Reduction of intramembranous particles in the periacrosomal plasma membrane of boar spermatozoa during in vitro capacitation: a statistical study

Membrane remodeling in the periacrosomal plasma membrane (PAPM) of boar spermatozoa during incubation in capacitation medium was examined by the freeze-fracture technique. In the preservation medium (PM) group, the major small (about 8 nm) intramembranous particles (IMP) and the minor large (> 10 nm) IMP were distributed evenly in the PAPM. The IMP-free area increased during capacitation. To correct the IMP-free area, arithmetically redistributed (ARD)-IMP density was used for statistical analysis. In the PM group, the mean density +/- SD of large IMP was 379 +/- 64 and 266 +/- 58/microm2, and that of small IMP was 1450 +/- 155 and 672 +/- 252/microm2 in protoplasmic (P) and external (E) faces, respectively. During capacitation, the significant (P < 0.01) reduction of large IMP density was encountered only in the E face of a few incubation groups, while that of the small IMP density occurred in the P face by 2 h. Consequently, reduction of the total IMP density of both faces was not significant in the large IMP, but it was significant (P < 0.01) in the small IMP. One-fifth of the total small IMP density reduced by 2 h. Filipin-sterol complexes (FSC) were numerous in the PAPM, and FSC-free areas also increased during capacitation. The mechanism of IMP-free area formation and the behavior of the small IMP in the PAPM during capacitation were discussed in relation to membrane stability.     

A molecular membrane model of sperm capacitation and the acrosome reaction of mammalian spermatozoa

The abundance of data pertaining to the metabolism of lipids in relation to mammalian fertilization has warranted an effort to assemble a molecular membrane model for the comprehensive visualization of the biochemical events involved in sperm capacitation and the acrosome reaction. Derived both from earlier models as well as from current concepts, our membrane model depicts a lipid bilayer assembly of space-filling molecular models of sterols and phospholipids in dynamic equilibrium with peripheral and integral membrane proteins. A novel feature is the possibility of visualizing individual lipid molecules such as phosphatidylcholine, phosphatidylethanolamine, lysophospholipids, fatty acids, and free or esterified cholesterol. The model illustrates enzymatic reactions which are believed to regulate the permeability and integrity of the plasma membrane overlying the acrosome during interactions between the male gamete and capacitation factors present in fluids of the female genital tract. The use of radioactive lipids as molecular probes for monitoring the metabolism of cholesterol and phosphatidylcholine revealed the presence of (1) steroid sulfatase in hamster cumulus cells, (2) lecithin: cholesterol acyltransferase in human follicular fluid, (3) phospholipase A₂, and (4) lysophospholipase in human spermatozoa. These enzymatic reactions can be integrated into a pathway that provides a link between the concepts of lysophospholipid accumulation in the sperm membranes and alteration of the cholesterol/phospholipid ratio as factors involved in the preparation of the membranes for the acrosome reaction. Capacitation is viewed as a reversible phenomenon which, upon completion, results in a decrease in negative surface charge, an efflux of membrane cholesterol, and an influx of calcium between the plasma and outer acrosomal membranes. Triggered by the entry of calcium, the acrosome reaction involves phospholipase A₂ activation followed by a transient accumulation of unsaturated fatty acids and lysophospholipids implicated in membrane fusion which occurs during the formation of membrane vesicles in spermatozoa undergoing the acrosome reaction.     

Zinc does not inhibit the capacitation of human spermatozoa in vitro

The effects of zinc on human sperm motility, fertilizing capacity (as assessed by penetration of human spermatozoa into the zona pellucida-free hamster oocyte), and nuclear chromatin decondensation were investigated using spermatozoa from four fertile donors. Both sperm motility and the penetration of sperm into zona-free hamster ova were consistently impaired in media containing 1,000 μM zinc. Spermatozoa from one man were similarly affected at a concentration of 500 μM zinc, but no adverse effects were noted at this zinc concentration in experiments with other donors. Since decreased fertilizing capacity in response to zinc was always accompanied by a significant decline in both the percentage of motile cells and mean swimming speeds, it appears that all of these results reflect a general toxic effect on the cells. At lower concentrations (125–250 μM), zinc had no effect on human sperm motility nor their ability to undergo capacitation and penetrate zona-free hamster ova in vitro. For some donors, zinc (125–500 μM) stimulated both the attachment of spermatozoa to the hamster vitellus and the incorporation of spermatozoa into the hamster ooplasm. The decondensation of human sperm nuclear chromatin in sodium dodecyl sulfate was largely inhibited when zinc was added to the medium, but no significant changes in nuclear stability were apparent after capacitation in zinc-free medium. We conclude that zinc, when present in subtoxic concentrations, does not adversely affect the ability of human spermatozoa to undergo capacitation and penetrate zona-free hamster ova in vitro.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

