Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Control of Pig Reproduction IX
    Rodriguez-Martinez, H. ; Soede, N.M. ; Flowers, W.L. - \ 2013
    Leicestershire, United Kingdom : Context Products Ltd (Society of Reproduction and Fertility volume 68) - ISBN 9781899043484 - 345
    varkens - geslachtelijke voortplanting - gameten - embryo's - kunstmatige inseminatie - embryotransplantatie - zwangerschap - partus - pasgeborenen - biggen - overleving - biotechnologie - metabolomica - eiwitexpressieanalyse - kunstmatige selectie - pigs - sexual reproduction - gametes - embryos - artificial insemination - embryo transfer - pregnancy - parturition - neonates - piglets - survival - biotechnology - metabolomics - proteomics - artificial selection
    The use of 2n gametes for introgression breeding in Oriental x Asiatic lilies
    Barba Gonzalez, R. - \ 2005
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Jaap van Tuyl. - Wageningen : S.n. - ISBN 9789085042686 - 111
    lilium - plantenveredeling - introgressie - gameten - hybriden - ploïdie - genetische variatie - chromosoomtransmissie - lilium - plant breeding - introgression - gametes - hybrids - ploidy - genetic variation - chromosome transmission
    Besides being an important horticultural crop, lily ( Lilium ) also serves as an interesting model plant for molecularcytogeneticresearch for several reasons. a) The crop includes cultivars of different taxonomic species each of which possess valuable horticultural traits that need to be combined in the new cultivars. b) The genomes of different species are so well differentiated genetically that the individual chromosomes can be clearly identified in the F 1 hybrids as well as in the progenies through DNA in situ hybridization techniques. c) The chromosomes are so large that the number and position of homoeologous recombinant segments can be clearly detected. d) Through careful selection, or through nitrous oxide treatment, 2n gametes can be obtained in sterileinterspecifichybrids. Taking advantage of thesefavourableattributes of lily, a molecularcytogeneticinvestigation was conducted on the possibilities of introgression of characters between Oriental and Asiatic lilies through the use of 2n gametes.

    For this purpose, more than 700 genotypes of F 1interspecifichybrid between Oriental × Asiatic lilies were produced by using special techniques of ovule and/or embryo culture techniques. All the diploidinterspecificOA hybrids (2n=2x=24) were highly sterile but 12 genotypes were found to produce 2n gametes in notable frequencies. Six of these were analyzed through GISH forintergenomicrecombination as well as meiotic nuclear restitution. In all cases there was evidence for the occurrence of first division restitution (FDR) gametes through three different types of cytological abnormalities, viz., post metaphase I division (PMI); post metaphase II (PMII) and asymmetric cytokinesis (Chapter 2). The 12 genotypes of OA hybrid that produced 2n gametes were used as parents, both as male and female, in crosses with both parental species, i.e., AA and OO, as well as the allotetraploid4x-OA(derived from doubling the chromosome number of OA hybrids withoryzalin). From these crosses, 246 triploid and 14 tetraploid progenies were obtained. The chromosome constitution of some of theallotriploidBC1 progenies wasanalysedthrough GISH. This confirmed the presence of both O and A genome chromosomes with some of them possessing recombinant segments. Theserecombinationshad obviously occurred during the development of 2n gametes in the F 1 hybrid (Chapter 3). A selected population from the BC 1 progenies (38 plants) was analyzed by GISH and FISH techniques in order to identify chromosomes as well as to determine Intergenomic recombination. These analyses indicated that most of the progeny plants had originated through FDR. However, a small proportion of the progeny originated through IMR. Three kind of plants where identified when considering the presence of the number of homoeologous chromosomes and the recombinant segments: a) plants in which both reciprocal products from a crossover were present ( O / A , A / O , where O represents the centromere of the O genome and A, the recombinant segment of Asiatic chromosome and vice versa); (b) plants in which one normal chromosome of the O genome and a recombinant chromosome where present (O, A / O ) and (c) plants in which one normal chromosome of the A genome and a recombinant chromosome where present (A, O / A ) (Chapter 4). Furthermore, with the aim of utilizing allotriploids (2n=3x=36) for introgression breeding two kinds of crosses were made, a) diploid Asiatic cultivars were used as female parent and allotriploids AOA hybrids as male parents and vice versa (2x - 3x and 3x - 2x); b) allotriploids were crossed with allotetraploids (4x-OA) and other 2n gametes producers. In all these crosses two kinds of allotriploids were used, those that had originated through crosses of 4x-OA with diploid Asiatic cultivars and those that had originated from crosses using 2n gametes of OA hybrids with diploid Asiatic cultivars. Flowcytometry analyses were used for ploidy determination and these results were confirmed by chromosome counting in some cases showing concurrence. In the case of the 2x - 3x and 3x - 2x crosses, diploid and near-diploid progeny was obtained. In the 3x - 4x crosses near-tetraploid, tetraploid, near-pentaploid and pentaploid progenies were obtained. These results indicate that the allotriploids produced aneuploid as well as euploid (x, 2x,3x) pollen grains. Through GISH analysis the identification of the parental as well as recombinant chromosomes was possible. Those allotriploids which originated from 2n gametes transmitted their recombinant chromosomes to the progeny achieving introgression of the recombinant segments.(Chapter 5).

