Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5‐(octa‐decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity. Mol. Reprod. Dev. 52:207–215, 1999. © 1999 Wiley‐Liss, Inc.     

Nuclear maturation and embryo development of porcine oocytes vitrified by cryotop: Effect of different stages of in vitro maturation

The present study was designed to evaluate the viability, meiotic competence and subsequent development of porcine oocytes vitrified using the cryotop method at different stages of in vitro maturation (IVM). Cumulus–oocyte complexes (COCs) were cultured in IVM medium supplemented with 1 mM dibutyryl cAMP (dbcAMP) for 22 h and then for an additional 22 h without dbcAMP in the medium. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase I (AI/TI) and metaphase II (MII) were found to occur predominantly at 0–22, 26, 32, 38 and 44 h of IVM, respectively. Oocytes were exposed to cryoprotectant (CPA) or vitrified after different durations of IVM (0, 22, 26, 32, 38 and 44 h). After CPA exposure and vitrification, surviving oocytes that were treated before completion of the 44 h maturation period were placed back into IVM medium for the remaining maturation period, and matured oocytes were incubated for 2 h. CPA treatment did not affect the viability of oocytes matured for 26, 32, 38 or 44 h, but significantly decreased survival rate of oocytes matured for 0 or 22 h. CPA treatment had no effect on the ability of surviving oocytes to develop to the MII stage regardless of the stage during IVM; however, blastocyst formation following PA was severely lower (P < 0.05) than that in the control. At 2 h post-warming, the survival rates of oocytes vitrified at 26, 32, 38 and 44 h of IVM were similar but were higher (P < 0.05) than those of oocytes vitrified at 0 or 22 h of IVM. The MII rates of surviving oocytes vitrified at 0 and 38 h of IVM did not differ from the control and were higher (P < 0.05) than those of oocytes vitrified at 22, 26 or 32 h of IVM. After parthenogenetic activation (PA), both cleavage and blastocyst rates of vitrified oocytes matured for 22, 26, 32, 38 and 44 h did not differ, but all were lower (P < 0.05) than those matured 0 h. In conclusion, our data indicate that survival, nuclear maturation and subsequent development of porcine oocytes may be affected by their stage of maturation at the time of vitrification; a higher percentage of blastocyst formation can be obtained from GV oocytes vitrified before the onset of maturation.     

Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages

Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 microM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 microM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 microM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 microM cysteamine to the maturation medium improved IVM of canine oocytes.     

Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro

Fully grown germinal vesicle-stage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle-stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin-stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6-16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum-containing medium. In the third experiment, medium conditioned by FSH-stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH-stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH- or LH-stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.     

Effects of in utero and in vitro incubation on the lipid-bound fatty acids and sterols of porcine spermatozoa

Sperm-rich semen and washed porcine spermatozoa were incubated for up to 2 hr either in utero in the presence of oviduct fluid or in vitro at 37°C. Sperm lipids were extracted and separated into phospholid and neutral lipid fractions. Eleven phospholipid and five neutral lipid fatty acids were identified and quantified using GC and GC-MS. The percentage of 22:5n6, the major phospholipid fatty acid, decreased slightly but significantly during 1.5 hr of in utero incubation (41.2–38.0%), but after 2.0 hr of in utero incubation no significant difference was observed (40.0%). None of the phospholipid fatty acids changed in concentration during in vitro incubation. The mole ratio of phospholipid to phospholipid fatty acid (1.00:1.27) did not change during incubation. The levels of neutral lipid-bound 14:0 decreased (43.5% to 31.8%) and that of 18:0 increased (11.1% to 18.2%) during in utero incubation. Similar but less pronounced changes were observed during in vitro incubation. (43.5% to 36.0%; and 11.1% to 15.8%, respectively). Two major sterols, cholestrol (73%) and desmosterol (27%) were identified by gas chromatography–mass spectrometry. The mole ratio of phospholipid to sterol (2.47:1:00) did not change during incubation.     

雌牛生殖道内游离氨基酸种类及含量分析

根据黄体的大小和形态，判断屠宰母牛的发情时期，分别收集发情第3天(D3)和第7天(D7)的输卵管液(OF)和子宫液(UF)，通过HPLC分析游离氨基酸种类及含量。总共检测到23种氨基酸，包括除Cys外的19种必需和非必需氨基酸，以及另外四种(βAla,Tau,Orm和Cit)非蛋白质氨基酸。OFD3、UFD3、OFD7和UFD7的游离氨基酸总量分别为31．6、46．9、26．3和17．2mmol/L，其中，Gly含量最高，分别为14．7、14．4、11．1和4．4，其次为Glu、Gln和A1a，其他氨基酸的含量均接近或低于lmmol/L。结果表明：氨基酸总量及个别氨基酸含量在不同体液及不同发育时期均存在一定程度的差异.     

