Bull spermatozoa selected by thermotaxis exhibit high DNA integrity, specific head morphometry, and improve ICSI outcome.

BACKGROUND: Sperm migration by thermotaxis is a guidance mechanism that operates along the oviduct and it has proved to be a valid method for selecting spermatozoa with low DNA fragmentation (SDF) in mice, humans, and stallions. This study aimed to analyse if bull spermatozoa could be selected by thermotaxis and to assess their quality in terms of SDF as well as determine the presence of a specific sperm subpopulation based on sperm morphometry and assess their fertilizing capacity by ICSI. METHODS: We used frozen-thawed sperm from 6 bulls and sperm selection by thermotaxis was performed with TALP medium supplemented with 25 mmol/L of HEPES and 5 mmol/L of caffeine. In these conditions, sperm selection was achieved, obtaining a net thermotaxis of 3.6%. Subsequently, we analysed the SDF of the migrated and not-migrated spermatozoa using the neutral COMET assay, and we evaluated the size of the sperm head using Hemacolor® staining with Motic Images Plus 3 software. Additionally, migrated and not-migrated spermatozoa by thermotaxis were used to fertilize bovine in vitro matured (IVM) oocytes by ICSI, a very inefficient procedure in cattle that is only successful when the oocyte is artificially activated. RESULTS: The results showed lower SDF (χ², P < 0.001, 13.3% reduction, n = 8) and lower head size parameters (length and width, P < 0.01; and perimeter and area, P < 0.001; n = 4) in those spermatozoa migrated in comparison to those not-migrated. The distribution of sperm subpopulations structure varied between groups, highlighting cluster 2, characterized by spermatozoa with small head size, and high ellipticity and elongated heads, as the most abundant in the thermotaxis migrated group. When performed ICSI (without oocyte artificial activation) with the thermotactic sperm, the blastocyst rate was 32.2% ± 9.3% in the group microinjected with the thermotactic spermatozoa vs. 8.3% ± 7.8% in the group of not-migrated sperm (χ², P < 0.05). CONCLUSION: Our results showed that bull sperm selection by thermotaxis has a much higher DNA integrity, small and elongated head size parameters, and different sperm subpopulation structure than the not-selected spermatozoa. Additionally, we evidenced that thermotactic spermatozoa improve ICSI success rates.

Protein Identification of Spermatozoa and Seminal Plasma in Bottlenose Dolphin (Tursiops truncatus).

Proteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins (Tursiops truncatus) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies.

Sperm Metabolomics through Nuclear Magnetic Resonance Spectroscopy.

This report reviews current knowledge of sperm metabolomics analysis using proton nuclear magnetic resonance spectroscopy (1 H-NMR) with particular emphasis on human and farm animals. First, we present the benefits of NMR over other techniques to identify sperm metabolites and then describe the specific methodology required for NMR sperm analysis, stressing the importance of analyzing metabolites extracted from both the hydrophilic and lipophilic phases. This is followed by a description of advances produced to date in the use of NMR to diagnose infertility in humans and to identify metabolic differences among the sperm of mammalian herbivore, carnivore, and omnivore species. This last application of NMR mainly seeks to explore the possible use of lipids to fuel sperm physiology, contrary to previous theories that glycolysis and oxidative phosphorylation (OXPHOS) are the only sources of sperm energy. This review describes the use of NMR to identify sperm and seminal plasma metabolites as possible indicators of semen quality, and to examine the metabolites needed to maintain sperm motility, induce their capacitation, and consequently, to predict animal fertility.

A drastic shift in the energetic landscape of toothed whale sperm cells.

Mammalian spermatozoa are a notable example of metabolic compartmentalization.1 Energy in the form of ATP production, vital for motility, capacitation, and fertilization, is subcellularly separated in sperm cells. While glycolysis provides a local, rapid, and low-yielding input of ATP along the flagellum fibrous sheath, oxidative phosphorylation (OXPHOS), far more efficient over a longer time frame, is concentrated in the midpiece mitochondria.2 The relative weight of glycolysis and OXPHOS pathways in sperm function is variable among species and sensitive to oxygen and substrate availability.3-5 Besides partitioning energy production, sperm cell energetics display an additional singularity: the occurrence of sperm-specific gene duplicates and alternative spliced variants, with conserved function but structurally bound to the flagellar fibrous sheath.6,7 The wider selective forces driving the compartmentalization and adaptability of this energy system in mammalian species remain largely unknown, much like the impact of ecosystem resource availability (e.g., carbohydrates, fatty acids, and proteins) and dietary adaptations in reproductive physiology traits.8 Here, we investigated the Cetacea, an iconic group of fully aquatic and carnivorous marine mammals, evolutionarily related to extant terrestrial herbivores.9 In this lineage, episodes of profound trait remodeling have been accompanied by clear genomic signatures.10-14 We show that toothed whales exhibit impaired sperm glycolysis, due to gene and exon erosion, and demonstrate that dolphin spermatozoa motility depends on endogenous fatty acid ß-oxidation, but not carbohydrates. Such unique energetic rewiring substantiates the observation of large mitochondria in toothed whale spermatozoa and emphasizes the radical physiological reorganization imposed by the transition to a carbohydrate-depleted marine environment.

Characterization of Bottlenose Dolphin (Tursiops truncatus) Sperm Based on Morphometric Traits.

