Long-term correction of diabetes in rats after lentiviral hepatic insulin gene therapy.

AIMS/HYPOTHESIS: Type 1 diabetes results from the autoimmune destruction of pancreatic beta cells. Exogenous insulin therapy cannot achieve precise physiological control of blood glucose concentrations, and debilitating complications develop. Lentiviral vectors are promising tools for liver-directed gene therapy. However, to date, transduction rates in vivo remain low in hepatocytes, without the induction of cell cycling. We investigated long-term transgene expression in quiescent hepatocytes in vitro and determined whether the lentiviral delivery of furin-cleavable insulin to the liver could reverse diabetes in rats. MATERIALS AND METHODS: To improve transduction efficiency in vitro, we optimised hepatocyte isolation and maintenance protocols and, using an improved surgical delivery method, delivered furin-cleavable insulin alone or empty vector to the livers of streptozotocin-induced diabetic rats by means of a lentiviral vector. Rats were monitored for changes in body weight and blood glucose, and intravenous glucose tolerance tests were performed. Expression of insulin was determined by RT-PCR, immunohistochemistry and electron microscopy. RESULTS: We achieved long-term transgene expression in quiescent hepatocytes in vitro (87 +/- 1.2% transduction efficiency), with up to 60 +/- 3.2% transduction in vivo. We normalised blood glucose for 500 days-a significantly longer period than previously reported-making this the first successful study using a lentiviral vector. This procedure resulted in the expression of genes encoding several beta cell transcription factors, some pancreatic endocrine transdifferentiation, hepatic insulin storage in granules, and restoration of glucose tolerance. Liver function tests remained normal. Importantly, pancreatic exocrine transdifferentiation did not occur. CONCLUSIONS/INTERPRETATION: Our data suggest that this regimen may ultimately be employed for the treatment of type 1 diabetes.

Study of demembranated, reactivated human spermatozoa with decondensed nuclei.

Reactivated movement of the axonemes in demembranated spermatozoa with decondensed nuclei allows decondensation to be monitored in vitro with minimal disruption, and provides access to the nucleus for ultrastructural investigation and experimental manipulation. In the present study, fresh liquefied semen samples with sperm concentrations > or = 13 x 10(6)/ml were diluted 1:10 with a demembranating solution containing 0.01-0.022% Triton X-100. Inter-sample variation in the concentration of Triton X-100 required to permeabilize the sperm membrane was observed as judged by the ability of the spermatozoa to be reactivated by ATP but not by an ATP-free control solution, with the extent of demembranation being checked by transmission electron microscopy. After exposure to DTT and heparin, coordinated and sometimes progressive movement of partially decondensed spermatozoa occurred in a reactivating solution. Unlike ram, human sperm heads required decondensation with heparin. An unusual ultrastructural feature of the decondensing human sperm nuclei, not previously reported, was the appearance of dense globular material extruding from the nucleus. Enzymatic treatment of the sections with protease but not with deoxyribonuclease removed this material, which was presumably protamine.

The development of smoothing-independent kinematic measures of capacitating human sperm movement.

The kinematic values which are determined relative to the average path by computer-aided sperm analysis (CASA) instruments are reliant upon the algorithm used for track smoothing, and therefore may differ depending upon the shape and complexity of the trajectories analysed. To overcome this potential source of error in the identification of particular trajectory patterns, kinematic values must be derived so that they are independent of the average path. In this study a number of novel kinematic measures were developed and tested for their ability to differentiate between hyperactivated and non-hyperactivated human spermatozoa. A definition for hyperactivation which relied solely upon smoothing-independent kinematic values was developed. This definition was tested on two groups of trajectories reconstructed at 60 Hz by a CASA instrument. An overall agreement of 99% between the classification of tracks as hyperactivated or non-hyperactivated was observed between the established 60 Hz definition validated for that particular CASA instrument and the new, smoothing-independent definition. Because the new values are only reliant upon (x,y) coordinates for their calculation, and do not require smoothing, it should not be difficult to incorporate them into existing CASA instruments.

Effect of image sampling frequency on established and smoothing-independent kinematic values of capacitating human spermatozoa.

