Fertilization Competence Research Articles

Lysine acetylation modulates mouse sperm capacitation

Mammalian sperm are unable to fertilize the egg immediately after ejaculation. To gain fertilization competence, they need to undergo a series of modifications inside the female reproductive tract, known as capacitation. Capacitation involves several molecular events such as phosphorylation cascades, hyperpolarization of the plasma membrane and intracellular Ca2+ changes, which prepare the sperm to develop two essential features for fertilization competence: hyperactivation and acrosome reaction. Since sperm cells lack new protein biosynthesis, post-translational modification of existing proteins plays a crucial role to obtain full functionality. Here, we show the presence of acetylated proteins in murine sperm, which increase during capacitation. Pharmacological hyperacetylation of lysine residues in non-capacitated sperm induces activation of PKA, hyperpolarization of the sperm plasma membrane, CatSper opening and Ca2+ influx, all capacitation-associated molecular events. Furthermore, hyperacetylation of non-capacitated sperm promotes hyperactivation and prepares the sperm to undergo acrosome reaction. Together, these results indicate that acetylation could be involved in the acquisition of fertilization competence of mammalian sperm.

Ion Channel Function During Oocyte Maturation and Fertilization.

The proper maturation of both male and female gametes is essential for supporting fertilization and the early embryonic divisions. In the ovary, immature fully-grown oocytes that are arrested in prophase I of meiosis I are not able to support fertilization. Acquiring fertilization competence requires resumption of meiosis which encompasses the remodeling of multiple signaling pathways and the reorganization of cellular organelles. Collectively, this differentiation endows the egg with the ability to activate at fertilization and to promote the egg-to-embryo transition. Oocyte maturation is associated with changes in the electrical properties of the plasma membrane and alterations in the function and distribution of ion channels. Therefore, variations on the pattern of expression, distribution, and function of ion channels and transporters during oocyte maturation are fundamental to reproductive success. Ion channels and transporters are important in regulating membrane potential, but also in the case of calcium (Ca2+), they play a critical role in modulating intracellular signaling pathways. In the context of fertilization, Ca2+ has been shown to be the universal activator of development at fertilization, playing a central role in early events associated with egg activation and the egg-to-embryo transition. These early events include the block of polyspermy, the completion of meiosis and the transition to the embryonic mitotic divisions. In this review, we discuss the role of ion channels during oocyte maturation, fertilization and early embryonic development. We will describe how ion channel studies in Xenopus oocytes, an extensively studied model of oocyte maturation, translate into a greater understanding of the role of ion channels in mammalian oocyte physiology.

IntelliGen technology creates sexed semen that exhibits physiological markers associated with fertilization competency and concomitant positive field fertility

IntelliGen technology creates sexed semen that exhibits physiological markers associated with fertilization competency and concomitant positive field fertility

Insights into pH regulatory mechanisms in mediating spermatozoa functions.

Regulation of pH in spermatozoa is a complex and dynamic process as sperm cells encounter different pH gradients during their journey from testes to the site of fertilization in female genital tract. The precise regulations of pH in sperm cells regulate the sperm functions such as motility, hyperactivity, capacitation, and acrosome reaction. Electrophysiological, pharmacological, and molecular studies have revealed the presence of different ion channels and exchanger systems which regulate intracellular pH in sperm cells as well as regulate sperm functions. Recent studies also have shown the potential involvement of pH in the regulation of fertility competence of sperm cells, and alterations in pH have shown to impede sperm functions. This mini-review discusses the probable mechanisms involved in pH regulation in sperm cells and how pH is involved in regulation of various sperm functions.

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure.

Conception in mammals is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum can be divided into the midpiece, the principal piece and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility; however, the actin organization in sperm remains elusive. In the present work, we show that there are different types of actin structures in the sperm tail by using three-dimensional stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.

Mouse quiescin sulfhydryl oxidases exhibit distinct epididymal luminal distribution with segment-specific sperm surface associations.

Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization; however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development.

