Viable diploid progeny induced from sperm of Chinese tetraploid pond loach by cold‐shock androgenesis

Natural tetraploid loach (Misgurnus anguillicaudatus) living in the Changjiang River basin and adjacent area in China has been considered a true genetic tetraploidy (4n = 100) with four sets of homologous chromosomes. Here, we reported its further supporting evidence provided by the cold‐shock androgenesis. Viable androgenetic progeny appeared when eggs were cold‐shocked at 3°C for 60 min, starting 5 min after fertilization with sperm of natural tetraploid males, although the survival rate was significantly lower than control group. The majority (87%) of androgenetic embryos were diploid (2n = 50) and all‐paternal inheritance was verified in larval stage by microsatellite genotypes. Microscopic observations confirmed the elimination of both egg nucleus and second polar body from a fertilized egg, followed by the cleavage exclusively with sperm nucleus. Thus, the appearance of normal diploid androgenetic progeny from sperm of natural tetraploid loach revealed the presence of four chromosome sets in tetraploid males.     

Disruption of normal meiosis in artificial inter-populational hybrid females of <Emphasis Type="Italic">Misgurnus</Emphasis> loach

Artificial cross between two genetically different populations of Japanese Misgurnus loach was made to examine the reproductive capacity of the artificial inter-populational hybrid females. Ploidy status and microsatellite genotypes of the eggs laid by these hybrids were inferred from those determined in progenies developed by normal fertilization with haploid loach sperm, induced gynogenesis with UV-irradiated goldfish sperm and/or hybridization with intact goldfish sperm. Some hybrid females laid unreduced diploid eggs genetically identical to the mother. However, these diploid eggs could not develop by spontaneous gynogenesis, but grow to triploid by incorporation of a sperm nucleus. Other hybrid females laid haploid eggs together with diploid eggs and/or various aneuploid and polyploid eggs. Thus, a disruption of normal meiosis occurred in inter-populational hybrid females. The results suggested that the two populations should be so distant as to give rise to atypical formation of unreduced and other unusual eggs in their hybrids.     

Simultaneous formation of haploid, diploid and triploid eggs in diploid–triploid mosaic loaches

In the loach Misgurnus anguillicaudatus , very few diploid–triploid mosaic individuals, which are generated by accidental incorporation of the sperm nucleus into diploid eggs produced by clonal diploid loach, occur in nature. Ploidy examination of gynogenetic progeny induced by activation with ultraviolet-irradiated goldfish sperm indicated that diploid–triploid mosaic females laid haploid, diploid and triploid eggs, simultaneously. In addition, triploid eggs exhibited larger egg sizes. Microsatellite genotyping of diploid–triploid mosaics revealed that triploid genotypes of mosaic mothers possessed two alleles specific to the clonal diploid and one allele from normal diploid male. Diploid eggs from a mosaic mother had genotypes absolutely identical to the diploid clone. Most genotypes of triploid eggs were identical to the mosaic mother, and one of the three alleles of the mosaic mother was transmitted to haploid eggs. These results suggested that diploid germ cells, which had a clonal genome, were differentiated into clonal diploid eggs, and triploid and haploid eggs were produced from triploid germ cells in the same ovary of mosaic individuals.     

