Sex selection of sperm in farm animals: status report and developmental prospects.

Pre-selection of spermatozoa based on the relative DNA difference between X- and Y-chromosome bearing populations by flow cytometry is an established method that has been introduced into commercial cattle production. Although several important improvements have increased the sort efficiency, the fertilising ability of sexed spermatozoa based on offspring per insemination is still behind farmers' expectations. The main stress factors, especially on mitochondria, that reduce the lifespan of spermatozoa are described, and new technical as well as biological solutions to maintain the natural sperm integrity and to increase the sorting efficiency are discussed. Among these methods are the identification of Y-chromosome bearing spermatozoa by bi-functionalised gold nanoparticles and triplex hybridisation in vivo as well as new laser-controlled deflection system that replaces the deflection of spermatozoa in the electrostatic field. Additionally, as well as a new nonsurgical transfer system of spermatozoa into the oviduct of cows has been developed and allows a significant reduction of spermatozoa per transfer. Altogether, the improvements made in the recent years will allow a broader use of sex-sorted spermatozoa even in those species that require more cells than cows and sheep.

Design, synthesis, biochemical studies, cellular characterization, and structure-based computational studies of small molecules targeting the urokinase receptor.

The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.

Targeting multiple conformations leads to small molecule inhibitors of the uPAR·uPA protein-protein interaction that block cancer cell invasion.

Interaction of the urokinase receptor (uPAR) with its binding partners such as the urokinase-type plasminogen activator (uPA) at the cell surface triggers a series of proteolytic and signaling events that promote invasion and metastasis. Here, we report the discovery of a small molecule (IPR-456) and its derivatives that inhibit the tight uPAR·uPA protein-protein interaction. IPR-456 was discovered by virtual screening against multiple conformations of uPAR sampled from explicit-solvent molecular dynamics simulations. Biochemical characterization reveal that the compound binds to uPAR with submicromolar affinity (K(d) = 310 nM) and inhibits the tight protein-protein interaction with an IC(50) of 10 µM. Free energy calculations based on explicit-solvent molecular dynamics simulations suggested the importance of a carboxylate moiety on IPR-456, which was confirmed by the activity of several derivatives including IPR-803. Immunofluorescence imaging showed that IPR-456 inhibited uPA binding to uPAR of breast MDA-MB-231 tumor cells with an IC(50) of 8 µM. The compounds blocked MDA-MB-231 cell invasion, but IPR-456 showed little effect on MDA-MB-231 migration and no effect on adhesion, suggesting that uPAR mediates these processes through its other binding partners.

Virtual screening targeting the urokinase receptor, biochemical and cell-based studies, synthesis, pharmacokinetic characterization, and effect on breast tumor metastasis.

Virtual screening targeting the urokinase receptor (uPAR) led to (±)-3-(benzo[d][1,3]dioxol-5-yl)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4-phenylbutan-1-amine 1 (IPR-1) and N-(3,5-dimethylphenyl)-1-(4-isopropylphenyl)-5-(piperidin-4-yl)-1H-pyrazole-4-carboxamide 3 (IPR-69). Synthesis of an analogue of 1, namely, 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC(50) near 30 µM. Both compounds blocked angiogenesis with IC(50) of 3 µM. Compounds 2 and 3 inhibited cell growth with IC(50) of 6 and 18 µM and induced apoptosis. Biochemical assays revealed leadlike properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 µM with a half-life of about 2 h. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.

Endothelin B receptor deficient transgenic rescue rats: a rescue phenomenon in the brain.

