New Techniques for Creating Parthenogenetic Larvae of the Sea Urchin Lytechinus pictus for Gene Expression Studies

Abstract

Sea urchins are model organisms for studying the spatial-temporal control of gene activity during development. The Southern California species, Lytechinus pictus, has a sequenced genome and can be raised in the laboratory from egg to egg in 4–5 months. Here, we present new techniques for generating parthenogenetic larvae of this species and include a gallery of photomicrographs of morphologically abnormal larvae that could be used for transcriptomic analysis. Comparison of gene expression in parthenogenotes to larvae produced by fertilization could provide novel insights into gene expression controls contributed by sperm in this important model organism. Knowledge gained from transcriptomics of sea urchin parthenogenotes could contribute to parthenogenetic studies of mammalian embryos.

1.0. INTRODUCTION

Between 1887 and 1940, studies of parthenogenetic activation of sea urchin eggs and their development into embryos and larvae were a significant endeavor in developmental biology. The goal of this early work was to discover how sperm activate eggs and set them on a path leading to mitosis and cellular differentiation. More than 50 physical and chemical treatments were found to activate parthenogenetic development^1–3. Eggs of various sea urchin species have different requirements for parthenogenetic activation, and years were spent developing a protocol applicable to most species. That protocol became known as “Loeb’s Double Treatment”, which involved exposure of unfertilized eggs for 1–3 min to weak butyric acid, followed by washing in normal seawater for 20 to 40 min, followed by a 15 min hypertonic treatment, and finally followed by washing and culturing in normal seawater to observe embryogenesis¹

Butyric acid activated the “cortical granule reaction”, evidenced by elevation of the fertilization envelope. The hypertonic treatment triggered the condensation of the cytaster, which nucleated microtubule polymerization to form the mitotic apparatus. In sea urchins the centrosome generating the cytaster is brought into the egg by the sperm^4–7. Success of the hypertonic treatment is independent of increased inorganic ion concentration, because hypertonic solutions of sucrose in seawater induce parthenogenetic cleavage and embryogenesis^1,8.

Lytechinus pictus is a preferred sea urchin species for mutigenerational studies because of a generation time of 4–6 months and ease of laboratory culture^9–11. Since publication of the first paper on L. pictus parthenogenotes¹¹, molecular methods have advanced to the point where transcriptional analysis such as RNAseq can be applied to single embryos, larvae, and cells^12–15. The purpose of this paper is to present new egg activation and culturing techniques to raise parthenogenic embryos and larvae of this species. To date, no molecular biology research has been applied to parthenogenetic sea urchins, probably because until recently, techniques had not advanced to the point where reasonable experimental questions could be asked. Application of transcriptome analysis to parthenogenetic sea urchins could yield new insights into the contribution of the sperm genome to control of gene expression during development.

2.0. TECHNIQUES

2.1. Spawning sea urchins

Adult Lytechinus pictus were collected near La Jolla, California, kept in flow-through aquaria and fed on kelp (Macrocystis pyrifera). Adults are spawned into seawater by injection of 0.1 ml 0.55 M KCl. Utmost care was taken to ensure that sperm do not contaminate eggs. If semen extrudes from the gonopores, syringes and hands that contacted the male are soaked for at least 1 min in excess tap water to osmotically inactivate sperm. The seawater over settled eggs is removed by aspiration and the eggs resuspended once in fresh Millipore filtered seawater (FSW) and allowed to settle. Egg jelly coats are not removed. All procedures are at 20–23C. For comparison of transcriptomes of parthenogenotes to fertilized individuals, a portion of eggs can be set aside, fertilized and cultured in petri dishes as described below.

