We previously demonstrated that sperm heads from amphibians (Xenopus và Rana) & zebrafish (Danio) could size giant lampbrush chromosomes when injected into the nucleus of amphibian oocytes. However, similar experiments with mammalian sperm heads were unsuccessful. Here we describe a slightly modified procedure and demonstrate that human sperm heads can size giant lampbrush chromosomes when injected into the oocyte nucleus of the frog Xenopus laevis or the newt Notophthalmus viridescens. Human & other mammalian chromosomes do not form recognizable lampbrush chromosomes in their own oocytes or in any somatic cells. These experiments thus demonstrate that the lampbrush condition is an inducible state & that the amphibian oocyte nucleus contains all factors required to remodel the inactive chromatin of a mammalian sperm into a transcriptionally active state. They also demonstrate that absence of lampbrush chromosomes from human oocytes must relate to lớn specific features of mammalian oogenesis, not to permanent genetic or epigenetic changes in the chromatin.
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Keywords: chromatin, oocyte, reprogramming, RNA polymerase II, sperm, transcription, Xenopus
Introduction
Lampbrush chromosomes (LBCs) are giant meiotic chromosomes first described more than 100 years ago from the oocyte nucleus or germinal vesicle (GV) of the axolotl (Flemming 1882) and a shark (Rückert 1892). LBCs occur in many species, including vertebrates, invertebrates, & even the single-celled alga Acetabularia, but they are best characterized from amphibian & avian oocytes (Callan and Lloyd 1960; Morgan 2002; Gall et al. 2004; Gaginskaya et al. 2009). Each homologue has an axis of chromomeres corresponding lớn transcriptionally inactive chromatin, from which transcriptionally active pairs of loops extend laterally. A coating of nascent ribonucleoprotein (RNP) fibrils makes these lateral loops visible by conventional light microscopy. Because of their enormous size, LBCs provide an ideal system in which khổng lồ study transcription, RNA processing & other general features of chromosome organization. The overall organization and significance of LBCs were summarized in the classic monograph by Mick Callan (Callan 1986) (see also www.projects.exeter.ac.uk/lampbrush).
In a previous study, we demonstrated that demembranated sperm heads from the frogs Xenopus and Rana, & the zebrafish Danio can size LBCs when injected into the GV of Xenopus laevis (Gall & Murphy 1998). Although they are unreplicated single chromatids, sperm LBCs are similar to lớn endogenous lampbrush bivalents in morphology & immunofluorescent staining properties. The induction of sperm LBCs in amphibian GVs provides a useful system for identifying cis- & trans-acting factors required for converting condensed chromatin into a transcriptionally active form. In our earlier experiments we failed to lớn induce LBCs from mammalian sperm heads. It was unclear whether the failure was due to lớn technical issues or lớn more fundamental differences between amphibian & mammalian chromatin. Because mammalian chromosomes vị not size recognizable LBCs during meiosis or in any somatic cells, one could postulate that mammalian chromatin is unable to assume the LBC condition. Here we describe new experiments under slightly different conditions in which human sperm heads give rise khổng lồ transcriptionally active LBCs when injected into the GV of the frog X. Laevis or the newt Notophthalmus viridescens. These experiments demonstrate that the amphibian GV contains all factors required to lớn reprogram inactive mammalian chromatin into a transcriptionally active state. Thus the absence of LBCs from mammalian oocytes must relate to lớn specific aspects of mammalian oogenesis và not to lớn permanent genetic or epigenetic features of mammalian chromatin.
Materials and Methods
Oocytes & LBC spreads
Adult frogs X. Laevis were purchased from Xenopus 1 (Dexter, MI) và adult newts N. Viridescens from the Sullivan Company (Nashville, TN). Oocytes were held at 16–18°C or 24°C in a small Petri dish of OR2 saline (Wallace et al. 1973). Chromosome spreads were prepared from individual GVs as described previously (Gall và Wu 2010).
