Actin and actin-binding proteins in bovine spermatozoa: potential role in membrane remodeling and intracellular signaling during epididymal maturation and the acrosome reaction.

The actin cytoskeleton influences a wide range of functions in nonmuscle somatic cells, including shape, movement, and interactions with extracellular matrices. The role of actin in mammalian male germ cells, however, particularly during post-testicular development, is not well understood. In this paper, we examine 1) the distribution of 3 actin-regulatory proteins (thymosin beta10, destrin, and a testis-specific actin capping protein) involved in controlling the balance between actin monomers (G-actin) and actin filaments (F-actin), and 2) the distribution and polymerization status of actin in bull spermatozoa during epididymal maturation and following acrosomal exocytosis. Results show that in fixed, permeabilized testicular spermatozoa all 3 regulatory proteins (as determined by binding of specific antibodies) are localized primarily to the acrosomal domain but during epididymal maturation they become confined to the equatorial segment. Following ejaculation, however, they extend back into the acrosomal region. In spermatozoa induced to undergo an acrosome reaction with the calcium ionophore, A23187, further rearrangement occurs with destrin, thymosin beta10, and TS-ACP appearing in the postacrosomal domain. Actin is also found over the acrosome of testicular spermatozoa with both G- and F-actin present, although the 2 forms show slightly different patterns of distribution. Subsequently, actin in the sperm head is largely confined to the equatorial segment until F-actin appears in the postacrosomal domain of acrosome-reacted spermatozoa. This redistribution of actin and actin-regulatory proteins, coupled with changing levels of actin polymerization, suggest a continuing role for actin in both post-testicular sperm maturation and acrosomal exocytosis.

Cellular distribution and molecular heterogeneity of MAC393 antigen (clusterin, beta-chain) on the surface membrane of bull spermatozoa.

The distribution and size of a surface membrane antigen identified by a monoclonal antibody (MAC9393) have been examined in testicular and epididymal bovine sperm preparations. Western blots indicated a substantial decrease in molecular mass of the antigen during epididymal maturation from approximately 87 kDa in the testis to approximately 35 kDa in the cauda epididymidis. This was accompanied by a change in its cellular localization from the neck and whole head to the acrosomal region. N-terminal microsequencing identified MAC393 antigen as the beta-chain of clusterin. A polyclonal antiserum to the alpha-chain of clusterin recognized both testicular and epididymal forms and revealed that the heterodimer was present on the sperm tail as well as the acrosome. These findings are explained by the co-existence of dimeric and monomeric pools of clusterin on spermatozoa. The polyclonal antiserum recognizes both testicular and epididymal forms of the heterodimer and although the monoclonal antibody binds to the testicular heterodimer, it only recognizes the beta-chain monomer of epididymal clusterin. These findings support previous observations made on human spermatozoa that two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the surface membrane and in seminal plasma.

Expression of a testis-specific putative actin-capping protein associated with the developing acrosome during rat spermiogenesis.

Actin-capping proteins are ubiquitous components of mammalian cells. They are known to regulate the polymerization state of actin and hence indirectly control the activity of the cytoskeleton and cell shape. As part of our investigation into the molecular mechanisms that direct differentiation of a round spermatid into an elongating spermatozoa, we report on a testis-specific 1.7-kb transcript from rat testis with sequence similarities to the alpha subunit of actin-capping proteins (ACPs) from somatic cells. The transcript contains a putative cAMP-responsive motif (CREM) upstream of the initiation codon in the DNA sequence and is expressed postmeiotically, first appearing between 20 and 30 days of postnatal development. The primary amino acid sequence is 90% identical to that of a previously identified testis-specific mouse protein, gsg3, both showing approximately 40% homology to the alpha subunit of somatic ACPs. An affinity-purified polyclonal antibody to a synthetic peptide derived from the rat transcript identified a 32-kDa protein on Western blots of testicular extracts. Indirect immunofluorescent localization of the protein on frozen sections of adult rat testis showed that it is intracellular and accumulates asymmetrically in the cytoplasm of round spermatids coincident with the position of the developing acrosome. This spatial expression parallels the distribution of F-actin during sperm differentiation, supporting the hypothesis that testis-specific ACPs have an important role in determining the final shape of mature sperm heads. A disturbance in the expression of these ACPs may underlie many of the abnormalities in sperm morphology observed in infertile semen.