Clonal diploid sperm of the diploid-triploid mosaic loach, Misgurnus anguillicaudatus (Teleostei:Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid, triploid and diploid-triploid mosaic individuals in a wild population of the Hokkaido island, Japan. Previous studies revealed the presence of a cryptic clonal lineage among diploid loaches, which is maintained by uniparental reproduction of genetically identical diploid eggs. In the present study, we analyzed distribution and genetic status of diploid and triploid cells in infrequent mosaic males. Flow cytometry, microsatellite genotyping and DNA fingerprinting verified that mosaic males consisted of diploid cells with genotypes identical to the natural clone and triploid cells with diploid genomes of the clonal lineage plus haploid genome from sperm nucleus of the father. Thus, the occurrence of diploid-triploid mosaicism might be caused by accidental fertilization of a diploid blastomere nucleus with haploid sperm after the initiation of clonal development of unreduced eggs. Such mosaic males produced fertile sperm with diploid DNA content. The experimental cross between normal diploid female and diploid-triploid mosaic male gave rise to the appearance of triploid progeny which exhibited two microsatellite alleles identical to the clonal genotype and one allele derived from the normal female. In DNA fingerprinting, such triploid progeny gave not only all the DNA fragments from the clone, but also other fragments from the normal female. Induced androgenesis using UV irradiated eggs and sperm of the mosaic male gave rise to the occurrence of diploid individuals with paternally derived microsatellite genotypes and DNA fingerprints, absolutely identical to the natural clonal lineage. These results conclude that the diploid-triploid mosaic male produced unreduced diploid sperm with genetically identical genotypes. The spermatogenesis in the clonal diploid cells under the mosaic condition suggests that triploid male somatic cells might transform genetically all-female germ cells to differentiate into functionally male gametes. The discovery of the mosaic male producing unreduced sperm suggests the theoretical occurrence of triploids and other polyploids by the syngamy of such paternally derived diploid gametes.     

Induction of Haploid Androgenesis in Pacific Oyster by UV Irradiation

Androgenesis, development from paternal but not maternal chromosomes, can be induced in some organisms including fish, but has not been induced previously in mollusk. In this study we investigated the induction of haploid androgenesis in the Pacific oyster by ultraviolet irradiation and observed nuclear behavior in the androgenetic eggs. Irradiation for 90 seconds at a UV intensity of 72 erg/mm² per second (6480 erg/mm²) was the optimal dose to achieve haploid androgenesis. The fertilization and development rates of D-shaped larvae decreased with increasing exposure time, and the development of the genetically inactivated eggs terminated before reaching the D-shaped stage. Cytologic observations showed that UV irradiation did not affect germinal vesicle breakdown or chromosomal condensation but caused various nuclear behavioral patterns during meiosis and first mitosis: 21.7% of eggs extruded all maternal chromosomes as 2 or 3 polar bodies, and 59.1% of eggs formed one female pronucleus. The maternally derived nucleus did not participate, or partially participated, in the first karyokinesis. The cytologic evidence demonstrates that the male genome is directing development in haploids produced by UV irradiation.     

Production of androgenetic diploid rainbow trout

Haploid androgenesis was induced in rainbow trout (Salmo gairdneri) when eggs were irradiated with 60Co gamma radiation prior to fertilization. Diploidy was restored to the androgenetic haploid zygotes by suppression of first cleavage division using hydrostatic pressure. Peak survival in the androgenetic diploid lots (32.5-38.9 percent of control) occurred when a pressure shock of 9000 pounds per square inch lasting from one to three minutes was applied to the eggs 345 minutes post-fertilization. Chromosomal analysis confirmed diploidy in the androgenetic individuals and suggested that YY rainbow trout are viable to at least the "eyed stage" of development. Inheritance patterns at two loci confirmed all-paternal inheritance. The relatively high yields of completely homozygous androgenetic rainbow trout and the potential for the use of androgenesis in the production of inbred lines and in genetic studies indicate that androgenesis may become a valuable tool in fish research and breeding.     

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.     

L’irradiation des spermatozoïdes au Laser HeNe à faible énergie chez la souris et chez l’homme

Introduction

The effects of low energy He?Ne 630 laser (LEL) irradiation on various aspects of cell metabolism, including proliferation, respiration, ATP synthesis and Ca²⁺ uptake, have been recently recognized in somatic cells, animal spermatozoa and embryonic cells.

Objectives

1- To analyze thein vitro effects of LEL irradiation on the mouse IVF model. 2- To assess the effects of experimental LEL irradiation on human spermatozoa in the sperm-zona free hamster egg penetration (SPA) model.