Oocyte injections
Sperm heads of Xenopus laevis were prepared from testes as described earlier (Newmeyer and Wilson 1991). A sample of normal human sperm was obtained from the Johns Hopkins University Medical School. The research was deemed by the Johns Hopkins Institutional đánh giá Board to qualify for exemption under category (4) of 45 CFR 46.101(b). The sperm tails were removed by sonication & the heads were concentrated by centrifugation. The technique for injection of human sperm heads into the GV was basically as described previously for experiments with Xenopus, Rana, và Danio (Gall & Murphy 1998) with four relatively minor changes. Although the effect of any one of these changes is difficult to lớn assess, the new protocol now gives a higher survival rate and allows us lớn hold oocytes for longer periods before observing the GV contents. A major difference between Xenopus and human sperm heads is the longer time needed for the human sperm heads lớn generate recognizable LBCs.
First, in our previous study we injected sperm heads that had been demembranated with lysolecithin, as originally described by Gurdon (Gurdon 1976). In the experiments described here, we found that intact sperm heads started swelling and eventually resolved into individual LBCs at a pace comparable khổng lồ that of demembranated sperm heads. For this reason, we omitted the lysolecithin step.
Second, we did not defolliculate the oocytes with collagenase before injection. Defolliculated oocytes are softer & easier to penetrate with the injection needle, but are more fragile & prone to lớn contamination. By using sharp needles with smaller tips, we managed to inject oocytes successfully without defolliculation.
Third, we did not centrifuge the oocytes before injection. The major purpose of centrifugation is lớn bring the GV khổng lồ the surface, where its position can be detected as a depigmented area. With practice, one can inject sperm heads into the GV even when it lies deeper within the oocyte.
Finally, in earlier experiments we found that the number of sperm heads actually injected decreased dramatically during the course of injecting multiple oocytes. We reasoned that this might be due khổng lồ adherence of sperm heads khổng lồ the inside wall of the needle. Khổng lồ prevent such sticking, we added 2–5% polyvinylpyrrolidone (PVP) khổng lồ the sperm suspension. Subsequently, we found that the number of sperm actually injected remained relatively constant during the course of an experiment. PVP has been used routinely in intracytoplasmic sperm injections involving mammalian sperm and oocytes.
After improving the injection and oocyte handling protocol, we were able lớn keep injected oocytes alive for several days. The longer incubation time is critical because human sperm heads expand more slowly than Xenopus sperm heads. In our previous experiments most of the injected oocytes were beginning khổng lồ degenerate before the human sperm heads had expanded.
Immunofluorescence staining and microscopy
GV spreads were stained with antibodies as described previously (Gall và Murphy 1998) with the following modifications. Samples were blocked with 10% horse serum for 30 min before incubation in primary antibody for 4–12 h at room temperature. Primary antibodies used in this study include mouse m
Ab H14 against Pol II (Bregman et al. 1995); mouse m
Ab Y12 against the Sm “epitope” (symmetric dimethylarginine) (Lerner et al. 1981; Brahms et al. 2000); & mouse m
Ab H1 against Xenopus coilin (Tuma et al. 1993). Secondary antibodies were Alexa 488- or Alexa 594-conjugated goat anti-mouse Ig
G or Ig
M (Molecular Probes, Eugene, OR). They were used together with 0.01 μg/ml 4′,6 diamidino-2-phenylindole (DAPI) for 4–12 h at room temperature. Slides were mounted in 50% glycerol. Specimens were examined with a Zeiss 63X 1.25 N.A. Planapo lens on the Zeiss Axioplan fluorescence microscope. Images were captured with a Micromax charge-coupled device camera (Princeton Instruments, Trenton, NJ) using the IPLab (3.5.5) image acquisition and analysis program (Scanalytics, Fairfax, VA).
