Isolation of oogonia from ovaries of the sea urchin Strongylocentrotus nudus

Abstract

The presence of oogonia in the ovaries of adult females is typical in species with a broadcast spawning reproductive strategy, including invertebrates and lower vertebrates. In sea urchins, difficulties in the study of oogonia arise from the small number of these cells and the lack of specific markers for their identification. Therefore, more reliable methods are needed for identifying and manipulating oogonial cells in quantities sufficient for experimentation. Homologs of the DEAD-box RNA helicase vasa expressed in germline cells have been proposed for use as markers to detect germline cells in diverse species. We have developed a method for the isolation of sea urchin oogonia by using immunocytochemistry with vasa antibodies, together with reverse transcription and the polymerase chain reaction to detect the expression of Sp-vasa and Sp-nanos2 homologs and a morphological approach to identify germline cells in sea urchin ovaries and cell fractions isolated from the ovarian germinal epithelium. This method has allowed us to obtain 15%-18% of small oogonia with 70%-75% purity from the total amount of isolated germ cells. Our findings represent the first methodological basis for obtaining cell populations containing sea urchin oogonia; this method might be useful as a tool for further investigations of the early stages of sea urchin oogenesis.

Molecular Cloning and Sexually Dimorphic Expression Analysis of nanos2 in the Sea Urchin, Mesocentrotus nudus

“…The efficiency of primers discovery through standard curve formulation reached 96%. Ubiquitin was used as an internal control [41]. The target genes relative expression was calculated with the 2 —ΔΔ C t method.…”

Localization of germ plasm‐related structures during sea urchin oogenesis

“…Vasa is one of components of germ plasm in many species. To confirm morphological data that described electron‐dense structures belong to germ plasm, I used antibodies specific to the sea urchin Vasa (Yakovlev et al, ). The presence of Vasa was identified by using western blot analysis on the spawned eggs and ovarian cell fractions that were enriched with either oogonia or oocytes.…”

“…Nevertheless, the molecular composition and the key players and their functions are still unknown in sea urchins. However, it is known that Vasa, one of key players in germ plasm, is present at both the RNA and protein levels during oogenesis, from oogonia to eggs, in sea urchins of the genus Strongylocentrotus (Juliano et al, ; Yakovlev et al, ) and in the sea cucumber A. japonicus (Yan et al, ). Coincidence of position of both electron‐dense organelles and Vasa suggest that the latter is a component of germ plasm‐related structures of female germ cells of sea urchins.…”

“…Cell fractions containing oogonia or oocytes were prepared as previously described (Matsumoto et al, ; Yakovlev et al, ). Eggs were washed with a Ca 2+ , Mg 2+ ‐free salt solution (CMFSS) (12 mM HEPES, pH 7.6–7.8, 385 mM NaCl, 10 mM KCl, 21 mM Na 2 SO 4 , 17 mM glucose), and settled by centrifugation (200 g for 2–3 min).…”

“…The samples were resolved on by 8% (Vasa) or 10% (α‐tubulin) SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dry milk in washing buffer (0.02% Tween 20 in PBS) for 1 hr and then incubated with 150 ng/ml affinity purified rabbit anti‐Vasa (Yakovlev et al, ) or mouse anti‐α‐tubulin (1:5,000, clone DM1A, Sigma‐Aldrich, St. Louis, MO) antibodies in washing buffer with 0.5% nonfat dry milk overnight at 4 °C. The blots were washed with washing buffer and incubated with goat anti‐rabbit or anti‐mouse antibodies conjugated to alkaline phosphatase (dilution 1:25,000, Sigma‐Aldrich, St. Louis, MO).…”