It is known that image sampling frequency affects sperm kinematic values, although no study has considered the relative effect upon hyperactivated and non-hyperactivated spermatozoa. We determined the relative effect of image sampling frequency on the classification of capacitating human spermatozoa, using the established kinematic measures as well as a series of new, smoothing-independent kinematic measures. Spermatozoa were prepared by direct swim-up from semen and sperm movement was recorded using a video system which gave 201 images/s on freeze-frame playback. Trajectories were reconstructed manually and kinematics were determined using the (x, y) co-ordinates of each track point. Lower image sampling frequencies were approximated by considering every second, third, fourth, sixth and eighth track point for each trajectory. Of the 22 kinematic values investigated, only three were not significantly affected by sampling frequency in both hyperactivated and non-hyperactivated spermatozoa. Also, significant differences were observed between hyperactivated and non-hyperactivated tracks at all image sampling frequencies studied for all but four kinematic measures.

Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa.

While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.

Improved preservation of the ram spermatozoan plasma membrane using betaine in the primary fixative.

Improved preservation of ram spermatozoa was obtained by filtration onto a Millipore filter followed by fixation in a fixative containing 23 mM betaine, which raised fixative osmolality only slightly. In samples fixed in control preparations with betaine omitted the plasma membrane had a ruffled appearance, while the betaine-containing fixative gave the plasma membrane a smooth contour which closely followed underlying structures. Betaine is known to function as an osmoprotectant in other situations, and may protect the cells from osmotic damage during the initial stages of fixation.

Quantitative observations of flagellar motility of capacitating human spermatozoa.

For technical reasons sperm head movement is assessed in kinematic analysis, while flagellar movement is the determining factor of head movement, not vice versa. It follows then that the development of new kinematic values to describe the movement of capacitating human spermatozoa should include the analysis of their flagellar movement. The aim of this study was to establish quantitative differences between flagellar movement patterns of hyperactivated and non-hyperactivated spermatozoa which could then be used in the evaluation of new centroid-based kinematic values. Spermatozoa were prepared by swim-up from semen into culture medium supplemented with 30 mg/ml human serum albumin. Sperm movement was recorded in 50 microm-deep chambers using a 200 Hz video system. Sperm movement was classified based on flagellar movement, with 24 non-hyperactivated and 26 hyperactivated spermatozoa included in the study. Flagellar analysis was performed using both a semi-automated analysis system (SIAM FLAG; 30 images at 200 Hz) and manual methods (100 Hz). Hyperactivated spermatozoa had significantly larger flagellar beat angles (> or = 87 degrees) and significantly lower flagellar beat frequencies (< or = 29.4 Hz) than non-hyperactivated human spermatozoa. In addition, the flagellar wave amplitude was significantly greater and the bend diameter significantly smaller for hyperactivated spermatozoa in the proximal region of the flagellum (up to 20 microm from the head-midpiece junction). The velocity of the hyperactivated wave was low in this region, although it was significantly slower than the non-hyperactivated wave in all regions of the sperm tail.

Improved preservation of ultrastructural morphology in human spermatozoa using betaine in the primary fixative.

Improved preservation of the ultrastructural morphology of human spermatozoa was obtained by filtration onto a Millipore filter (0.45 micron pore size) followed by fixation for 1 h in a fixative containing 3% glutaraldehyde, 1% formaldehyde and 21 mM betaine in Hepes or cacodylate buffer. Betaine raised fixative osmolality only slightly but markedly improved the ultrastructural appearance of the spermatozoa; it is known to function as an osmoprotectant in other situations, and may protect the cells from osmotic damage during the initial stages of fixation. The method is suitable for oligozoospermic samples as they are concentrated when the seminal plasma passes through the filter. Utilizing fixation after swim-up, it is suitable also for highly viscous semen. The fixative gave equally good preservation of sperm heads and tails and can be stored prior to use. The Millipore filter did not dissolve until placed in acetone prior to embedding, simplifying the exchange of solutions during processing. Improved preservation of rat epididymal ultrastructure was obtained by addition of betaine to the fixative of Ito & Karnovsky (1968), using immersion fixation.

Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g).

In order to design a feasible somatic cell gene delivery system for the treatment of type I diabetes, a suitable cell type needs to be determined. We have previously shown that the stable transfection of the full-length insulin cDNA into the human liver cell line, (HEP G2ins) resulted in synthesis, storage and acute regulated release of insulin to analogues of cAMP, but not to the physiological stimulus glucose. In attempting to explain the lack of glucose responsiveness of the HEP G2ins cells we have stably transfected these cells with the human islet glucose transporter GLUT 2 (HEP, G2ins/g cells). The HEP G2ins/g cell clones exhibit glucose-stimulated insulin secretion and glucose potentiation of the secretory response to nonglucose secretagogues. While glucose responsiveness commenced at a lower concentration than normal islets, a secretion curve approaching normal physiological conditions was generated. Immunoelectron microscopy revealed the presence of insulin-containing granules, similar in size and appearance to those of the normal beta cell. These results demonstrate that while it is most likely that the HEP G2ins/g cell line predominantly secretes insulin via the constitutive pathway, significant acute regulated release was seen in response to glucose, and thus represents significant progress in the creation of a genetically engineered 'artificial beta cell' from a human hepatocyte cell line.