Effects of melatonin administration on seminal plasma metabolites and sperm fertilization competence during the non-reproductive season in ram

The purpose of this study was to investigate the effects of ram melatonin treatment on the sperm quality and metabolite composition of the seminal plasma in the non-breeding season. Four mature rams were treated with 54 mg melatonin in March subcutaneous implants and four untreated rams served as control. At 0, 30, 90 and 120 days semen samples were collected and sperm, separated from seminal plasma, was evaluated for its capacity to fertilize and produce embryos in vitro. Seminal plasma metabolites were extracted and analyzed by capillary electrophoresis/mass spectroscopy. In the resulting electropherograms, the area corresponding to selected metabolites was extracted and quantified. Ram melatonin treatment affected the in vitro fertilization competence of sperm. Blastocyst output increased until 90 days after treatment (27.20 ± 7.35 vs 54.7 ± 4.4% at 0 and 90 days respectively; p < 0.05) while the untreated group did not show statistical differences.In treated rams, the concentration of melatonin in seminal plasma increased from 3.34 ± 1.70 at day 0–9.65 ± 2.89 AU (Arbitrary Units) after 90 days, then decreased to reach the level of the untreated ram after 120 days (p < 0.05). During 90 days after melatonin treatment, an increase (p < 0.05) in seminal plasma concentrations of glutamic acid (6.28 ± 1.53 vs 14.93 ± 1.53 AU at 0 and 90 days respectively), glutamine (16.89 ± 4.65 vs 54.51 ± 4.65 AU), carnitine (22.97 ± 9.81 vs 104.30 ± 9.81 AU), acetyl-carnitine (48.15 ± 17.32 vs 217.69 ± 17.32 AU), choline (1.82 ± 1.55 vs 14.16 ± 1.55 AU) and arginine (1.31 ± 1.08 vs 14.25 ± 1.08 AU) was detected. Tyrosine concentration increased during 30 days from melatonin treatment (12.79 ± 3.93 vs 27.08 ± 3.04 AU) but at 90 days its levels were similar to the untreated group. In conclusion, melatonin treatment during the non-breeding season improves the concentration of several metabolites in seminal plasma and sperm fertilization competence in Sarda breed ram.

The Different Calcium+2 Intensity Profile and Quality of Oocyte and Goat Sperms after Cryopreservation

This research aims were to study the effect of the oocyte and sperms cryopreservation of Indonesian local goat on the post-thawing quality and profile or characters of Calcium+2 intensity in relating with their fertility capacity. A study was conducted to test the freezing method and post-thawing viability both stock cells stored in the deep freezer and liquid nitrogen (-80°C of vs -196°C). A fertility test of sperms has been conducted through in vitro of sperm quality, while the oocytes cryopreserved test was done by in vitro maturation (IVM) rate (%). The profile of Calcium 2+ was performed and analysis by Confocal Laser Scanned Microscope (CLSM). The result showed that IVM rate of goat oocyte is considered lower when cryopreserved in –80°C than in –196°C. Meanwhile, sperm is considered having a good quality in 2 methods of cryopreservation with post-thawing motility > 40 % (SNI 2014). There is an important difference between Calcium intensity of fresh and post-thawing both for oocyte and spermatozoa. Calcium +2 profiles is varied individually on the peak of intensity, but it considered expressed the same profile of each fresh and post-thawing cell. In vitro fertilization test need to be performed to complete the viability and fertility competence of these sperm and oocyte freezing stocks.