Homozygous endothelin B receptor deficiency leads to congenital aganglionosis of the gut in rats and mice, equivalent to human Hirschsprung disease. Homozygous endothelin B receptor deficient rats (spotting lethal rats, sl/sl) are characterized not only by this developmental disorder of the enteric nervous system, which limits their life span to 3-4 weeks, but exhibit an increased rate of apoptosis in the dentate gyrus compared to wildtype (+/+) rats. Recently, endothelin B receptor deficient transgenic rescue rats (sl/sl, tg/tg) were created to further investigate the role of the endothelin B receptor in mature animals. Linkage of the human dopamine-beta-hydroxylase promoter to the rat endothelin B receptor gene and expression of this transgenic construct results in normal development of the enteric nervous system. We investigated the expression pattern of this transgenic construct in the brain by using reverse transcriptase polymerase chain reaction. Unexpectedly, transgene mRNA expression was not restricted to the brain stem where adrenergic and noradrenergic nuclei are known to be present but, in addition, was also detectable in hippocampus and cortex. Using in situ tailing technique, cleaved caspase-3 immunohistochemistry and analysis of hematoxylin-eosin-stained serial sections, we found that all studied transgenic animals were rescued from the increased rate of apoptosis in the dentate gyrus characteristic for non-transgenic sl/sl rats. This finding supports our previous observation that the endothelin B receptor might be an important regulatory element supporting cellular survival in the hippocampus during postnatal development. The endothelin B receptor deficient transgenic rescue rats used here are rescued from developmental disorders both in the gut and in the brain.

Expression patterns of erythropoietin and its receptor in the developing midbrain.

The expression patterns of erythropoietin (EPO) and its receptor (EPOR) were investigated in the midbrain and in adjacent parts of the synencephalon and hindbrain of embryonic C57Bl mice. On embryonic (E) day 8 (E8), virtually all neuroepithelial cells expressed EPOR. After neural tube closure, subsets of these cells downregulated EPOR. In contrast, radial glial cells were EPOR-immunolabeled from E11 onwards. Simultaneously, subpopulations of early developing neurons upregulated EPO and expressed HIF-1, known to transcriptionally activate EPO. Three-dimensional reconstructions revealed subpopulations of EPO-expressing neurons: (1) in the trigeminal mesencephalic nucleus (TMN), (2) at the rostral transition of the midbrain and synencephalon, (3) in the basal plate of the midbrain, (4) in the trigeminal motor nucleus, and (5) in the trigeminal principal sensory nucleus. In the rostral midbrain and synencephalon, EPO-immunoreactive neurons were attached to EPOR-expressing radial glial cells. The identity of radial glial cells was proven by their immunoreactivity for antibodies against astrocyte-specific glutamate transporter, brain lipid-binding protein, and nestin. From E12.5 onwards EPOR was downregulated in radial glial cells. Viable neurons of the TMN continued to express EPO and upregulated EPOR. Our findings provide new evidence that components of the EPO system are present in distinct locations of the embryonic brain and, by interactions between neurons and radial glial cells as well as among clustered TMN neurons, may contribute to its morphogenesis. Whether the observed expression patterns of EPO and EPOR may reflect EPO-mediated trophic and/or antiapoptotic effects on neurons is discussed.

Hypoxia and hypoxia-inducible factor modulated gene expression in brain: involvement in neuroprotection and cell death.

Hypoxia, due to impaired cerebral blood flow, has hazardous effects on brain structure and function. Therefore, mechanisms should exist to meet the needs for hypoxic adaptation via regulation of gene expression. Signaling between the O2 sensor and the regulator(s) of transcription is only partially characterized and requires regulatory transcription factors. Among these regulatory proteins, hypoxia-inducible factor-1 (HIF-1) appears to have a key role. HIF-1 modulates gene activity in response to low O2 tensions in the developing and in the adult brain. Moderate hypoxia may elicit autoprotective mechanisms or hypoxia-induced regulators can contribute to mechanisms leading to cell death. Moreover, reactivation of embryonic gene expression may occur after injury-induced hypoxia. Thus, analyses of embryonic and pathogenic models should help to understand how hypoxia-mediated proliferative/cell death processes are involved in brain development and in the pathogenesis of acute or chronic neurodegenerative brain diseases.

To what extent are the retinal capillaries ensheathed by Müller cells? A stereological study in the tree shrew Tupaia belangeri.