    Finally, through N2O treatments the formation of 2n gametes was induced in some of the genotypes of OA hybrids. Out of eight genotypes that were treated, six were totally sterile and two were known to produce 2n gametes in low frequencies. In the case of the totally sterile genotypes, three of them were able to produce progeny and in one of the known 2n gamete producers the production of 2n pollen was considerably increased from 0-30% to 55-80%. GISH analysis in the progeny revealedintergenomicrecombination between the parental genomes, indicating that FDR and IMR restitution mechanisms were responsible for the 2n pollen formation.(Chapter 6).

    These results show the advantages of the 2n gametes to generate genetic variation and the possibility to use them to produce progeny. It also shows the possibility of using triploid hybrids for further breeding and that introgression of chromosome segments can be achieved through chromosome assortment and recombination among the parental genomes. Furthermore, ploidy levels of the progeny can be manipulated depending upon the ploidy level of the parents. The induction of 2n gametes by N2O treatments in sterileinterspecificlily hybrids provides many possibilities to generate completely new hybrids. This investigation shows that by using sexual polyploidization and by monitoring the progenies trough FISH and GISH techniques, more accurate insights into the process of introgression can be obtained.
    Herkomst en differentiatie van sex-cellen bij gewervelde dieren, speciaal bij vissen.
    Timmermans, L.P.M. - \ 1996
    Wageningen : Landbouwuniversiteit Wageningen - 35
    gewervelde dieren - vissen - gameten - celdifferentiatie - geslachtsdifferentiatie - vertebrates - fishes - gametes - cell differentiation - sex differentiation
    Germ cell development in larval and juvenile carp (Cyprinus carpio L.)
    Winkoop, A. van - \ 1995
    Agricultural University. Promotor(en): L.P.M. Timmermans. - S.l. : Van Winkoop - ISBN 9789054854067 - 168
    kiemcellen - gameten - cyprinodontidae - karper - germ cells - gametes - cyprinodontidae - carp

    This thesis describes the development of larval and juvenile gonads of a teleost fish, the common carp, with special attention to the differentiation of the primordial germ cells. The early gonadal development has received relatively little attention, hitherto, as the research on fish reproduction has been focussed mainly on germ cell maturation in adult fish species. The early development of gonads and germ cells, however, is highly important, as in that period a.o. sex determination occurs.

    In the early larval period the gonadal tissue is gradually formed around the primordial germ cells (PGCs) which are already located at the sites of the future gonadal primordia. The PGCs in the developing larval gonads increase in size between week 2 and week 4 after fertilization. Concomitantly, ultrastructural changes occur in the cytoplasm and nuclei of these cells, which is supposed to be a preparation for the rapid proliferation of PGCs after week 6.