A search for sex-specific antigens on bovine spermatozoa using immunological and biochemical techniques to compare the protein profiles of X and Y chromosome-bearing sperm populations separated by fluorescence-activated cell sorting.

Currently, the only successful method for separating X and Y chromosome-bearing spermatozoa is fluorescence-activated cell sorting. Although effective, this technique is of limited usefulness to the animal breeding industry as it cannot produce the large volumes of sexed spermatozoa needed for artificial insemination. An attractive alternative would be to identify an immunological marker confined to one sperm type and, therefore, significant scientific effort has been expended in examining antibodies that appear to recognize approximately 50% of spermatozoa in an ejaculate. However, no sex-specific antigens have yet been identified from spermatozoa. Using the opportunity afforded by the development of sperm separation by fluorescence-activated cell sorting, we have made a thorough search for differences between X and Y chromosome-bearing bull spermatozoa using both biochemical and immunological methods. Techniques for radiolabelling surface membrane proteins, in conjunction with SDS-PAGE, failed to show any differences between populations. Similarly, a wide range of monoclonal antibodies raised to ejaculated, cauda epididymidal and testicular spermatozoa failed to distinguish between the X and Y chromosome-bearing spermatozoa. Only after analysis by high resolution two-dimensional SDS-PAGE was an indication obtained that X-specific proteins occur. However, these proteins are not associated with the surface membrane and further work is necessary to confirm their association with the X chromosome and to characterize them more fully. Our inability to detect sex-specific differences in sperm surface antigenicity suggests that further work on this immunological approach to semen sexing is unlikely to be profitable.

Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death.

Compartmentalization of surface membrane antigens into discrete regions or domains is a characteristic feature of differentiated cells. In mammalian spermatozoa at least 5 surface domains are known, implying the presence of barriers or boundaries within the plasma membrane. Using the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusibility of fluorescent lipid analogues 1,1'-dihexadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiIC[16]) and 5-(N-octa-decanoyl) aminofluorescein (ODAF), we have investigated lipid topology and dynamics in the plasma membrane of ejaculated bull spermatozoa. Contrary to reports in the literature, we have found that DiIC(16) stains only dead or damaged spermatozoa whereas ODAF intercalates into the plasma membrane of both live and dead cells, each type showing a distinctive staining pattern. FRAP analysis with ODAF revealed that diffusion coefficients on live spermatozoa are significantly faster on the acrosome and postacrosome (29.3x10(-9) cm2/second) than on the midpiece and principal piece (11.8x10(-9) cm2/second). Recovery (R) is >90% in all domains. ODAF diffusion also shows regionalized temperature-sensitivity with a 4-fold increase over the sperm head and a 1.8-fold increase on the tail between 20 degrees C and 37 degrees C. Remarkably, dead or permeabilized spermatozoa rapidly develop a large immobile phase (R<25%) over the whole plasma membrane. This rigidification is temperature insensitive and irreversible suggesting major changes in the physical state of membrane lipids. It is concluded that lipid diffusion in the plasma membrane of live bull spermatozoa is rapid and varies significantly between surface domains. Following permeabilization or cell death, however, a large immobile phase develops indicating substantial changes in membrane lipid disposition.

Long-term culture of fully differentiated adult insect neurons.

An identified group of cells, dorsal unpaired median (DUM) neurons, isolated from the central nervous system of adult cockroaches can be grown in vitro for extended periods. These cells often develop morphological characteristics that differ from their in situ appearance but physiological experiments demonstrate that they retain their distinctive in vivo membrane properties. A method for culturing insect CNS neurons, which includes the use of a haemolymph-derived growth-enhancing factor is described, and this technique is compared with other attempts to develop an efficient system for producing in vitro preparations of well-defined, identifiable neurons that would provide a model system for aspects of neuron developmental growth, repair and function.