2.2. Ionophore activation

Eggs are resuspended in 30–50 ml FSW and fields of several hundred examined with phase contrast at 100X magnification to be certain no eggs have undergone spontaneous activation as judged by elevation of the fertilization envelope. A portion of the egg suspension is poured into a 12 ml conical graduated glass tube and the eggs sedimented by gentle hand centrifugation. A settled egg pellet of 0.2–0.3 ml is resuspended in 10 ml FSW and mixed by tube inversion. The egg suspension is rapidly poured into a 100 ml glass beaker containing a 10 ul drop of Ionomycin (1 mM; dissolved in 100% dimethylsulfoxide; Calbiochem Cat.No. 407952). All times reported in this paper refer back to zero time, which is the moment of mixing eggs with ionophore. The beaker is rapidly swirled 2–3 min and poured into a 50 ml graduated conical plastic tube, which is gently hand centrifuged to settle the eggs and the FSW aspirated away. At 3–4 min the egg pellet is resuspended in 40 ml FSW. At 8, 12 and 16-min, the eggs are washed in 40 ml fresh FSW by hand centrifugation and aspiration of the FSW over the egg pellet. After the final wash the tube is capped with parafilm and mixed by hand inversion once a minute.

2.3. Hypertonic treatment

At 18 min the activated eggs in 40 ml FSW are gently sedimented, the FSW removed, and at 20 min the egg pellet resuspended in 40 ml FSW containing an extra 15g NaCl per liter (increasing the NaCl concentration of FSW by 267 mM)¹. The eggs are swirled in a beaker and at 32 min gently sedimented and the supernatant removed. At 35 min (after 15 min in hypertonic FSW) the egg pellet is resuspended in 40 ml normal FSW. The eggs are then washed twice in 40 ml FSW and resuspended to 100 ml total volume FSW.

2.4. Culturing

The 100 ml activated egg suspension is divided into five 20 ml portions that are poured into 9 cm diameter petri dishes that are covered and held at 20–23C without agitation. In 24 h, swimming mesenchyme blastulae and early gastrulae are present and are isolated using a hand operated Pasteur pipette and dissecting microscope. At least 50% of the eggs have degenerated into a mass of cytoplasmic blebs held within their fertilization envelopes. After the swimmers are collected they are resuspended in 100 ml fresh FSW and 20 ml portions poured into five petri dishes that are covered and kept at 20–23C. In approximately 2.5 days the prism/early pluteus stages have complete guts and are ready to feed on the single cell alga, Rhodomonas lens, provided at previously specified concentrations^9–11. At 2-day intervals the surviving larvae are collected using a Pasteur pipette and dissecting microscope and recultured in a new petri dish in 20 ml fresh FSW. Almost all feeding late gastrulae develop to the 4-arm early pluteus stage. The biggest die off begins about day 8–10, when the plutei transition from the 4- to the 6-arm stage^9,16. The feeding parthenogenote larvae can be distinguished from non-feeding larvae because the feeders are larger size and slightly yellowish from incorporation of algal pigments, and their midguts are light green (Fig. 1).

Fig. 1.

Photomicrographs of morphologically abnormal parthenogenetic Lytechinus pictus plutei. Number on each frame is the days in culture. Red cells contain the pigment echinochrome. C-E are the same individual. The brown inclusion in E is the dead urchin rudiment. Developmental arrest at the 4-arm stage as seen in F is common. Shapes such as those seen in J-M and O, P are also common. The guitar shaped individual (Q) and R-T plutei all came from the same egg lot. In these four, the first two large posterior arms had fused to form one large arm with two side-by-side skeletal rods. Diameter of an individual larva varies from 200–300 um.

2.5. Photography

All observations, manipulations and photography utilized a Leica M165 FC microscope with an 1.6X planapo objective lens and a Canon EOS 60D camera. To repetitively photograph the same pluteus on consecutive days, the individual is collected using a mouth or hand pipette with a minimum internal diameter of 3 times the larval diameter. The larva is released into a 200 ul drop of FSW in a 3 mm thick well slide with a well diameter of 1.8 cm and a center depth of 1.5 mm. Approximately 150 ul FSW is withdrawn. If needed, a thin glass needle is used to turn the larva over. The gradual slope of the well bottom finally stops the larva from swimming. Photographs are immediately taken and 200 ul of FSW added to the well, the larva recovered and replaced in the culture dish. Such careful treatment does not damage the larva.

