Protective effects of a SIRT1 inhibitor on primordial follicle activation and growth induced by cyclophosphamide: insights from a bovine in vitro folliculogenesis system.

PURPOSE: Although oncological advances have improved survival rates of female cancer patients, they often suffer a reduced fertility due to treatment side effects. In the present study, we evaluated the potential fertoprotective effects of the specific inhibitor of SIRT1, EX-527, on the gonadotoxic action exerted by cyclophosphamide (CPM) on loss of primordial follicles (PFs). METHODS: The effects of the CPM metabolite phosphoramide mustard (PM) on follicle activation, growth and viability and the protective action of EX-527 against PM effects were evaluated on bovine ovarian cortical strips in vitro cultured for 1 or 6 days. To understand whether PFs exposed to PM plus EX-527 were able to activate and grow to the secondary stage after suspension of the treatment, strips cultured for 3 days in PM plus EX-527 for 3 days were transferred to plain medium until day 6. Follicle growth and health were evaluated through histology and viability assay at a confocal microscope. In order to investigate the molecular pathways underlying the ovarian response to PM in the presence of EX-527, we analysed the protein level of SIRT1, HuR, PARP1 and SOD2 after 1 day of in vitro culture. RESULTS: We found that (1) PM, the main CPM active metabolite, promotes PF activation; (2) the ovarian stress response induced by PM includes a SIRT1-dependent pathway; and (3) EX-527 reduces PF activation and growth induced by PM. CONCLUSION: SIRT1 can represent a candidate molecule to be targeted to protect ovarian follicles from alkylating agents and EX-527 could represent a potential fertoprotective agent for cancer patients.

Fertility preservation during the COVID-19 pandemic: mitigating the viral contamination risk to reproductive cells in cryostorage.

Reopening fertility care services across the world in the midst of a pandemic brings with it numerous concerns that need immediate addressing, such as the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on the male and female reproductive cells and the plausible risk of cross-contamination and transmission. Due to the novelty of the disease the literature contains few reports confirming an association of SARS-CoV-2 with reproductive tissues, gametes and embryos. Cryobanking, an essential service in fertility preservation, carries the risk of cross-contamination through cryogenic medium and thus calls for risk-mitigation strategies. This review aims to address the available literature on the presence of SARS-CoV-2 on tissues, gametes and embryos, with special reference to the possible sources of cross-contamination through liquid nitrogen. Strategies for risk mitigation have been extrapolated from reports dealing with other viruses to the current global crisis, for safety in fertility treatment services in general, and specifically for oncofertility.

Germinal stage vitrification is superior to MII stage vitrification in prepubertal mouse oocytes.

This study investigated if in vitro maturation (IVM) before or after vitrification would be more successful for prepubertal oocytes. To mimic prepubertal conditions in an experimental setup, oocytes were collected from healthy 14, 21 and 28day old Swiss albino mice. The germinal vesicle (GV) stage oocytes and in vitro matured MII oocytes were subjected to vitrification-warming. Both structural (meiotic spindle morphology, mitochondrial integrity, cortical granules) and functional (sperm zona binding, fertilization) characteristics were assessed in oocytes after warming. This study demonstrated that IVM was more detrimental to prepubertal oocytes than to young adults. Further, vitrification of the IVM oocytes resulted in an increase in the number of abnormal meiotic spindles, a change in the cortical distribution pattern, a reduction in sperm zona binding and the fertilization rate. Importantly, oocyte integrity was better when prepubertal oocytes were vitrified before, rather than after, IVM. The above observations support GV stage vitrification for prepubertal oocytes requiring fertility preservation. Understanding the mechanisms behind the differing outcomes for oocytes from immature females will help in refining current protocol, thereby retaining the oocytes' maximum structural and functional integrity Further investigation is necessary to determine whether human prepubertal oocytes also behave in a similar way. It is to be noted here, with great emphasis, that a major limitation of this study is that the oocytes' abilities were tested only until fertilisation, as a consequence of which the study cannot reveal the developmental potentials of the embryos beyond fertilisation.

Supplementation of sperm media with zinc, D-aspartate and co-enzyme Q10 protects bull sperm against exogenous oxidative stress and improves their ability to support embryo development.