Aflatoxin B1 impairs sperm quality and fertilization competence

Aflatoxins are poisonous byproducts of the soilborne fungus Aspergillus, involved in the decomposition of plant materials. Aflatoxins can be found in various food products, such as maize, sorghum, millet, rice and wheat. AFB1 is the most toxic of these, classified as a carcinogen and mutagen for both humans and animals. AFB1 has been detected in human cord blood and placenta; however, its toxic effect on sperm is less known. The current study examines sperm responses associated with AFB1 exposure. These included acrosome integrity and function, mitochondrial polarity, DNA fragmentation, fertilization competence and early embryonic development. Spermatozoa were obtained from bull ejaculate and epididymis and capacitated in vitro for 4h with 0, 0.1, 1, 10 and 100μM AFB1. Following capacitation, acrosome reaction (AR) was induced by Ca2+ ionophore. The integrity and functionality of sperm were examined simultaneously by florescent staining. A Halosperm DNA fragmentation kit was used to evaluate DNA integrity. An in-vitro culture system was used to evaluate fertilization competence and blastocyst formation rate, using bovine oocytes.Findings indicate dose-responsive variation among compartments to AFB1 exposure. Sperm viability, expressed by integrity of the plasma membrane, was lower in sperm isolated from ejaculate or epididymis after culturing with AFB1. Exposure to AFB1 reduced the proportion of sperm from the epididymis tail undergoing acrosome reaction induced by Ca2+ ionophore. AFB1 impaired mitochondrial membrane potential (ΔYm) in sperm isolated from ejaculate and the epididymis tail. Exposing ejaculated sperm to AFB1 increased the proportion of sperm with fragmented DNA and reduced the proportion of embryos that cleaved to the 2- to 4-cell stage, 42h postfertilization, however, the proportion of embryos that developed to blastocysts, 7days postfertilization, did not differ among groups. The findings explore the harmful effects of AFB1 on sperm viability, ΔΨm and DNA integrity associated with fertility competence. We postulate that AFB1-induced fragmentation in paternal DNA might have a carryover effect on the quality of developing embryos. Further evaluation for the quality of blastocysts derived from sperm exposed to AFB1 is warranted.

Deficiency of peroxiredoxin 6 or inhibition of its phospholipase A2 activity impair the in vitro sperm fertilizing competence in mice

Prdx6−/− male mice are subfertile, and the deficiency or inactivation of Peroxiredoxins (PRDXs) is associated with human male infertility. We elucidate the impact of the lack of PRDX6 or inhibition of its calcium-independent phospholipase A2 (Ca2+-iPLA2) activity by MJ33 on fertilization competence of mouse spermatozoa. Sperm motility, viability, fertilization and blastocyst rates were lower in Prdx6−/− spermatozoa than in C57BL/6J wild-type (WT) controls (p ≤ 0.05). MJ33 inhibited the PRDX6 Ca2+-iPLA2 activity and reduced these parameters in WT spermatozoa compared with controls (p ≤ 0.05). Levels of lipid peroxidation and of superoxide anion (O2•─) were higher in Prdx6−/− than in WT spermatozoa (p ≤ 0.05). MJ33 increased the levels of lipid peroxidation and mitochondrial O2•─ production in treated versus non-treated WT spermatozoa. Acrosome reaction, binding to zona pellucida and fusion with the oolemma were lower in Prdx6−/− capacitated spermatozoa than WT capacitated controls and lower in WT spermatozoa treated with the PRDX6 inhibitor. In conclusion, the inhibition of the PRDX6 Ca2+-iPLA2 activity promotes an oxidative stress affecting viability, motility, and the ability of mouse spermatozoa to fertilize oocytes. Thus, PRDX6 has a critical role in the protection of the mouse spermatozoon against oxidative stress to assure fertilizing competence.

Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa

ABSTRACTPost-testicular sperm maturation and storage within the epididymis is a key determinant of gamete quality and fertilization competence. Here we demonstrate that mouse spermatozoa possess a complex small non-protein-coding RNA (sRNA) profile, the composition of which is markedly influenced by their epididymal transit. Thus, although microRNAs (miRNAs) are highly represented in the spermatozoa of the proximal epididymis, this sRNA class is largely diminished in mature spermatozoa of the distal epididymis. Coincident with this, a substantial enrichment in Piwi-interacting RNA (piRNA) abundance in cauda spermatozoa was detected. Further, features of cauda piRNAs, including; predominantly 29–31 nts in length; preference for uracil at their 5' terminus; no adenine enrichment at piRNA nt 10, and; predominantly mapping to intergenic regions of the mouse genome, indicate that these piRNAs are generated by the PIWIL1-directed primary piRNA production pathway. Accordingly, PIWIL1 was detected via immunoblotting and mass spectrometry in epididymal spermatozoa. These data provide insight into the complexity and dynamic nature of the sRNA profile of spermatozoa and raise the intriguing prospect that piRNAs are generated in situ in maturing spermatozoa. Such information is of particular interest in view of the potential role for paternal sRNAs in influencing conception, embryo development and intergenerational inheritance.