Sperm production and steroidogenesis in testes of the common carp, Cyprinus carpio L., at different stages of maturation

Testes from carp, Cyprinus carpio L., at five different maturational stages from immature through to spermiation and regression were incubated with or without addition of carp hypophysial homogenate (chh) for 8 or 20 h. Concentrations of steroids and spermatozoa were measured in the medium and the residual tissue examined histologically. There was an increase in the area of the germinal cysts containing spermatozoa, the percentage of the testis which they occupied and in the production of spermatozoa as the gonadosomatic index (GSI) increased, but this was unaffected either by incubation or by pretreatment with chh. The major steroid in plasma and in in vitro testicular cultures from all of the maturing fish captured in winter was 1 I-ketotestosterone. The production rate of this steroid in virro was unaffected by GSI, while plasma levels tended to increase with GSI. 17.20β-Dihydroxy-4-pregnen-3-one was detectable in significant amounts in only a few spermiating fish in summer, but was stimulated more in incubations with chh in maturing winter than in summer spermiating or post-spawning fish. 17,20a-Dihydroxy-4-pregnen-3-one was not detectable in incubations, but plasma concentrations tended to increase towards spermiation and were positively correlated with the size of the cyst. After spawning, fish had low plasma steroid levels and failed to respond in vitro to pituitary extract, indicating a testicular post-spawning refractoriness.     

A study on the amount of high-mobility-group chromatin proteins in T-cells at different stages of differentiation

The amounts of high-mobility-group proteins (HMG) 1 and 2 in different mouse T-cell populations are studied. It is shown that the quantity of HMG 1 and 2 is different in functionally distinct T-cells. The level of these proteins in thymus cells is higher than in cortisone-resistant thymocytes and peripheral T-cells; it increases in the cytotoxic cells generated in mixed lymphocyte culture. The quantity of HMG is negligible in memory T-cells and increases when the latter cells are stimulated again. The differences found in the levels of HMG 1 and 2 could be related to the rate of cell proliferation and to the changes in chromatin structure at each functional stage of differentiating T-cells.     

Differences in the rate of redistribution of receptors for concanavalin A in vivo and in vitro on spermatozoa from normal mice and from sterile mice carrying different T/t locus haplotypes

The redistribution of receptors for fluorescein isothiocyanate-conjugated concanavalin A (FITC-con A) on mouse spermatozoa during maturation has been studied in vivo and in vitro using normal mice and sterile mice carrying two different lethal T-locus haplotypes (t^x/t^y). Receptors for FITC-con A, uniformly distributed on the head of functionally immature sperm within the proximal epididymis, become localized as the cells acquire functional maturity within the distal portion of the epididymis. Examining samples of epididymal sperm incubated for short periods of time in vitro, a similar increase in the proportion of discretely labeled cells is observed with time. Sperm from sterile t^x/t^y males also show redistribution of receptors for FITC-con A in vivo and in vitro; however, a significantly smaller proportion of cells of the population achieve a discrete distribution of receptors comparable to that displayed on sperm from fertile mice. It is concluded that mouse sperm undergo redistribution of receptors for con A normally in acquiring functional maturity within the epididymis and upon liberation from the male urogenital tract; the sterility of t^x/t^y mice may be due to a genetically based impairment of plasma membrane reorganization reflected in sluggish redistribution of receptors for con A shown by these cells in vivo and in vitro.     

The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation

In a previous study we have shown that the addition of growth hormone (GH) during in vitro maturation accelerates nuclear maturation, induces cumulus expansion, and promotes subsequent cleavage and embryonic development. The aim of this study was to investigate whether the promotory effect of GH on subsequent cleavage and blastocyst formation is due to an improved fertilization and whether this effect is caused by an improved cytoplasmic maturation of the oocyte. Therefore, bovine cumulus oocyte complexes (COCs) were cultured for 22 hours in M199 supplemented with 100 ng/ml bovine GH (NIH-GH-B18). Subsequently the COCs were fertilized in vitro. Cultures without GH served as controls. To verify whether the promoted fertilization is caused by the effect of GH on cumulus expansion or oocyte maturation, cumulus cells were removed from the oocytes after in vitro maturation (IVM) and denuded MII oocytes were selected and fertilized in vitro. Both IVM and in vitro fertilization (IVF) were performed at 39°C in a humidified atmosphere with 5% CO₂ in air. At 18 hours after the onset of fertilization, the nuclear stage of the oocytes was assessed using 4,6-diamino-2-phenylindole (DAPI) staining. Oocytes with either an metaphase I (MI) or MII nuclear stage and without penetrated sperm head were considered unfertilized; oocytes with two pronuclei, zygotes, and cleaved embryos were considered normally fertilized; and oocytes with more than two pronuclei were considered polyspermic. To evaluate cytoplasmic maturation, the distribution of cortical granules 22 hours after the onset of IVM, and sperm aster formation 8 hours after the onset of fertilization were assessed. In addition, to assess the sperm-binding capacity, COCs were fertilized in vitro, and 1 hour after the onset of fertilization the number of spermatozoa bound to the oocytes was counted. The addition of GH during IVM significantly (P < 0.001) enhanced the proportion of normal fertilized oocytes. Removal of the cumulus cells prior to fertilization and selection of the MII oocytes did not eliminate the positive effect of GH on fertilization. No effect of GH on the sperm-binding capacity of the oocyte was observed. In addition, GH supplementation during IVM significantly (P < 0.001) enhanced the migration of cortical granules and sperm aster formation. It can be concluded that the promotory effect of GH on the developmental competence of the oocyte is due to a higher fertilization rate as a consequence of an improved cytoplasmic maturation. Mol. Reprod. Dev. 49:444–453, 1998. © 1998 Wiley-Liss, Inc.     