Sperm volumetric dynamics during in vitro capacitation process in bovine spermatozoa

Previous studies have demonstrated that sperm head morphometry can be used as a potential diagnostic tool for detecting biophysical changes associated with sperm viability in bovine spermatozoa. In this study, sperm head morphometry was used to investigate its value as a biophysical marker for detecting volumetric changes in bovine spermatozoa under in vitro capacitating and non-capacitating incubation conditions. To further test this hypotesis, aliquots of pooled, washed bovine sperm were incubated in either Tyrode’s complete medium with heparin (TCMH; a capacitating medium containing Ca²⁺, NaHCO₃ and heparin), Tyrode’s complete medium heparin-free (TCM; a medium containing just Ca²⁺ and NaHCO₃) or Tyrode’s basal medium (TBM; a non-capacitating medium free of Ca²⁺, NaHCO₃ and heparin, used as control). Aliquots of sperm were processed for morphometric analysis at different incubation-time intervals (0, 3 and 6 h at 38°C), and the chlortetracycline assay was used simultaneously to confirm the ability of the sperm to undergo capacitation (B pattern) and the acrosome reaction (AR pattern) status in each medium. After 3 h of incubation under TCMH conditions, a significant increase was observed in the percentage of B and AR patterns and a significant decrease was found in all sperm morphometric parameters (P<0.01). Interestingly, after 6 h of incubation in TCMH, the percentage of B and AR patterns increased drastically over time and marked differences were found in the dimensional and shape parameters, which were significantly smaller compared with TBM or TCM media (P<0.001). Significant correlations were observed between sperm size and AR pattern (r=−0.875, P<0.01). In conclusion, sperm head morphometry can be used as a potential biophysical marker for detecting volumetric changes during capacitation process in bovine spermatozoa.     

In vitro effect of gamma-aminobutyric acid on bovine spermatozoa capacitation

Sperm capacitation is defined as the maturational changes that render a sperm competent for fertilization and occurs in the female reproductive tract. Identification of the factor/s that regulate sperm capacitation would allow the understanding of these phenomena. Among these factors, gamma-aminobutyric acid (GABA) has recently become as a putative modulator of sperm function. The aim of this study was to explore the presence of a GABAergic regulation of bovine sperm capacitation as well as the possible intracellular mechanisms involved. GABA was detected in fresh semen by a sensitive radioreceptor assay (spermatozoa, 0.064 +/- 0.003 nmoles/10(6) cells; seminal plasma, 23.21 +/- 1.16 nmoles/ml). Scatchard analysis of [(3)H]-muscimol binding to sperm membranes yielded a linear plot consistent with a single population of binding sites (K(d) = 3.87 nM, B(max) = 417 fmol/mg prot.). [(3)H]-muscimol specific binding to sperm membranes was significantly inhibited by the GABA A receptor (GABA A-R) antagonist bicuculline and by the agonists muscimol and isoguvacine. Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of capacitated spermatozoa (chlortetracycline assay). We observed a significant increment on intracellular calcium and cyclic 3',5' adenosine monophosphate (cAMP) concentrations induced by GABA, being the cation influx abolished when the cell suspensions were coincubated with the antagonists bicuculline or picrotoxin. It is concluded that GABA induces sperm capacitation through an intracellular mechanism dependent on calcium influx and cAMP accumulation mediated by a specific GABA A-R.     