    An important part of the investigations was devoted to the production of monoclonal antibodies (MAbs). In general, MAbs may recognise differentiation stages accurately. This enables the investigation of the factors which may influence such differentiation processes. The MAbs were raised against primary and early secondary spermatogonia. The procedures are presented how germ cells were isolated from testis tissue and how the percentage of these cells (diameter>10 μm) could be enriched with a percoll gradient from an initial 4% in the early spermatogenic testis to more than 70%.

    One of the produced MAbs recognised antigens, appearing first in the cytoplasm and subsequently on the surface membrane of the enlarging PGCs in the larval gonads between week 2 and week 4. This supports the hypothesis that the enlarging PGCs are differentiating cells, possibly in preparation for the fast proliferation of PGCs after week 6. Four of the produced MAbs reacted with PGCs from the onset of PGC proliferation (after week 6) up to and including early (primary) spermatogonia in the adult testis (or oogonia in ovaries). This indicates that specific differentiation antigens are present on these cells. These MAbs may be used for the recognition of primary spermatogonia after isolation of germ cells for cell culture which is necessary for the investigation of factors which stimulate or inhibit specific steps, a.o. the onset of meiosis.

    Furthermore, the possible stimulation of gonadal development in larval or juvenile carp by injection with homologous pituitary extract containing a.o. the gonadotropic hormone (GTH) was investigated. It was found in juvenile carp that the gonads were greatly enlarged and that precocious spermatogenesis occurred after injection of pituitary extract (confirming data obtained in other fish species after injection of GTH). However, also gonadal development and PGC proliferation in larval fish was stimulated. Such effect of pituitary extract on larval gonads and germ cells has not been reported previously. The evidence obtained indicates a function of GTH in this stimulation. The question, however, whether one of the MAb-recognised determinants is involved awaits elucidation.

    Isolation and characterization of Spinacia oleracea L. sperm cells
    Theunis, C.H. - \ 1992
    Agricultural University. Promotor(en): J.L. van Went; H.J. Wilms. - S.l. : Theunis - 89
    spinacia oleracea - spinazie - chenopodiaceae - gameten - spermatozoön - eicellen - cytologie - histologie - spinacia oleracea - spinach - chenopodiaceae - gametes - spermatozoa - ova - cytology - histology

    Gametes are specialized cells with the natural capacity to fuse in a well determined way. The fusion products are intended to develop into new individuals. Basic knowledge of gametes is of great importance for both traditional plant breeding as well as for modem biotechnology and gene manipulation. For applications in these fields, more knowledge is necessary of the characteristics of gametes and the mechanisms involved in the process of gamete recognition and fusion. Isolated gametes form ideal material to investigate this. The present study was focused on the isolation and characterization of the male gametes, the sperm cells.

    In chapter 1 an introduction is given. The current information we have on in situ sperm cells, and on the subject of sperm cell isolation, is summarized in this chapter.

    In chapter 2 the ultrastructure is described of the pollen grains of Spinacia oleracea and the sperm cell pair therein. The pollen grain is trinucleate and consists of a vegetative cell and two sperm cells. The pollen grain wall is tectate, with many germination pores, which have a hexagonal distribution. The vegetative nucleus together with the sperm cells are located in the periphery of the pollen grain and are organized in a "male germ unit". The cytoplasm of the vegetative cell contains vacuoles and electron dense vesicles. The mitochondria have a size of 0.3 μm to 0.5 μm. The ER is often organized in single elements, and bears ribosomes. The plastids are filled with starch and only the outer membrane is visible. The high amount of starch may be used in an autotrophic way of germination, or for osmotic stabilization during germination. Microtubules are not found in the vegetative cytoplasm.

    The sperm cell inside the pollen grain contains a heterochromatic nucleus, mitochondria, dictyosomes, and ER. The two sperm cells are attached to each other. They form a pair which is surrounded by a vegetative plasma membrane. Only a few microtubules have been shown in the sperm cell cytoplasm. In previous studies, microtubules have been clearly demonstrated inside the sperm cells. Therefore, it was concluded that the used method of freeze substitution does not completely stop the breakdown of microtubules.

