Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success

Palabras clave extraídas del contenido del artículo (título y materias)[EN] Mammalian spermatozoa undergo a strong selection process along the female tract to guarantee fertilization by good quality cells, but risks of fertilization with DNA-damaged spermatozoa have been reported. In contrast, most external fertilizers such as fish seem to have weaker selection procedures. This fact, together with their high prolificacy and external embryo development, indicates that fish could be useful for the study of the effects of sperm DNA damage on embryo development.We cryopreserved sperm from rainbow trout using egg yolk and low-density lipoprotein as additives to promote different rates of DNA damage. DNA fragmentation and oxidization were analyzed using comet assay with and without digestion with restriction enzymes, and fertilization trials were performed. Some embryo batches were treated with 3-aminobenzamide (3AB) to inhibit DNA repair by the poly (ADP-ribose) polymerase, which is an enzyme of the base excision repair pathway. Results showed that all the spermatozoa cryopreserved with egg yolk carried more than 10% fragmented DNA, maintaining fertilization rates of 61.1G2.3 but a high rate of abortions, especially during gastrulation, and only 14.5±4.4 hatching success. Furthermore, after 3AB treatment, hatching dropped to 3.2±2.2, showing that at least 10% DNA fragmentation was repaired. We conclude that trout sperm maintains its ability to fertilize in spite of having DNA damage, but that embryo survival is affected. Damage is partially repaired by the oocyte during the first cleavage. Important advantages of using rainbow trout for the study of processes related to DNA damage and repair during development have been reportedSIThis work was supported by grant LE007A06 from the Junta de Castilla y León (Spain)

Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss)

Palabras clave extraídas del título[EN] Zygotic repair of the paternal genome is a key event after fertilization. Spermatozoa accumulate DNA strand breaks during spermatogenesis and can suffer additional damage by different factors, including cryopreservation. Fertilization with DNA-damaged spermatozoa (DDS) is considered to promote implantation failures and abortions, but also long-term effects on the progeny that could be related with a defective repair. Base excision repair (BER) pathway is considered the most active in zygotic DNA repair, but healthy oocytes contain enzymes for all repairing pathways. In this study, the effects of the inhibition of the BER pathway in the zygote were analyzed on the progeny obtained after fertilization with differentially DDS. Massive gene expression (GE; 61 657 unique probes) was analyzed after hatching using microarrays. Trout oocytes are easily fertilized with DDS and the high prolificacy allows live progeny to be obtained even with a high rate of abortions. Nevertheless, the zygotic inhibition of Poly (ADP-ribose) polymerase, upstream of BER pathway, resulted in 810 differentially expressed genes (DEGs) after hatching. DEGs are related with DNA repair, apoptosis, telomere maintenance, or growth and development, revealing a scenario of impaired DNA damage signalization and repair. Downregulation of the apoptotic cascade was noticed, suggesting a selection of embryos tolerant to residual DNA damage during embryo development. Our results reveal changes in the progeny from defective repairing zygotes including higher malformations rate, weight gain, longer telomeres, and lower caspase 3/7 activity, whose long-term consequences should be analyzed in depthSIThis work was supported by the Junta de Castilla y León (Spain) (project LE365A11-2) and the Spanish Ministry of Economy and Competitiveness (project AGL2011-27787

TMEM95 is a sperm membrane protein essential for mammalian fertilization.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization. © 2020, Lamas-Toranzo et al

Behavioral mechanisms of mammalian sperm guidance

In mammals, sperm guidance in the oviduct appears essential for successful sperm arrival at the oocyte. Hitherto, three different potential sperm guidance mechanisms have been recognized: thermotaxis, rheotaxis, and chemotaxis, each of them using specific stimuli - a temperature gradient, fluid flow, and a chemoattractant gradient, respectively. Here, we review sperm behavioral in these mechanisms and indicate commonalities and differences between them

Comparison of four methods to evaluate sperm DNA integrity between mouse caput and cauda epididymidis

It is well known that transit through the epididymis involves an increase in the compaction of sperm chromatin, which acquires fully condensed status at the caput epididymidis. The purpose of this study was to compare the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay, the comet assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test by analysing spermatozoa from the caput and cauda epididymidis in order to demonstrate the ability of each technique to discriminate between different degrees of sperm maturity related to chromatin compaction and DNA fragmentation. Our results suggest that some populations of DNA-fragmented spermatozoa associated with immature sperm can only be identified using the comet assay and the SCSA but not with the SCD test or the TUNEL assay. © 2012 AJA, SIMM & SJTU. All rights reserved

Progesterone effects on mouse sperm kinetics in conditions of viscosity

The spermatozoa delivered to the female genital tract need to swim towards the oocyte through viscous secretions. Once close to the oocyte, the spermatozoa are guided by a gradient of progesterone (P4 ) and other unknown chemoattractants via a process known as chemotaxis. Using polyvinylpyrrolidone to establish the conditions of viscosity, we examined the response of mouse spermatozoa to P4 . Herein, we show that in low-viscous media, P4 induces hyperactive-like motility whereby sperm show erratic trajectories and non-progressive movement. However, an opposite response is produced in viscous medium in that trajectories are linear and motility is more progressive and less erratic. Our observations provide a behavioural explanation for the chemotaxis of spermatozoa swimming under viscous conditions in a spatial gradient of the chemoattractant P4 . They also highlight the importance of using viscous solutions to mimic in vivo conditions when analysing sperm behaviour in response to any stimulus. © 2016 Society for Reproduction and Fertility

Altered gene transcription and telomere length in trout embryo and larvae obtained with DNA cryodamaged sperm

Sperm cryopreservation could entail DNA damage, promoting base oxidization and strand breaks. In a previous work we showed that trout DNA damaged sperm is able to fertilize leading to embryo loss when the repair system of the oocyte is inhibited. Here we have analysed the later effects on embryo and larvae of fertilizing trout oocytes with cryopreserved DNA-damaged spermatozoa. Fish have weak sperm selection mechanisms, are very prolific and have external embryo development, being convenient models for this type of study. We cryopreserved rainbow trout semen using extenders containing egg yolk or their low density lipoprotein fraction to obtain samples with different degrees of DNA damage. DNA fragmentation was evaluated using the Comet assay and telomere length using quantitative-PCR. Fertilization trials were performed and the transcription at different developmental stages of telomerase reverse transcriptase (Tert) and eight genes related with embryo growth and development (Igf1, Igf2, Igfr1a, Igfr1b, Gh1, Gh2, Ins1 and Ins2) were analyzed using quantitative-PCR in surviving embryos and larvae. Results showed an increase in sperm DNA fragmentation after both cryopreservation procedures as well as a decrease in sperm telomere length. Larvae obtained with damaged sperm showed longer telomeres and Tert overexpression. The transcription of the analyzed genes in these embryos and larvae was also modified with respect to the control, most of them as an increase at hatch. We conclude that fertilization with cryopreserved DNA-damaged spermatozoa significantly affects offspring performance, detectable as an increase in telomere length as well as some alterations in gene expression in surviving embryo and larvae. © 2011 Elsevier Inc

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

14 Pág.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.This research was funded by the Spanish Ministry of Science, Innovation and Universities, Grant RTI2018-096736-A-I00 and by the Community of Madrid, Grant IND2018/BIO-9610. S.R.D. was supported by “doctorados industriales 2018” fellowship of the Community of Madrid (IND2018/BIO-9610). S.P.C. was supported by a Ramón y Cajal contract from the Spanish Ministry of Science, innovation and Universities (RYC-2016-20147).Peer reviewe

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