Embryo-endometrial interaction associated with the location of the embryo during the mobility phase in mares.

Embryo-maternal crosstalk is essential to establish pregnancy, with the equine embryo moving throughout the uterus on days 9-15 (ovulation = day 0) as part of this interaction. We hypothesized that the presence of a mobile embryo induces local changes in the gene expression of the endometrium. On Day 12, the endometrial transcripts were compared among three groups: uterine horn with an embryo (P+, n = 7), without an embryo (P-, n = 7) in pregnant mares, and both uterine horns of nonbred mares (NB, n = 6). We identified 1,101 differentially expressed genes (DEGs) between P+ vs. NB and 1,229 DEGs between P- vs. NB. The genes upregulated in both P+ and P- relative to NB were involved in growth factor pathway and fatty acid activation, while downregulated genes were associated with oxytocin signaling pathway and estrogen receptor signaling. Comparing the transcriptome of P+ to that of P-, we found 59 DEGs, of which 30 genes had a higher expression in P+. These genes are associated with regulating vascular growth factors and the immune system, all known to be essential in early pregnancy. Overall, this study suggests that the mobile embryo influences the endometrial gene expression locally.

Germ cell recovery, cryopreservation and transplantation in the California white sturgeon, Acipenser transmontanus.

The white sturgeon (Acipenser transmontanus) is the largest freshwater fish in North America. Because of the unique life history characteristics of sturgeon, including longevity, late maturation and long spawning intervals, their aquaculture can be a significant investment of resources. As a result of habitat loss and overharvesting, natural populations of white sturgeon are threatened and there is a growing effort to improve conservation aquaculture programs. Germ cell transplantation is an innovative technology previously demonstrated in a variety of fish species to be able to produce a surrogate broodstock. The technique relies upon optimal donor germ cell recovery and transplantation into a recipient fish. In this study, we developed and optimized the harvest of donor cells for germline transplantation and evaluated methods for ovary cryopreservation for the first time in the white sturgeon. We found that harvesting gonads from juveniles between the ages of 1.5 and 2.5-years resulted in reliably high proportions of pre-meiotic cells regardless of sex, a critical feature for using white sturgeon for transplantation studies since the species shows no distinguishing external sex characteristics. From the viable cells, we identified germline cells using immunolabeling with the antibody DDX4, a marker specific to the germline. For in vivo tracking of donor cells during transplantations, gonadal cells were stained with a long half-life non-toxic cell membrane dye, PKH26, and microinjected into the peritoneal cavity of newly hatched white sturgeon larvae. Larvae were reared until 3 months post-transplantation to monitor for colonization and proliferation of PKH26-labeled cells within the recipient larval gonads. Furthermore, viable cell detection, assessment of germline-specificity, and transplantation was determined for cells recovered from cryopreserved ovarian tissue from sexually immature females. Transplantations using cells cryopreserved with media supplemented with dimethyl sulfoxide (DMSO) rather than ethylene glycol (EG) demonstrated the highest number of PKH26-labeled cells distributed along the gonadal ridges of the larval recipient. Determining optimal methods of tissue cryopreservation, and germ cell recovery and transplantation are foundational to the future development of germ cell transplantation as a strategy to improve the aquaculture and conservation of this species. Our study demonstrates that conservation actions, such as surrogate breeding, could be utilized by hatcheries to retain or improve natural gamete production without genetic modification, and provide an encouraging approach to the management of threatened sturgeon species.

An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section I.

As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed.

An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II.

As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmic sperm injection (ICSI), as well as current and future developments in sperm sorting technologies. Ultimately, novel methods of semen selection will be assessed based on their efficacy in other species and their relevance and future application towards ARTs in the horse.

A Review of OCT4 Functions and Applications to Equine Embryos.