Fractal analysis of capacitating human spermatozoa.

While head centroid-derived kinematic values have been determined for the trajectories of hyperactivated human spermatozoa, the definitions are not robust with respect to image sampling frequency and track analysis methods. The determination of the fractal dimension of a trajectory has been suggested as an alternative descriptive parameter for hyperactivated motility. Here, we have investigated two methods for the determination of the fractal dimension of a trajectory. A simple but useful equation was found to be: D = log(n)/[log (n + log (d/L)], where the n is the number of intervals in the trajectory, d is the planar extent of the curve and L is the length of the trajectory. This equation was not influenced by scaling of the trajectory. A fractal dimension (D) >=1.30 was found to define hyperactivated trajectories, and D <= 1.20 defined non-hyperactivated trajetories, reconstructed at both 30 and 60 Hz. However, when circling tracks were studied, all had D > 1.30, even though they were classified as non-hyperactivated by curvilinear velocity and/or amplitude of lateral head displacement values. An analysis of a series of non-ideal track segments suggested a relationship between a track's linearity and its fractal dimension. It was determined by a linear regression analysis that the fractal dimension of a trajectory was inversely proportional to its linearity (r = -0.77, P < 0.001). Although the fractal dimension of a trajectory is a good indicator of its regularity (describing its space-filling properties), it should not be used as the sole criterion for the classification of a trajectory as hyperactivated.

Variable kinematics of capacitating human spermatozoa.

Human spermatozoa were prepared by swim-up from semen into in-vitro fertilization (IVF) culture medium, and their movement recorded by videomicrography using NTSC video to give 60 images/s on freeze-frame playback. Trajectories in which spermatozoa appeared to switch from one type of motility pattern to another (from non-hyperactivated to transitional or star-spin hyperactivated), were reconstructed manually and the movement characteristics determined for short consecutive sequences along each track. The spermatozoa were able to switch between all of these motility patterns, apparently at random. These observations illustrated that hyperactivated motility is a reversible state in human spermatozoa, with phase changes from hyperactivated to non-hyperactivated motility patterns along trajectories. It was not necessary for spermatozoa to pass through the transitional hyperactivated motility phase when switching from non-hyperactivated to star-spin hyperactivated motility or vice versa.

Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2).

To develop a model somatic gene therapy system for diabetes, a human hepatoma cell line (HEP G2) was transfected with a mammalian expression vector carrying the full-length human insulin cDNA. More proinsulin than insulin was released daily by the stably transformed cell line (HEP G2ins). However, on acute stimulation with 5mM 8-Br-cAMP and 10mM theophylline the HEP G2ins cells released predominantly insulin into the medium. The cells did not secrete insulin in response to glucose. Examination of acid-ethanol extracts confirmed insulin was preferentially being stored. Immunohistochemical analysis of the cells also showed (pro)insulin was being stored. Electron microscopy revealed large membrane-bound vacuoles, containing electron-dense material, which were not seen in control cells. Glucokinase activity and albumin secretion of the transfectants were unaltered from the controls. Five-minute pulse-chase labelling of the HEP G2ins cells with 3H-leucine confirmed insulin synthesis in the presence of 20mM glucose and 5mM 8-Br-cAMP. A dose-response curve for insulin synthesis was also generated to increasing concentrations of glucose with a half Vmax of 4.9mM. Our results show that the introduction of insulin cDNA into a human hepatoma cell line results in synthesis, storage and acute regulated insulin release and lend credence to the possibility of engineering a liver cell to secrete insulin acutely in response to physiological stimuli.

Kinematics of capacitating human spermatozoa analysed at 60 Hz.