The cellular ensheathment of capillaries in the 3 outer capillary layers of the central retina of the adult tree shrew Tupaia belangeri was studied quantitatively by transmission electron microscopy. Using a stereological approach, the relative surface of capillary basal lamina ensheathed by Müller cells and by nonmacroglial cells (collectively termed non-Müller cells) was estimated in 5 animals. The participation of Müller cells was distinctly different in the 3 capillary layers studied. In the outermost capillary layer 1, the mean (standard deviation) percentage surface coverage by non-Müller cell processes was 46.8 (15.3)%. Much less of the capillary basal lamina was ensheathed by non-Müller cells in capillary layers 2 and 3 (3.0 (2.1)% and 0.3 (0.3)% respectively). The observed total variation of the stereological estimates for the surface fraction of Müller cells (expressed as the between-subject coefficient of variation) was significantly higher in capillary layer 1 (28.8%) compared with capillary layers 2 (2.2%) and 3 (0.3%). In capillary layer 1, the high observed total variation was due to a high biological variation among animals for the fractions of both Müller cell and non-Müller cell ensheathment. The rare occurrence of direct contacts between the capillary basal lamina and the perikarya of either microglial cells (capillary layer 3) or amacrine cells (capillary layer 2) corresponded well to the low stereological values obtained for the relative capillary surface ensheathed by non-Müller cells in these capillary layers. Previously, extensive and frequent contacts between the basal lamina of capillaries belonging to capillary layer 1 and horizontal cells had been observed in single sections. The present study quantitatively demonstrates a marked paucity of macroglial investment of capillaries located in capillary layer 1 of Tupaia. It can be concluded that horizontal cells ensheath most of the capillary surface not invested by Müller cells.

Pattern of calretinin immunoreactivity in the main olfactory system and the vomeronasal system of the tree shrew, Tupaia belangeri.

The distribution of the calcium-binding protein calretinin was studied in peripheral and central parts of the main olfactory system (MOS) and the vomeronasal system (VNS) of adult tree shrew Tupaia belangeri. The calretinin immunoreaction was carried out with a peroxidase-coupled polyclonal antibody. In the VNS, complete labeling of all receptor cells and vomeronasal nerve fibers was observed, whereas only a subset of the somata and dendrites of receptor cells and of the olfactory nerve fibers of the MOS was immunoreactive. From the immunoreactive dendritic clubs of vomeronasal receptor cells, calretinin-labeled structures, presumably clumps of microvilli, arose that terminated within immunopositive portions of the mucus. In the main olfactory bulb, the neuropil of some of the glomeruli was immunoreactive. All periglomerular and many mitral cells were labeled. The external plexiform layer was subdivided into a faintly immunoreactive superficial half and a strongly immunoreactive deep half. Immunoreactive basal dendrites of mitral cells could be followed into either the deep half or the superficial half. In the laminated internal granular layer, a subset of immunopositive granule cells extended dendrites into the external plexiform layer. Mitral cells and granule cells with dendrites ascending to different levels of the external plexiform layer may represent functional subclasses. In the accessory olfactory bulb, all vomeronasal nerve fibers, glomeruli, and mitral/tufted cells were labeled, whereas immunoreactive periglomerular cells and internal granule cells were only scattered. In Tupaia, calretinin immunoreactivity is a more general property of the primary projecting neurons of the VNS than of the MOS and possibly indicates the involvement of calretinin in the perception of certain of the olfactory qualities.

Capillary-contacting horizontal cells in the retina of the tree shrew Tupaia belangeri belong to the mammalian type A.

