudación de la meiosis, la resistencia de la ZP a la digestión enzimática y parámetros relacionados con la FIV. Los resultados mostraron que los tres inhibidores ejercían un efecto sobre la expansión de las células del cúmulus y que la reanudación de la meiosis solamente se vio detenida al añadir el inhibidor aminoguanidina. Posteriormente, se planteó la posibilidad de que la inhibición de las NOS afectara a la S-nitrosilación de proteínas del ovocito. Para ello se realizó nitrosilación in situ cuantificándose la intensidad de fluorescencia. De este modo, se demostró que la S-nitrosilación de proteínas es una de las vías por la que el NO ejerce su efecto en el ovocito, revelando la importancia del NO en la MIV. The porcine species, owing to their genetic, anatomic, and physiologic similarities with humans, is considered as the ideal animal model for studying and developing novel therapies against human diseases. However, the efficiency of porcine embryo in vitro production (IVP) is nowadays still unacceptably low, because the problem of polyspermic fertilization has still not been adequately worked out and the embryo in vitro culture conditions are still considered to be suboptimal. For these reasons, the present doctoral thesis aims to deepen the understanding of the different factors related to the physiology of the pig oviduct that could be used to modulate the in vitro fertilization (IVF) process and contribute to improve pig embryo IVP systems. Therefore, we investigate three different ways of improvement, in terms of complexity and physiological implications, such as: 1) the oviductal fluid (OF), 2) α-l-fucosidase, 3) nitric oxide (NO). The aim of the first study was to determine the optimal conditions for the OF inclusion in the IVF protocols. For this purpose, in vitro matured oocytes were pre-incubated in OF collected before or after ovulation and subsequently used for IVF. Steroids concentrations in OF were measured and used in IVF experiments. Finally the IVF efficiency was compared between fresh or frozen semen with oocytes preincubated in preovulatory OF. Results showed that pig IVF efficiency improved with preincubation in preovulatory OF. When frozen-thawed semen was used with preovulatory OF incubated oocytes, penetration ability and polyspermy increased. The estradiol (E2) concentration in preovulatory OF was greater than that determined in blood serum at the same phase of the estrous cycle. We conclude that preincubation of oocytes in preovulatory OF or the addition of E2 can be considered suitable tools to increase the efficiency of IVF, only with fresh semen, in the pig. In the second study the role of α-L-fucosidase in pig IVF was investigated. Therefore, the enzyme was added to the IVF medium and a significant increase in sperm binding to the zona pellucida (ZP) and penetration was observed. Thereafter, the sperm-associated α-L-fucosidase was detected by indirect immunofluorescence. In this case, neither the intensity of fluorescence nor the patterns of sperm-associated α-L-fucosidase distribution were affected. Finally, experiments focused on the study of the capacitation events were conducted. The results showed that the addition of exogenous α-L-fucosidase increased the sperm intracellular [Ca 2+] and P-tyrosine phosphorylation, suggesting a role in promoting capacitation and at the same time protecting the sperm cells from premature acrosome reaction. This sequence of events would, in turn, explain the increased ability of this population of treated spermatozoa to bind ZP and penetrate the oocyte. The third study arose to determine the effect of NO during pig oocyte maturation. In vitro maturation (IVM) medium was supplemented with three NOS inhibitors, and a NO donor. The effects on cumulus cells expansion, meiotic resumption, ZP resistance to enzymatic digestion and various parameters related to IVF were studied. The results showed that after IVM, the three NOS inhibitors exerted an inhibitory effect on cumulus cells expansion, while meiotic resumption was suppressed only when the inhibitor aminoguanidine was added. Subsequently, to determine whether the NOS inhibition affect the S-nitrosylation of oocyte proteins, we performed in situ S-nitrosylation and the fluorescence intensity was quantified. Thus, it was shown for the first time that the S-nitrosylation of proteins is one of the ways by which NO exerts its effect on porcine maturation, revealing the importance of NO in both IVM and subsequent fertilization

Response to ovarian stimulation by FSH (Folltropin®) and yield of OPU in adult heifers obtained by different assisted reproduction techniques