The evolution of hamster sperm motility during capacitation and interaction with the ovum vestments in vitro

Movement characteristics of golden hamster spermatozoa were studied upon collection from the cauda epididymis, during an incubation which capacitates the spermatozoa in vitro, during penetration of the cumulus, and during attachment to and penetration of the zona pellucida. High-speed videomicrography was employed to quantitate flagellar beat frequency and shape. The status of the acrosome was also assessed. During capacitation, hamster spermatozoa become increasingly invigorated before the onset of hyperactivated motility. Within the cumulus, beat frequency and curvature are reduced, apparently in response to the physical resistive properties of the matrix material. These properties appear to vary within the cumulus. Initial attachment to the zona precedes completion of the acrosome reaction, is non-rigid, and is accompanied by increased beat frequency and curvature. Subsequently, the onset of rigid binding to the zona, completion of the acrosome reaction, and increased flagellar beat frequency are very closely associated in time. The latter produces an increase in thrust against the zona. Preliminary results indicate that ensuing zona penetration requires not more than five minutes, is at oblique angles, and is associated with a continuation of vigorous flagellar beating.     

Changes in tyrosine phosphorylation associated with true capacitation and capacitation-like state in boar spermatozoa

Capacitation is defined as a series of events that render boar sperm competent to fertilize, either in vivo or in vitro. Moreover, preliminary stages of cryopreservation of spermatozoa involving cooling to 5 degrees C have been shown to induce capacitation-like changes in boar spermatozoa. Capacitation of boar spermatozoa is accompanied by protein phosphorylation, however the relationship between both processes is poorly understood. Capacitation status was assessed by chlortetracycline (CTC) staining. Changes in protein tyrosine phosphorylation were examined in pre-cleared whole cell lysates using a specific anti-phosphotyrosine monoclonal antibody. Our results in boar spermatozoa show a significant positive correlation between p32 tyrosine phosphorylation levels and percentage of capacitated (CTC pattern B) spermatozoa. Moreover, incubation of boar spermatozoa with two unrelated tyrosine kinase inhibitors induces a significant reduction in the percentages of capacitated and acrosome-reacted (AR) boar spermatozoa and a reduction in the p32 tyrosine phosphorylation. In our conditions, cooling boar spermatozoa to 5 degrees C and rewarming to 39 degrees C in a noncapacitating medium results in similar CTC staining patterns to those obtained after incubation of boar sperm for 1 or 4 hr at 39 degrees C in a capacitating medium. However, cooled-rewarmed fails to induce an increase in p32 tyrosine phosphorylation in boar spermatozoa. Moreover, CTC staining patterns of cooled-rewarmed spermatozoa do not change after incubation with a tyrosine kinase inhibitor. In conclusion, our results show a direct relationship between capacitation and tyrosine phosphorylation and suggest that p32 tyrosine phosphorylation levels could be used as a marker of the true capacitation changes observed in boar spermatozoa. Moreover, our results show that true capacitation and capacitation-like changes induced after cooling involve alternative intracellular tyrosine phosphorylation pathways in boar spermatozoa.     

Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.     

Surface alterations during the capacitation of mammalian spermatozoa

Capacitation is the process by which mammalian sperm acquire the ability to undergo the acrosome reaction which, in turn, is a prerequisite for sperm-egg fusion and penetration. Until recently, it was thought that capacitation involved subtle physiological and chemical changes which had no morphological counterparts even at the electron microscopic level. However, it has now been shown by a number of investigators that material associated with the plasma membrane surface is either lost or extensively redistributed during in vitro or in vivo capacitation. We have made use of lectins and antibodies as probes of the sperm surface during capacitation and the acrosome reaction. Concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) have been used in conjunction with fluorescent tags (FITC) and ultrastructural markers (ferritin, hemocyanin) to study the surface of golden hamster, guinea pig, mouse and human spermatozoa. Con A and WGA label the plasma membrane overlying the acrosomal region quite uniformly on these species. After capacitation there is a specific loss (or masking) of lectin binding sites over the acrosomal region of the sperm head in all species examined. Antibodies prepared against sperm and specific antibodies to a cell surface protein (fibronectin) were also tagged with fluorescent or ultrastructural markers and used to label the surfaces of sperm before and after capacitation. These probes also indicate a specific loss of surface associated material over the acrosomal surface after capacitation. These results are consistent with the notion that there is a general removal of surface components during capacitation and that this denuding of the surface is a prerequisite for the following membrane fusion events involved in the acrosome reaction and sperm-egg fusion.     