High levels of reactive oxygen species in the semen of infertile patients or spontaneously generated during in vitro sperm handling may impair sperm quality, fertilization and embryo developmental competence. We recently reported that zinc, d-aspartate and co-enzyme Q10, contained in the dietary supplement Genadis® (Merck Serono), have protective effects on human and bull sperm motility, lipid peroxidation and DNA fragmentation in vitro; furthermore, in bovine, treated spermatozoa had an improved ability to support embryo development. However, only a few studies have investigated the protective role of antioxidants during in vitro sperm handling in the presence of an exogenous oxidative stress. Herein, to simulate such conditions in an animal model, we induced exogenous oxidative stress on spermatozoa through the xanthine-xanthine oxidase system and investigated its effects on sperm function and subsequent embryo developmental competence in the presence of zinc, d-Asp and CoQ10 protection. The main results showed that exogenous oxidative stress decreased sperm motility, increased sperm DNA fragmentation, and reduced fertilization and blastocyst rates and quality. Pre-treatment with zinc, d-aspartate and co-enzyme Q10 before exogenous oxidative stress was able to prevent these effects. Supplementation of sperm culture media with zinc, d-aspartate and co-enzyme Q10 could protect sperm from oxidative stress damage during in vitro handling in assisted reproductive technologies.

Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures.

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

Individually Cultured Bovine Zygotes Successfully Develop to the Blastocyst Stage in an Extremely Confined Environment.

The possibility of detecting the developmental competence of individually cultured embryos through analysis of spent media is a major current trend in an ART setting. However, individual embryo culture is detrimental compared with high-density group culture due to the reduced concentration of putative embryotropins. The main aim of this study was to identify an individual culture system that is not detrimental over high-density group culture in the bovine model. Blastocyst rates and competence were investigated in a conventional (GC) group, semi-confined group (MG), and individual culture (MS) in a commercial microwell device. Main findings showed that: (1) individual embryos can be continuously cultured for 7 days in ~70 nL microwells (MS) without detrimental effects compared with the GC and MG; (2) MS and MG blastocysts had a reduced number of TUNEL-positive cells compared to GC blastocysts; (3) though blastocyst mean cell numbers, mitochondrial activity, and lipid content were not different among the three culture conditions, MS blastocysts had a higher frequency of small-sized lipid droplets and a reduced mean droplet diameter compared with GC and MG blastocysts. Overall, findings open the way to optimize the development and competence of single embryos in an ART setting.

Advanced Maternal Age Affects the Cryosusceptibility of Ovulated but not In Vitro Matured Mouse Oocytes.

Oocyte cryopreservation is offered to women of various age groups for both health and social reasons. Oocytes derived from either controlled ovarian stimulation or in vitro maturation (IVM) are cryopreserved via vitrification. As maternal age is a significant determinant of oocyte quality, there is limited data on the age-related susceptibility of oocytes to the vitrification-warming procedure alone or in conjunction with IVM. In the present study, metaphase II oocytes obtained from 2, 6, 9, and 12 month old Swiss albino mice either by superovulation or IVM were used. To understand the association between maternal age and oocyte cryotolerance, oocytes were subjected to vitrification-warming and compared to non vitrified sibling oocytes. Survived oocytes were evaluated for mitochondrial potential, spindle integrity, relative expression of spindle checkpoint protein transcripts, and DNA double-strand breaks. Maturation potential and vitrification-warming survival were significantly affected (p < 0.001 and p < 0.05, respectively) in ovulated oocytes from the advanced age group but not in IVM oocytes. Although vitrification-warming significantly increased spindle abnormalities in ovulated oocytes from advanced maternal age (p < 0.01), no significant changes were observed in IVM oocytes. Furthermore, Bub1 and Mad2 transcript levels were significantly higher in vitrified-warmed IVM oocytes (p < 0.05). In conclusion, advanced maternal age can have a negative impact on the cryosusceptibility of ovulated oocytes but not IVM oocytes in mice.

In Vitro Culture of Mammalian Embryos: Is There Room for Improvement?