Injection of human sperm heads into the Xenopus GV
In earlier experiments we injected sperm heads from Xenopus, Rana, & Danio into Xenopus GVs và saw the formation of morphologically typical LBCs from the originally highly condensed chromatin. The entire process usually required about 24 h for completion. Similar experiments with mouse và human sperm heads were unsuccessful for unknown reasons. To chạy thử whether the failure of human sperm heads to form LBCs was due to lớn technical issues or lớn more fundamental incompatibility between mammalian and amphibian species, we performed new injections but modified the conditions of the experiment. Specifically, we extended the time period over which the observations were made from about 24 hr khổng lồ several days. Oocytes injected with human sperm heads were incubated in OR2 medium at 16–18°C. Similar lớn Xenopus sperm, the human sperm heads swelled within 3–6 h after injection. At this time Xenopus sperm heads begin to lớn stain with m
Ab H14 against the phosphorylated C-terminal domain name of pol II, indicative of pol II uptake from the nucleoplasm. However, the human sperm heads did not stain at this time, but instead began lớn vacuolate. Over the next 15–20 h, the swollen human sperm heads took on a more open configuration, often with prominent nuclear bodies on the surface. At this stage, we could detect staining with m
Ab H14 (Fig. 1a–c). Thus, it seems that a major difference between the Xenopus and human sperm heads is the time needed for physical expansion & uptake of pol II. Eventually human sperm heads resolved into loose clusters of fuzzy threads about 40–48 h after injection (Figure 1d–f). At still later stages these loose clusters fell apart to khung individual DAPI-positive chromosomes with intense pol II staining (Fig. 1g–i). In favourable cases the number of individual chromosomes approximated the human haploid number of 23 (Fig. 1h). Although these chromosomes did not display obvious loops, their fuzzy appearance và staining with an antibody against pol II strongly suggested that they were transcriptionally active.
Expansion of human sperm heads injected into the X. Laevis GV. A–c Two sperm heads 19 h after injection have swollen và begun lớn take up pol II. Khổng lồ the left of the sperm heads is part of one endogenous Xenopus LBC. The DAPI stain is much brighter in the human sperm heads, because each contains a complete haploid chromosome mix (3.5 pg DNA). The entire endogenous X. Laevis LBC phối contains roughly 4X as much DNA but spread over 18 extended bivalents. D–f Three sperm heads 42 h after injection are slightly more swollen. An occasional chromosome (arrow) can be found separate from the clusters. G–i A single sperm head 63 h after injection has now resolved into a group of separate chromosomes, presumably the haploid number (23) of univalent chromosomes. Although the chromosomes are covered with pol II, individual transcription loops are not readily visible.
Temperature accelerates the time for formation of LBCs
Because the formation of LBCs from human sperm heads was slow, we reasoned that we could tốc độ up the process by increasing the temperature at which the oocytes were incubated after injection. We injected human sperm heads into Xenopus GVs, và then divided the injected oocytes into two groups for incubation at two different temperatures, 16°C–18°C & 24°C. At 44 hr after injection, sperm heads in oocytes held at 16°C–18°C were still condensed (Fig. 2a–c). In contrast, in the group incubated at 24°C, long transcription loops extended from the sperm clusters, even though the clusters as a whole were still compact và had not resolved into individual chromosomes (Fig. 2d–f). These loops occasionally exhibited three stereotypical features of LBC loops: first, the RNP matrix had a thin-to-thick morphology indicative of the direction of transcription (arrows, Fig. 2d); second, the pol II staining appeared as a line of uniform thickness from one over of the loop lớn the other (Fig. 2e); and finally the loop was DAPI-negative because its DNA axis is so highly extended (Fig. 2f). The thickness of the pol II line is about 0.4 μm & presumably represents the diffraction-limited image of polymerase molecules closely spaced along the DNA axis of the loop (Miller & Hamkalo 1972)
Effect of temperature on expansion of sperm heads. A–c A single sperm head 44 h after injection showed minimal expansion when the recipient oocyte was held at 16°C. Lưu ý the associated nuclear bodies with a relatively unstained core & a surrounding shell that stains strongly with m
Ab Y12 against the “Sm” epitope (symmetric dimethylarginine). D–f A single sperm head 42 h after injection was more expanded when the recipient oocyte was held at 24°C. One very long transcription loop extends out from the central cluster. The phase contrast image (a) shows the characteristic “thin-to-thick” loop matrix of ribonucleoprotein, which indicates the direction of transcription (arrows). The pol II antibody (b) shows uniform staining along the entire loop, presumably due khổng lồ close packing of pol II molecules on the DNA axis. The axis itself is not detectable by DAPI staining (c) because of its extreme attenuation.