Structural and rheological properties conferring fertilization competence to Xenopus egg-coating envelope

The extracellular egg-coating envelope that comprises a meshwork of filaments polymerized by glycoproteins plays a pivotal role in species-selective sperm recognition and subsequent fertilization; however, the structural and rheological properties conferring fertilization competence to the egg-coating envelope remain poorly unveiled. Here we show several nanoscale-structural and viscoelastic properties of the egg-coat using the transmission electron microscopy and the quartz crystal microbalance experiments, following clamp of the egg-coat at either fertilization-competent or -incompetent statuses by short-term pretreatment with synthetic peptides. Individual filament of approximately 4.8 nm diameter crossed one another, forming several types of intersections. Higher competence-inducing treatment changed the proportion of V-, Y-, and T-type intersections, and induced more randomly deflected angles at intersections. Incompetence-inducing treatment increased the median of a Gaussian distribution of filament lengths that had a peak of 10–20 nm under control conditions; furthermore, this treatment created bumps in the 30–40 and 50–60 nm windows. Quartz crystal microbalance study revealed that viscoelasticity of the competent VE suspension was lower than that of incompetent VE, indicating that viscoelastic property required for successful fertilization resides within a specific range. These findings indicated that the architecture of the egg-coat is capable of rapid and dynamic remodeling, which determines fertilization efficiency.

TCTP regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality

If no fertilization occurs for a prolonged time following ovulation, oocytes experience a time-dependent deterioration in quality both in vivo and in vitro due to processes called postovulatory aging. Because the postovulatory aging of oocytes has marked detrimental effects on embryo development and offspring, many efforts have been made to unveil the underlying mechanisms. Here we showed that translationally controlled tumor protein (TCTP) regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality. Spindle dynamics decreased with reduced TCTP level during aging of mouse oocytes. Knockdown of TCTP accelerated the reduction of spindle dynamics, accompanying with aging-related deterioration of oocyte quality. Conversely, overexpression of TCTP prevented aging-associated decline of spindle dynamics. Moreover, the aging-related abnormalities in oocytes were rescued after TCTP overexpression, thereby improving fertilization competency and subsequent embryo development. Therefore, our results demonstrate that TCTP-mediated spindle dynamics play a key role in maintaining oocyte quality during postovulatory aging and overexpression of TCTP is sufficient to prevent aging-associated abnormalities in mouse oocytes.

Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.

Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte? The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity. ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC. This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation. Single oocytes and their correspondingCC were analysed. Oocytes were examined usingPLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics. Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smallerIL-ZP area and thickness in mature oocytes than in immature oocytes (all P < 0.05). In mature oocytes, IL-ZP retardance was significantly correlated with the expression of all four ZP mRNAs (all P < 0.05). The oocyte ZP3 expression was the main predictor of the fertilization capacity, next to IL-retardance and IL-thickness. Using stepwise regression analysis, IL-thickness combined with EFNB2 expression in CC and the patient's ovarian response resulted in a noninvasive oocyte fertilization prediction model. Not applicable. This is a retrospective study and the relation of oocyte mRNA levels to fertilization capacity is indirect as oocyte gene expression analysis required lysis of the oocyte. Overall relations between PLM observations, mRNA expression changes and intrinsic oocyte competence were successfully documented. As such PLM and CC gene expression are confirmed as valuable noninvasive techniques to evaluate oocyte competence. This study was funded by University of Torino, Italy, WFWG UZ-Brussel and Agentschap voor Innovatie door Wetenschap en Technologie IWT 110680, Belgium. All authors declare that their participation in the study did not involve actual or potential conflicts of interests.

Analysis of gene expression in granulosa cells post-maturation to evaluate oocyte culture systems in the domestic cat.

