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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Sperm mitochondrial integrity is not required for hyperactivated motility, zona binding, or acrosome reaction in the rhesus macaque.

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired.

The macaque sperm actin cytoskeleton reorganizes in response to osmotic stress and contributes to morphological defects and decreased motility.

Sperm undergo extreme variations in temperature and osmolality during cryopreservation, resulting in cell damage that includes plasma membrane defects, changes in cell volume, decreased motility, and flagellar defects. However, the fundamental biologic mechanisms underlying these events are poorly understood. We investigated the effects of osmotic stress and cytochalasins b (CB) and d (CD), naturally occurring toxins that disrupt actin organization, on the actin cytoskeleton and motility of Rhesus macaque sperm (Macaca mulatta).

Effects of environmental tobacco smoke in vitro on rhesus monkey sperm function.

The objective of this study was to use a non-human primate model to examine the effect of ETS on sperm function. Sperm samples were collected from adult rhesus monkeys (Macaca mulatta) and treated with different levels of ETS exposed medium. ETS treatment decreased the percentage of motile sperm and motion parameters.

Changes in membrane lipid order with capacitation in rhesus macaque (Macaca mulatta) spermatozoa.

Lipophilic fluorescent dye merocyanine 540 is believed to stain cell membranes with increasing affinity as the lipid components become more disordered and has been associated with changes in membrane fluidity. The aim of this study was to determine whether membrane lipid disorder is associated with capacitation of macaque spermatozoa. To induce capacitation, spermatozoa from 5 rhesus macaques were incubated at 37 degrees C (5% CO(2) in air) for 2 hours in a modified Biggers-Whitten-Whittingham medium containing 30 mg/mL bovine serum albumin and 36 mmol/L NaHCO(3).

Dry storage of sperm: applications in primates and domestic animals.

Cryopreservation of spermatozoa, oocytes and embryos, as well as somatic cells or cell lines for cloning from cells, are all options for the long-term storage of unique genotypes and endangered species. Spermatozoal cryopreservation and storage currently require liquid nitrogen or ultralow refrigeration-based methods for long- or short-term storage, which requires routine maintenance and extensive space requirements. The preservation of stem cells also has strict requirements for long-term storage to maintain genetic integrity.