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Injection of human sperm heads into the newt GV
The most characteristic feature of LBCs is the presence of lateral loops, which consist of either single transcription units (one “thin-to thick” region) or multiple transcription units (two or more such regions)(Gall et al. 1983). Although it was clear from injection of human sperm heads into Xenopus oocytes at 24°C that transcriptionally active LBC loops could be formed, most of the loops were quite short, manifested primarily as a general fuzziness along the chromosome axis. In our earlier experiments with Xenopus sperm heads we noted that the loops on induced LBCs were remarkably large when a heterologous injection was made into the GV of the newt Notophthalmus. Therefore, we carried out a similar experiment by injecting human sperm heads into newt oocytes. The results were dramatic. Not only did the induced human LBCs form more quickly in the newt nuclei, but they were larger và their loops were especially prominent (Fig. 3).
Formation of human LBCs from sperm heads injected into the GV of the newt Notophthalmus. A–c A single sperm head 25 h after injection has resolved into a loose cluster of individual chromatids that show active transcription (pol II stain in c). D–f A single human chromatid with an overall length of about 60 μm. Note the long transcription loops (arrow) visible by phase contrast (d) và by staining with an antibody against pol II (f). DAPI staining reveals condensed chromatin along the chromosome axis (e).
Association of nuclear bodies with human LBCs
At an early stage in their expansion human sperm heads become associated with one or more spherical, phase-dark bodies up to lớn 6–7 μm in diameter (Fig. 2a và and4a).4a). An antibody against the protein coilin (m
Ab H1) showed preferential staining of a thin rim on the periphery of these bodies (Fig. 4g), as did an antibody against symmetric dimethylarginine (m
Ab Y12) (Figs. 2b and and4c).4c). In favourable cases where the human LBCs were individually recognizable, one could see that the spherical bodies were attached directly to the DAPI-positive axis of the chromosomes (Fig. 4e and g, arrowheads). Both the staining pattern và the attachment khổng lồ the LBCs is highly reminiscent of the newly described “pearls” of X. Laevis and X. Tropicalis (Nizami & Gall, this volume). Pearls have a coilin-positive rim surrounding a core that contains U85 sca
RNA & U3 sno
RNA. Pearls are attached to chromosomal loci that stain with antibodies against pol III và they disappear when oocytes are treated with inhibiters of pol III activity. Further study will be needed khổng lồ confirm that the bodies associated with human LBCs are the same as pearls.
Association of nuclear bodies with human LBCs. A–c At an early stage of expansion the human sperm heads are often associated with spherical nuclear bodies (a) whose periphery stains strongly with m
Ab Y12 against the “Sm” epitope (symmetric dimethylarginine) (c). D–g When the sperm head is more expanded and individual chromosomes are distinguishable, one can see that the nuclear bodies are attached to the DNA axis of the chromosomes (arrowheads in e và g). An antibody against the protein coilin preferentially stains the cortex of the bodies (g).
It is also significant that the bodies associated with the human LBCs stain with anti-coilin m
Ab H1. This antibody is highly species-specific for Xenopus, not staining coilin in either newt or human cells. Thus it is clear that the coilin in the nuclear bodies associated with the human LBCs is Xenopus coilin. In our earlier injection experiments we showed that the loops on Xenopus LBCs derived by injection of Xenopus sperm heads into newt GVs stained strongly with an antibody against a newt-specific protein. Both examples demonstrate that endogenous proteins are used for assembly of the induced LBCs & associated bodies.