Hyperactivation is a concomitant of eutherian sperm capacitation, characterized by the development of high amplitude flagellar waves with a corresponding increase in velocity. In humans, kinematic values have been derived which describe the movement characteristics of spermatozoa analysed at 30 images/s. However, these values are frame rate-dependent, and modern computer-aided sperm analysis (CASA) instruments used for studying sperm movement now use 60 images/s. This study used first-principles manual track analysis to derive the range of movement characteristics which describe hyperactivated motility of human spermatozoa at 60 images/s. Standard terminology for centroid-derived movement characteristics, as recommended by the World Health Organization, was used. US-standard (NTSC) video recordings of capacitating human sperm populations were replayed using a non-interlaced freeze-frame video cassette recorder, and individual tracks reconstructed on acetate overlays. Tracks were classified as either forward progressive or hyperactived based upon flagellar beating patterns, then reconstructed manually at x3540 and analysed using both manual methods and basic geometric calculations from (x, y) coordinates (Cartesian methods) similar to those used by CASA instruments. In all, 40 hyperactivated and 40 forward progressive tracks were studied. A set of Boolean arguments defining hyperactivated motility was derived, and there was generally good agreement between the limits derived by manual and Cartesian methods. The limits for the definition of hyperactivated motility of human spermatozoa at 60 Hz derived by Cartesian methods were: curvilinear velocity > or = to 180 micrograms/s and linearity < or = to 45% and wobble < 50% and amplitude of lateral head displacement ALHmean > 6.0 micrograms or ALHmax > 10.0 micrograms.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivated movement of decondensed rat sperm models and a description of their ultrastructure.

This study aimed at finding optimal conditions to decondense rat sperm nuclear chromatin with minimal damage. This was judged by the ability of the sperm-tail axoneme in the partially decondensed sperm models to be reactivated. Decondensation was assessed by phase contrast microscopy. Partial decondensation was judged to occur when the bright refractive appearance of the sperm nucleus turned black, and full decondensation when the nucleus turned pale and increased in volume. Demembranation was shown to have occurred by electron microscopy. With 0.03% Triton X-100 rat caudal epididymal sperm were partially demembranated to produce sperm models. Demembranation using a 0.1% solution of Triton X-100 was complete, but as with the solution of 0.05% Triton X-100, resulted in poorer reactivation of the partially decondensed sperm models. Reactivated movement of decondensed sperm models was used to assess the effect of the decondensing agents DTT and heparin. We were only able to achieve reactivation of sperm models that had undergone partial decondensation. Optimal reactivation was obtained after rat sperm models had decondensed in the decondensation solution containing 5 mM DTT, 6 mM EDTA, and 27.3 or 34.1 USP/ml heparin. Concentrations of heparin above or below these values resulted in a decrease in the number of sperm models reactivated. Ultrastructurally, sperm partially decondensed with 5 mM DTT, 6 mM EDTA, and 34.1 USP/ml heparin had their plasma membrane further extracted compared with sperm treated with 0.03% Triton X-100 alone. Decondensation was greatest in the peripheral regions of the nucleus with extraction of the acrosome but not of the perforatorium. The decondensed regions had a filamentous appearance. This procedure will allow access to sperm nuclear chromatin for experimental manipulation in rat sperm models.

Effects of the lipid peroxidation product (E)-4-hydroxy-2-nonenal on ram sperm function.

(E)-4-hydroxy-2-nonenal (HNE) is a lipid peroxide end-product which exerts powerful biological effects in a variety of cell and tissue systems. The effects of exogenous HNE on ram spermatozoa were examined in vitro. HNE inhibited the motility of diluted ram spermatozoa in a dose-dependent (100-400 mumol l-1) manner (P < 0.05). The extent of motility loss varied with sperm concentration as well as with HNE concentration, and was manifested as a progressive decrease in mean sperm velocity. The suppressive effect of 250-500 mmol HNE l-1 on the motility of reactivated ram sperm models (P < 0.05) was prevented by the addition of 1 mmol reduced glutathione l-1 to the reactivation medium, suggesting that HNE inhibits ram sperm motility via oxidation of sulfhydryl groups in the axoneme. Oxygen uptake by ram spermatozoa was inhibited (P < 0.05) by the addition of 100 or 200 mumol HNE l-1. Glucose utilization was maintained in the presence of 200 mumol HNE l-1, suggesting that fructolysis was unaffected by HNE. As was the case with motility, the inhibition of oxidative metabolism by HNE was not reversed by washing the spermatozoa. The activity of ram sperm acrosomal enzymes released by cold shock, as measured by hydrolysis of N-benzoyl-DL-arginine p-nitroanilide (BAPNA), was reduced in the presence of 100 mumol HNE l-1 (P < 0.05). No evidence was found of disruption of the acrosomal outer membrane or the sperm plasma membrane as a result of exposure to HNE.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for reactivation of the movement of demembranated ram sperm models with decondensed nuclei and a description of their ultrastructure.

