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

Soriano‐Úbeda, Cristina; Romero-Aguirregomezcorta, Jon; Matás, Carmen; Visconti, Pablo E.; García–Vázquez, Francisco Alberto

doi:10.1186/s40104-019-0324-y

Cited by 28 publications

(35 citation statements)

References 54 publications

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“…Mouflon sperm clusters described here are in accordance with those described in domestic ram 86 and boar sperm 87 during capacitation. Our results show that kinematic parameters followed an hyperactivated pattern in ejaculated sperm under capacitating conditions but not in epididymal sperm which supports the immunoblot and IFF results.…”

Section: Discussionsupporting

confidence: 88%

Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon)

Martínez-Fresneda

Castaño

Bóveda

et al. 2019

Sci Rep

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Spermatozoa must undergo the process of capacitation to fertilize the egg which involves a cell destabilizing process. Capacitation-like changes such as protein tyrosine phosphorylation (PTP) are associated with cryopreservation. The aim of this study was to compare the cryoresistance and capacitation response of epididymal and ejaculated sperm of European mouflon (Ovis musimon). Post-thaw sperm parameters were analysed from epididymal and ejaculated samples cryopreserved by slow-freezing or ultrarapid-freezing for comparison. Sperm capacitation status was assessed by the semiquantification of PTP levels, cell localization of PTP and kinematic clustering. Epididymal sperm had higher cryoresistance than ejaculated sperm in both freezing techniques, and slow-freezing rendered better results than ultrarapid-freezing in both sperm samples. Ejaculated sperm had higher PTP levels than epididymal sperm and, additionally, ejaculated sperm showed higher phosphorylation in capacitating (CA) than in non-capacitating (NCA) conditions while there was no effect of medium in epididymal sperm. There was a higher tail PTP in CA than in NCA conditions in both types of sperm. Kinematic analysis revealed that the cluster associated with hyperactivated movement increased in ejaculated sperm incubated in CA whereas no effect of medium was observed in epididymal sperm clusters. In conclusion, epididymal sperm showed better freezability and lower capacitation status compared to ejaculated sperm.

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Section: Discussionsupporting

confidence: 88%

Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon)

Martínez-Fresneda

Castaño

Bóveda

et al. 2019

Sci Rep

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“…Total protein was isolated from 2 × 10 6 spermatozoa, immunoblotted and stripped as previous studies of our group described . The primary polyclonal antibodies were antiphospho‐PKA substrates (9624, Cell Signaling Technology, 1:2000), antiphosphotyrosine (4G10, Millipore, 1:10,000), and anti‐β‐tubulin (β‐tub) (T0198, 1:5000).…”

Section: Methodsmentioning

confidence: 99%

“…Sperm suspensions were added to the IVF wells giving a final concentration of 25 × 10 3 cells/mL. After 18 hours coculture, the putative zygotes were fixed with 0.5% glutaraldehyde in phosphate‐buffered saline (PBS), stained with 1% (w/v) Hoechst 33342 in PBS, and examined under an epifluorescence microscope at 200× and 400× (Leica® DMR) as has been previously described . The parameters analyzed were percentage of penetrated oocytes by one or more spermatozoa (Pen, %), percentage of oocytes penetrated by only one spermatozoon or monospermy percentage of penetrated oocytes (Mon, %), mean number of spermatozoa per penetrated oocyte (Spz/O), and number of spermatozoa bound to zona pellucida (Spz/ZP) (Supporting Information).…”

