Head Membranes Research Articles

Maturation of sperm volume regulation in the rat epididymis

Sperm maturation in the epididymis may involve differences between mature and immature spermatozoa in their volume regulatory osmolyte response. Spermatozoa obtained from the rat caput and cauda epididymidis were examined for their ability to regulate volume after transfer from in situ epididymal osmolality (measured to be 343 +/- 13 and 365 +/- 19 mmol kg(-1), respectively) to that of the female tract in single- and multiple-step protocols. Cells withstood the single-step treatment better than the multistep protocol. Sperm volume estimates by flow cytometric measurements of forward scatter of cells with intact head membranes was more sensitive than those by assessing cell coiling microscopically. At osmolalites below 210 mmol kg(-1) both caput and cauda cells ruptured, limiting the use of flow cytometry. Above this critical value, the use of quinine showed that both caput and cauda cells could regulate volume, but cauda cells were the more effective. Of several organic osmolytes studied, myo-inositol, glutamate and KCl caused only temporary and slight swelling of spermatozoa cells in hypotonic medium. Spermatozoa of both maturities seemed to use potassium as the preferred osmolyte for regulating volume.

Effects of cryoprotectant treatments on bovine sperm function and osmolyte content

The hypothesis that addition and removal of cryoprotectants to and from spermatozoa would initiate regulatory volume decrease, and lead to osmolyte loss and reduced sperm function, was tested. Common cryoprotectants, in the absence of freezing and thawing, affected bovine ejaculated spermatozoa by lowering their total and progressive motility in medium, reducing their migration through surrogate cervical mucus, damaging sperm head membranes and inducing sperm tail coiling. Sperm function was slightly better maintained after cryoprotectants were added and removed in multiple small steps rather than in a single step. The intracellular content of the polyol osmolytes, D-sorbitol and myo-inositol, exceeded that of the zwitterion osmolytes, L-carnitine and L-glutamate. Certain cryoprotectants reduced intracellular L-carnitine and L-glutamate concentration but not that of myo-inositol or D-sorbitol. Multistep treatments with some cryoprotectants had advantages over one-step treatments in mucus penetration depending on the original amount of intracellular carnitine and glutamate in the spermatozoa. Overall, sperm quality was best maintained by multistep treatment with glycerol and propanediols that were associated with decreased intracellular glutamate concentration. Bovine spermatozoa seem to use glutamate to regulate cryoprotectant-induced cell swelling.

Novel mode of action of spinosad: Receptor binding studies demonstrating lack of interaction with known insecticidal target sites

The ability of spinosyn A to either enhance or displace binding to selected insecticidally-relevant receptors was investigated using a number of radioligands including, [ 3H]imidacloprid and [ 3H]ivermectin in tissues from the ventral nerve cord (VNC) membranes of the American cockroach, Periplaneta americana and head membranes from the housefly, Musca domestica. In these insect neural tissues, spinosyn A does not appear to alter the binding of a number of radioligands suggesting that spinosyn A does not interact directly with a variety of known receptors, including nicotinic or γ-aminobutyric acid (GABA)-based insecticidal target sites. However, available data are consistent with spinosyn A interacting with a site distinct from currently known insecticidal target sites, thus supporting a novel insecticidal mechanism of action for the spinosyns.

Zinc inhibits protein phosphorylation in isolated sperm head membranes in Spisula solidissima

Zinc, when added to the media in concentrations as low as one micromolar, decreased protein phosphorylation in the isolated sperm head membranes in Spisula solidissima, 70-75%. Other divalent cations barium, strontium, cobalt, and copper, in similar concentrations exerted no effect. Sodium fluoride, a phosphatase inhibitor, had no effect on the zinc induced inhibition on protein phosphorylation and, when zinc was added to the media after the phosphorylation reaction of proteins, the inhibitory effect of zinc was lost. The autoradiography of -32P labeled proteins on SDS gel confirmed the inhibitory effect of zinc on protein phosphorylation. The inhibitory role of zinc on protein phosphorylation in the sperm membranes suggests a physiological role of this cation on membrane activity in the process of fertilization.