Preimplantation embryo culture, pivotal in assisted reproductive technology (ART), has lagged in innovation compared to embryo selection advancements. This review examines the persisting gap between in vivo and in vitro embryo development, emphasizing the need for improved culture conditions. While in humans this gap is hardly estimated, animal models, particularly bovines, reveal clear disparities in developmental competence, cryotolerance, pregnancy and live birth rates between in vitro-produced (IVP) and in vivo-derived (IVD) embryos. Molecular analyses unveil distinct differences in morphology, metabolism, and genomic stability, underscoring the need for refining culture conditions for better ART outcomes. To this end, a deeper comprehension of oviduct physiology and embryo transport is crucial for grasping embryo-maternal interactions' mechanisms. Research on autocrine and paracrine factors, and extracellular vesicles in embryo-maternal tract interactions, elucidates vital communication networks for successful implantation and pregnancy. In vitro, confinement, and embryo density are key factors to boost embryo development. Advanced dynamic culture systems mimicking fluid mechanical stimulation in the oviduct, through vibration, tilting, and microfluidic methods, and the use of innovative softer substrates, hold promise for optimizing in vitro embryo development.

Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review.

Over the past 40 years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

Enhanced solute transport and steady mechanical stimulation in a novel dynamic perifusion bioreactor increase the efficiency of the in vitro culture of ovarian cortical tissue strips.

Introduction: We report the development and preliminary evaluation of a novel dynamic bioreactor to culture ovarian cortical tissue strips that leverages tissue response to enhanced oxygen transport and adequate mechanical stimulation. In vitro multistep ovarian tissue static culture followed by mature oocyte generation, fertilization, and embryo transfer promises to use the reserve of dormant follicles. Unfortunately, static in vitro culture of ovarian tissue does not promote development of primordial to secondary follicles or sustain follicle viability and thereby limits the number of obtainable mature oocytes. Enhancing oxygen transport to and exerting mechanical stimulation on ovarian tissue in a dynamic bioreactor may more closely mimic the physiological microenvironment and thus promote follicle activation, development, and viability. Materials and Methods: The most transport-effective dynamic bioreactor design was modified using 3D models of medium and oxygen transport to maximize strip perifusion and apply tissue fluid dynamic shear stresses and direct compressive strains to elicit tissue response. Prototypes of the final bioreactor design were manufactured with materials of varying cytocompatibility and assessed by testing the effect of leachables on sperm motility. Effectiveness of the bioreactor culture was characterized against static controls by culturing fresh bovine ovarian tissue strips for 7 days at 4.8 × 10-5 m/s medium filtration flux in air at -15% maximal total compressive strain and by assessing follicle development, health, and viability. Results and Conclusions: Culture in dynamic bioreactors promoted effective oxygen transport to tissues and stimulated tissues with strains and fluid dynamic shear stresses that, although non-uniform, significantly influenced tissue metabolism. Tissue strip culture in bioreactors made of cytocompatible polypropylene preserved follicle viability and promoted follicle development better than static culture, less so in bioreactors made of cytotoxic ABS-like resin.

Protective effects of in vitro treatment with zinc, d-aspartate and coenzyme q10 on human sperm motility, lipid peroxidation and DNA fragmentation.

BACKGROUND: Spermatozoa are extremely vulnerable to oxidative stress caused by the unbalance between concentrations of reactive oxygen species and antioxidant scavenging systems present inside the male reproductive tract. In spite of a large number of clinical studies that claimed the beneficial effects of antioxidant oral administration on sperm physiology and fertility, only a few studies were addressed to evaluate their effects on spermatozoa in vitro. Main aims of the present study were to assess the influence of zinc, D-aspartate and coenzyme Q10, included in the dietary supplement Genadis (Merck Serono), on human sperm motility, DNA fragmentation and lipid peroxidation. METHODS: Semen samples, obtained from forty-four patients (23-30 years of age) were enrolled in this study, twenty-four were normospermic and twenty patients were oligospermic. Semen samples were analysed for sperm progressive motility and kinetics through computer assisted analysis, DNA fragmentation and lipid peroxidation. RESULTS: Main results showed that in both normo and oligospermic samples, total and progressive sperm motility is maintained by in vitro treatment with zinc, D-aspartate and coenzyme Q10, whereas a significant decrease of these parameters occurs in parallel samples incubated in medium alone. Zinc, D-aspartate and coenzyme Q10 also prevented the decrease of sperm kinetics but such an effect was highly significant only in oligospermic samples. Moreover, they also protected spermatozoa by the increase of DNA fragmentation and lipid peroxidation. CONCLUSIONS: Zinc, D-aspartate and coenzyme Q10 exert a direct protective effect on human spermatozoa preventing the decrease of motility and the increase of DNA fragmentation and lipid peroxidation during in vitro culture.

Replacement of sodium with choline in slow-cooling media improves human ovarian tissue cryopreservation.