3.0. RESULTS AND DISCUSSION

3.1. Ionophore activation

Sea urchin eggs are normally activated by an increase in intracellular Ca²⁺ triggered by fusion with sperm^17–19. Brandriff et al.,¹¹ were the first to use the Ca²⁺ ionophore A23187 for parthenogenetic activation of L. pictus eggs and also the first to make adult parthenogenotes of this species. During normal sea urchin egg fertilization, the increase in cytoplasmic free Ca²⁺ comes from intracellular membrane enclosed vesicles. A23187 increases intracellular Ca²⁺ by transporting this ion into eggs through the plasma membrane and also releasing it from cytoplasmic vesicles. We prefer to use Ionomycin, because unlike A23187, Ionomycin releases Ca²⁺ from intracellular stores and transports only minor amounts through the plasma membrane²⁰. At a final Ionomycin concentration of 1uM, 100% of morphologically normal fresh eggs elevate fertilization envelopes. Washing the activated eggs several times in FSW is important to remove traces of Ionomycin.

3.2. Hypertonic seawater treatment

The osmotic pressure of seawater must be increased by 50% to induce the condensation of cytasters that will form the poles of the mitotic apparatus for parthenogenetic cell division¹. The exact time following Ionomycin activation and the duration of time in hypertonic media are both important and would have to be experimentally determined for other species. Our optimal treatment with hypertonic FSW is for 15 min, from 20 to 35 min after beginning Ionomycin activation. That is a similar optimal time found for Arbacia punctulata¹. If the hypertonic treatment is only for 5 or 10 min, that is, from 20 to 25 min, or 20 to 30 min after the beginning of ionophore exposure, almost no hatched blastulae are recovered after one day of culture. If the 15 min window begins at 5, 10 or 15 min after beginning Ionomycin treatment, few parthenogenotes are recovered, in fact most show cytoplasmic fragmentation similar to that seen by Loeb¹ (in his Figures 19, 20). If the hypertonicity begins at 20 min and ends at 45 or 55 min, few live embryos are found after 1 day in culture.

We hypothesize that most morphological abnormalities of early development of parthenogenote sea urchins of this species result from the number and positioning of hypertonically-induced cytasters as they grow into one or more mitotic spindles^4–7. At the time of the first cleavage, multipolar division figures are most probably nucleated from the condensation of multiple cytasters. Parthenogenetic cytaster formation has been found to be easier and more reproducible in L. pictus eggs than in S. purpuratus⁴. The entire cytoplasm of parthenogenetic activated eggs can contain more than 100 cytasters, some of which can possess as many as eight centrioles⁴. If cytaster formation could be controlled in number and location a greater success in parthenogenetic normal development might be possible.

3.3. Development

As previously reported by all others working on parthenogenetic sea urchins, we also found that the first division of activated eggs takes approximately twice the normal time (2 hours at 21C). Division can appear completely normal, or produce blastomeres of unequal size. Division furrows can progress abnormally from only one side of the metaphase plate¹¹. Intense blebbing of the cytoplasm occurs in a high percentage of eggs, and blebs can completely bud off from the remaining egg cytoplasmic. Some eggs attempt to divide into four or more fragments identically as seen in polyspermic zygotes. As cleavage continues, a small percentage of morphologically normal 4, 8, 16-cell stages are observed with a dissecting microscope. Developmental synchrony is not high as compared to fertilized zygotes. The most frequent abnormality of hatched blastulae is the presence of loose cells in the blastocoel. Such abnormalities of cleavage in parthenogenotes of this species were previously described¹¹. As previously observed for this species, and parthenogenotes of all other sea urchin species, the percentage of morphologically normal hatched blastulae and gastrulae varies greatly with egg lot^{1–3, 8–11, 16}. We estimate that in our experiments probably only 2–5% of activated eggs become 4- armed plutei.