Bottlenose dolphin (Tursiops truncatus) males follow many reproductive strategies to ensure their paternity. However, little is known about the sperm traits, including morphometric features, that contribute to their reproductive success. Our aim was to study dolphin sperm morphometry (a total of 13 parameters) in two adult males to evaluate (i) presumptive sperm subpopulations, (ii) the correlation of sperm morphometry with testosterone levels and (iii) the effect of refrigerated storage on the sperm morphometry. Sperm populations were classified into four principal components (PCs) based on morphometry (>94% of cumulative variance). The PCs clustered into two different sperm subpopulations, which differed between males. Furthermore, the levels of serum testosterone were positively correlated with the length of the midpiece but negatively correlated with head width and the principal piece, flagellum and total sperm lengths. Most of the sperm morphometric parameters changed during the storage period (day 1 vs. day 7), but only the principal piece length was affected by the storage temperature (5 °C vs. 15 °C). This is the first study to identify dolphin sperm subpopulations based on morphometry and the influence of serum testosterone and refrigeration on sperm morphometry.

The Presence of D-Penicillamine during the In Vitro Capacitation of Stallion Spermatozoa Prolongs Hyperactive-Like Motility and Allows for Sperm Selection by Thermotaxis.

Assisted reproductive technologies (ARTs) in the horse still yield suboptimal results in terms of pregnancy rates. One of the reasons for this is the lack of optimal conditions for the sperm capacitation in vitro. This study assesses the use of synthetic human tubal fluid (HTF) supplemented with D-penicillamine (HTF + PEN) for the in vitro capacitation of frozen/thawed stallion spermatozoa by examining capacitation-related events over 180 min of incubation. Besides these events, we explored the in vitro capacity of the spermatozoa to migrate by thermotaxis and give rise to a population of high-quality spermatozoa. We found that HTF induced higher levels of hyperactive-like motility and protein tyrosine phosphorylation (PTP) compared to the use of a medium commonly used in this species (Whitten's). Also, HTF + PEN was able to maintain this hyperactive-like motility, otherwise lost in the absence of PEN, for 180 min, and also allowed for sperm selection by thermotaxis in vitro. Remarkably, the selected fraction was enriched in spermatozoa showing PTP along the whole flagellum and lower levels of DNA fragmentation when compared to the unselected fraction (38% ± 11% vs 4.4% ± 1.1% and 4.2% ± 0.4% vs 11% ± 2% respectively, t-test p < 0.003, n = 6). This procedure of in vitro capacitation of frozen/thawed stallion spermatozoa in HTF + PEN followed by in vitro sperm selection by thermotaxis represents a promising sperm preparation strategy for in vitro fertilization and intracytoplasmic sperm injection in this species.

Changes in the Cellular Distribution of Tyrosine Phosphorylation and Its Relationship with the Acrosomal Exocytosis and Plasma Membrane Integrity during In Vitro Capacitation of Frozen/Thawed Bull Spermatozoa.

During sperm capacitation, intracellular signaling leads to protein tyrosine phosphorylation (PTP) of multiple cellular structures. However, the connection of this molecular signaling to the physiology of capacitated spermatozoa is not completely understood. This is the case of the short lifespan of capacitated spermatozoa and their increased susceptibility to initiate acrosomal exocytosis (AE) during incubation. Herein, by employing frozen/thawed bull spermatozoa, we aimed to study the relationship between PTP with AE and with plasma membrane integrity (PMI) at the cellular level. For this, we employed double staining following immunofluorescence for PTP combined with fluorescence probes for the acrosome (PNA-FITC) and PMI (LIVE/DEAD Fixable Dead Cell Stain Kit). Our results revealed that the presence of PTP at sperm head was less abundant in the sperm fraction that triggered the AE after 3 h of incubation under capacitating conditions, or by its induction with calcium ionophore, compared to the unreacted fraction. Furthermore, PTP at the equatorial region of the head (PTP-EQ) was enriched in the fraction showing damaged membrane while induction of AE with calcium ionophore did not alter the PMI and its relation to PTP-EQ. These results suggest that spontaneous AE and induced AE trigger similar cellular events regarding PTP and the spermatozoa showing PTP-EQ are more prone to suffer plasma membrane damage.

Novel Techniques of Sperm Selection for Improving IVF and ICSI Outcomes.

Almost 50% of the infertility cases are due to male factors. Assisted reproductive technologies (ARTs) allow to overcome the incapacity of these patients' spermatozoa to fertilize the oocyte and produce a viable and healthy offspring, but the efficiency of the different techniques has still the potential to improve. According to the latest reports of the European Society of Human Reproduction and Embryology (ESHRE) and the Centers for Disease Control and Prevention of the United States (CDC), the percentages of deliveries per ART cycle in 2014 and 2016 were 21 and 22%, respectively. Among the reasons for this relatively low efficiency, the quality of the spermatozoa has been pointed out as critical, and the presence of high percentages of DNA-damaged spermatozoa in patients' ejaculates is possibly one of the main factors reducing the ARTs outcomes. Thus, one of the main challenges in reproductive medicine is to ensure the highest quality of the spermatozoa used in ARTs, and specifically, in terms of genetic integrity. The latest techniques for the preparation and selection of human spermatozoa are herein discussed focusing on those proven to improve one or several of the following parameters: sperm genetic integrity, fertilization capacity, embryo production, and in vitro survival, as well as pregnancy and delivery rates following in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In addition, we discuss the potential of techniques developed in non-human mammals that could be further transferred to the clinic.