Ovarian tissue cryopreservation is a promising technique for fertility preservation in young female cancer patients and efforts have been made to improve its effectiveness. During cooling and thawing, sodium ions significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, should not contribute to the intracellular solute load. The present study assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles (fresh control n=266; cryopreserved n=363), collected from ovarian biopsies of 11 women (22-40years) during laparoscopic surgery, were studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. The results demonstrate that choline substitution leads to: (i) an improved preservation of oocytes and follicular cells; (ii) the recovery of a higher percentage of grade-1 follicles negative for p53, p21 and Apaf-1 apoptotic markers; (iii) a reduced mitochondrial damage as observed at an ultrastructural level; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media may represent a valuable tool to improve human ovarian tissue cryopreservation. Ovarian tissue cryopreservation is a promising fertility preservation approach for cancer patients before undergoing treatments that irreversibly reduce the ovarian reserve. Autotransplantation of ovarian cortical strips has resulted in viable offspring in animal models and human. Worldwide, 20 live births have been reported thus far following autotransplantation of frozen-thawed ovarian tissue. However, currently the success rate of this technology is far from being satisfactory. This could be due to inappropriate cryopreservation procedures that might impair the physiology of ovarian follicles. Sodium ions contained in freezing media significantly contribute to the 'solute effect' that plays a major role in disrupting cell membranes. Choline ions, which do not cross the cell membrane, would not be expected to contribute to the intracellular solute load. In the present study we assessed the effects of sodium substitution with choline in slow-cooling freezing media on human ovarian cortical strip cryopreservation. A total of 629 follicles, collected from ovarian biopsies of 11 women (aged 22-40years) during laparoscopic surgery, have been studied by light microscopy, immunohistochemistry and transmission electron microscopy to evaluate their morphology, apoptosis and ultrastructure. Results demonstrated that choline substitution allowed: (i) a better preservation of oocytes and follicular cells; (ii) the recovery of an higher percentage of healthy follicles negative for apoptotic markers; (iii) a lower mitochondria ultrastructural damage; and (iv) a better preservation of ovarian tissue stroma. In conclusion, the use of choline-based media could represent a valuable tool to cryopreserve human ovarian tissue for fertility preservation.

Impact of prepubertal bovine ovarian tissue pre-freeze holding duration on follicle quality.

Ovarian tissue cryopreservation prior to gonadotoxic treatment is the only recommended option for fertility preservation in prepubertal girls. Due to the technical complexity of this technique, limited number of centres across the world are equipped to offer the facility. Hence, the retrieved ovarian tissue needs to be maintained at hypothermic temperature (4 °C) for long time during shipment. The time taken between tissue retrieval and cryopreservation could influence the functionality of cells during fertility restoration. This study explored the tissue integrity and follicle quality of ovarian cortical slices subjected to pre-freeze holding for various time durations in vitro. Prepubertal bovine ovarian tissue from < 12 months old animals were handled at hypothermic holding (4 °C) for 0, 24, 48 and 72 h. The tissues were assessed for follicle viability through confocal analysis of live-dead labelled samples, and follicle quality and tissue integrity through histology. Results have shown that follicle viability, and overall follicle quality were not significantly affected at the end of 72 h hypothermic holding. Though, the observation reassures extended hypothermic holding prior to freezing, findings need to be validated in human tissue prior to use in clinical fertility preservation programs.

Dynamic in vitro culture of bovine and human ovarian tissue enhances follicle progression and health.

In vitro ovarian cortical tissue culture, followed by culture of isolated secondary follicles, is a promising future option for production of mature oocytes. Although efforts have been made to improve the culture outcome by changing the medium composition, so far, most studies used static culture systems. Here we describe the outcome of 7 days cultures of bovine and human ovarian cortical tissue in a dynamic system using a novel perifusion bioreactor in comparison to static culture in conventional and/or gas permeable dishes. Findings show that dynamic culture significantly improves follicle quality and viability, percentage and health of secondary follicles, overall tissue health, and steroid secretion in both species. Model predictions suggest that such amelioration can be mediated by an enhanced oxygen availability and/or by fluid-mechanical shear stresses and solid compressive strains exerted on the tissue.

Mitochondrial Dysfunction and Oxidative Stress Caused by Cryopreservation in Reproductive Cells.