Section: Methodsmentioning

confidence: 99%

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig

et al. 2020

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Background: Molecules from the female reproductive tract modulate capacitation and function of sperm cells in vivo. These molecules vary in a quantitative and qualitative manner throughout the estrous cycle. Objectives: This work evaluates the effect of using various female reproductive fluids on capacitation and fertilization of pig spermatozoa in vitro. Material and methods: The effects of culturing spermatozoa in different fluids on the levels of sperm protein kinase A (pPKA), tyrosine phosphorylation, acrosome reaction, and in vitro fertilization (IVF) were evaluated. The fluids tested were as follows: oviductal fluid (OF) from five phases of the estrous cycle, namely early and late follicular (OF-EF, OF-LF), early and late luteal (OF-EL, OF-LL) and periovulatory (pOF), follicular fluid from medium-sized follicles, and secretions of cumulus-oocyte complexes (conditioned medium). Results:The pPKAs and tyrosine phosphorylation were decreased by OF-EF, OF-LF, OF-EL, and pOF but not by follicular fluid and conditioned medium. OF-EF, OF-LF, and pOF also decreased the sperm acrosome reaction. Moreover, the effect of pOF on pPKAs and tyrosine phosphorylation was reversible. In in vitro fertilization, OF-EF, OF-LF, OF-EL, and pOF reduced the percentage of penetrated oocytes, the mean number of spermatozoa per penetrated oocyte, and increased monospermy.Conclusion: OF from follicular, early luteal, and periovulatory phases of the estrous cycle modulates the sperm protein phosphorylation as well as the acrosome reaction involved in capacitation and increases monospermic fertilization in in vitro fertilization. Our findings suggest that fluids from the female reproductive tract could be used as additives in porcine IVF systems to modulate sperm-oocyte interaction. K E Y W O R D Sboar, fluid, in vitro fertilization, oviduct, capacitation

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“…However, despite some clear evidences found in vitro, little attention has been paid to mimicking the in vivo sperm transit, specially through the uterine horns, for further use in ARTs. Thus, pH, CO 2 or HCO 3 − variations in culture media during in vitro gamete preparation and/or interaction showed relevant consequences in the fertilization output [49,50]. Also, an in uence of the uterine uid composition on sperm selection and consequent embryo culture was observed in vitro in several species including porcine [51][52][53].…”

Section: Discussionmentioning

confidence: 99%

In vivo measurement of pH, CO2 and HCO3- levels in the uterus of non-anesthetized sows through the estrous cycle and after insemination

López-Albors¹,

Llamas-López²,

Ortuño³

et al. 2020

Preprint

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Background The pH-CO2-HCO3− system is a ubiquitous biological regulator with important functional implications for reproduction. Knowledge of the physiological values of its components is relevant for reproductive biology and the optimization of Assistant Reproductive Techniques (ARTs). In vivo pH of the oviduct and uterus has been estimated by direct in situ measurements in a few species. However, regarding the levels of CO2 and HCO3−, information is very scarce and, when available, it comes from fluid samples instead of in vivo estimations. This study describes a non-invasive method to measure pH and % of CO2 in the uterus of sows with cutting-edge technology and no medication. Sows were at three different reproductive conditions, estrous with no insemination E(-)AI and after insemination E(+)AI, and diestrous (non-estrous, NE). From pH and CO2 data, HCO3− concentration was estimated. Results The designed methodology allowed for in situ time-lapse recording of pH and % of CO2 within the uterus of non-anesthetized sows. Variable oscillatory patterns of pH, CO2 and HCO3− were found independently of the estrous condition. Insemination did not changed the levels of uterine pH, % of CO2 and HCO3− concentration, -E(-)AI = E(+)AI-, but all the values were affected by the estrous cycle in a way that decreased significantly at diestrous condition - E(-)AI and E(+)AI > NE-. Conclusions A non-invasive approach to the porcine uterus with novel optical probes allowed the obtaining of in situ physiological values of pH, CO2, and HCO3− at different reproductive conditions. While the short-time presence of sperm in the uterus did not change the physiological milieu, the whole pH-CO2-HCO3− system was affected by the estrous cycle. This study contributes to a better understanding of the in vivo regulation of the pH/CO2/HCO3− system in the uterus and may help to optimize the protocols of sperm treatment for in vitro fertilization.

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

Cited by 28 publications

References 54 publications

Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon)

Epididymal and ejaculated sperm differ on their response to the cryopreservation and capacitation processes in mouflon (Ovis musimon)

Periovulatory oviductal fluid decreases sperm protein kinase A activity, tyrosine phosphorylation, and in vitro fertilization in pig

In vivo measurement of pH, CO2 and HCO3- levels in the uterus of non-anesthetized sows through the estrous cycle and after insemination

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