Pyrethroids cypermethrin,deltamethrin and fenvalerate have different effects on in vitro maturation of pig oocytes at different stages of growth

Pesticides can significantly harm reproduction in animals and people. Pyrethroids are often used as insecticides, and their toxicity for mammals is considered to be low. However, cypermethrin, deltamethrin and fenvalerate – as potent specific inhibitors of protein phosphatase calcineurin – can influence the meiosis of mammalian oocytes. The objective of this study was to evaluate the effects of these pyrethroids on the in vitro maturation of pig oocytes at different levels of meiotic competence. Under the tested concentrations, cypermethrin, deltamethrin and fenvalerate neither had a significant effect on the viability of oocytes nor did they induce significant degeneration of oocytes. However, these pyrethroids significantly affected meiotic maturation. The effects depended on the stage of meiotic competence of the oocytes. Maturation of growing pig oocytes with partial meiotic competence was induced. On the other hand, in fully grown pig oocytes with full meiotic competence, maturation in vitro was delayed. The specificity of these effects was further supported by the same effect of non-pyrethroidal inhibitors of calcineurin – cyclosporin A or hymenistatin I – on the maturation of oocytes with different levels of meiotic competence. However, pyrethroids, which do not inhibit calcineurin – allethrin or permethrin – had no effect on pig oocyte maturation. We demonstrated a significant effect of pyrethroids on the maturation of mammalian oocytes under in vitro conditions. This indicates that exposure to these substances could affect the fertility of people or animals.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.     

Changes of elemental concentrations around and on the surface of fowl sperm membrane during maturation in the male reproductive tract and after in vitro storage

X-ray microprobe analysis was performed to investigate the changes of elemental concentrations around or on the membrane of the head, midpiece, and principal piece regions of individual fowl spermatozoa during maturation in the male reproductive tract and after storage in vitro at 4°C. The pattern of change of elemental concentrations during maturation and postejaculation was, in general, similar in the three different subcellular regions; i.e., concentrations of sodium, potassium, chlorine, and calcium decreased gradually during sperm passage through the male reproductive tract and after storage. Phosphorus concentration remained almost constant in the male tract and decreased gradually after storage. In contrast, magnesium, zinc, and copper concentrations showed an interesting pattern: concentrations increased significantly during maturation to a maximum at ejaculation and decreased again after storage. The ratios of sodium to potassium in the midpiece region showed patterns similar to those of magnesium, zinc, and copper concentrations.     

Using RT-PCR and glutathione level to study the effect of follicular fluid on in vitro maturation and gene expression of sheep oocytes

The aim of this study is to evaluate the effect of sheep follicular fluid (SFF) supplementation of the in vitro maturation (IVM) media of sheep oocytes on the resumption of meiosis, glutathione (GSH) level, and expression of apoptosis (Bax, Bcl-2) as well as heat shock protein beta-1 (HSPB1) genes. Sheep ovaries were collected from the central slaughterhouse of Riyadh city, KSA. Oocytes were aspirated from 3 to 8 mm follicles. Sheep oocytes were cultured in maturation medium with different concentrations of sheep follicular fluid: 0% (control), 10%, 20% and 40% for 24 h. The results indicated that the maturation rate of oocytes was significantly (p ≤ .05) decreased in 40% SFF (36.87%) versus the control (61.3%), 10% SFF (63.95%) and 20% SFF (64.08%). The supplementation of the IVM medium with 10% SFF induced an intra-oocyte GSH concentration that was significantly higher than in sheep oocytes cultured with 20% and 40% SFF and similar to the GSH content in oocytes cultured without SFF. Real-time polymerase chain reaction analysis of gene expression revealed no significant differences in the Bax and HSPB1 genes between the control and 10% SFF, whereas they were significantly higher in 40% FF (p ≤ .05) compared to the control. The expression of Bax:Bcl-2 was significantly higher in 20% and 40% SFF compared to the control group. In conclusion, the addition of SFF to the IVM culture of sheep oocytes is recommended to support nuclear maturation and increase oocyte competence.     