Mitochondria, fundamental organelles in cell metabolism, and ATP synthesis are responsible for generating reactive oxygen species (ROS), calcium homeostasis, and cell death. Mitochondria produce most ROS, and when levels exceed the antioxidant defenses, oxidative stress (OS) is generated. These changes may eventually impair the electron transport chain, resulting in decreased ATP synthesis, increased ROS production, altered mitochondrial membrane permeability, and disruption of calcium homeostasis. Mitochondria play a key role in the gamete competence to facilitate normal embryo development. However, iatrogenic factors in assisted reproductive technologies (ART) may affect their functional competence, leading to an abnormal reproductive outcome. Cryopreservation, a fundamental technology in ART, may compromise mitochondrial function leading to elevated intracellular OS that decreases sperm and oocytes' competence and the dynamics of fertilization and embryo development. This article aims to review the role played by mitochondria and ROS in sperm and oocyte function and the close, biunivocal relationships between mitochondrial damage and ROS generation during cryopreservation of gametes and gonadal tissues in different species. Based on current literature, we propose tentative hypothesis of mechanisms involved in cryopreservation-associated mitochondrial dysfunction in gametes, and discuss the role played by antioxidants and other agents to retain the competence of cryopreserved reproductive cells and tissues.

Sperm Oxidative Stress during In Vitro Manipulation and Its Effects on Sperm Function and Embryo Development.

Reactive oxygen species (ROS) generated at low levels during mitochondrial respiration have key roles in several signaling pathways. Oxidative stress (OS) arises when the generation of ROS exceeds the cell's antioxidant scavenging ability and leads to cell damage. Physiological ROS production in spermatozoa regulates essential functional characteristics such as motility, capacitation, acrosome reaction, hyperactivation, and sperm-oocyte fusion. OS can have detrimental effects on sperm function through lipid peroxidation, protein damage, and DNA strand breakage, which can eventually affect the fertility of an individual. Substantial evidence in the literature indicates that spermatozoa experiencing OS during in vitro manipulation procedures in human- and animal-assisted reproduction are increasingly associated with iatrogenic ROS production and eventual impairment of sperm function. Although a direct association between sperm OS and human assisted reproductive techniques (ART) outcomes after in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI) is still a matter of debate, studies in animal models provide enough evidence on the adverse effects of sperm OS in vitro and defective fertilization and embryo development. This review summarized the literature on sperm OS in vitro, its effects on functional ability and embryo development, and the approaches that have been proposed to reduce iatrogenic sperm damage and altered embryonic development.

Reduced ovarian response to controlled ovarian stimulation is associated with increased oxidative stress in the follicular environment.

Serum estradiol (E2) level is routinely used to monitor the ovarian response during controlled ovarian hyperstimulation (COH) and the concentration of serum E2 may influence the oocyte quality and pregnancy outcome. However, the knowledge on the association between COH induced serum E2 level, oocyte quality and embryo development is limited. Therefore we investigated the association between serum E2 level, oxidative stress in the follicular fluid and granulosa cells (GCs) response to elucidate the association between E2 level and embryological outcome. In this study, patients (n = 30) undergoing ART were categorized as 'normal responders' (NR, n = 10), 'poor responders' (PR, n = 10) and hyper responders (HR, n = 10). The follicular fluid malondialdehyde (MDA) level was determined. The total RNA extracted from GCs was subjected to analyse the relative abundance of transcripts of stress response genes (P53, caspase 3,8-oxoguanine DNA glycosylase, OGG1 and heat shock protein 70; HSP70) and embryological outcome was noted. Follicular fluid MDA level was significantly higher in PR (p < 0.01) compared NR and HR whereas number of top-quality embryos were significantly lower in PR and HR compared to NR (p < 0.01). The relative expression of P53, HSP70, and OGG1 in GCs was significantly elevated in PR (p < 0.05-0.01). An inverse relationship was established between serum E2 level vs follicular MDA level (r = -0.45; p < 0.01) and follicular MDA level vs. number of top-quality embryos (r = -0.45; p < 0.01). Hence, patients with low serum E2 had elevated oxidative stress in their follicular environment and poor quality embryos implicating the risk of oxidative stress in patients with poor ovarian response.

Dynamic Characterization of the Biomechanical Behaviour of Bovine Ovarian Cortical Tissue and Its Short-Term Effect on Ovarian Tissue and Follicles.

