Impact of ram sperm cryopreservation on the dynamic changes associated with in vitro capacitation over time

Sperm cryopreservation is an important tool for preserving genetic diversity in endangered species. This technique is also relevant for accelerating and disseminating genetic progress when used in combination with artificial insemination or embryo transfer. However, the application of sperm cryopreservation in sheep breeding programs is still limited. In most ruminant species, sperm are particularly susceptible to suffer cryodamage, especially ram sperm. Changes in sperm structure or function during cryopreservation can affect sperm quality, the physiological process by which sperm acquire their fertilizing ability known as sperm capacitation, and ultimately sperm fertility. Therefore, understanding the functional, structural, and molecular damages caused by the freezing-thawing process is essential to prevent or reduce these changes. In the first part of this Thesis, a comprehensive review of the molecular changes induced by sperm cryopreservation and their effect on different sperm structures and functions was carried out to better understand how sperm functionality and fertility is affected after cryopreservation. Moreover, those factors that may influence sperm cryotolerance as well as the new strategies developed to reduce sperm cryodamage were also discussed in this review from a molecular point of view. After reviewing diverse molecular aspects of sperm cryobiology, different studies were carried out in the present Thesis to elucidate a relevant topic that is not fully understood: ''how cryopreservation of ram sperm affects the dynamic changes associated with sperm capacitation over time.'' In the initial chapter of this Thesis, we investigated the effect of the cryopreservation process on ram sperm functionality during in vitro capacitation. Diverse sperm parameters (viability, mitochondrial activity, acrosome integrity, tyrosine phosphorylation, ROS production, and kinematics) were evaluated in fresh and frozen-thawed ram sperm incubated under capacitating conditions at 1, 5, 15, 30, 60, 120, 180 and 240 min and under non-capacitating conditions at 0, 15 and 240 min. Flow cytometry and Computer Assisted Sperm Analysis System (CASA) were used for different analyses. After a short incubation period under capacitating conditions (from 5 to 30 min), mitochondrial activity and tyrosine phosphorylation significantly increased in cryopreserved ram sperm. Simultaneously, the motility pattern of cryopreserved sperm also changed, showing low linearity (LIN) and high curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH), which might be related to hyperactivation. Similar modifications were found in fresh sperm but only after an incubation period of 180 to 240 min under capacitating conditions. In both types of samples (fresh and cryopreserved sperm), tyrosine phosphorylated proteins showed a different distribution during the time course of in vitro capacitation. While tyrosine phosphorylation remained practically constant in the head in fresh and cryopreserved sperm, phosphorylation progressively increased in the midpiece (at 15-30 min in cryopreserved samples and 180-240 min in fresh samples) and then in the principal piece (at 30 min in cryopreserved samples and 240 min in fresh samples). These findings suggest that cryopreserved ram sperm may undergo capacitation-associated changes similar to fresh sperm, but after a brief exposure to capacitating conditions (from 5 to 30 min). On the contrary, incubations exceeding that period of time severely impaired sperm quality in cryopreserved samples, being statistically significant after 30 min for acrosome integrity, 60 min for sperm motility and 120 min for sperm viability and mitochondrial activity. In the second chapter, we explored how cryopreservation alters ram sperm proteome during in vitro capacitation to have a better understanding of the effect of cryopreservation on sperm capacitation at the molecular level. The protein profile of ram sperm was studied and compared at different time points of incubation under capacitating conditions (1, 15 and 240 min) and non-capacitating conditions (0, 15 and 240 min) in fresh and frozen-thawed samples using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). Most of the identified proteins were located at the plasma membrane (34%), cytoplasm (25%), and mitochondria (18%). The comparison between fresh and cryopreserved sperm proteins showed that cryopreservation increased biological processes related to apoptosis-stress response and redox status, while the comparison between sperm proteins from sperm incubated in capacitating and non-capacitating conditions revealed that capacitation increased biological processes associated with signaling, metabolism, motility, and reproduction. Twelve proteins showed quantitative changes between fresh and cryopreserved sperm at 0 and 15 min of incubation under capacitating and non-capacitating conditions. Furthermore, three proteins in fresh samples and six proteins in cryopreserved samples underwent significant changes in abundance during the time course of in vitro capacitation. These differentially abundant proteins were involved in oocyte recognition, sperm motility, metabolism, signaling, apoptosis-stress response and spermatogenesis. These results showed that cryopreservation altered diverse proteins required for an optimal sperm functionality and fertility, which may explain why fresh and cryopreserved ram sperm showed different responses to the capacitation process. Moreover, this study allowed the identification of several ram sperm proteins altered by cryopreservation that are believed to play a key role during capacitation. In the third and last chapter of this Thesis, we investigated whether cryopreservation affected the dynamic changes that take place in the chromatin structure of ram sperm during in vitro capacitation. Variations in the chromatin structure are closely related to sperm fertility and previous studies in other species have shown that the chromatin structure of sperm can be modified during capacitation. Therefore, we hypothesized that in vitro capacitation of ram sperm could also produce changes in the chromatin structure and these changes could be affected by cryopreservation. Diverse nuclear parameters, including sperm chromatin decondensation, high DNA stainability (HDS), DNA fragmentation (%DFI) and DNA methylation were evaluated in fresh and frozen-thawed ram sperm after 1, 5, 15, 30, 60, 120, 180 and 240 min under capacitating conditions and after 0, 15 and 240 min under non-capacitating conditions. Flow cytometry (Sperm Chromatin Structure Assay; SCSA), immunocytochemistry (5-Methylcytosine) and a cytochemical stain (aniline blue) were used for different analyses. Despite cryopreserved sperm showed a higher DNA fragmentation than fresh sperm, chromatin stability was not affected in any type of sample (fresh and cryopreserved Summary 5 sperm) over the incubation period under capacitating and non-capacitating conditions(%DFI remained constant). In contrast, chromatin decondensation and HDS significantly increased at specific time points during in vitro capacitation of ram sperm but not under non-capacitating conditions, reaching the highest values from 5 to 30 min in cryopreserved samples and from 180 to 240 min in fresh samples. Prolonged incubations (60-240 min) under capacitating conditions also induced hypermethylation in cryopreserved ram sperm. These findings revealed that in vitro capacitation reduced sperm chromatin condensation in fresh and cryopreserved ram sperm without adversely affecting DNA integrity. Such changes in chromatin condensation appeared in cryopreserved sperm earlier than in fresh sperm, while prolonged incubations under capacitating conditions altered the DNA methylation pattern of cryopreserved ram sperm. In conclusion, the present Thesis provides an extensive characterization of the multiple effects that sperm cryopreservation has on the numerous events that occur progressively and simultaneously during in vitro capacitation of ram sperm. Most of these events were similar between fresh and cryopreserved ram sperm but occurred faster after sperm cryopreservation, probably as a result of those functional, structural and protein changes observed in ram sperm after the cryopreservation process