Previously, ultrastructural evidence has been presented that, in the retina of adult Tupaia belangeri, the perikarya and processes of horizontal cells extensively ensheath the basal lamina of capillary cross sections located between the inner nuclear layer and the outer plexiform layer. The present study tests whether these horizontal cells can be further characterized by applying a polyclonal antibody against glial fibrillary acidic protein (GFAP). GFAP-immunoreactivity was noted in the astrocytic plexus ensheathing retinofugal axons in the nerve fiber layer. The vitreal endfeet and parts of the trunks of M*uller cells were also labelled. Moreover, a large subpopulation of vessel-contacting horizontal cells was strongly GFAP-immunoreactive. Immunoreactivity was found in the perinuclear cytoplasm and in the sturdy primary dendrites of these cells. The somata of GFAP-immunoreactive horizontal cells were unevenly distributed. These cells had three to seven primary dendrites that showed considerable overlap with the dendrites of neighbouring horizontal cells. For these reasons, GFAP-immunoreactive horizontal cells were classified as belonging to the mammalian type A. Whether the simultaneous occurrence of two glial features, viz. extensive ensheathment of retinal capillaries and immunoreactivity for a polyclonal antibody towards GFAP, supports the view that retinal horizontal cells represent a cell type intermediate between neurons and glial cells is discussed.

The patterns of cell death and of macrophages in the developing forebrain of the tree shrew Tupaia belangeri.

The patterns of cell death and of macrophages were investigated in the forebrain and eyes of the tree shrew Tupaia belangeri during five phases of optic cup formation. Seventeen embryos were studied. Three- dimensional reconstructions were made from one embryo of each phase. In phase 1 (V-shaped optic evagination) a midline band of cell death passes through the closing anterior neuroporus. From phases 2 (optic vesicle) to 5 (far-advanced invagination) the midline band of cell death extends in the dorsal wall of the forebrain to its rostral pole and, further, into its ventral wall. At the approximate future position of the optic chiasm this ventral pycnotic area, predicted but so far unidentified by others, is connected to a previously described second band of cell death passing through the optic anlagen. Recently, evidence has been presented that chicken embryos develop holoprosencephaly and cyclopia when ventral forebrain structures are lost secondary to experimentally induced apoptosis. Our findings in Tupaia suggest that, in cases of spontaneous malformations of this kind, such an atypical pycnotic area in the ventral telencephalon might result from the defective regulation of cell death processes during optic cup formation. In the forebrain and eyes of Tupaia, the occurrence of bands of cell death precedes the appearance of the earliest intraepithelial macrophages. From phase 3 (onset of invagination) onwards almost all of them are concentrated along the band of cell death.

Horizontal cells invest retinal capillaries in the tree shrew Tupaia belangeri.

Retinal capillaries were studied by transmission electron microscopy, immunohistochemistry and lectin histochemistry in the adult tree shrew Tupaia betangeri. In capillaries from all four vascular layers, adjacent endothelial cells were connected by tight junctions. Up to three layers of pericyte processes were embedded in the subendothelial basal lamina. However, pericytes frequently contacted the endothelial cells. In the innermost vascular layer (capillary layer 4), S-100-immunopositive astrocytes and the vitreal processes of S-100-immunopositive Muller cells entirely ensheathed the capillary basal lamina. However, capillaries revealing an incomplete macroglial investment were observed in the outer vascular layers, predominantly in capillary layers 1 and 2. In sections of capillaries located between the inner nuclear layer and the outer plexiform layer (capillary layer 1), these "gaps" were filled with the perikarya and electron-lucent processes of horizontal cells that ensheathed up to approximately nine tenths of the capillary circumference. Horizontal cells were identified by ultrastructural criteria. They were distinct from microglial cells by not being reactive for Griffonia simplicifolia isolectin-B4. In Tutpaia, vessel-contacting horizontal cells reside in a position reported to be occupied by the processes of Müller cells in other mammals. Current concepts of retinal function and pathology, which are based on the assumption that retinal vessels are strictly isolated from retinal neurons, at least in Tupaia, might deserve reconsideration.

The earliest invasion of macrophages into the developing brain and eye of the tree shrew Tupaia belangeri.