A blood-derived attachment factor enhances the in vitro growth of two glial cell types from adult cockroach.

Reactive glial cells from chemically-lesioned areas of the central nervous system (CNS) of adult cockroaches (Periplaneta americana) have been grown in vitro on a substrate of fibronectin. This paper reports the enhancement of growth that is achieved when blood cells, serum, or medium conditioned by a 2-h incubation with blood cells are used as an alternative substrate. Glial cells rapidly grew out from connective explants to form extensive radial mats of cells linking up with those from adjacent explants on each of the blood-derived substrates. In addition to supporting the growth of reactive glial cells, characterised by their long, thin, branching morphology, these substrates also revealed the presence of a second type of glial cell, not previously found on fibronectin. Such cells, derived from ganglionic explants, behaved in a very different way to the reactive glia, initially spreading out to form a flattened sheet of phase-bright cells, before migrating away over the culture surface. The growth-enhancing effects of this blood-derived factor may play a role in the events following damage to the insect CNS, where it is known that the entry of blood cells into the lesion site is an important precursor to the rapid and structured repair seen in this system.

Cell specific DNA-labelling in the repairing blood-brain barrier of the insect Periplaneta americana.

This study uses a recently developed technique for preserving the ultrastructure of cells in the insect CNS during immunohistochemical processing for 5-bromo-2-deoxyuridine incorporation into newly synthesised DNA. The results allow us to identify the proliferating cell classes in the regenerating blood-brain barrier. High resistance barrier cells do not label with the antibody but sheath cells clearly do. Intermediate cell types appearing during repair are identified. It is hypothesised that these cells generate matrix molecules for neural lamella repair and may represent transitional forms as invasive blood cells transdifferentiate into functional sheath cells.

Long-term growth in vitro of isolated, fully differentiated neurones from the central nervous system of an adult insect.

A method is described for the isolation and growth in vitro of fully differentiated neurones from the thoracic ganglia of adult cockroaches. The presence of insect blood in the culture system is shown to promote growth. The morphology of the growing neurones and the plasticity of the branching processes are described and growth rates are measured. Using a fluorescent Ca2+ indicator dye, changes of intracellular calcium levels in the growing neurones in response to K+ depolarization have been measured. The results, indicating the presence of voltage-dependent Ca2+ channels on neuronal processes in vitro, show that neurones can be maintained in a functional state for several weeks by this technique. Such preparations could prove useful for studying a variety of physiological and pharmacological properties of neurones, including the mechanisms controlling growth, synapse formation and neuronal interactions with other cell types.

Cell proliferation in the repairing adult insect central nervous system: incorporation of the thymidine analogue 5-bromo-2-deoxyuridine in vivo.

Uptake of the thymidine analogue 5-bromo-2-deoxyuridine into non-neuronal cells of the insect central nervous system has been examined following a controlled lesioning of the glial elements. The pattern of BUdR labelling along the penultimate abdominal connective was examined over a period of 17 days. Cell proliferation occurred in and immediately around the site of damage in both perineurial and subperineurial glial cells but at different times post-lesion for the two regions. Proliferation in the perineurial zone was maximal at 6-8 days post-lesion but continued for at least 17 days. Subperineurial proliferation was less dramatic and peaked between days 8-11 post-lesion. In both areas division appears to be confined to the reactive glial cells. These results are discussed in the context of past research on this system, particularly with regard to the restoration of the blood-brain barrier.

Adult insect glial culture: Activation, substrate effects and proliferation.