In vitro toxic effects of metal compounds on kinetic traits and ultrastructure of rabbit spermatozoa

Metal compounds have been associated with male reproductive toxicity in vivo. The aim of the present study was to investigate the in vitro effects of 20 metal compounds using rabbit ejaculated spermatozoa as a study model for spermiotoxicity. Five of the metals tested (arsenic, cadmium, chromium, mercury and vanadium) reduced sperm motility and curvilinear velocity. Ultrastructural analyses revealed three types of damage to sperm head membranes in relation to the metal used: acrosome breakage with formation of various sized microvesicles (arsenic, cadmium, mercury and platinum); a large round hole (arsenic, cadmium and chromium), and numerous folds in the acrosome membrane (vanadium). The vanadium compound, followed by chromium and mercury compounds, determined a higher number of damaged spermatozoa. In conclusion, all the studied metal compounds, at levels higher than 1 μM, may reduce sperm kinetic characteristics and probably fertilizing capacity by triggering specific morphological damages to the head and/or by inhibiting motility.

Method-Related Estimates of Sperm Vitality

Comparison of methods that estimate viability of human spermatozoa by monitoring head membrane permeability revealed that wet preparations (whether using positive or negative phase-contrast microscopy) generated significantly higher percentages of nonviable cells than did air-dried eosin-nigrosin smears. Only with the latter method did the sum of motile (presumed live) and stained (presumed dead) preparations never exceed 100%, making this the method of choice for sperm viability estimates.

Sperm head membrane reorganisation during capacitation

The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Junctional protein ring structures seal the surface of the mid-piece from the head and the tail respectively and probably prevent random diffusion of membrane molecules over the protein rings. Despite the absence of such lateral diffusion-preventing structures, the sperm head surface is also highly heterogeneous. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood. Nevertheless, it is known that each of the sperm head surface regions has specific roles to allow sperm to fertilize the oocyte: a specific region is devoted to zona pellucida binding, a larger area of the sperm head surface is involved in the acrosome reaction (intracellular fusion), while yet another region is involved in egg plasma membrane binding and fertilization fusion (intercellular membrane fusion). All three events occur in the area of the sperm head where the plasma membrane covers the acrosome. Recently, lipid ordered microdomains (lipid rafts) were discovered in membranes of many biological specimens including sperm. In this review, we cover the latest insights about sperm lipid raft research and discuss how sperm lipid raft dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction.

Operational Review of the North Head Membrane Bioreactor in Sydney, Australia

Operational Review of the North Head Membrane Bioreactor in Sydney, Australia

Comparative studies with six extenders for sperm cryopreservation in the cynomolgus monkey (Macaca fascicularis) and rhesus monkey (Macaca mulatta)

Ejaculated spermatozoa from cynomolgus monkeys and rhesus monkeys were frozen in straws with six different extenders (TTE, DM, mDM, LG-DM, G-DM, and TCG) containing glycerol. Sperm motility and head membrane and acrosomal integrity were evaluated after freezing and thawing, and the cryoprotective effects were compared among the extenders and the two species studied. The results showed that sperm motility and motility recovery with the six extenders were comparable for the cynomolgus and rhesus monkeys. There was no significant difference in sperm motility and head membrane integrity among the six extenders in either the cynomolgus or rhesus monkeys (P>0.05). However, a slightly but statistically lower percentage of acrosomal integrity was found with TCG in both species compared to the other extenders (P<0.05). These findings demonstrate that TTE, DM, mDM, LG-DM, G-DM, and TCG are equally suitable extenders for the cryopreservation of spermatozoa from cynomolgus and rhesus monkeys.

Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa

A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2–4 h Giemsa staining at 25–40° C is recommended for stallion semen.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa in a chemically defined extender

To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender. Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris mTTE synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %, 3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P0.05) for 5 % glycerol (42.95 +/- 2.55 and 50.39+/- 2.42, respectively) than those of the other groups (1%: 19.19 +/- 3.22 and 24.84 +/- 3.64; 3%: 34.23 +/- 3.43 and 41.37 +/- 3.42; 10%:15.68 +/- 2.36 and 21.39 +/- 3.14; 15%: 7.47 +/- 1.44 and 12.90 +/- 2.18). The parameters for 30 min equilibration(42.95 +/- 2.55 and 50.39 +/- 2.42) were better (P0.05) than those of the other groups (10 min: 31.33 +/- 3.06 and 38.98 +/- 3.31; 60 min: 32.49 +/- 3.86 and 40.01 +/- 4.18; 90 min: 31.16 +/- 3.66 and 38.30 +/- 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.