Material & Methods

1- In a mouse IVF model, pooled oocytes were incubated for 24 h. with LEL-irradiated and non-irradiated epididymal mouse spermatozoa. The percentage of fertilized eggs, intracellular Ca²⁺ and Ca²⁺ uptake as well as the effects of reactive oxygen scavengers were compared in the two groups. 2- In the enhanced SPA model, with overnight TEST-Yolk preincubation, LEL-irradiated and non-irradiated ejaculated spermatozoa from 43 infertile men were incubated for 3 h with fresh zona-free hamster eggs. The percentage of eggs penetrated (SPA%) by irradiated or control sperm was compared. Acrosome reaction (AR) and Hemizona Assay (HZA) were also performed on the same ejaculates.

Results

1- In mice, brief LEL irradiation enhanced intracellular Ca²⁺ influx and increased thein vitro fertilization capacity of spermatozoa. Mitochondrial Ca²⁺ transport mechanisms and H₂O₂ appeared to be involved in these LEL effects. 2- In the 43 male patients, AR and SPA were correlated, while SPA and HZA were poorly correlated. In the whole group, the mean SPA% of LEL-irradiated and control sperm were not significantly different: 49.8±39.9 and 51.0±34.9, respectively. No significant change was observed in 50% of cases. Following irradiation, SPA% increased in 25% of cases and decreased in 25% of cases. However, classifying all cases according to the SPA cut-off point of 30% as poor and good sperm in this enhanced SPA model, showed that LEL irradiation significantly increased SPA% only in the poor sperm subgroup (n=16) from 5.5.±8.9 to 23.1±25.2 (p<0.0001). This increase was observed in 50% of these patients. In the good sperm category (n=27), no improvement of SPA% was observed. On the contrary, LEL irradiation decreased SPA% from 76.1±24.4 to 67.6±28.

Conclusion

In a mouse IVF model, LEL irradiation of spermatozoa improved intracellular Ca²⁺influx and egg fertilization. H₂O₂ seems to participate in the biochemical cascade transforming light signals into a biological response. In man, using the SPA model, LEL sperm irradiation might improve the penetration capacity only in the case of poor quality sperm, by increasing sperm egg penetration in one half of cases, otherwise LEL is ineffective. Further studies are required to confirm these results, but LEL should not be considered for clinical use until its safety has been proven. (Supported by grants from the Chief Scientist Office, Ministry of Health)     

Experimental analysis of a paternally inherited extrachromosomal factor

Virtually all known cases of extrachromosomal inheritance involve cytoplasmic inheritance through the maternal line. Recently, a paternally transmitted factor that causes the production of all-male families has been discovered in a parasitic wasp. The wasp has haplodiploid sex determination: male offspring are haploid and usually develop from unfertilized eggs, whereas females are diploid and usually develop from fertilized eggs. It has been postulated that this paternal sex-ratio factor (psr) is either (1) an infectious agent (a venereal disease) that is transmitted to the female reproductive tract during copulation with an infected male and, subsequently, causes all-male families or (2) a male cytoplasmic factor that is transmitted by sperm to eggs upon egg fertilization and, somehow, causes loss of the paternal set of chromosomes.—Experimental evidence is presented which shows that the factor requires egg fertilization for transmission to the next generation; therefore, it is likely to be a cytoplasmic factor. Significant potential intragenomic conflict results from the presence of this factor and two other sex-ratio distorters in this wasp species.     

Disturbances in the ploidy level in the gynogenetic sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>

Artificial mitotic gynogenesis, a chromosome set manipulation, is applied to provide the homozygous progeny with only maternal inheritance. Here, gynogenetic development was induced in the sterlet Acipenser ruthenus L. (Acipenseridae) by activation of the eggs originating from albino females with the UV-irradiated spermatozoa from wild-coloured males, followed by the heat shock applied to suppress the first mitotic division in the haploid zygotes. All experimentally obtained gynogenetic offspring possessed recessive albino coloration. Moreover, the genetic verification, based on three microsatellite DNA markers, confirmed the only maternal inheritance in the albino progeny. Cytogenetic screening enabled identification of the aneuploids, haploids, diploids, triploids, tetraploids and mosaic individuals among the gynogenetic larvae that hatched from the eggs subjected to the heat shock. Furthermore, 40% of the larvae from the haploid variants of the research that were not exposed to the temperature shock showed the diploid chromosome number. A variation of the ploidy level observed in the gynogenetic sterlets may be the consequence of the spontaneous polyploidisation that occurred in the haploid zygotes. Moreover, observation during embryogenesis showed varied stages of eggs development and the asynchronous cell cleavages that may have resulted in the chromosomal disturbances observed in the gynogenetic sterlets here.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Variations in the breeding behavior of cichlids and the evolution of the multi-functional seminal plasma protein,seminal plasma glycoprotein 120