The earliest occurrence of macrophages was investigated in the brain and optic anlagen of the tree shrew Tupaia belangeri. Nineteen serially sectioned embryos, belonging to five phases of programmed neuroepithelial cell death previously found during optic cup formation, were used. Macrophages were identified by structural criteria and by labelling with the lectin Griffonia simplicifolia I-B(4). Macrophages, most probably derived from the yolk sac, are present in the perineural vessels of the phase 1 embryo (V-shaped optic evagination). Within this compartment, their number increases up to phase 4 (advanced invagination) and drops during phase 5. This first wave of macrophages is followed by a second one occurring within the perineural mesenchyme and within the neuroepithelium of the brain and eyes from phase 3 onwards. In the phase 4 embryos, a considerable rise in the number of intraventricular macrophages is noted. During phase 5 (far advanced invagination), marked vascularization of the brain starts, and a peak of macrophages is noted in the neuroepithelium and in the ventricular lumen of the brain. This spatiotemporal pattern suggests that, in Tupaia, the earliest macrophages are simultaneously shifted from perineural vessels into the neuroepithelial walls of the developing brain and, at earlier stages than previously described in other vertebrate species, of the eye anlagen.

Pattern of cell death during optic cup formation in the tree shrew Tupaia belangeri.

Developmental cell death during optic cup formation was investigated in the tree shrew Tupaia belangeri. Twenty-six embryos from days 12 to 16 of prenatal ontogenesis were studied by light microscopy. Prior to the optic vesicle stage, a dorsal area of cell death surrounded the lumen of the V-shaped optic evagination (phase 1). A ventral band of dead cells, found in the optic vesicle (phase 2), preceded a dorsal focus of cell death (phase 3) previously described as a characteristic avian feature. During further invagination (phase 4), a peak of cell death was represented by a ventrodorsal band extending from the diencephalon over the complete optic anlage. The main areas of cell death found in phases 2 to 4 were, topographically, segments of this band. Also, the distinct areas of cell death reported in the literature for the vertebrate species studied so far fit well into this ventrodorsal band found in Tupaia. Thus, most probably, a common spatio-temporal sequence of cell death exists in all of them. In Tupaia, dead cells concentrated at the diencephalic insertion of the optic stalk, the suboptic necrotic center (SONC) reported by several authors, were part of the early ventral band of cell death originating from the median floor of the prosencephalon (phase 2). During optic cup formation, the SONC was part of the ventrodorsal band and, thus, was not secondarily formed by the subdivision of a pre-existing distal ventral area of cell death as reported for several other vertebrates.

Disk formation in retinal cones of Tupaia belangeri (Scandentia).

Existing hypotheses on the mode of disk formation in the photoreceptor cells of mammals appear to be incompatible: (1) plasma membranes of adjacent evaginations form a disk which, subsequently, is internalized by a disk rim; (2) pinocytotic vesicles are pinched off from the plasma membrane and fuse into a larger vesicle, which flattens and forms a disk. We have studied the development of the cone outer segment and the disk formation in Tupaia belangeri by transmission electron microscopy. During the first two postnatal weeks, the distal part of the single cilium, which is inserted apically on the inner segment, becomes balloon-shaped. Apical to the axoneme, it contains tubular and vesicular material, which, most probably, has been detached from the axonemal microtubules. These tubules and vesicles do not contribute to disks. The balloon-shaped expansion, later retained as the ciliary backbone, establishes the contact with the pigment epithelium. Formation of disks, from the 12-day-old Tupaia onwards, occurs between adjacent evaginations at the outer segment base. The initial disk rims are "hooked" to the ciliary axonemal microtubules. The axonemal microtubules are involved in the initiation and in the alignment of the disks. Disk rim formation and, thus, internalization of disks proceeds from the base to the apex of the outer segment, that is, from the younger to the older disks. In the adult Tupaia, an uneven progression of disk rim formation on both sides of the axoneme is found among consecutive disks. The seemingly incompatible hypotheses on the mode of disk formation reflect a heterochrony of the internalization of membranes and of the disk formation among different mammals and, possibly, between cones and rods.

Ciliogenesis in photoreceptor cells of the tree shrew retina.