OCT4 is a core transcription factor involved in pluripotency maintenance in the early mammalian embryo. The POU5F1 gene that encodes the OCT4 protein is highly conserved across species, suggesting conserved function. However, studies in several species including mice, cattle, and pigs, suggest that there are differences in where and when OCT4 is expressed. Specifically, in the horse, several studies have shown that exposure to the uterine environment may be necessary to induce OCT4 expression restriction to the inner cell mass (ICM) of the developing embryo, suggesting that there may be equine-specific extrinsic regulators of OCT4 expression that have not yet been investigated. However, an alternative hypothesis is that this restriction may not be evident in equine embryos because of our inability to culture them to the epiblast stage, preventing the observation of this restriction. In vitro studies have identified that OCT4 is expressed in the immature equine oocyte and in the early equine embryo, but OCT4 expression has not been studied after the formation of the ICM in the equine embryo. Despite the gaps in knowledge about equine-specific functions of OCT4, this factor has been used in studies assessing equine embryonic stem cells and to induce pluripotency in equine somatic cells. This review describes the role of OCT4 in the equine embryo and its applications in equine stem cell research.

Transcriptome profiling of individual rhesus macaque oocytes and preimplantation embryos.

Early mammalian embryonic transcriptomes are dynamic throughout the process of preimplantation development. Cataloging of primate transcriptomics during early development has been accomplished in humans, but global characterization of transcripts is lacking in the rhesus macaque: a key model for human reproductive processes. We report here the systematic classification of individual macaque transcriptomes using RNA-Seq technology from the germinal vesicle stage oocyte through the blastocyst stage embryo. Major differences in gene expression were found between sequential stages, with the 4- to 8-cell stages showing the highest level of differential gene expression. Analysis of putative transcription factor binding sites also revealed a striking increase in key regulatory factors in 8-cell embryos, indicating a strong likelihood of embryonic genome activation occurring at this stage. Furthermore, clustering analyses of gene co-expression throughout this period resulted in distinct groups of transcripts significantly associated to the different embryo stages assayed. The sequence data provided here along with characterizations of major regulatory transcript groups present a comprehensive atlas of polyadenylated transcripts that serves as a useful resource for comparative studies of preimplantation development in humans and other species.

The relationship between mitochondrial DNA copy number and stallion sperm function.

Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P < 0.05). Varying copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly influenced by data from individual stallions despite the low number of stallions sampled with low sperm motility. Further genome sequencing is necessary to investigate if mutations or deletions are the underlying causes of inconsistent copy numbers across mitochondrial genes. In conclusion, we show, for the first time, that increased mtDNA copy number is associated with decreased total sperm motility in stallions. We therefore suggest that mtDNA copy number may be an indicator of defective spermatogenesis in stallions.

Mitochondrial oxygen consumption is a unique indicator of stallion spermatozoal health and varies with cryopreservation media.

Mitochondrial oxygen consumption is a sensitive indicator of spermatozoal health in the context of cryopreservation. We investigated oxygen consumption of equine sperm mitochondria during incubation in four commercially available sperm cryopreservation extenders: modified INRA 96, BotuCrio, EZ Freezin-"LE" and "MFR5", in addition to several other parameters including motility, reactive oxygen species (ROS) production and viability. All experimental endpoints, with the exception of average path velocity, were affected significantly by freezing extender type after freezing and thawing. Sperm in INRA 96 had the lowest average progressive motility after thawing (24 ± 4.8%, P < 0.05). Sperm in EZ Freezin-"LE" had the highest post thaw viability (79 ± 3.1%, P < 0.05) and lowest post thaw ROS production (13 ± 2.4%), but sperm in BotuCrio had the highest maximal oxygen consumption levels, while also demonstrating similar ROS production and viability. This difference would not have been detected using conventional sperm analytical methods. In addition, sperm in BotuCrio had the highest average total motility (49 ± 7.4%), progressive motility (41 ± 6.4%), and velocity (VAP, 90 ± 3.6 µm/s) indicating that this medium preserved mitochondrial function optimally after cryopreservation. Mitochondrial oxygen consumption was positively correlated with traditional measures of sperm function including motility and viability (r = 0.62 and r = 0.49, respectively, P < 0.05), thus making it a sensitive method for determining cryopreservation success and mitochondrial function in stallion sperm.

Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.

Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity.

Relationship between paternal somatic health and assisted reproductive technology outcomes.