    In order to release the sperm cells, the vegetative cells has to be opened. The osmotic shock method, which has been used in some species, does not work for pollen grains of Spinacia oleracea. Even in pure water only a small percentage of the pollen grains bursts. For this reason, a mechanical method has been developed, using a glass roller to squash large quantities of pollen grains. This method is described in chapter 3.

    Because of the squashing of the grain, the sperm cells are released from the pollen grain together with most of the vegetative cytoplasm. Since physical breaking is applied, any medium can be chosen in which the breaking is performed. After the squashing, the mixture of pollen grains, free sperm cells, vegetative nuclei, vegetative organelles, and pollen grain fragments is filtered over a 25 μm nylon filter. Subsequently, the filtrate is centrifuged on a 20% percoll layer for further elimination of small debris. With this method, a fraction is obtained which contains numerous sperm cells, but which is still contaminated with small vegetative organelles, and small pollen fragments. The yield is approximately 5-10% with a final concentration of 4x10 6sperm cells/ml.

    The free sperm cells are elongated just after squashing, but become spherical after a short time. The originally paired sperm cells separate. The close association of the sperm cell pair with the vegetative nucleus is not maintained during squashing and is therefore, not a firm binding. The sucrose concentration of the medium does not influence the change in shape of the free sperm cells. The volume, however, is influenced by the osmotic value of the medium. The 25 % sucrose concentration was chosen for the rest of the experiments in order to avoid damage caused by osmotic swelling. The diameter of the isolated sperm cells can vary from 4 μm to 9 μm, depending on the sucrose concentration.

    Immediately after isolation, more than 90% of the sperm cells is viable (tested with the fluorescein diacetate test). Soon after isolation, however, some of the cells loose their viability. After 18 h, only 50% of the isolated cells is still viable. Storage of the isolated sperm cells at low temperatures (0°C) doubles the lifespan. Addition of 1 % vitamin C also enlarges the lifespan. It is concluded that depletion of energy is not the cause of the loss of viability, since addition of 0.1 M ATP makes no difference for the lifespan.

    With histochemical tests, using calcofluor white MR2 for cellulose, analine blue for callose, and the PAS reaction for carbohydrates, no cell wall material was observed around the isolated sperm cells. This indicates that the cells are true protoplasts.

    In chapter 4 the results are presented of the analysis of the numbers of mitochondria in isolated sperm cells. To visualize the mitochondria, two staining methods have been used. The DiOC 6 (3) staining (in a concentration of 0.1 μg/ml) gives better results with less background staining, than the Rhodamine 123 staining.

    The analysis was carried out on individual sperm cells, as well as on sperm cell pairs. If individual sperm cells were used, two populations of sperm cells seemed to be present, with an average of respectively 10.3 and 17.8 mitochondria per sperm cell. However, by counting the mitochondria in sperm cell pairs, it is found that there is only one population of sperm cells. The average is 12.4 ± 4.6 mitochondria per sperm cell. The number of mitochondria per sperm cell varies from 2 to 25, which is a high variation. This high variation can be explained in two ways. It is possible that already after the division of the microspore, a high variation exists in number of mitochondria per generative cell. The second explanation can be, that during the development of the generative cell and/or sperm cells, mitochondria are produced or lost.

    With the technique of freeze-fracturing, the plasma membranes of the isolated sperm cells were examined, including the intra-membrane particles (IMP's). In chapter 5 the results are presented. Also with this method, no remnants of the vegetative plasma membrane were found around the sperm cells. Only incidentally, transverse fracture planes through whole sperm cells are found. Most of the fracture planes of sperm cells follow the sperm cell plasma membrane exposing either the PF or the EF face. Neither the ES or the PS faces are found. Both the PF as well as the EF face show IMP's. These IMP's are randomly distributed, and no pattern can be recognized. The PF face has a density of 719 IMP's/μm 2. The EF face has a density of 2088 of IMP's/μm 2. Evidently, the EF half of the sperm cell membrane contains approximately 3 times more IMP's than the PF half. For sporophytic protoplast it has been reported that the PF half contains more IMP's than the EF half. The specific IMP distribution in the sperm cell plasma membrane may be related to the process of gamete recognition and subsequent fusion. With respect to IMP's density, only one type of sperm cells was observed and therefore for this character, no dimorphism could be established.