Background

Seminal plasma proteins are associated with successful fertilization. However, their evolutionary correlation with fertilization mechanisms remains unclear. Cichlids from Lake Tanganyika show a variety-rich spawning behavior that is associated with the transfer of the sperm to the egg for fertilization. One of these behaviors, called “oral fertilization,” emerged during their speciation. In oral fertilization, females nuzzle the milt from male genitalia and pick up the released eggs in their mouths, which are then fertilized inside the oral cavity. Thus, the success of the fertilization is dependent on the retention of sperm in the oral cavity during spawning. Sperm aggregation and immobilization in viscous seminal plasma may help retain the sperm inside the oral cavity, which ultimately determines the success of the fertilization. Seminal plasma glycoprotein 120 (SPP120) is one of the major seminal plasma proteins present in cichlids. SPP120 has been implicated to immobilize sperm and increase the milt viscosity. However, the functional linkage between oral fertilization and seminal plasma proteins has not been investigated.

Results

During trials of simulated oral fertilization, it was observed that milt viscosity contributed to fertilization success by facilitating longer retention of the milt inside the mouth during spawning. Glycosylation of SPP120 was associated with high milt viscosity. Its glycosylation was specifically present in the milt of cichlid species exhibiting oral fertilization. Moreover, recombinant SPP120 from several the oral fertilization species strongly immobilized/aggregated sperm. Therefore, the functions of SPP120 (immobilization/aggregation and its glycosylation) may contribute to success of oral fertilization, and these functions of SPP120 are more prominent in oral fertilization species. In addition, comparative phylogenetic analyses showed a positive evolutionary correlation between SPP120 function and oral fertilization. Hence, these evolutions may have occurred to keep up with the transition in the mode of fertilization. In addition, rapid evolution in the molecular sequence might be associated with functional modifications of SPP120.

Conclusion

These results suggest that SPP120 might be associated with oral fertilization. In other words, reproductive traits that define the interaction between sperms and eggs could be the evolutionary selective force that cause the rapid functional modification of the fertilization-related reproductive protein, SPP120.

     