OBJECTIVE: To study the association between paternal medical comorbidities and the outcomes of assisted reproductive technology (ART). DESIGN: Retrospective cohort study. SETTING: Academic reproductive medicine center. PATIENT(S): We analyzed fresh ART cycles uszing freshly ejaculated sperm from the male partner of couples undergoing ART cycles from 2004 until 2014. We recorded patient and partner demographic characteristics. The cohort was linked to hospital billing data to obtain information on selected male partners' comorbidities identified using ICD-9-CM codes. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Fertilization, clinical pregnancy, miscarriage, implantation, and live-birth rates as well as birth weights and gestational ages. RESULT(S): In all, we identified 2,690 men who underwent 5,037 fresh ART cycles. Twenty-seven percent of men had at least one medical diagnosis. Men with nervous system diseases had on average lower pregnancy rates (23% vs. 30%) and live-birth rates (15% vs. 23%) than men without nervous system diseases. Lower fertilization rates were also observed among men with respiratory diseases (61% vs. 64%) and musculoskeletal diseases (61% vs. 64%) relative to those without these diseases. In addition, men with diseases of the endocrine system had smaller children (2,970 vs. 3,210 g) than men without such diseases. Finally, men with mental disorders had children born at an earlier gestational age (36.5 vs. 38.0 weeks). CONCLUSION(S): The current report identified a possible relationship between a man's health history and IVF outcomes. As these are potentially modifiable factors, further research should determine whether treatment for men's health conditions may improve or impair IVF outcomes.

Progesterone Accelerates the Completion of Sperm Capacitation and Activates CatSper Channel in Spermatozoa from the Rhesus Macaque.

During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 µM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 µM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa "priming" as they move into a gradient of progesterone in search for the oocyte.

Flagellar ion channels of sperm: similarities and differences between species.

Motility and fertilization potential of mammalian sperm are regulated by ion homeostasis which in turn is under tight control of ion channels and transporters. Sperm intracellular pH, membrane voltage and calcium concentration ([Ca(2+)]i) are all important for sperm activity within the female reproductive tract. While all mammalian sperm are united in their goal to find and fertilize an egg, the molecular mechanisms they utilize for this purpose are diverse and differ between species especially on the level of ion channels. Recent direct recording from sperm cells of different species indicate the differences between rodent, non-human primate, ruminant, and human sperm on the basic levels of their ion channel regulation. In this review we summarize the current knowledge about ion channel diversity of the animal kingdom and concentrate our attention on flagellar ion channels of mammalian sperm.

Oxidative damage to rhesus macaque spermatozoa results in mitotic arrest and transcript abundance changes in early embryos.

Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO2 in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development.

Oxidative stress in zebrafish (Danio rerio) sperm.

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.

Antioxidant treatment in the absence of exogenous lipids and proteins protects rhesus macaque sperm from cryopreservation-induced cell membrane damage.

Osmotic stress caused oxidative stress in rhesus macaque sperm, which was alleviated by antioxidant supplementation. The objective of the present study was to demonstrate that cryopreservation of rhesus macaque sperm also induces reactive oxygen species (ROS) production, and to determine whether ROS have an important role in cryopreservation-induced membrane. Additionally, we evaluated the antioxidant capacity of TEST (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-Tris) buffer (with 20% egg yolk and 13% skim milk) and supplementation with antioxidants, superoxide dismutase (SOD), catalase (CAT), and α-tocopherol. There was a substantial level of ROS production in both the presence (15% increase in superoxide, P < 0.01; 14% increase in hydrogen peroxide, P < 0.01) and absence of egg yolk (EY) and skim milk (SM; 33% increase in superoxide, P < 0.001; 48% increase in hydrogen peroxide, P < 0.001). Superoxide dismutase provided little membrane protection against ROS, but increased postthaw total and progressive motility by 10% (P < 0.01) and 15% (P < 0.05), respectively. Supplementation with CAT and α-tocopherol in the presence of EY and SM decreased H(2)O(2) by 55% (P < 0.01) and 49% (P < 0.001), whereas supplementation with CAT and α-tocopherol in the absence of EY and SM reduced the level of lipid peroxidation by 61% (P < 0.05) and 28% (P < 0.01). In conclusion, this is apparently the first report that cryopreservation of rhesus macaque sperm induced a significant increase in ROS and that antioxidant supplementation (N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid-Tris) can significantly decrease the extent of ROS-induced membrane damage.

Loss of fertilization potential of desiccated rhesus macaque spermatozoa following prolonged storage.