The influence of different parental combinations and incubation temperature on the RNA and DNA content of herring larvae at hatching: a pilot study

In four half-sib pairings, herring mothers affected the standard length, dry weight, and RNA and DNA content of their progeny, while fathers affected larval RNA and DNA content. The amounts of RNA and DNA in offspring from one male were also influenced by temperature in that the highest RNA contents were found at the lowest temperature and highest DNA contents at the highest temperature. The results indicate that both environmental and genetic factors influence nucleic acid contents of young herring larvae, and this may limit the suitability of RNA : DNA ratio as a condition measure of newly hatched herring larvae.     

A case study: looking at the effects of fragmentation on genetic structure in different life history stages of old-growth mountain hemlock (Tsuga mertensiana)

We examined fine-scale genetic structure of mountain hemlock (Tsuga mertensiana) in an old-growth stand and an adjacent seedling population, with the goal of detecting the effects of fragmentation. Three hundred and six old-growth trees and 195 naturally regenerating seedlings were genotyped at 5 microsatellite loci. Genetic diversity was similar across old-growth life stages and within the clear-cut seedlings. Significant inbreeding was found in the adult class (30+ cm diameter at breast height) of old-growth seedlings and in the adjacent natural regeneration. Relatedness was significantly associated with physical distance for both the oldest age class and for regenerating seedlings in the adjacent clear-cut, whereas intermediate classes showed no such association. As intermediate classes show no isolation by distance, the associations that arise probably occur from single cohort regeneration that clearly has taken place in the clear-cut, and possibly when the oldest old-growth trees were established. Parentage analysis suggested that large-scale fragmentation, such as this clear-cut, allowed for increased long-distance seed dispersal. We conclude that long-lived tree populations can consist of a cohort mosaic, reflecting the effects of fragmentation, and resulting in a complex, age-dependent, local population structure with high levels of genetic diversity.     

Influence of copper on the early post-implantation mouse embryo: An in vivo and in vitro study

Summary Female mice were injected intravenously with copper sulphate on either the 7th day (early egg cylinder stage of development), the 8th day (late egg cylinder stage), or the 9th day (early somite stage of development), and examined on the 10th day of gestation. Injection on the 7th day was found to be embryo-lethal; when females were injected on the 8th day, the majority of the surviving embryos exhibited anomalies of the neural tube and/or the heart, while injection on the 9th day resulted in a very low incidence of anomalies. The most common malformations seen on the 10th day involved failure of closure of the neural tube in the head region of the embryo, and various types of anomalies of cardiac rotation and shape. When additional females injected on the 8th day were examined on the 12th day, a high proportion of the fetuses examined had developed exencephaly.A further group of embryos from untreated females were explanted on the 9th day and cultured in vitro in various concentrations of copper sulphate. The lowest levels tested had little obvious effect on neural tube closure. Intermediate doses resulted in, retarded and anomalous embryonic development, while the highest levels employed resulted in neural tube and cardiac anomalies similar to those produced in vivo.The results demonstrate both the direct toxic effect of copper on embryonic development and that the stage of embryonic development at the time of exposure determines both the nature and the extent of the effect.     

稻虱红单节螯蜂寄生不同虫龄白背飞虱对二者发育表现的影响

【目的】为了解稻虱红单节螯蜂Haplogonatopus apicalis与白背飞虱Sogatella furcifera间的互作关系,开展了稻虱红单节螯蜂在白背飞虱不同龄期寄生时,对寄主及其自身发育表现影响的研究。【方法】在室内25℃条件下,观察了被寄生的白背飞虱各龄若虫及其寄生蜂稻虱红单节螯蜂的发育表现。【结果】白背飞虱2, 3, 4和5龄若虫被寄生后,当龄及其后各龄的历期均显著延长;2和3龄若虫被寄生后,成虫羽化率仅分别为54.29%和60.95%,显著低于在4和5龄若虫被寄生后的成虫羽化率（分别为96.20%和100%）。稻虱红单节螯蜂寄生白背飞虱5龄若虫后的发育历期（23.77 d）显著短于寄生2龄若虫后的发育历期（27.77 d）;寄生3龄若虫的成蜂羽化率最高,为56.19%;而寄生5龄若虫的羽化雄蜂比例最高,为77.12%。【结论】稻虱红单节螯蜂寄生可使白背飞虱若虫发育历期显著延长,白背飞虱2和3龄若虫是稻虱红单节螯蜂发育的适宜寄主。