Real-Time Observation of Germinal Vesicle Migration During Oocyte Meiotic Cell Division Using Ovarian Fluorescent Transgenic Zebrafish

Introduction

Meiotic cell division of oocytes (oocyte maturation) in fish is triggered by luteinizing hormone (LH) secreted from the pituitary gland, which acts on follicle cells to induce the production of maturation-inducing hormone (MIH), 17,20β-DHP, in follicle cells (Fig. 1). In zebrafish, LH secretion occurs at midnight before spawning. ¹ It is predicted that the induced production of 17,20β-DHP will be completed around 2–3AM under our laboratory conditions, while the zebrafish are sleeping (in our lab we turn off the lights at 11PM and turn on the lights at 9AM). Then, 17,20β-DHP activates ovulation-inducing pathway in follicle cells by binding to nuclear progesterone receptor (Pgr) through autocrine or paracrine signaling. Fish follicle cell layer consists of an outer theca cell layer and an inner granulosa cell layer, where theca cells proceed to synthesize 17β-hydroxyprogesterone, the precursor of 17,20β-DHP, and granulosa cells catalyze the conversion of 17β-hydroxyprogesterone transferred from theca cells to 17,20β-DHP to complete the production of MIH, 17,20β-DHP. ²

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FIG. 1.

Induction pathway of oocyte maturation and ovulation in zebrafish. When LH secreted from the pituitary acts on follicle cells, it induces the production of MIH, 17,20β-DHP, in follicle cells. Then, 17,20β-DHP activates the ovulation-inducing pathway in follicle cells by binding to the nuclear Pgr. Secreted 17,20β-DHP also act on mPR on the cell membrane of oocytes to induce oocyte maturation. The ovulation induction pathway in follicle cells is called as the genomic pathway because Pgr bound to MIH acts as a transcription factor and activate the transcription. Contrary, oocyte maturation-inducing pathway in oocyte called as nongenomic pathway because that is activated without transcription of genes by mPR mediated intracellular signal transduction pathway. LH, luteinizing hormone; MIH, maturation-inducing hormone; mPR, membrane progesterone receptor; Pgr, progesterone receptor.

We hypothesize that the same thing happened in zebrafish. Secreted 17,20β-DHP from granulosa cells acts on membrane progesterone receptors (mPRs) on the cell membrane of oocytes to induce oocyte maturation.^3–5 Oocyte maturation is completed by 5AM on the morning of the spawning day. Then ovulation of oocytes is completed by 8AM. After light at 9AM, fish wake up and start sexual behavior for mating if females have ovulated eggs in their abdomen. After oocyte maturation, the name oocyte changes to egg. Mating continues for 1–2 h (9–11AM), so fertilization of eggs occurs during this period. ¹

Ovulation-inducing pathway in follicle cells called as genomic pathway because that is activated by transcription of genes by MIH-bound Pgr acting as transcription factor. ⁶ In contrast, oocyte maturation-inducing pathway in oocyte called as nongenomic pathway because that is activated without transcription of genes by mPR mediated intracellular signal transduction pathway. ⁷ The nucleus of the oocyte germinal vesicle (GV) is located midway between the animal pole and the center of the oocyte. Upon stimulation with 17,20β-DHP, GVs begin to move toward the animal pole (Fig. 2). After reaching at animal pole, GV will disappear by nuclear breakdown (germinal vesicle breakdown, [GVBD]) during first meiotic cell division. Oocyte continues meiotic cell division until metaphase of second meiotic cell division and then division will be stopped. During these steps, the oocyte becomes transparent as shown in the photo on the bottom right (Fig. 2).

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FIG. 2.

GV migration during oocyte maturation. The GV is located at the center of the oocyte. After stimulation by MIH, GV starts moving to the animal pole. After lysis at the animal pole, GV will disappear by nuclear breakdown GVBD onset of first meiotic cell division. Oocyte continues meiotic cell division until metaphase of second meiotic cell division and then division will be stopped. During these steps, the oocyte becomes transparent as shown in the photo on the bottom right. GV, germinal vesicle; GVBD, germinal vesicles breakdown.

The transgenic (TG) technique is well established in zebrafish. ⁸ For example, a TG with germ-specific expression of green fluorescent protein (GFP) is a useful tool to visualize germ cells produced by fusing the GFP gene to the regulatory regions of the vasa gene. ⁹

Hsiao et al. established a TG zebrafish line with a fluorescent ovary. ¹⁰ The cDNA is integrated into this strain to express enhanced green fluorescent protein (EGFP) driven by the medaka β-actin promoter; however, EGFP expression is restricted to oocytes and gills in adult fish. The nuclei (GVs) of oocytes in the TG line are fluorescent because EGFP is loosely bound to the nucleus. ¹¹

The bodies of wild zebrafish are naturally opaque and their ovaries are not externally visible. This makes noninvasive studies of the ovaries difficult, even in the TG line. Therefore, we tried to produce transparent zebrafish with fluorescent ovaries by crossing the wild-type TG fish and transparent strain, roy.