Desiccation provides a novel spermatozoal preservation technique because it eliminates the need to store spermatozoa in liquid nitrogen, resulting in decreased opportunities for cross-contamination of samples and lower costs of spermatozoal banking, storage, and shipping. The objective of this study was to desiccate rhesus macaque spermatozoa and to evaluate the fertility following storage. Semen from four male rhesus macaques (Macaca mulatta) were collected using electroejaculation, washed through a Percoll gradient, and resuspended to 100×10(6) spermatozoa/mL in a simple vacuum drying buffer containing the disaccharide trehalose (10mM HEPES, 5mM KCl, 65mMNaCl, 150mMtrehalose, 5.7% bovine serum albumin, BSA). Cells were desiccated in 50µl drops under vacuum (22inHg) at ambient temperature until the water content was less than 1g H(2)O/g dry weight. Initial motility was high (90-70%) and was reduced by desiccation (0%). Membrane integrity was investigated using the two fluorochromes, SYBR 14 and propidium iodide (PI, Molecular Probes, Inc.), with flow cytometry. After desiccation, 100% of the spermatozoa were stained red with PI indicating plasma membrane compromise. Samples were stored in air-tight polyvinyl plastic bags purged with N(2) gas for 10s and vacuum sealed. The samples were protected from light and either stored at room temperature (treatment 1) or at -80°C (treatment 2). Samples were rehydrated 7-10 days post desiccation in 150µl BWW containing 0.5% BSA and used for intracytoplasmic spermatozoa injection (ICSI) to compare fertilization and embryo development to freshly collected samples. For control embryos, one freshly ejaculated motile sperm with normal morphology was immobilized by scoring a small incision in the plasma membrane over the sperm tail before injecting into an oocyte. For the dried sperm treatments, one sperm with normal morphology was selected and scored before injecting into an oocyte. After injection, the embryos were individually cultured in CMRL medium with 10% fetal bovine serum (FBS) media on pre-plated buffalo rat liver (BRL) cells at 37°C and 5% CO(2). Fertilization was assessed at 14, 16, 22, and 24h. Embryo development was evaluated daily from day 3 to day 11. The fertilization rate was 68%, 73%, and 45% for the control, treatment 1, and treatment 2 groups, respectively. The blastocyst rate was 40%, 5%, and 0% for control, treatments 1, and 2, respectively. Treatment group 1 had comparable fertilization rates with control group (73% vs. 68%) and was not significantly different (P<0.05), but as development progressed, fewer embryos developed beyond the morula stage. Treatment 2 had a lower fertilization rate than control (45% vs. 68%), although not significantly different, and embryos did not develop past the morula stage. This study demonstrates that while the vacuum dried spermatozoa were immotile and had compromised plasma membrane integrity, they were capable of fertilization using ICSI and could support embryo development to the morula stage.

Osmotic stress stimulates phosphorylation and cellular expression of heat shock proteins in rhesus macaque sperm.

The cryosurvival of sperm requires cell signaling mechanisms to adapt to anisotonic conditions during the freezing and thawing process. Chaperone proteins heat shock protein 70 (HSP 70) and heat shock protein 90 (HSP 90; recently renamed HSPA and HSPC, respectively) facilitate some of these cell signaling events in somatic cells. Sperm were evaluated for their cellular expression and levels of phosphorylation of both HSP 70 and HSP 90 under anisotonic conditions as a potential model for cell signaling during the cryopreservation of macaque spermatozoa. In order to monitor the level of stress, the motility and viability parameters were evaluated at various time points. Cells were then either prepared for phosphoprotein enrichment or indirect immunocytochemistry. As controls, the phosphoserine, phosphothreonine, and phosphotyrosine levels were measured under capacitation and cryopreservation conditions and were compared with the phosphoprotein levels expressed under osmotic conditions. As expected, there was an increase in the level of tyrosine phosphorylation under capacitation and cryopreservation conditions. There was also a significant increase in the level of all phosphoproteins under hyperosmotic conditions. There was no change in the level of expression of HSP 70 or 90 under osmotic stress conditions as measured by Western blot. The enrichment of phosphoproteins followed by Western immunoblotting revealed an increase in the phosphorylation of HSP 70 but not HSP 90 under osmotic stress conditions. Indirect immunofluorescence localized HSP 70 to the postacrosomal region of sperm, and the level of membrane expression of HSP 70 was significantly affected by anisotonic conditions, as measured by flow cytometry. Taken together, these results suggest a differential role for HSP 70 and HSP 90 during osmotic stress conditions in rhesus macaque sperm.