Glial cells from an adult insect, Periplaneta americana, have been grown in neurone-free cultures. No growth occurred from freshly-excised fragments of abdominal nervous connectives. Vigorous growth was obtained, however, from explants of connectives induced to proliferate by prior exposure to a toxin, ethidium bromide, applied selectively to glial cells in vivo. Glial growth in vitro is dependent upon the initiation of early stages of repair in vivo: this supports the idea that haemocytes which invade the lesion zone immediately after damage are involved in directing proliferation of perineurial and sub-perineurial glia. In contrast, both glial and neuronal cells grew in vitro from explanted abdominal ganglia without prior glial lesioning, indicating that different factors may determine cellular regeneration in this domain. The morphology of the proliferating cells was influenced by the substrate; extensive glial migration was restricted to areas of close contact between cell and substrate surface.

Cell recruitment during glial repair: the role of exogenous cells.

Selective disruption of the neuroglia in penultimate abdominal connectives of the cockroach nerve is followed by a rapid accumulation of cells in the perineurial layer of the lesion. Subsequently, there is an abrupt, secondary, rise in cell numbers in the undamaged perineurial tissues, anterior to the lesion and adjacent to the 4th abdominal ganglia. By 7 days the increased cell numbers are again effectively confined to the original lesion zone. The initial rise in cell numbers is postulated to result from an invasion by blood-borne haemocytes and the subsequent increase, in undamaged perineurial tissues, from the mobilization of endogenous reactive cells. Recruitment of the endogenous cells is inhibited if the haemocytes are excluded from the lesion. There is a slower mobilization of sub-perineurial cells, which, again, is inhibited following exclusion of haemocytes from the lesion zone. It is postulated that the recruitment of the endogenous reactive cells is initiated by the invading haemocytes which transform to granule-containing cells and release diffusible morphogenic and/or mitogenic factors.

Mechanisms of glial regeneration in an insect central nervous system.

As in other repairing systems, glial regeneration in insect central nervous connectives, following selective chemical lesioning, involves both exogenous and endogenous elements. Our current evidence, including that obtained with monoclonal antibodies, indicates that the reactive, granule-containing cells are derived from a sub-population of circulating haemocytes which, within 24 h, invade, and are restricted to, the lesion zone. The granule-containing cells are involved in the initial repair of the perineurial region. They also contribute to the first stage in the restoration of the blood-brain barrier and are responsible for recruiting reactive endogenous glia, apparently from the vicinity of the anterior abdominal ganglion. The granule-containing cells transform into or are replaced by functional glia between 3 and 5 days after selective glial disruption, coincident with the appearance in the lesion zone of dividing reactive cells.

Blood cells contribute to glial repair in an insect.

Using antibodies specific for haemocytes, we have shown that these blood cells penetrate the abdominal nervous connectives of the cockroach following selective disruption of the glia using the DNA-intercalating drug, ethidium bromide, as a glial toxin. Within 4 days post-lesion, the labelled cells formed a mosaic beneath the neural lamella and penetrated deeply among the disrupted subperineurial glia. These observations confirm that exogenous cells are involved in glial repair and support a previous hypothesis that they play critical roles in both structural repair and the recruitment of endogenous reactive cells.

Glial repair in an insect.

The repair of cockroach central nervous connectives, following selective glial disruption, involves an initial invasion of the lesion by a novel cell class. The available evidence, including that obtained using monoclonal antibodies, shows that these cells arise from circulating haemocytes. These invasive exogenous cells are restricted to the lesion zone. They are not only involved in initial repair of the peripheral glial elements, but may also be responsible for initiating recruitment and division of endogenous reactive cells. There is a clear anterior polarity in this recruitment, with significantly higher numbers of cells appearing anterior to, and then within, the lesion area. Characteristically, recognizable exogenous cells decline in number after 3 days, although there is no overall reduction in cell numbers within the lesion at this stage, nor has significant cell division begun. This suggests that the haemocyte-derived cells transform into, or are replaced by, functional perineurial glia, between 3 and 5 days, coincident with the restoration of the blood-brain barrier and the onset of endogenous cell division. Glial repair in the insect CNS can thus be divided into three phases which show striking similarities to the repair sequence in vertebrate brain. These include: an initial invasion of the lesion by exogenous cells, subsequent glial proliferation and then longer term fluxes in cell numbers and distribution.