Figure 1 presents photomicrographs of morphologically abnormal feeding, L. pictus parthenogenote larvae. The number on each frame represents days after ionophore activation. To our knowledge there is no gallery of photomicrographs of its kind in the sea urchin literature. Many parthenogenetic blastulae, gastrulae and plutei appear almost morphologically normal, such as seen in frames C-E, which is the same individual on days 12, 16 and 18. The dark structure close to the midgut in frame E is the dead urchin rudiment. On day19 this individual was dead. Frames A and B show larvae with relatively normal morphology with one arm missing (A28) and one arm short (B12). Frame F shows a developmentally arrested 4-arm pluteus at day 37. Those surviving to the 6- and 8-arm stage can differ greatly in their rate of developmental progression, and careful observation shows that almost all have some discernable morphological abnormality⁸. The quasi-normal and grossly abnormal individuals can be picked out using a hand or mouth pipette, photographed and their RNA extracted. In parthenogenetic larvae the most frequent abnormality is loss of the first two oral arms as seen in several frames (some examples being frames A,B,F,G,&K). One egg lot produced many parthenogenote larvae in which the first pair of large posterior arms were fused together to form one large arm as seen in Fig.1, frames Q to T.

The development of this species has been well studied at the light^{9, 10} and electron microscopic^{21, 22} levels. The most morphologically advanced 8-arm parthenogenote pluteus we produced is shown with its dead rudiment in Fig1E. This individual was abnormal in that it had only one of the three pedicellaria that advanced fertilized plutei possess^{21, 22}. One technical advantage we rely on for observation of all individuals is that the FSW depth in the petri dish is so shallow as to allow dissecting microscope visual inspection of hundreds of individuals in little time.

In the history of parthenogenetic sea urchins, only three groups have reported parthenogenetic sea urchin plutei that completed metamorphosis, and two of these groups grew juvenile adults to a size where sex could be determined. Delage²³, grew six parthenogenotes of Paracentrotus lividus through metamorphosis, four died soon afterward, but two lived for months and both were males. Shearer and Lloyd⁸ spent years attempting to grow adult parthenogenotes of Echinus esculentus and succeeded in raising 15 individuals through metamorphosis. However, all their juvenile urchins died within weeks after beginning to feed and their sex remained undetermined. Their paper⁸ is significant for the beautiful drawings of 15 parthenogenetic morphologically abnormal plutei of this species, however, none are as abnormal as those in our Fig.1. Brandriff et al.¹¹ grew six L. pictus parthenogenotes through metamorphosis, and at 16 months determined that all were females. In the above examples there is no mention of using the parthenogenote gametes in backcross experiments. We made no attempt to raise parthenogenotes through metamorphosis, because it is not an objective of this techniques paper. Also, Hinegardner¹⁶, estimated that with this species, only one egg out of 10 million can be raised into a post-metamorphic feeding juvenile parthenogenote.

4.0. CONCLUSION

Cytological studies of chromosomes²⁴, biochemical studies of the activation of DNA replication²⁵ and amino acid transport²⁶ would also be interesting to study in these parthenogenotes. Although we do not present quantitative data on the percentages of parthenogenote blastula, gastrula, prism, and larval stages, every experiment we have done in the past two years has produced enough parthenogenotes to allow the isolation of individuals with specific abnormalities for transcriptome analysis^12–15. There are few approaches available for studying the sperm contribution to gene control during development in sea urchins that lack the application of classical genetics. Our techniques to create parthenogenotes of this species could lead to a deeper understanding of the sperm contribution to gene control. This is the singular overwhelming reason to promote the molecular analysis of sea urchin parthenogenotes.

There are more than 1,000 publications listed in PubMed on the subject of “parthenogenetic activation of mammalian oocytes”. These studies can be divided into two lines, one intentionally inducing parthenogenetic activation in the creation of embryonic stem cell lines, and the other dealing with spontaneous activation during in vitro fertilization procedures. Sea urchins are basal deuterostomes, at the base of the evolutionary tree leading to the vertebrates. Unhindered by ethical and practical considerations of mammalian embryo experimentation, sea urchin parthenogenotes could inform a deeper understanding of gene activation in higher organisms.

Abbreviations used:

FSW

filtered seawater