Maturation of oocytes is a prerequisite for successful embryo development. The fertilization competence of in vivo derived oocytes is significantly higher than that of oocytes matured in vitro. Commonly evaluated morphological criteria for oocyte maturation do not reflect the complexity and quality of maturation processes. Oocytes and granulosa cells are communicating closely in a bidirectional way during follicular growth and maturation. Assessing the mRNA expression of specific genes in granulosa cells could be a non-invasive way to evaluate the conditions of in vitro oocyte maturation. The objective of this study was to elucidate the influence of two different FSH additives on the in vitro maturation rate and gene expression of cumulus-oocytes complexes in domestic cat. Feline oocytes were matured in a medium, supplemented with LH and 0.02 IU/ml porcine FSH versus 0.02 IU or 1.06 IU/ml human FSH. Granulosa cells were separated from oocytes directly after 24hr of maturation or after additional 12hr of in vitro fertilization. Gene expression levels were analysed by quantitative PCR for aromatase, antimullerian hormone, follicle stimulating hormone receptor (FSHR), luteinizing hormone/choriogonadotropin receptor (LHCGR) and prostaglandin E synthase. Neither oocyte maturation rate nor gene expression levels differed after 24 or 36hr in all three groups. However, variations were discovered in correlations of expression levels, for instance for FSHR and LHCG, indicating differences in the fine-tuning of in vitro maturation processes under varying FSH supplementations. We suppose that correlation between gene expressions of selected genes suggests a superior maturation quality of feline oocytes.

Effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 inhibitor on the meiotic and developmental competence of feline oocytes.

Oocyte cryopreservation is the technique of choice for the long-term storage of female gametes. However, it induces an irreversible loss of oocyte viability and function. We examined the effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitor (ROCKi) on the meiotic and developmental competence of feline oocytes. We examined the expression of LIM kinase (LIMK) 1 and 2, with and without ROCKi treatment. Cumulus oocyte complexes (COCs) were matured in vitro with 0, 10, 20, and 40 µM ROCKi. The oocytes were subsequently assessed for maturation rate and embryo development following in vitro fertilization. We repeated the COC experiment, but vitrified and warmed the COCs prior to culture. We detected LIMK1 and LIMK2 expression in feline oocytes, which could be downregulated by ROCKi treatment. The ROCKi at 10 µM affected neither meiotic nor developmental competence (P > 0.05, versus control). However, high concentrations of ROCKi during maturation induced meiotic arrest at metaphase I. Appropriate concentrations of ROCKi significantly improved the normal fertilization rate of vitrified warmed oocytes (49.4 ± 3.4%) compared with that of the control (42.8 ± 8.6%, P < 0.05). The ROCKi also significantly improved the embryo cleavage rate (36.1 ± 3.8%) as compared with the non-treated control (27.4 ± 2.5%, P < 0.05). Thus, this study revealed that the main mediators of the ROCK cascade (LIM kinases) are expressed in feline oocytes. The ROCKi (10 µM) did not compromise the meiotic or developmental competence of feline oocytes. In addition, 10 µM ROCKi improved the cytoplasmic maturation of vitrified–warmed oocytes as indicated by their fertilization competence.

New approaches regarding the in vitro maturation of oocytes: manipulating cyclic nucleotides and their partners in crime.

Several discoveries have been described recently (5-10 years) about the biology of ovarian follicles (oocyte, cumulus cells and granulosa cells), including new aspects of cellular communication, the control of oocyte maturation and the acquisition of oocyte competence for fertilization and further embryo development. These advances are nourishing assisted reproduction techniques (ART) with new possibilities, in which novel culture systems are being developed and tested to improve embryo yield and quality. This mini-review aims to describe how the recent knowledge on the physiological aspects of mammalian oocyte is reflecting as original or revisited approaches into the context of embryo production. These new insights include recent findings on the mechanisms that control oocyte maturation, especially modulating intraoocyte levels of cyclic nucleotides during in vitro maturation using endogenous or exogenous agents. In this mini-review we also discuss the positive and negative effects of these manipulations on the outcoming embryo