Osmotic stress induces oxidative cell damage to rhesus macaque spermatozoa.

Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa survival. Additionally, evidence indicates that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa as well. Our objective was to determine the effect of reactive oxygen species (ROS) on rhesus macaque (Macaca mulatta) sperm function and to determine whether osmotic stress elicits the production of ROS. In the first experiment, the xanthine-xanthine oxidase (X-XO) system was used to generate the ROS superoxide anion (O(2)(-.)) and hydrogen peroxide (H(2)O(2)) in the presence or absence of the ROS scavengers superoxide dismutase and catalase, respectively. In the second experiment, osmotic stress was introduced by incubation of spermatozoa in a series of anisosmotic media ranging from 100 to 1000 mOsmol/kg in the presence or absence of the antioxidant alpha-tocopherol. Treatment with the X-XO system resulted in a significant increase in the generation of O(2)(-.) and H(2)O(2) that was detectable using flow cytometry. The ROS generated by the X-XO system was dose dependent, and as the concentration of ROS increased, motility decreased and lipid peroxidation increased while no affect was observed on viability. Incubation of spermatozoa in anisosmotic media also resulted in an increase in O(2)(-.) generation and lipid peroxidation that was significantly decreased in the presence of the powerful antioxidant alpha-tocopherol. These results clearly indicate that osmotic stress causes oxidative stress in rhesus macaque spermatozoa, which strongly supports the hypothesis that cryopreservation-induced osmotic stress may lead to oxidative cell damage.

New insights into the mechanisms of fertilization: comparison of the fertilization steps, composition, and structure of the zona pellucida between horses and pigs.

The mechanism of fertilization remains largely enigmatic in mammals. Most studies exploring the molecular mechanism underlying fertilization have been restricted to a single species, generally the mouse, without a comparative approach. However, the identification of divergences between species could allow us to highlight key components in the mechanism of fertilization. In the pig, in vitro fertilization (IVF) and polyspermy rates are high, and spermatozoa penetrate easily through the zona pellucida (ZP). In contrast, IVF rates are low in the horse, and polyspermy is scarce. Our objective was to develop a comparative strategy between these two divergent models. First, we compared the role of equine and porcine gametes in the following five functions using intraspecific and interspecific IVF: ZP binding, acrosome reaction, penetration through the ZP, gamete fusion, and pronucleus formation. Under in vitro conditions, we showed that the ZP is a determining element in sperm-ZP attachment and penetration, whereas the capacity of the spermatozoa is of less importance. In contrast, the capacity of the spermatozoa is a key component of the acrosome reaction step. Second, we compared the composition and structure of the equine and porcine ZP. We observed differences in the number and localization of the ZP glycoproteins and in the mesh-like structure of the ZP between equine and porcine species. These differences might correlate with the differences in spermatozoal attachment and penetration rates. In conclusion, our comparative approach allows us to identify determining elements in the mechanism of fertilization.

Rhesus macaque blastocysts resulting from intracytoplasmic sperm injection of vacuum-dried spermatozoa.

BACKGROUND: Sperm desiccation is an attractive approach for sperm preservation. In this study, we examined the feasibility and efficiency of intracytoplasmic sperm injection using vacuum-dried rhesus macaque sperm in CZB medium supplemented with 10% fetal bovine serum. METHODS: A total of 109 MII oocytes were injected with 69 fresh ejaculated sperm and 40 vacuum-dried sperm. RESULTS: Cleavage occurred in 97% of oocytes injected with fresh, motile sperm and in 88% of oocytes injected with vacuum-dried sperm. Of the cleaved oocytes, 68% fresh sperm-injected oocytes and 74% of dried sperm-injected oocytes developed to the compact morula stage. Blastocyst development was comparable between fresh-injected (16%) and vacuum-dried-injected (17%) oocytes. Differences between treatment groups were not significant. Transmission electron microscopic observation of the blastocysts indicated no detectable differences between fresh sperm and dried sperm-derived embryos. CONCLUSIONS: We conclude that vacuum-dried rhesus macaque sperm are capable of inducing fertilization and development of pre-implantation embryos when sperm were dried under vacuum and microinjected into normal viable oocytes.