THE PRODUCTION OF PARTHENOGENETIC MAMMALIAN EMBRYOS AND THEIR USE IN BIOLOGICAL RESEARCH

1. The eggs of many mammalian species show signs of early parthenogenetic development as they age after ovulation and oocytes may form transplantable terato-carcinomas. These cases of apparently spontaneous parthenogenetic development suggest that the cells of the female germ line have an inherent tendency to divide and differentiate. 2. The ovulated eggs of virgin female mammals may be stimulated to start parthenogenetic development by a wide variety of treatments. Most of these damage the egg so that it does not develop beyond the 4 cell stage. However if the eggs are exposed to electrical activation, hyaluronidase treatment, or temperature shock then in many cases they will develop into blastocysts. 3. These blastocysts may be either haploid or diploid. Haploid blastocysts may be formed either by the egg extruding the nucleus of the second polar body or by the egg dividing in half, so that the female pronucleus is in one cell and the nucleus of the second polar body is in another cell. Diploid blastocysts are formed by the retention of the nucleus of the second polar body within the egg. The way in which the egg develops may be controlled by altering the osmolarity of the culture medium, the age of the egg at the time of activation, or the strain of animal used. 4. The action of the sperm on the egg can be defined by comparing the events of normal fertilization and parthenogenetic activation. Both these stimuli cause the egg to expose binding sites for Concanavalin A to synthesize DNA and to divide. However, the release of cortical granules, which occurs after fertilization, does not appear to be induced by parthenogenetic activation, and it is significant that parthenogenones lack the sperm nucleus and mitochondria. 5. The majority of parthenogenones die soon after implantation. Death at this time occurs with parthenogenones obtained from the activated eggs of both inbred and outbred stocks. Death might be caused by recessive lethal mutations or by extra-genetic effects of the maternal chromosomes. 6. Parthenogenones contain endogenous A-type particles which shows that these bodies are inherited through the female germ line. 7. Parthenogenones may in the future provide both a method for chromosome mapping and a source of haploid cells. At present the use of mammalian parthenogenones in biological research is restricted by the heavy embryonic losses which occur around the time of implantation. This means that the role of the sperm, gene activity and virus expression must be studied during a very limited period. Part of the mortality before implantation is the consequence of the damage which the egg suffers during activation and it should be possible to reduce this loss by improving the techniques for activation. It may also be possible to increase the quantity of cells derived from haploid and diploid mammalian embryos by deriving teratocarcinomas from them.     

An intermediate state of fertilization involved in internalization of sperm components

The pathway of sperm entry during sea urchin fertilization was analyzed by using sperm covalently labeled with fluorescent and radioactive tracers. Sperm that have been covalently labeled on their surfaces with fluorescein isothiocyanate (FITC) or a radioactive congener, diiodofluorescein isothiocyanate (¹²⁵IFC), transfer labeled components to the egg that persist throughout early development. In order to study the transfer of sperm components and their fate after fertilization, cytochalasin B-dependent inhibition of fertilization, previously shown to permit the cortical reaction of sea urchin eggs but block sperm pronuclear incorporation, was investigated. Under certain conditions cytochalasin B or D (CB or CD) results in about half of the activated eggs having both the sperm nucleus and the fluorescently labeled sperm components arrested apparently at the level of the egg plasma membrane. This arrest of internalization was reversed by removal of CB or CD, and the sperm derivatives entered the egg. When sperm were labeled noncovalently with ethidium bromide or rhodamine 123, fluorescence was transferred to the egg in the cytochalasin-inhibited state in a fashion similar to that found in normal fertilization; in both cases the sperm fluorescence disappeared within a few minutes of fertilization, due to the repartitioning of the noncovalent dyes into the egg cytoplasm. It is concluded that cytochalasin arrests fertilization at an intermediate step in which the sperm has fused with the egg to achieve cytoplasmic continuity, but in which the subsequent internalization of sperm components is inhibited. After removal of cytochalasins the fluorescent sperm components move from the egg surface to an internal site, a process that can be monitored by time-lapse video microscopy with an image intensifier to permit extended observations of sperm fluorescence. The cytoplasmic location of labeled sperm components was substantiated by autoradiography of early embryos fertilized with ¹²⁵IFC-labeled sperm; transfer of sperm components to an internal site was seen after fertilization of either sea urchin or mouse eggs. Taken together, the data suggest that the fate of the labeled sperm surface components, as well as that of the sperm nucleus, is to be transferred to the egg cytoplasm, and that this transfer is mediated by the actin-dependent cytoskeleton of the egg.     

Alteration of the cortical actin cytoskeleton deregulates Ca2+ signaling, monospermic fertilization, and sperm entry

Background

When preparing for fertilization, oocytes undergo meiotic maturation during which structural changes occur in the endoplasmic reticulum (ER) that lead to a more efficient calcium response. During meiotic maturation and subsequent fertilization, the actin cytoskeleton also undergoes dramatic restructuring. We have recently observed that rearrangements of the actin cytoskeleton induced by actin-depolymerizing agents, or by actin-binding proteins, strongly modulate intracellular calcium (Ca²⁺) signals during the maturation process. However, the significance of the dynamic changes in F-actin within the fertilized egg has been largely unclear.