Transmission electron microscopy of the retinal cones from several prenatal, young postnatal and adult tree shrews (Tupaia belangeri) reveals that the centrioles, from which the ciliary precursors of the outer segments grow out, are not transported into a pre-existing inner segment, but are positioned under the apical plasma membrane of cone precursor cells all through the inner segment formation. Ciliogenesis starts before or on embryonic day 20 and thus precedes initial formation of the inner segment by 20 days, which is half the gestation period. Thus, the maturation of the outer segment covers a considerably longer period than has been previously described. Published observations from other mammals can be interpreted as conforming with the situation in Tupaia. In other vertebrates, compared to mammals, marked heterochronies do occur. In Tupaia, the centrioles and the cilium are located close to the central longitudinal axis of the photoreceptor precursor cell from the 20-day-old embryo to the 5-day-old juvenile. In this position the microtubule apparatus originating from the centrioles should be most effective in transporting the mitochondria into the inner segment. In the 12-day-old tree shrew, when transport of the mitochondria into the inner segment has been completed, centrioles and cilium have shifted into an eccentric position and the light-collecting megamitochondria have approached the disks of the outer segment. This eccentric position is maintained in all later developmental stages. In certain of the retinal areas of the adult Tupaia, the connecting cilia of neighbouring cones are always positioned on the same side of the inner segments.

"Lens mitochondria" in the retinal cones of the tree-shrew Tupaia belangeri.

In all mammals, the mitochondria of the cones of the retina are concentrated in the inner segment. Uniquely in tree-shrews (Tupaia, Scandentia, Mammalia), a "megamitochondrion" exhibiting highly specialized systems of densely packed cristae and a very electron dense matrix, is located apically in the inner segment. The ellipsoid is a solid body containing several megamitochondria and, towards its base, a large number of smaller mitochondria. The refractive index of isolated, but not oriented, inner segments of Tupaia belangeri is higher (XA = 1.405) than in any other mammal studied so far. The consistent geometrical pattern of the multilamellar crista-matrix systems, oriented longitudinally towards the outer segment, suggests an additional optical function of the megamitochondria.

The role of microtubules and microtubule-organising centres during the migration of mitochondria.

The translocation of mitochondria towards the primitive inner segment of the cones in the tree shrew Tupaia belangeri was investigated by transmission electron microscopy. Throughout ontogeny the migrating mitochondria were codistributed with cytoplasmic microtubules which were preserved after the application of conventional preparation techniques for transmission electron microscopy. Both the basal body of the connecting cilium and the second centriole located in the vicinity of the basal body were demonstrated to act as microtubule-organising centres (MTOCs) from which axonemal and cytoplasmic microtubules originated. The megamitochondria in the inner segment of the retinal cones of Tupaia are unique among mammals with respect to their extraordinary size and to their ordered distribution characterised by longitudinal and radial size-gradients within developing and mature cone inner segments. Thus the consistent finding of microtubules and MTOCs in the structurally polarised cones represents an extreme example of the capacity of cells to regulate the transport and distribution of organelles.

Morphogenesis of megamitochondria in the retinal cone inner segments of Tupaia belangeri (Scandentia).

The morphogenesis of the megamitochondria in the retinal cones of prenatal, young postnatal and adult tree shrews (Tupaia belangeri) was studied by transmission electron microscopy and three-dimensional reconstruction techniques. The initial assembly of the supranuclear cone mitochondria and their subsequent migration towards the developing inner segment conform to the morphogenetic pattern known from other mammals. Within the first postnatal week, however, a marked increase in both the number of the cristae and the matrix density occurs in the inner segment mitochondria of Tupaia. These mitochondria then grow, initially exhibiting a basal-to-apical size-gradient. In the 17-day-old Tupaia, this gradient is superseded by a radial size-gradient that, in addition to the single apical megamitochondrion, is characteristically found in the adult Tupaia. The number of megamitochondria remains almost constant from day 12 of postnatal ontogenesis to the adult stage. Each megamitochondrion consists of an apically located body from which several long processes project towards the base of the inner segment. In the older stages, the number of small mitochondria that most probably have budded off from the megamitochondrial processes clearly increases. We consider that megamitochondria in the cone inner segments of Tupaia arise by the growth of a single mitochondrion and not by the fusion of smaller mitochondria.