In the procedure described, demembranated ram sperm models were treated to decondense their nuclei, and the movement of their sperm tails was then reactivated. The duration of movement was sustained for more than 30 min, with a tail-wave frequency of up to 14 Hz. Decondensed sperm models adhered to each other and to the slide; but adherence was prevented by addition of an anti-agglutination factor derived from dialyzed, freeze-dried ram seminal plasma, which allowed models to swim freely. Electron microscopy showed that most areas of the sperm model nuclei were decondensed and contained fine interconnected filaments with structures resembling nucleosomes at anastomosing regions. Some central, lateral, and basal regions of the nuclei were not fully decondensed. The axoneme, outer dense fibers, and fibrous sheath were not affected ultrastructurally by the extraction procedure; the mitochondria of the middle piece appeared to be extracted, while the acrosome but not the perforatorium was removed. Use of decondensed, reactivated sperm models provides a means to monitor the decondensation of sperm in vitro while maintaining minimal disruption to sperm tail components. This will allow access to the nucleus for experimental manipulation in sperm models.

Effect of ethanol and methanol on the motility of Saccostrea commercialis sperm and sperm models.

The organic solvents methanol and ethanol at concentrations of 2.5% and 5% (v/v), respectively, were found to significantly (P less than 0.001) decrease the radius of curvature and track velocity of S. commercialis sperm. To observe the effects of the solvent directly on the axoneme, S. commercialis sperm models were prepared by extraction with Triton X-100 and reactivation with ATP in media containing acetate anions, DTT, magnesium, and cAMP. Concentrations of 0.1% Triton X-100 demembranated sperm while 0.01% and 0.05% Triton X-100 permeabilized sperm. Sperm models were successfully produced after reactivation with 1 mM ATP. At pH 8.25, 1% (v/v) ethanol or methanol was observed to increase waveform asymmetry and significantly (P less than 0.001) decrease track velocity of 0.1% Triton X-100 demembranated sperm models. Similarly 1% (v/v) ethanol increased tail-wave asymmetry and decreased track velocity of 0.01% and 0.05% Triton X-100 permeabilized sperm models. Reactivated motility of 0.05% Triton X-100 permeabilized sperm models prepared at pH 7.8 were poor and improved after treatment with 7% (v/v) ethanol, which increased waveform asymmetry and doubled the track velocity of sperm. This stimulatory effect of ethanol was unchanged in the presence of the alcohol dehydrogenase inhibitor pyrazole. Concerning the precise mechanism of action of ethanol on the axoneme, we conclude that a stimulatory or inhibitory effect of ethanol is dependent on the pH of the sperm model system used.

Electron microscopic observations on the effect of gossypol on rat cauda epididymis.

Gossypol administered orally to male rats at a daily dose of 20 mg/kg body weight for 63 days caused hypertrophy of the cauda epididymal epithelium, with more than fourfold increase in height of the cells. The principal cells lost most of their microvilli and formed apical blebs which appeared to produce the dense secretory material which was found in the lumen. Less dramatic but similar changes also occurred after 9 days on the same regimen, with the height of the epithelium doubling. However after 19 days on this regimen, with the height of the epithelium doubling. However after 19 days on this regimen, the epithelium looked fairly normal apart from a maintained hypertrophy. As reported in other studies, the cauda epididymal sperm were severely damaged and immotile; many were decapitated and the oxygen uptake was low. Ultrastructural defects were abnormal or absent mitochondria, absence of plasma membranes and axonemal components and accessory fibres.

A model for swimming unipolar spirilla.

A mathematical model employing slender body theory is constructed for a unipolar Spirillum volutans cell with the model cell allowed to move unconstrainedly in the fluid. The results are compared with observation and previous studies and the effects of varying cell dimensions are investigated.

Studies of the mechanism of action of gossypol as a male antifertility agent.

Gossypol administered orally to male rats at a daily dose of 20 mg/kg body weight for 62 days caused infertility. There were changes in the epididymal epithelium and the sperm were severely damaged and immotile. The sperm head was often detached; other defects were abnormal mitochondria, absence of plasma membranes and axonemal and accessory fibres and a lower oxygen uptake. To study the effect of gossypol on the motor apparatus of sperm, ram sperm were demembranated with the detergent, Triton-X-100. Such sperm models can normally be reactivated with ATP but gossypol (2.5-12.5 microM) decreased reactivation and must have a direct effect on the axoneme. Gossypol also inhibited ram sperm adenyl cyclase which is essential for maintaining high levels of cAMP in sperm and, in turn, motility. Ram sperm adenyl cyclase required Mn2+ for activity and high Mn2+ concentrations protected the enzyme from gossypol inhibition. Electron spin resonance studies proved that gossypol chelated Mn2+ with the formation of a 2:1 complex.