Los objetivos de este trabajo han sido realizar un seguimiento ecográfico de los ovarios bovinos para comprobar la respuesta folicular de vacas adultas nacidas por diferentes técnicas de reproducción asistida (TRA), tras aplicar un protocolo de estimulación ovárica con FSH (Folltropin®); y determinar la eficacia reproductiva de los animales estudiados, analizando la cantidad y calidad de los ovocitos obtenidos por aspiración folicular (Ovum Pick-Up, OPU) antes y después del protocolo de estimulación empleado. La experiencia se ha llevado a cabo en las instalaciones del santuario bovino de la Granja Docente Veterinaria de la Universidad de Murcia. Se han utilizado 4 hembras bovinas obtenidas por distintas TRA: inseminación artificial (IA), transferencia de embriones (TE) producidos por TRA con ovocitos cultivados in vitro en medios convencionales y por TE obtenidos por TRA con ovocitos cultivados con fluidos naturales (folicular y oviductal).Los animales fueron estimulados durante 5 semanas mediante un protocolo que consistía en la aplicación de GnRH y posteriormente, FSH (Folltropin®, 350 UI/animal) durante 3 días en dosis decrecientes. Una vez a la semana, al finalizar el protocolo de estimulación ovárica, se realizaba una ecografía de los ovarios y una aspiración folicular ecoguiada (OPU) para comprobar la respuesta al protocolo de estimulación y determinar el número de folículos y de ovocitos obtenidos tras la OPU y su calidad ovocitaria. Previamente al periodo de estimulación con FSH, las vacas fueron ecografiadas y sometidas a OPU 3 semanas sin estimulación ovárica como grupo control.A lo largo del trabajo realizado, se ha comprobado la respuesta al protocolo de estimulación ovárica ensa-yado, y se ha determinado la eficacia reproductiva de los animales, según el número de folículos aspirados y la cantidad y calidad de los ovocitos obtenidos por OPU en cada una de ellas.The aims of this study were to perform an ultrasound monitoring of bovine ovaries to check the follicular response of adult heifers born by different assisted reproduction techniques (ARTs), after applying an ovarian stimulation protocol with FSH (Folltropin®); and determine the reproductive efficiency of the animals included in the study, analyzing the quantity and quality of the oocytes obtained by ultrasound guided follicular aspira-tion (Ovum Pick-Up, OPU) before and after the stimulation protocol used.The experiment was performed in the facilities of the bovine sanctuary of the Veterinary Teaching Farm of the University of Murcia. The four heifers included in the study were obtained by different ARTs: 1) artificial insemination (AI) and, 2) embryo transfer. The embryos used for ET were either produced in vitro in either conventional media or in media supplemented with natural fluids (follicular and oviductal).The animals were stimulated for 5 weeks by means of a protocol that consisted of the administration of GnRH and later, FSH (Folltropin®, 350 IU/animal) for 3 days in decreasing doses. Once a week, at the end of the ovarian stimulation protocol, an ultrasound exam of the ovaries and an OPU were performed to check the response to the stimulation protocol and to determine the number of follicles and oocytes obtained after the OPU and their quality. Prior to the stimulation with FSH, the cows were echographied and underwent OPU for 3 weeks without ovarian stimulation as a control group.In this study, we were able to evaluate the response to the ovarian stimulation protocol tested, and to deter-mine the reproductive efficiency of the animals in terms of the number of follicles aspirated, the quantity and the quality of the oocytes obtained by OP

Progesterone induces the release of bull spermatozoa from oviductal epithelial cells

The mechanism that causes the detachment of spermatozoa from the oviductal reservoir around the time of ovulation remains to be elucidated. Because the cumulus cells of the bovine oocyte are known to secrete progesterone (P4), and P4 has been shown to act upon cation channels of spermatozoa (CatSper) in human spermatozoa, it was hypothesised that P4 could induce hyperactivation due to an influx of extracellular calcium, and this would facilitate detachment of spermatozoa from oviductal epithelial cells. Therefore, this study aimed to investigate the role and mechanism of action of P4 in the release of spermatozoa from bovine oviduct epithelial cells (BOEC). Initial dose–response assessments on sperm hyperactivation determined the optimum concentration of P4 (10 nM), mibefradil (a non-specific Ca2+ channel antagonist; 5 µM), NNC 55-0396 dihydrochloride (NNC; a CatSper antagonist; 2 µM), mifepristone (a classical and membrane P4 receptor antagonist; 400 nM) and AG205 (a membrane P4 receptor antagonist; 10 μM). BOEC explants were incubated with frozen–thawed bovine spermatozoa for 30 min, following which loosely bound spermatozoa were removed. Two experiments were completed. In Experiment 1, BOECs were treated for 30 min with either no treatment, P4, NNC, mibefradil, P4 + mibefradil, P4 + NNC, P4 + mibefradil + NNC or P4 + EGTA. In Experiment 2, BOECs were treated for 30 min with either no treatment, P4, mifepristone, AG205, mifepristone + AG205, P4 + mifepristone, P4 + AG205 or P4 + mifepristone + AG205. The number of spermatozoa remaining bound per millimetre squared of BOEC explant was determined. Progesterone stimulated the release of bound spermatozoa from BOEC explants, whereas NNC, mibefradil and EGTA inhibited this release. The release of spermatozoa by P4 was inhibited in the presence of both mifepristone and AG205, whereas the combination of both had the greatest inhibitory action on P4 release of spermatozoa. These findings suggest the presence of a P4 membrane receptor on bovine spermatozoa and that P4-induced release of spermatozoa from BOECs is likely mediated by extracellular Ca2+