Results
N: number of dissected individuals in the respective week
0 20 40 60 80 100
2 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks 9 weeks 10 weeks 11 weeks Adult Age (pf)
Gonad type (%)
Indifferent Gonads Ovaries Altered Ovaries Testes N= 22 N= 31 N= 26 N= 32
N= 33 N= 32 N= 30 N= 35 N= 153 N= 12
Figure 1.7: Developing testes with male germ cells in different developmental stages
Fig. 1.7a: Very early testis of an 11-week-old fish with lumen formation (lu), spermatogonian cysts (cyst) and spermatogonia (ga)
Fig. 1.7b: Testis of an 8-week-old zebrafish with various male germ cells.
ga: spermatogonia lu: lumen; sc : spermatocytes , sz: spermatozoa (Page 33)
Mature gonads
Spermatogenesis:
Cells in various stages of spermatogenesis were seen in separate cysts within a single tubule along the gonadal axes of the testis of mature zebrafish (Figure 1.9).
Oocyte maturation:
The development of zebrafish oocytes is divided into five stages, based on prominent morphological features. Perinucleolar oocytes, identifiable by a few peripherally located nucleoli as well as by small, localised areas of intense basophilia in the cytoplasm, mark the beginning of the primary growth stage. The beginning of oocyte maturation is identifiable by the appearance of cortical alveoli marks (yolk vesicles). Another prominent event occurring within this cortical alveolus stage is the formation of a vitelline envelope. During the vitellogenic stage, the oocyte increases in size, primarily due to the accumulation of yolk. In histological sections the main yolk bodies appear brighter than the surrounding matrix.
During oocyte maturation, the germinal vesicle migrates toward the oocyte periphery. The nuclear envelope breaks down and the chromosomes proceed to the second meiotic prophase, at which point progress is arrested. In mature oocytes the nucleus is dissolved and the ooplasm consists of yolk bodies only. The oocyte is ready to be ovulated into the ovarian lumen. In a mature zebrafish ovary, oocytes at all stages are present, without dominant populations. After ovulation the follicle layer remains in the ovary. These postovulatory follicles are clear signs of recent spawning. Postovulatory follicles are irregularly shaped structures composed of columnar follicle cells. Those mature oocytes that have not been not ovulated are absorbed by follicle cells. The early phase of this process is characterised by the disintegration of some yolk globules and by a less regular shape. The zona radiata slowly dissolves, as indicated by a loss of striations and uneven diameter. In subsequent phases of atresia, granulosa cells enlarge and, upon rupture of the zona radiata, invade the degenerate oocyte. Figure 1.10 summarises the findings and characterisation. For a detailed description of zebrafish oocyte development see Selman et al. (1993).
9a
SD SD
200 µm swb
9b
sz
50 µm
9d
ga
spd
sz
spd
gb Lc
sc a
20 µm 9c
ga
sz
sc a spd sc b
ga
20 µm
Figure 1.9: Transverse section (3µm) through the testes of mature male zebrafish to illustrate the normal testicular organisation. The single lobules are divided by connective tissue. Figure 9a shows an overview. 9b shows a single lobule with different developmental stages in the lobule wall and spermatozoa in the lobule lumen. 9c and 9d: Transverse section through a lobule to illustrate the different spermatogenetic cell types. The solid white arrow in Fig 9c marks the breakdown of lobule structure to release the spermatozoa into the lobule lumen.
SD: sperm duct, swb: swim bladder; ga: spermatogonia A; gb: spermatogonia B; sc a:
spermatocytes A; sc b, spermatocytes B; spd: spermatids, sz: spermatozoa.
pay attention to the different scales
Figure 1.10.1 – 1.10.10: Zebrafish oocytes in different stages of development.
Fig. 1.10.1: Perinucleolar stage of primary oocyte growth. Several nucleoli appear at the periphery of the germinal vesicle (GV). During the primary growth stage, the oocyte grows from a diameter of approximately 10-20 µm to a diameter of ca. 100 µm. Although this is approximately a thousand-fold increase in oocyte volume, it is important to realise that ovaries containing oocytes in the primary growth stage only are still relatively small, having gonadosomatic indices of <2, and thus these ovaries are generally perceived as immature (Selman and Wallace 1989). Ovaries remain in this stage of oocyte development until shortly before maturing.
Fig. 1.10.2: Oocyte in advanced cortical alveolus stage. Cortical alveoli (ca) or yolk vesicles fill the oocyte cytoplasm. The germinal vesicle enlarges and becomes irregular in shape. ve:
vitelline envelope or zona radiata
Fig. 1.10.3: Enlargement of Fig. 10.2. The vitelline envelope is in turn surrounded by follicle cells.
Follicle cells are organised into an inner monolayer of granulosa cells and an outer layer of theca cells (TC). The pore channels through the zona radiata are visible. Through those pore canals oocyte microvilli contact granulosa cells (Takashima and Hibiya 1995). (ca) Cortical alveoli
Fig. 1.10.4: Vitellogenic oocyte (voc). During this major growth stage the oocyte increases in size primarily due to the accumulation of yolk. As yolk bodies accumulate centripetally within the oocyte, cortical alveoli are progressively displaced towards the periphery On both sides, oocytes in a less developed stage are seen. Coc: Cortical alveolus stage, Poc: Primary growth stage
Fig. 1.10.5: Oocyte maturation. During this stage, meiosis is reinitiated. The nucleus migrates towards the oocyte periphery (future animal pole?). The nuclear envelope breaks down. ca:
cortical alveoli, N: nucleus, ve: vitelline envelope or zona radiata Fig. 1.10.6: Enlargement of Fig.10.5. ne: nuclear envelope
Fig. 1.10.7: Mature oocyte (moc). At the end of maturation the yolk bodies lose their crystalline main bodies and develop a homogeneous interior. Prior to ovulation the follicle cells retract from the oocyte and their microvillar processes withdraw from the pore canals of the vitelline envelope (Selman et al. 1993).
Fig. 1.10.8: Postovulatory follicle (POF). The follicle layer is easy to identify. After ovulation the follicle layer remains in the ovary. These postovulatory follicles are clear signs of recent spawning. Postovulatory follicles are irregularly shaped structures composed of columnar follicle cells and an underlying connective tissue theca, and degenerate rapidly. 24h after spawning the postovulatory follicle has greatly shrunken or collapsed on itself. The prominent underlying connective tissue theca is still present but not as abundant
Fig. 1.10.9: á-stage atretic oocyte (A1): the vitelline envelope slowly dissolves, as indicated by the uneven diameter In subsequent phases of the alpha atresia, granulosa cells enlarge and, upon rupture of the vitelline envelope, invade the degenerate oocyte The alpha stage ends when the resorption of the oocyte is complete
Fig. 1.10.10: Advanced phase of á-stage atretic oocyte (A2) pay attention to the different scales. (Pages 38 and 39)
Figure 1.10, part 1: Legend see page 37
10.1
10.3 TC
ve ca
10.6
yp N
ve
ne 10.4
Voc Coc
Poc
10.5
ca
yp N
ve
100 µm 20 µm
20 µm
10.2
N ve
ca
100 µm
100 µm 50 µm
Figure 1.10, part 2: Legend see page 37 10.7
Moc
10.8
POF
10.9
A1
10.10
A2
100 µm
50 µm 50 µm
100 µm