    Morphometrical and ultrastructural characterization of the isolated sperm cells has been reported in chapter 6. Experiments with various fixations demonstrate that sperm cells are fragile, and difficult to fixate. The osmotic value of the fixation media appear to be of great importance. Also with this method it is clear that the isolated sperm cells are separate and completely spherical. The surrounding vegetative plasma membrane has disappeared. No cell wall material is observed. The sperm cell contains a large nucleus which can be either heterochromatic or euchromatic. The mitochondria are spherical and frequently appear to be clustered in groups of 5 to 10 mitochondria, but also individual mitochondria have been observed. The dictyosomes have 4 to 5 cisterns with associated small vesicles. Small vacuoles are present. The endoplasmatic reticulum is sparse, and often dilated. Ribosomes are sometimes grouped in polysomes. No microtubules have been observed. From these observations it is clear that isolated cells contain a similar set of organelles as the in situ sperm cells. After measuring the section diameters, the average diameter of the complete cell is calculated to be 3.66 μm with the used fixation conditions. From surface area's in the sections is calculated that 50 % of the sperm cell is occupied by its nucleus, 2.5 % of the cell is mitochondria, and 0. 6 % of the cell is dictyosome. The ultrastructural analysis did not give any indication that in Spinacia oleracea sperm cell dimorphism in regard of sperm cell size, exists.

    In chapter 7 the results of the present thesis are discussed in general sense and in a broader context. Results which have already been discussed in previous chapters are not further discussed in this general discussion.

    The isolation technique of "physical breaking", developed for Spinacia oleracea is compared with the "osmotic shock" technique, used for other species. The advantages and disadvantages of both techniques are presented and discussed. The usefulness of the two techniques in further research of isolated gametes are explained. Preferences for one of the two technique clearly depends on the aims of the further research.

    The general discussion highlights the two major phenomena sperm cells show when isolated: the changing of a spindle shaped cell to a spherical cell, and the loss of viability. The change in shape occurs in all species observed thus far. Likely, the change in shape is a natural process which also occurs during fertilization. The loss of viability can be slowed down with low temperature, and anti-oxidantia. During the natural fertilization process, extended viability is not necessary, since the free sperm cells fuse rapidly with the female partners. So also for this character it may be a natural phenomenon.

    Sperm cell dimorphism, which has been reported in some species, is not found in Spinacia oleracea. The isolated sperm cells form good material to study dimorphism because of the large ~ties of cells which can be analyzed.

    De effecten van het rapen van legsels van zilvermeeuwen als beheersmaatregel op de voedselkeuze van de soort
    Wit, A.A.N. de - \ 1989
    Arnhem : RIN (Intern rapport / Rijksinstituut voor Natuurbeheer 89/1)
    dieren - verspreiding - uitsterven - voedingsgedrag - gameten - Laridae - eicellen - symbiose - zoölogie - Larus argentatus - diergemeenschappen - animals - dispersal - extinction - feeding behaviour - gametes - Laridae - ova - symbiosis - zoology - Larus argentatus - animal communities
    The egg of an assassin bug (Rhinocoris sp.) from the Ivory Coast
    Cobben, R.H. ; Henstra, S. - \ 1968
    Wageningen : [s.n.] (Mededeling / Laboratorium voor entolologie no. 155) - 2
    ivoorkust - gameten - miridae - eicellen - cote d'ivoire - gametes - ova
    Gametocidal action of dichloroacetic acid
    Kho, Y.O. ; Bruyn, J.W. de - \ 1963
    Wageningen : IVT (Mededeling / Instituut voor de Veredeling van Tuinbouwgewassen 195)
    dichloorazijnzuur - gameten - teratogenese - dichloroacetic acid - gametes - teratogenesis
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