Eosinophilic Dermatitis Associated With Trichosporon asahii in a Cockatiel (Nymphicus hollandicus)

Abstract A cockatiel (Nymphicus hollandicus) was presented with generalized pruritis and feather loss around the head and patagial membranes of the wings. Treatment with antiparasitic and antibiotic agents did not result in clinical improvement. The lesions progressed to a generalized crusty dermatitis of the skin in the areas of feather loss. Results of skin biopsy revealed acute eosinophilic folliculitis and pyoderma containing budding yeasts. A yeast, Trichosporon asahii, was identified on fungal culture of a skin biopsy sample. Skin lesions and pruritis rapidly resolved with oral ketoconazole therapy.

Drosophila nicotinic receptors: evidence for imidacloprid insecticide and α-bungarotoxin binding to distinct sites

The principal mammalian brain nicotinic acetylcholine receptors (nAChRs) are the α-bungarotoxin (α-BGT)-insensitive α4β2 and the α-BGT-sensitive α7 subtypes assayed with radiolabeled nicotinoids and α-BGT, respectively. Drosophila head membranes bind the insecticide radioligand [ 3H]imidacloprid ([ 3H]IMI) and [ 3H]α-BGT with K D 5.7 and 2.7 nM and B max 980 and 1400 fmol/mg protein, respectively. The hypothesis that [ 3H]IMI at 2.5 or 20 nM and [ 3H]α-BGT at 1 or 10 nM bind to distinct sites or subtypes is tested by using these radioligands alone and together in simultaneous dual binding experiments. These studies show no interference by one radioligand in the binding of the other one, i.e., independent binding, and that both unlabeled IMI and α-BGT give biphasic displacement curves. The pharmacological profiles of [ 3H]IMI and [ 3H]α-BGT suggest distinct binding sites for the two radioligands. These findings are consistent with those obtained with hybrid receptors assembled from Drosophila α subunits and a vertebrate β subunit and with immunological and protein biochemical approaches. This study, therefore, provides direct evidence for distinct IMI- and α-BGT-sensitive sites or subtypes in Drosophila brain.

Receptor assay-guided isolation of anti-GABAergic insecticidal alkaloids from a fungal culture.

The gamma-aminobutyric acid (GABA) receptor bears sites of action for insecticides. To discover GABA receptor-directed insecticides in natural products, fungal culture extracts were screened for their ability to inhibit specific binding of the radiolabeled noncompetitive antagonist [3H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane to housefly head membranes. The screening efforts led to the isolation of two alkaloids from Aspergillus terreus: PF1198A (alantrypinone) and PF1198B (serantrypinone), which had IC50 values of 0.34 and 2.1 microM, respectively, in this assay. These compounds were ca. 47-61-fold selective for housefly vs rat GABA receptors. Both compounds showed insecticidal activity against Myzus persicae in the range of 100-500 ppm. Binding assay-guided screening should provide significant opportunities for the identification of novel and selective insecticides.

Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen

Cryopreservation imposes irreversible damage to sperm membranes, such as swelling and disruption of plasma and acrosome membranes, changes in membrane fluidity, altered influx of calcium, and changes in enzyme activity. Morphological integrity of the sperm plasma membrane has been widely studied using different techniques, including exposure of spermatozoa to hypoosmotic solutions (provides information concerning the biochemical activity of the sperm tail membrane), supravital test using eosin stain (yields information regarding sperm head membrane integrity), and Trypan-blue Giemsa stain (TBG; reveals both sperm plasma membrane and acrosome integrity). The objective of this study was to combine these tests in order to provide information about the integrity of the whole sperm surface, as well as acrosome status, and determine if the results of these tests were associated with sperm in vitro fertilizing ability. Stepwise regression analyses yielded a model in which fertility (maintain variable) was expressed as a combination of the results of different spermatological parameters (independent variables). The results of a test combining supravital eosin staining of samples previously submitted to hypoosmotic swelling test (STHOS) accounted for the greatest proportion of variation in fertilization rates (78%). Inclusion of the results of dual staining with TBG increased the proportion of variation in fertility rate that could be accounted for to 82%. Therefore, sperm plasma membrane integrity and function, and acrosome integrity can be considered important variables for normal sperm function and STHOST and TBG could be used for the prognosis of the potential fertility of bovine semen samples used for IVF or AI.