Discussion
The major finding of this study is that condensed human chromatin from sperm heads is able to khung typical LBCs when placed in the environment of the amphibian oocyte nucleus. Although a formal definition of a LBC is difficult to lớn make, for purposes of the discussion here we mean a giant chromosome with transcriptionally active lateral loops visible by conventional light microscopy. Our earlier experiments involved a detailed analysis of the LBCs formed when X. Laevis sperm heads were injected into the GV of the same species. We showed that the induced LBCs were identical lớn endogenous LBCs in all essential respects, with the exception that they consisted of single, unpaired chromatids rather than meiotic bivalents. We also showed that sperm heads of the frog Rana pipiens và the zebrafish Danio rerio transformed into recognizable univalent LBCs when injected into the GV of X. Laevis. However, experiments with cricket (Acheta domesticus), mouse (Mus musculus), and human sperm heads were unsuccessful, leaving unanswered the question whether the source of the inactive chromatin is important. Our original experiments involved three species that normally have LBCs in their oocyte nuclei, whereas human chromosomes bởi not go through a LBC stage during oocyte development (or at any other time). Our experiments demonstrate that the absence of LBCs from mammalian oocytes must be due lớn specific features of mammalian oogenesis & not lớn permanent genetic or epigenetic features of mammalian chromatin.
Now that we have obtained positive results with human sperm heads, we feel confident in predicting that chromatin from essentially any sperm or germ line source can be converted to lớn the LBC state, so long as it can be made accessible to factors in the GV. Whether chromatin from fully differentiated somatic cells can be similarly converted lớn the LBC state remains lớn be demonstrated.
The importance of factors in the GV, as opposed khổng lồ the source of the chromatin, is underscored by comparing injections into frog and newt GVs. In our earlier experiments we showed that induced Xenopus LBCs were larger and had more prominent lateral loops when formed in the newt GV (Notophthalmus) than in the endogenous frog GV (Xenopus). The same is true in our current experiments with human LBCs. A comparison of the LBCs in Figs. 1 and and44 with those in Fig. 3 shows a dramatic difference. It is well known that LBCs of the newt are much larger than those of Xenopus, và it has frequently been assumed that this difference is related khổng lồ the large difference in total genomic DNA of the two species (3 pg versus 35 pg in the haploid genome). Our injection experiments raise the interesting possibility that at least some of the differences in LBC form size are related khổng lồ specific factors in the GV, not simply khổng lồ differences in genomic DNA content.
At present we have very little information concerning the endogenous factors in the GV that are involved. We know that pol II goes onto the chromatin at an early stage, but we assume that the chromatin itself must be modified before transcription can begin. The GV và its giant chromosomes provide a uniquely favourable system in which to lớn study both the changes that occur during reprogramming of the chromatin & the factors in the nucleoplasm that are responsible for the reprogramming.
The ideas presented here owe much khổng lồ discussions with Svetlana Deryusheva, Garry Morgan, Zehra Nizami, & Jun Wei Pek. This work was supported by research grant R01 GM33397 from the National Institute of General Medical Sciences of the National Institutes of Health. The nội dung is solely the responsibility of the authors và does not necessarily represent the official views of the National Institutes of Health. JGG is American Cancer Society Professor of Developmental Genetics.
Genetics includes the study of heredity, or how traits are passed from parents lớn offspring. The topics of genetics vary & are constantly changing as we learn more about the genome and how we are influenced by our genes.
Inheritance Patterns and Punnett Squares
Notes và Slides on Mendelian Genetics – basic lesson lớn introduce genetics và probability
Simple Genetics Practice– using mendelian genetics và Punnett squares
Peas, Please – practice setting up squares for basic Mendelian traits in pea plants (Key, Tp
T)
How lớn Solve Dihybrid Crosses – step by step guide on setting up 4×4 squares and determining ratios
Advanced Bunny Genetic Crosses with two traits – also includes fruit fly genetics
Bunny Genetics 2 – basic crosses, rabbits have gray or trắng hair, black or red eyes
Practice Punnett Squares with Skinny Pigs – hairless guinea pigs are the result of a recessive ren (Key, Tp
T)
Horse Genetics – basic practice worksheet on monohybrid and dihybrid crosses, using a gait trait found in horses
Fruit Fly Genetics (Vg) – practice worksheet on vestigial wing flies và eye color, which is sex-linked (Key, Tp
T)
Genetics Practice Problems – crosses involving guinea pigs, goats, and pea plants; monohybrid và dihybrid crosses
Explore the Genetics of Corn Snakes – dihybrid crosses with corn snakes, màu sắc is polygenic
Blood Disorder Genetics – a worksheet with genetics problems that relate khổng lồ specific disorders: sickle cell anemia, hemophilia, và Von Willebrand disease.