We succeeded in establishing a TG line with transparent bodies and ovarian fluorescence (named β-roy). ¹² Using the β-roy fish, we successfully monitored the changes in the ovaries during a sex reversal experiment. ¹³ In this study, we used oocytes of the strain to monitor the migration of GVs during meiotic cell division. As expected, we were able to observe GV migration in real time. We believe that the strain can be an instructive source for meiotic cell division and the dynamics of movement of materials in cells by microfilaments.

Materials and Methods

Results and Discussion

We tried real-time observation of GV migration using oocytes of ovarian fluorescent TG zebrafish, β-roy (Fig. 3). GVs in oocytes of β-roy grow more strongly that cytoplasm by GFP fluorescent. After stimulation by 17,20β-DHP, the migration of GV could be monitored under the microscope until the completion of GVBD. Fluorescence images were taken at 5-min intervals (Fig. 4). At high magnification, micropyle could be observed on the animal pole (Fig. 5). The micropyle is a tubular structure formed during oogenesis at the animal pole of fish oocytes as an entry point for spermatozoa. The micropyle is closed when a single sperm enters the oocyte and is thought to play a role in polyspermy rejection.

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FIG. 3.

GV of β-roy can be observed by fluorescence of EGFP. Ovaries of β-roy female grow by EGFP fluorescence in live zebrafish as shown in panel (A). When oocytes are dissected from ovaries, GVs of oocytes are observed by fluorescence (B). A GV in an oocyte is indicated by a white arrow. Scale bar = 0.5 mm. EGFP, enhanced green fluorescent protein.

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FIG. 4.

Real-time observation of GV migration during oocyte maturation. After stimulation with MIH, GV in the oocyte was photographed under GFP filter views at 5-min intervals. In this oocyte, GVBD occurred during 175 to 185 min after the addition of MIH. Photographs of the oocyte under bright field before and after incubation are also shown in the upper left and lower right. GFP, green fluorescent protein

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FIG. 5.

Enlarged image of EGFP-expressing oocyte of β-roy. GFP filter views of β-roy oocyte during GV migration were photographed. The micropyle can be seen at the center of the animal pole. GV are migrating toward micropyle. There is a gradient of contents inside the oocyte from vegetal pole to animal pole and vice versa as indicated by an arrow. A movie prepared by stacking photographs during GV migration is available as Supplemental Movie.

We prepared a short time movie by stacking the photos before and after GVBD (Supplementary Movie S1). In the movie, migration of GV toward the micropyle and GVBD after reaching the animal pole can be observed. We also tried to observe actin filaments, which are shown to make the force of GV movement in goldfish. ¹⁷ By injecting fluorescence-labeled phalloidin into oocytes during oocyte maturation, we were able to stain the actin filaments formed during GV migration. Actin fibers were formed as they surrounded the GV during migration (Fig. 6A). Also, spreading actin filaments surrounding GVs were observed immediately after GVBD (Fig. 6B). This localization of actin fibers suggests that bundles of actin fibers move the GV to the animal pole in zebrafish.

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FIG. 6.

Actin filaments surrounding GV during migration. Actin filaments in oocytes were visualized with Acti-stain™ 488 fluorescent phalloidin. GFP filter views of Roy's oocyte during GV migration were photographed. GV is surrounded by actin filaments during migration (A). Actin filaments spread around the animal pole after GVBD (B). A GV in the oocyte is indicated by the white arrow. The green fluorescence in this figure is from fluorescent phalloidin but not from GFP. Scale bar = 0.1 mm.

In this article, we presented the use of our TG strain to visualize the movement of the nucleus of zebrafish oocytes. The strain is fertile and capable of maintaining a population. In the β-roy fish, the ovaries in living fish are visible from the outside of the body with the naked eye or with a fluorescence microscope. Although not shown in this study, this fish model has useful research applications because it allows continuous observation of the internal organs of the same individual throughout its life, as demonstrated by the observation of EGFP expression during development. Thus, this strain provides a useful alternative model for noninvasive studies of normal and pathological changes in gonadal structures at later stages of life and the genesis of other diseases and changes caused by exposure to chemical substances. ¹³

Finally, the following tables show how to observe this GV migration in a teaching laboratory (Table 1) and tips for using the laboratory course in biology education (Table 2).