Organ culture of adult cockroach CNS: ultrastructural and physiological characteristics.

The abdominal nerve cord of adult Periplaneta americana has been maintained in culture for up to 5 weeks. The ultrastructural appearance of in vitro cords resembles that of in vivo cords although some degeneration of glia may occur during the first 2 weeks in culture. The glial cells most affected are those which make up the perineurium. The blood-brain barrier, formed by the perineurium in vivo, breaks down in vitro. Despite this, normal action potentials were recorded from giant axons in 1 month old cultured connectives.

Glial toxin effect on protein synthesis in an insect connective.

Analysis of electron autoradiographs from the nerve cord of the insect, Periplaneta americana (L.) shows a significant incorporation of 3H-labelled protein in the axons. The axonal activity is greatly reduced after treatment of the cord with the glial toxin ethidium bromide. This is interpreted as substantiating the possibility that adaxonal glia can transfer proteins to the axons.

Relationship between lipofuscin granules and polyunsaturated fatty acids in the housefly, Musca domestica.

The fractional volume occupied by lipofuscin granules in epithelial cells of the midgut or oenocytes of abdominal fat body of 3-day-old and 13-day-old male houseflies was determined in two groups of flies by electron microscopic morphometry. One group had developed from larvae reared on diets containing no added polyunsaturated fatty acids and the second from larvae reared on diets containing added linoleic acid. No polyunsaturated fatty acids could be detected in the lipids of the first group of flies using a method which would have detected their presence in amounts greater than 0.1% of the total esterified fatty acids. The second group contained at least two hundred times more than this minimal level. The volume of lipofuscin granules increased significantly (p less than 0.01) (about threefold for the fat body and twofold in midgut cells) between 3 days and 13 days of age but no statistically significant difference was seen between the two groups of flies at the same age. The results show that if lipofuscin formation depends on the oxidation of polyunsaturated fatty acids in the housefly, then extremely small amounts of the acids are involved which lie below the detection limit of the methods employed. The age-associated small increase of extractable fluorescence seen previously in the linoleic acid group of flies is not associated with an increase in the lipofuscin granules.

The concentration of choline into specific regions of the thoracic ganglion in B-methylcholine fed houseflies.

When larvae of the housefly, Musca domestica, are reared on a diet in which most of the choline has been replaced by a choline analogue, B-methylcholine, the small amount of choline available to the insect is largely taken up into the nervous system where it is incorporated into phosphatidylcholine and acetylcholine. Using autoradiographic techniques it is shown that, in the thoracic ganglion, the phosphatidylcholine component of this preferentially accumulated choline is not evenly distributed throughout the tissue but that a well-defined pattern of patches of silver grains is found over certain areas of the neuropile. Underlying this pattern are axons and glial processes structurally indistinguishable from the axons and glia of adjacent areas which show a much lighter autoradiographic reaction. It is concluded that the axons which produce this high density of silver graining are cholinergic and have a high affinity for choline.

The permeability to ions of the neural lamella and the extracellular spaces in the C.N.S. of Anodonta cygnea.

Rapid axonal depolarization follows the elevation of [K+]o in the Ringer bathing the surfaces of the connectives of both intact and desheathed cerebrovisceral connectives of Anodonta cygnea. Potassium movements between the blood or medium bathing the surface of the connectives and the axonal membranes can be accounted for in terms of a first-order diffusion process. No visible structural barriers to prevent free movements of materials through the neural lamella and the extracellular spaces can be detected in electron micrographs of the cerebro-visceral connectives. In desheathed preparations fast action potentials are conducted in sodium-free (dextran) Ringer. Mechanisms of axona .l function and ionic regulation in this and other invertebrate central nervous tissues are discussed in the light of these observations.