Heredity Wordsearch – fill in the blank, find the words on a puzzle, basic vocabulary
Genetics nhận xét Guide – focus on vocabulary, Mendel’s crosses, và practice genetics with Punnett squares
Simulations and Hands-on ActivitiesPenny Genetics – flip a coin khổng lồ compare actual outcomes versus predicted outcomes from a Punnett square
Heredity Simulation – use popsicle sticks khổng lồ show how alleles are inherited
Variations on a Human Face – toss a penny khổng lồ determine the features of a face, such as freckles, dimples; then draw that face.
Paper Pets – another simulation using paper models with traits for eyes, nose, mouth, và hair.
Codominance, Multiple Alleles, and Sex-Linkage
Codominance and Incomplete Dominance – practice genetics on roan cows and pink flowers
Cow Genetics – roan coloration và horns, also includes a two-trait cross
Slides and Notes on Sex-Linked Genes
Fruit Flies and X-Linked Traits – practice crosses that involve sex-linkage; fruit flies và calico cats
Sex Linked Traits – students practice doing crosses; tortoiseshell cats, colorblindness, and eye màu sắc in fruit flies
Drosophila (Fruit fly) Simulation – use a simulator lớn run crosses with flies khổng lồ determine phenotype ratios & perform bỏ ra square analysis
Calico Cats & X Linked Traits – more practice with sex-linked traits, see also advanced version that goes into tortoiseshell vs calico
Why Are There No Male Calico Cats – case study & slide presentation that explore X inactivation & coat color in cats
Epistasis in Labrador Retrievers – explore the black (B_E_) , yellow (B_ee) , và brown (bb
E_ ) màu sắc patterns in dogs
Sexy Chickens – inheritance patterns in birds, using the ZW sex determination concept
Blood Type Genetics – practice with blood type crosses và other ABO type alleles, multiple allele traits
Multiple Allele Traits in Chickens – shows how combs are inherited (rrpp x RRpp)
Frizzle Frazzled Chickens – incomplete dominance in feather types, with double dominant being unhealthy
How
Do Genes Determine Skin Color? – this case study uses presentation slides to explore a real-life scenario where a Nigerian mother has two very fair skinned children.
Design-a-Species – using the rules of inheritance (Mendel), create an organism; with dominant & recessive traits, multiple allele traits, và codominance
Genetics Project – Create a species and show how its traits are influenced by genetics
Genetics Overview for AP Biology – summarizes the various types of crosses students may encounter on the AP test
Genetics & Statistics
Hardy-Weinberg Problem mix – statistical analysis, using HW equation & some dragons
Hardy Weinberg Simulation – track an allele in population by simulating how parents pass alleles lớn offspring
Corn Genetics & Chi Square – statistical analysis, using preserved corn & counting kernels
Albino Corn Genetics – grow corn, 3:1 albino ratio, lab report analyzes F1, F2 crosses
Chi Square Modeling Using Candy – count the number of each color in a bag lớn determine if they occur in equal proportions
Drosophila Virtual Lab – choose fruit fly parents and analyze offspring in this virtual lab
Genetics of Wisconsin Fast Plants – grow plants from seeds và analyze phenotype; perform bỏ ra square analysis
Human Genetics
Case Study – Cystic Fibrosis Mutations
Cystic Fibrosis and Cell Membrane Transport
SRY not SRY – case study on the sex determining region of the Y chromosomes
Case Study – How vì Genes Determine Skin Color
Analyzing Human Pedigrees
Human Genetics Overview
Your Genes Your Choices– this is a more involved group assignment where groups read scenarios about genetic testing & ethics involved.
Genetic Science Ethics– survey as a group ethical questions involved genetics (cloning, gen therapy..)
Name the gen – Explore Genes with BLAST – submit gene sequences to determine the related human trait