Temperature dependence of capacitation in bat sperm monitored by zona-free hamster ova

The temperature dependence of capacitation in bat sperm (Myotis lucifugus lucifugus) was studied by monitoring fertilizations rates of zona-free hamster ova at different temperatures. Spermatozoa were cultured in BWW medium at temperatures 4°C, 24°C, 32°C, 42°C, and 55°C from 0–24 hr. Activation of sperm could be determined visually due to the change in movement seen through light microscopy. Activation was later confirmed by higher rates of fertilization. Preincubation of the bat sperm was found to have a direct effect on the success of penetration of the zona-free hamster ova. Holding bat spermatozoa at low temperature for long intervals allowed them to remain motile but unable to fertilize. Sperm are not irreversibly damaged, however, and activation, when the temperature is increased to 32°C, is faster than when sperm are intitially put at 32°C, resulting in good fertilization rates.     

Photic regulation of L-arginine uptake in the golden hamster retina

One of the limiting steps in the regulation of nitric oxide (NO) synthesis is the availability of its precursor, L-arginine, which depends on the presence of a specific uptake system. A characterization of the L-arginine uptake mechanism in the golden hamster retina was performed. This mechanism was stereospecific, saturable, and monophasic, with an apparent of 56.1 +/- 2.0 microM and a maximum velocity of 36.0 +/- 2.8 pmol/mg prot/min. The basic amino acids L-lysine and L-ornithine but not D-arginine or the nitric oxide synthase inhibitors, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine impaired L-arginine influx. Preincubation with L-lysine for 1 h prior to the transport assay significantly stimulated L-arginine uptake. Saturation studies of L-arginine uptake performed at 12.00 and 24.00 h indicated a higher value of Vmax at midnight than at midday. When the hamsters were placed under constant darkness or constant light for 48 h and killed at equivalent time points, representing subjective day and subjective night, the differences in L-arginine influx disappeared. Semiquantitative RT-PCR analysis showed that the levels of mRNAs for both CAT-1 and CAT-2B were significantly higher at midnight than at midday. L-Arginine significantly increased cGMP accumulation in a time-dependent manner, with maximal effects during the night. Based on these results, it might be presumed that hamster retinal L-arginine uptake is regulated by the photic stimulus.     

Changes in calmodulin compartmentalization throughout capacitation and acrosome reaction in guinea pig spermatozoa

Calmodulin has been postulated as a mediator in the calcium-dependent processes that culminate in the acrosome reaction. Changes in calmodulin compartmentalization as a consequence of the increased permeability to extracellular calcium during capacitation and acrosome reaction have been suggested. In the present study the temporal localization of calmodulin in guinea pig spermatozoa was studied during in vitro capacitation and acrosome reaction by indirect immunofluorescence. Capacitation was achieved by incubation in Tyrode medium supplemented with pyruvate, lactate, and glucose in the presence and in the absence of calcium. Acrosome reaction was elicited in three different conditions: 1) by transfer to minimal culture medium containing pyruvate and lactate (MCM-PL) after in vitro capacitation 2) by 0.003% Triton-X 100 treatment, and 3) by A 23187 addition to sperm samples incubated in MCM-PL. During capacitation, calmodulin was observed both in the acrosome and in the flagellum; this localization seemed to be independent of the presence of extracellular calcium and of exogenous substrates. Throughout the acrosome reaction, different stages of calmodulin compartmentalization were observed. It became clustered around the equatorial region just before or a little after the acrosome reaction had occurred. Later, it was observed around the postacrosomal region in the acrosome-reacted sperm. The changes in calmodulin distribution were found to be dependent on the stage in the acrosome reaction.     

