Isolation and characterization of Spinacia oleracea L. sperm cells

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.