5‐Amino‐1‐(2,6‐dichloro‐4‐trifluoromethyl‐phenyl)‐3‐ [3H3]‐methylsulfanyl‐1H‐pyrazole‐4‐carbonitrile (CTOM): Synthesis and Characterization of a Novel and Selective Insect GABA Receptor Radioligand.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Fluorometric study of rabbit sperm head membrane phospholipid asymmetry during capacitation and acrosome reaction using Annexin-V FITC.

This study was conducted to evaluate phosphatidylserine translocation in head plasma membrane of Percoll-gradient purified of rabbit cauda epididymal sperm during capacitation and acrosome reaction (AR) using Annexin-V. Propidium iodide was used as control to reject dead or dying cells. The presence and distribution of Annexin-V binding sites were analyzed using flow fluorocytometry and confocal microscopy. After 6 h of incubation of sperm in capacitation medium, the number of cells positively stained with Annexin-V showed a small but significant increment. The Annexin-V binding sites produced during capacitation were found mainly in the post-acrosomal region of the sperm head plasma membrane. After AR induction with progesterone, the localization of phosphatidylserine was changed and the Annexin-V binding sites were found almost only in the acrosomal region, but with higher number of binding sites in the equatorial area. On the contrary, after AR induction with A23187, phosphatidylserine translocation, although predominant over the acrosomal region, was also observed in the post-acrosomal region. Plasma membrane destabilization during capacitation and AR may be important for sperm-oocyte fusion.

Identification and Structural Characterization of the Neuronal Luteinizing Hormone Receptor Associated with Sensory Systems

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor involved in regulation of ovarian and testicular functions. Here we show that the receptor is present also in specific areas of the peripheral and central nervous system and may thus have a broader functional role than has been anticipated. Full-length LHR mRNA and two receptor protein species of M(r) 90,000 and 73,000, representing mature and precursor forms, respectively, were expressed in adult and developing rat nervous tissue, starting at fetal day 14.5. The receptor was capable of ligand binding because it was purified by ligand affinity chromatography, and human chorionic gonadotropin and LH were able to displace (125)I-labeled human chorionic gonadotropin binding to fetal head membranes in a dose-dependent manner. Finally, two 5'-flanking sequences ( approximately 2 and 4 kb) of the rat LHR gene were shown to direct expression of the lacZ reporter to specific areas of the peripheral and central nervous system in fetal and adult transgenic mice, especially to structures associated with sensory, memory, reproductive behavior, and autonomic functions. Importantly, the transgene activity was confined to neurons and colocalized with the cytochrome P450 side chain cleavage enzyme. Taken together, these results indicate that the neuronal LHR is a functional protein, implicating a role in neuronal development and function, possibly by means of regulating synthesis of neurosteroids.

Isocoumarin Derivative as a Novel GABA Receptor Ligand from Neosartorya quadricincta

For the purpose of discovering GABA receptor-directed insecticides in natural products, fungal culture extracts were screened for their ability to inhibit the specific binding of the noncompetitive antagonist [3H]EBOB to housefly head membranes. The screening efforts led to the isolation of a derivative of dihydroisocoumarin (PF1223) from the culture of Neosartorya quadricincta. This compound at 2.2 μM inhibited [3H]EBOB binding by 65%. This ligand might prove to be a lead compound for the identification of novel insecticides acting at the insect GABA receptor. © Pesticide Science Society of Japan

Studies on the Structure-Activity Relationship and Mode of Actions of Neonicotinoid Insecticides

The binding affinity of neonicotinoids for housefly head membranes was evaluated to elucidate the physicochemical features of the compounds involved in the ligand-nicotinic acetylcholine receptor (nAChR) interactions. Both steric and electrostatic factors of the substituents on the aromatic and imidazolidine rings were found to influence the binding affinity. [14C]Imidacloprid was employed to clarify the metabolism of imidacloprid in the flies. It was found that the compound was degraded, and the metabolites were then excreted from the flies in a short period after administration. Agonist actions of neonicotinoids on nAChRs were evaluated to examine their relationships with the binding and insecticidal activities. High correlations were observed among these activities, suggesting that the channel opening of nAChRs resulting from the neonicotinoid binding is likely to cause the insecticidal actions.