The status of acrosomal caps of hamster spermatozoa immediately before fertilization in vivo

Adult female golden hamsters were induced to superovulate. When they were mated several hours prior to ovulation or artificially inseminated about the time of ovulation, nearly 100% of their eggs were subsequently fertilized monospermically. During the progression of fertilization when the eggs were still surrounded by compact cumulus oophorus, the contents of the ampullary region of the oviducts were collected and spermatozoa moving in the ampullary fluid, within the cumulus and on/in the zonae pellucidae of unfertilized eggs, were examined by light and electron microscopy to evaluate the status of their acrosomal caps. Most spermatozoa swimming in the ampullary fluid had apparently intact acrosomal caps, while the vast majority moving within the cumulus had distinctly modified acrosomal caps. Most spermatozoa that had passed through the cumulus and reached the zona surfaces had remnants of their acrosomal caps (“acrosomal ghosts”). When the ghosts were present around the sperm heads on the zona, the heads pivoted about a point roughly corresponding to the places where the ghosts were located. The ghosts seemed to firmly attach to the zona surfaces, then were split open by the sperm heads and left behind as the sperm heads advanced into the zona. A few spermatozoa on the zona surfaces had no acrosomal ghosts (at least not detectable by light microscopy). In this case, the sperm head pivoted about either the inner acrosomal membrane or the equatorial segment of the acrosome. In no instance were spermatozoa with intact acrosomal caps found on zona surfaces. We infer from these observations that most spermatozoa in vivo initiate their acrosome reactions while they are advancing through the cumulus. When they arrive at the zona surfaces, acrosomal ghosts are generally present on the sperm heads. These ghosts appear to hold sperm heads to zona surfaces as well as to restrict the direction of advancement of sperm head through the zona. In a minority of cases, ghostless spermatozoa reach the zona surfaces. As these spermatozoa appear to be able to penetrate the zona successfully, structures other than the acrosomal ghost (ie, the inner acrosomal membrane and the plasma membrane over the equatorial segment of the acrosome) may also attach to zona surfaces before spermatozoa penetrate into the zona.     

Microsatellites for diversity studies in the golden hamster (Mesocricetus auratus)

Ten polymorphic microsatellites were developed for the golden hamster (Mesocricetus auratus), a widely used model organism in biological and medical researches. All loci were used to analyse the microsatellite variability in wild golden hamsters from Syria and in a sample of domestic animals comprising different strains. Average mean expected heterozygosity (H_E) and mean allele number (A) of domestic hamsters measured 0.279 ± 0.058 and 2.6 ± 0.306, respectively, compared to 0.809 ± 0.019 and 8.3 ± 1.075 found for wild hamsters. Cross‐species application in other Mesocricetus species proved conservation of most loci throughout the genus.     

Effect of neurosteroids on the retinal gabaergic system and electroretinographic activity in the golden hamster

It has been established that neurosteroids can either inhibit or enhance GABA(A) receptor activity. Although GABA is the main inhibitory neurotransmitter in the mammalian retina, the effects of neurosteroids on retinal GABAergic activity have not been investigated. The aim of this work was to study the neurochemical and electroretinographic effects of neurosteroids in the golden hamster. On one hand, pregnenolone sulfate inhibited and allotetrahydrodeoxycorticosterone increased GABA-induced [36Cl]- uptake in neurosynaptosomes. On the other hand, in whole retinas, pregnenolone sulfate increased, whereas allotetrahydrodeoxycorticosterone decreased high potassium-induced [3H]GABA release. The effect of both neurosteroids on GABA release was Ca2+-dependent, as in its absence release was not altered. The intravitreal injection of pregnenolone sulfate or vigabatrin (an irreversible inhibitor of GABA degradation) significantly decreased scotopic b-wave amplitude, whereas the opposite effect was evident when bicuculline or allotetrahydrodeoxycorticosterone were injected. A protein with a molecular weight close to that of hamster adrenal cytochrome P450 side-chain cleavage (P450scc) was detected in the hamster retina. P450scc-like immunoreactivity was localized in the inner nuclear and the ganglion cell layers. These results indicate that neurosteroids significantly modulate retinal GABAergic neurotransmission and electroretinographic activity. In addition, the selective localization of P450scc suggests that neurosteroid biosynthesis might occur only in some layers of the hamster retina.