Nucleoprotein cytochemistry during oogenesis in a unisexual fish, Poecilia formosa

Summary Cytochemical methods and electron microscopy were used to study changes in the chemical composition of nuclear, nucleolar and perinuclear bodies during the early stages of oocyte development inPoecilia formosa, an apomictic species of fish that produces only female offspring. Prominent accumulations of ribonucleoprotein (RNP) occur in nucleoli and appear on either side of the nuclear envelope during diplotene. In certain planes of section, RNP material seems to be in transit across this interface.En bloc acid extractions or RNAse treatment abolished basophilia and markedly reduced the electron density of both nucleoli and cytoplasmic nucleolar-like bodies. DNA-specific fluorescent probes such as mithramycin failed to reveal nucleolar cores in poeciliid oocytes, although the same procedures showed unequivocal localization of GC-rich DNA cores within multiple nucleoli of diplotene oocytes fromXenopus laevis or the rainbow trout,Salmo gairdneri. Also, cytological hybridization studies, utilizing [³H]rRNA as a probe for nucleolar oocytes. Feulgen-stained pachytene oocytes ofP. formosa have twice the number of chromosome strands seen in similar stages of oocytes from two, related bisexual species,P. mexicana andP. latipinna. Although the bivalent nature of these chromosomes could not be resolved with the light microscope, configurations resembling, but not identical to, synaptonemal complexes were identified by electron microscopy.     

Tritiated-histidine incorporation into Drosophila salivary chromosomes

An autoradiographic study of H³-histidine incorporation into nonhistone protein of explanted larval salivary gland chromosomes of D. virilis showed patterns of incorporation that were dependent upon the stage of larval development. The sequence of changes in the development of several puffs in a specific chromosomal region was followed using the appearance of pigment in the anterior spiracles as a means of larval staging. H³-histidine incorporation into these puffs in prepupae occurred as the puffs were regressing in size and protein staining. Acid extraction of histone and nucleic acid failed to alter the character of the autographs; presumably a non-histone protein is involved in the H³-histidine incorporation. Other puff sites in the same prepupal chromosomes showed various patterns of isotopic amino acid incorporation indicating that the pattern reported for a specific region may not be true for all puff sites.     

Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.     

Genetic architecture of the cryptic species complex of Acanthocyclops vernalis (Crustacea: Copepoda). II. Crossbreeding experiments, cytogenetics, and a model of chromosomal evolution

Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within-line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n = 8 and one with 2n = 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex.     

Structure,composition and properties of naturally occurring non-calcified crustacean cuticle

Crustaceans are known for their hard, calcified exoskeleton; however some regions appear different in colour and opacity. These include leg and cheliped tips in the grapsid crab, Metopograpsus frontalis. The chelipeds and leg tips contain only trace levels of calcium but a significant mass of the halogens, chlorine (Cl) and bromine (Br). In contrast, the carapace is heavily calcified and contains only a trace mass of Cl and no Br. In transverse section across the non-calcified tip regions of cheliped and leg the mass percent of halogens varies with position. As such, the exoskeleton of M. frontalis provides a useful model to examine a possible correlation of halogen concentration with the physical properties of hardness (H) and reduced elastic modulus (E_r), within a chitin-based matrix. Previously published work suggests a correlation exists between Cl concentration and hardness in similar tissues that contain a metal (e.g. zinc). However, in M. frontalis H and E_r did not vary significantly across cheliped or leg tip despite differences in halogen concentration. The non-calcified regions of M. frontalis are less hard and less stiff than the carapace but equivalent to values found for insect cuticle lacking metals. Cheliped tips showed a complex morphological layering that differed from leg tips.