142 POSSIBLE BENEFIT OF INTRACYTOPLASMIC SPERM INJECTION ON THE EFFICIENCY OF EMBRYO PRODUCTION IN AGED COWS

Reduction in oocyte quality is a major factor responsible for declining fertility with age. The abnormal fertilization rate of oocytes from aged cows was reportedly higher than that of oocytes from young cows (Iwata et al. 2011. Reprod. Fertil. Dev. 23, 424–432). We hypothesised that assisted fertilization by intracytoplasmic sperm injection (ICSI) might improve the developmental abilities of oocytes collected from aged females. The aims of the study were (1) to determine the effect of maternal aging on the ability of bovine oocytes to undergo cytoplasmic maturation, fertilization, and further embryo development; and (2) to determine whether ICSI would improve the efficiency of embryo production in aged cows. Cows aged 30 to 50 months or &gt;120 months were defined as young or aged, respectively. Cumulus-oocyte complexes were harvested from abattoir-derived ovaries of young (40 ± 7 months, n = 89) and aged (136 ± 12 months, n = 55) Holstein cows and matured for 23 h in TCM-199 supplemented with 5% fetal bovine serum (FBS) at 38.5°C under 5% CO2 with saturated humidity. Then, surrounding cumulus cells were removed, and cortical granules in the oocyte were stained with Lens culinaris–fluorescein isothiocyanate to evaluate the cytoplasmic maturation. Matured oocytes were inseminated by IVF or ICSI. At 15 h post-insemination, the numbers of pronuclei were determined to evaluate the fertilization rates. Presumptive IVF- or ICSI-derived zygotes were cultured for 5 days in CR1aa medium with 2% FBS and subsequently in USU6 with 5% FBS for 3 days at 38.5°C in 5% O2, 5% CO2, and 90% N2 with saturated humidity. Chromosome numbers of blastocysts were counted to evaluate the effect of maternal aging on ploidy. All experiments were performed with more than 4 independent runs, and data were analysed using chi-square tests. The distribution of matured oocytes into different cortical granule classes was affected by age, with a significantly lower (P &lt; 0.01) proportion of class III (mature cytoplasm) oocytes from aged cows (29%) compared with those from young cows (57%). Although fertilization rates following IVF did not differ between the groups, the proportion of abnormal fertilization (more than 2 pronuclei) was 32% in the aged group: higher than in the young group (15%; P &lt; 0.01). The rates of cleaved embryos following IVF were the same among groups, but the rate of development to the blastocyst stage of oocytes from aged cows (38%) was significantly (P &lt; 0.05) lower than in those from young cows (52%). Moreover, the proportion of diploid blastocysts with 2 sets of chromosomes (2n = 60) was lower (47%) in the aged than in the young groups (75%; P &lt; 0.05). However, in the ICSI embryos, the rates of development to the blastocyst stage did not differ significantly between groups (young 36%; aged 43%). Thus, maternal aging might impair the cytoplasmic maturation of bovine oocytes, which could be associated with abnormal fertilization or low developmental competence. Our results also indicate possible beneficial effects of ICSI on the efficiency of embryo production in aged cows.

186 SUPPLEMENTATION WITH LOW DOSES OF DIMETHYL SULFOXIDE DURING IN VITRO MATURATION RESULTS IN IMPROVED IN VITRO EMBRYO PRODUCTION IN CATTLE