The ovary is a dynamic mechanoresponsive organ. In vitro, tissue biomechanics was reported to affect follicle activation mainly through the Hippo pathway. Only recently, ovary responsiveness to mechanical signals was exploited for reproductive purposes. Unfortunately, poor characterization of ovarian cortex biomechanics and of the mechanical challenge hampers reproducible and effective treatments, and prevention of tissue damages. In this study the biomechanical response of ovarian cortical tissue from abattoir bovines was characterized for the first time. Ovarian cortical tissue fragments were subjected to uniaxial dynamic testing at frequencies up to 30 Hz, and at increasing average stresses. Tissue structure prior to and after testing was characterized by histology, with established fixation and staining protocols, to assess follicle quality and stage. Tissue properties largely varied with the donor. Bovine ovarian cortical tissue consistently exhibited a nonlinear viscoelastic behavior, with dominant elastic characteristics, in the low range of other reproductive tissues, and significant creep. Strain rate was independent of the applied stress. Histological analysis prior to and after mechanical tests showed that the short-term dynamic mechanical test used for the study did not cause significant tissue tear, nor follicle expulsion or cell damage.

Redox control of surface protein sulphhydryls in bovine spermatozoa reversibly modulates sperm adhesion to the oviductal epithelium and capacitation.

Oviductal fluid molecules, such as sulphated glycosaminoglycans and disulphide-reductants, may represent periovulatory signals for the release of spermatozoa from the oviductal reservoir in the bovine species. Disulphide-reductants release spermatozoa through the reduction of sperm-surface disulphides to sulphhydryls (SH). Herein, we studied sperm-surface protein SH through labelling with maleimidylpropionyl biocytin in the initial sperm suspension, in the subpopulations able and unable to adhere to the in vitro cultured oviductal epithelium, and in spermatozoa released either through the disulphide-reductant penicillamine (PEN) or the sulphated glycosaminoglycan heparin (HEP). Adhesion assays were performed to study the ability of released spermatozoa to readhere to the oviductal epithelium. Results showed that the level of SH in sperm-surface proteins was: 1) low in adhering spermatozoa; 2) high in spermatozoa unable to adhere; and 3) markedly increased in released spermatozoa. Adhesion assays showed that: 1) PEN-released spermatozoa promptly recovered adhesion after removal of the disulphide-reductant and could be released again in response to PEN; 2) conversely, a limited number of HEP-released spermatozoa was able to readhere to the oviductal epithelium and this ability was not affected by HEP removal. Recovery of adhesion was associated to reoxidation of sperm-surface protein SH and to the reversal of capacitation. In conclusion, redox modulation of sperm-surface protein SH is involved in the release of spermatozoa adhering to the oviduct in vitro; the reversible action of disulphide-reductants might be responsible for intermittent phases of adhesions and releases; and the irreversible action of HEP indicates that it may represent a terminal releasing signal.

In vitro exposure to CPF affects bovine sperm epigenetic gene methylation pattern and the ability of sperm to support fertilization and embryo development.

Several studies have demonstrated that overexposure to pesticides can reduce mammalian sperm quality, impairing male fertility. Chlorpyrifos (CPF), a widely used organophosphate pesticide, was shown to impair spermatogenesis by inducing the formation of highly reactive toxic intermediates. To gain further insight into the mechanisms underlying the cytotoxicity and genotoxicity of CPF, bovine spermatozoa were exposed in vitro to environmental CPF concentrations and the motility, in vitro fertilization rates, DNA fragmentation, chromatin alterations, and methylation patterns were assessed. Motility and in vitro fertilization rates were significantly reduced in spermatozoa exposed to CPF, while DNA fragmentation and putative chromatin deconstruction appeared to increase at higher pesticide concentrations. In situ hybridization was carried out with X and Y probes on sperm samples exposed to different CPF concentrations, and subsequent analysis highlighted a significant percentage of spermatozoa with a peculiar morphological malformation, in which a narrowing occurred at the level of the hybridization. Analysis of potential abnormalities in the methylation pattern of NESP55-GNAS and XIST promoters displayed no differentially methylated regions in GNAS promoter relative to the control, whereas spermatozoa exposed to 10 µg/mL CPF had increased methylation variance in one region of imprinted XIST promoter. Our results provide support that CPF can induce a genotoxic effect on spermatozoa, impairig their ability to fertilize and support preimplantation embryo development in vitro. These observations are worrying since altered levels of sporadic methylation in genes of male gametes may affect the success of reproduction and contribute to infertility. Environ. Mol. Mutagen. 60:85-95, 2019. © 2018 Wiley Periodicals, Inc.