Methodology/Principal Findings

We have measured changes in intracellular Ca²⁺ signals and F-actin structures during fertilization. We also report the unexpected observation that the conventional antagonist of the InsP₃ receptor, heparin, hyperpolymerizes the cortical actin cytoskeleton in postmeiotic eggs. Using heparin and other pharmacological agents that either hypo- or hyperpolymerize the cortical actin, we demonstrate that nearly all aspects of the fertilization process are profoundly affected by the dynamic restructuring of the egg cortical actin cytoskeleton.

Conclusions/Significance

Our findings identify important roles for subplasmalemmal actin fibers in the process of sperm-egg interaction and in the subsequent events related to fertilization: the generation of Ca²⁺ signals, sperm penetration, cortical granule exocytosis, and the block to polyspermy.     

Haploid plants carrying a sodium azide-induced mutation (fdr1) produce fertile pollen grains due to first division restitution (FDR) in maize (Zea mays L.)

Key message

We induced a fdr1 mutation in maize which makes haploid plants male fertile due to first division restitution; the optimum sodium azide treatment on maize kernels has been identified.

Abstract

Sodium azide mutagenesis experiments were performed on haploid and diploid maize plants. Kernels with haploid embryos of maize inbred line B55 were induced by pollinating with RWS pollen. These kernels were treated with 0.2, 0.5, or 1.0 mM sodium azide solution for 2 h. The 0.5 mM solution was optimal for inducing numerous albino sectors on the treated plants without significant damage. Kernels of a maize hybrid, Oh43 × B55, were treated with sodium azide solutions at concentrations of 1.5, 2.0, 2.5, and 3.0 mM. Haploids were generated by pollinating RWS pollen. The highest rate of chlorophyll mutations in seedlings (15.3 % [13/85]) was recorded with the 2.5 mM concentration. A mutated haploid plant (PP1-50) with higher pollen fertility was isolated during the experiments. This haploid plant produced four kernels on the ear after selfing. These kernels were germinated and produced ears with full seed set after selfing. The haploid plants induced from PP1-50 diploids also exhibited high pollen fertility. In situ hybridization studies showed that meiocytes in PP1-50 haploid anthers underwent first division restitution at a rate of 48 % and produced equally divided dyads. We designated the genetic factor responsible for this high pollen fertility as fdr1. PP1-50 haploid ears exhibited high levels of sterility, as seen for regular haploids. Diploid PP1-50 meiocytes in the anther underwent normal meiosis, and all selfed progenies were normal diploids. We concluded that the fdr1 phenotype is only expressed in the anthers of haploid plants and not in the anthers of diploid plants.     

Structural and developmental variability in the female gametophyte of Griffithella hookeriana,Polypleurum stylosum,and Zeylanidium lichenoides and its bearing on the occurrence of single fertilization in Podostemaceae

Key message

Reduced female gametophyte.

Abstract

Angiosperms are characterized by the phenomenon of double fertilization with Podostemaceae as an exception that appears to extend to the entire family. Our earlier work demonstrated the cause of failure of double fertilization and ascertained the occurrence of single fertilization in Dalzellia zeylanica (Tristichoideae, Podostemaceae). In continuation with this work, three more members, i.e., Griffithella hookeriana (Tul.) Warming, Polypleurum stylosum (Wight) Hall, and Zeylanidium lichenoides (Kurz) Engl. (Podostemoideae), have been investigated in the present work. We studied the ontogenetic development of female gametophyte and tracked the path of the two sperm cells from the time of their formation in the pollen tube through their entry into the synergid and gamete fusion. We report the occurrence of a remarkably reduced 3-nucleate, 3-celled mature female gametophyte consisting of an egg cell and two synergids in all the three genera. Interestingly, the central cell is formed during female gametophyte development, but exhibits a species-specific, limited life span, and eventually degenerates prior to the entry of the pollen tube into the synergid, resulting in a failure of double fertilization. Sperm dimorphism on the basis of fluorochrome stainability has been recorded in Z. lichenoides. Further, morphogenetic constraints on the part of male (sperm selection, functional reductionism) and female gametophyte (structural reductionism, inaccessibility of central cell) presumably ensure the failure of double fertilization in these species. Thus, loss of double fertilization in this family is likely a derived condition.     