Oocyte in vitro maturation (IVM) is crucial for subsequent in vitro embryo production. It involves acquisition of competence for fertilization and embryo development. Therefore, its optimization could have a direct impact on in vitro embryo development. Dimethyl sulfoxide (DMSO) is commonly used as solvent or vehicle, but also increases the membrane permeability and behaves as a scavenger of cytotoxic free radicals. The aim of this study was to evaluate the effect of DMSO supplementation during bovine oocyte maturation on subsequent in vitro embryo development and to determine the optimal usage dose with no toxic effect. To this aim, cumulus-oocyte complexes were collected from slaughterhouse ovaries and IVM in TCM 199 containing 10% fetal bovine serum, 10 µg mL−1 of FSH, 0.3 mM sodium pyruvate, 100 mM cysteamine, and 2% antibiotic-antimycotic. The oocytes were incubated for 24 h at 6.5% CO2 in humidified air at 38.5°C. For Experiment 1, IVM medium was supplemented with DMSO at concentrations of 0, 0.1, 0.5, 1, or 10% (vol/vol) DMSO (n = 241, 195, 42, 192, 172 oocytes) and IVM rate was determined by presence of the first polar body. For Experiment 2, 0, 0.1, 0.25, 0.5, 0.75, 1, or 10% (vol/vol) DMSO (n = 446, 322, 65, 194, 77, 250, 39 oocytes) was supplemented to IVM medium and cleavage and blastocyst rates were determined to establish the optimal usage dose. In vitro fertilization was performed according to Brackett and Oliphant (1975), with 16 × 106 spermatozoa/mL for 5 h. Afterwards, presumptive zygotes were cultured in SOF for 7 days at 38.5°C and 5% O2. Cleavage and blastocyst rates were determined on Days 2 and 7, respectively. Results were statistically analysed using Fisher’s exact test by GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Also, the percentage of blastocyst was adjusted to DMSO concentration using the R software quadratic regression model. The optimum usage dose was determined by calculating the maximum of the estimated predictive equation. In vitro maturation in 10% DMSO resulted in significantly lower first polar body extrusion rates (0% = 74%a, 0.1% = 73%a, 0.5% = 83%a, 1% = 66%a, and 10% = 8%b; different letters indicate statistical differences) and lower cleavage rates (0% = 75%a, 0.1% = 77%a, 0.25% = 80%a, 0.5% = 79%a, 0.75% = 78%a, 1% = 77%a, and 10% = 3%b) than the other treatments. Furthermore, blastocyst production was higher for the 0.25 and 0.5% (vol/vol) supplemented DMSO groups (0% = 26%b, 0.1% = 37%ab, 0.25% = 40%a, 0.5% = 41%a, 0.75% = 34%ab, 1% = 23%b, and 10% = 0%c). The predictive equation results indicate that the maximum percentage of blastocysts is obtained with a concentration of 0.458% (vol/vol) of DMSO. In conclusion, DMSO supplementation during IVM of bovine oocytes had a positive effect on in vitro development. Further studies will be carried out to elucidate its mechanism of action.

Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality

Atrazine (ATZ), one of the most extensively used herbicides, is considered a ubiquitous environmental contaminant. ATZ is a known endocrine disruptor, and deleterious effects on reproductive function have been shown, even at low, ecologically relevant doses (0.1–3μg/L). Once it enters the body, ATZ is metabolized to various metabolites, which are further detected in the urine, serum and tissues. In mammals, the major ATZ metabolite is diaminochlorotriazine (DACT). The current study focuses on direct effects of low doses of ATZ and DACT on bovine sperm isolated from ejaculates or epididymis compartments (head, body and tail). Sperm were incubated under capacitation conditions with or without 0.1–10μM ATZ or 1–100μM DACT. The integrity and functionality of sperm membranes (plasma, acrosomal and mitochondrial) were examined simultaneously by fluorescence staining at 0, 2 and 4h of incubation. Acrosome reaction (AR) was induced by Ca++ ionophore, after capacitation. The findings indicated that both ATZ and DACT adversely affect sperm, expressed by damaged sperm membranes. ATZ had a prominent effect on epididymal-tail sperm, expressed as disruption of all examined membranes, mostly at low (0.1 or 1μM) concentrations; pseudo-AR and that induced by Ca++ ionophore were both affected by exposure to 0.1μM ATZ (P<0.05 and P<0.00004, respectively). A similar pattern was documented for sperm isolated from ejaculates (P<0.002 and P<0.001, respectively). ΔYm was affected by ATZ in sperm isolated from the epididymis tail (1μM, P<0.0009), but not in that isolated from ejaculates. DACT reduced sperm viability at all examined concentrations and in all fractions. DACT at 1μM impaired ΔΨm in sperm isolated from the epididymis tail and ejaculate (P<0.005). DACT at 100μM did not induce pseudo-AR in sperm isolated from the ejaculate, but did in sperm isolated from the epididymis tail (P<0.05). Induction of AR by Ca++ ionophore was impaired in sperm isolated from ejaculate and exposed to 10 or 100μM DACT (P<0.05) and in sperm isolated from the epididymis tail and exposed to 1, 10 or 100μM DACT (P<0.0004). These findings reveal the harmful effect of exposure to ATZ and DACT, mainly at low ecologically relevant doses, on sperm viability, AR and mitochondrial function. We conclude that sperm at advanced stages of spermatogenesis, through its passage and storage in the epididymis compartments as well as in the ejaculate, is sensitive to herbicide. The results suggest that ATZ- or DACT-induced disruptions of sperm membranes might impair sperm fertilization competence.