Le sperme « inflammatoire »: ses relations avec la fertilité

Objectives

Sperm inflammation is caused by bacterial or viral infection of the male genitourinary tract; it is often clinically asymptomatic. There is a dilemma about the causal relationship between leukocytes as markers of inflammation and poor semen quality. We were interested in sperm changes at molecular level caused by inflammation.

Material and methods

This study was based on a literature review and personal data. In 200 male partners of infertile couples with normal semen analysis, the percentage of sperm with DNA denaturation and the level of reactive oxygen species (ROS) were determined by flow cytometric analysis, after acridine orange and dihydroethidium stainings, and correlated with seminal plasma elastase levels.

Results

In the literature, a positive relationship between inflammation and increased sperm apoptosis was found with increased necrosis and decreased mitochondrial membrane potential. We found a positive correlation between the percentage of sperm with denatured DNA and elastase levels. The percentage increased from 8.6% at elastase level 0–100 μg/l to 15.7% at elastase level 100–250 μg/l; this increase was not dependent on ROS production. The percentage of sperm with denatured DNA normalized at elastase levels above 600 μg/l.

Discussion and conclusion

Changes in sperm DNA or membranes do not necessarily affect classical semen characteristics or reduce fertility in males. They can, however, have a negative effect on capacitation and acrosomal reaction, resulting in failed fertilization or poor embryo development. Before treatment, we must take into account the location and the duration of the inflammation as well as the damage done to sperm.     

Changes in intracellular content of glutathione and thiols associated with gamma-glutamyl cycle during sperm penetration and pronuclear formation in rat oocytes

The content of glutathione and other thiols in rat eggs was examined during sperm penetration and pronuclear formation by high-performance liquid chromatography with fluorescence detection. Reduced glutathione (GSH) content was higher in unfertilized oocytes (8.50 +/- 0.29 pmol/egg) and penetrated eggs with a decondensed sperm nucleus (DSH eggs; 7.72 +/- 0.56 pmol/egg) than eggs at the pronuclear stage (PN eggs; 5.93 +/- 0.10 pmol/egg). The content of oxidised glutathione (GSSG) was not different among experimental groups (152.6 +/- 74.1 nmol/egg in unfertilized eggs, 146.0 +/- 50.0 nmol/egg in DSH eggs and 39.7 +/- 17.3 nmol/egg in PN eggs). The GSSG/GSH ratio did not change during fertilization. Although the reduced cysteinylglycine content of eggs did not change among experimental groups, the oxidised form of cysteinylglycine increased (p < 0.025) between sperm decondensation (6.9 +/- 1.5 nmol/egg in unfertilized oocytes and 10.1 +/- 2.1 nmol/egg in DSH eggs) and pronuclear formation (40.5 +/- 11.5 nmol/egg in PN eggs). Low contents of cystine were detected during fertilization but cysteine and gamma-glutamylcysteine were not detected in any treatment groups. These results demonstrate that GSH content in rat eggs decreases between sperm decondensation and pronuclear formation, probably due to the increased activity of gamma-glutamyl transpeptidase.     

Prematurely condensed human sperm chromosomes after in vitro fertilization (IVF)

Summary During an in vitro fertilization (IVF) program 122 inseminated eggs showing polar body extrusion, but neither formation of pronuclei nor cell cleavage were analysed cytogenetically. Nine of these eggs showed prematurely condensed sperm chromosomes of the G₁-phase (G₁-PCC) besides the haploid set of maternal metaphase II chromosomes. This phenomenon can be explained by the permanent arrest of the oocytes at metaphase II after sperm penetration and hence the continuing presence of cytoplasmic chromosome condensing factors which lead to the induction of PCC in the sperm nucleus. The overall frequency of this aberrant type of fertilization was calculated to be in the order of 3–4% of all in vitro fertilized eggs.