Oocyte Development and Staging in the Florida Bass, Micropterus floridanus (LeSueur, 1822), with Comments on the Evolution of Pelagic and Demersal Eggs in Bony Fishes
Oocyte development and staging is described from histological preparation of the ovaries of field collected specimens of the Florida Bass, Micropterus floridanus. Oocyte development in Florida Bass progresses through six stages beginning with oogonial proliferation and ending with ovulation, the release of eggs. The stages are based on mitosis and meiosis, the core processes of Adaptable Oocyte Staging. After ovulation, the postovulatory follicle complex produces a mucus-like fluid with unknown function; we speculate that it protects the eggs immediately upon ovulation and upon their release into freshwater during spawning. Atresia is rare in the ovaries of reproductive Florida Bass. The release of cortical alveoli after fertilization follows a unique, extended time-release sequence. Oocyte development in the freshwater perciform Florida Bass follows precisely a pattern that has been documented for marine perciform fishes that produce positively buoyant, pelagic, non-adhesive eggs. The eggs of Florida Bass are morphologically like these pelagic eggs in that they have a single large oil globule and no attachment filaments. Yet Florida Bass eggs have small, clear yolk globules and an adhesive egg envelope and are functionally demersal. Thus, classification of an egg as pelagic or demersal should consider its morphology as well as its function, especially if egg type is to be used to interpret phylogenetic relationships or evolutionary patterns. The clearing of yolk due to the uptake of water during the development of actinopterygian eggs, as they progress from a demersal to a potentially pelagic phase, may be a general mechanism of oocyte maturation in teleost fishes.
Micropterus floridanus. (A) Ovary transverse section to illustrate basic morphology, here in a regressed female collected in September. The ovary is suspended from the dorsal body wall by the mesovarium. The encompassing ovarian wall has multiple extensions (arrow) into the lamellae. Primary growth oocytes are on either side of these extensions. Spherical, clear spaces (one is marked *) are oocyte atresia remnants from the previous breeding season. PAS/MY, Bar = 500 μm. (B) The principal oocytes in the ovary have perinucleolar nuclei (nu); small PAS-positive “granules” in the ooplasm indicates that they are in primary growth and have developing cortical alveoli. Two primary growth oocytes, each with one nucleolus, are visible. A germinal epithelium (GE) lines the ovarian lamella and borders the ovarian lumen. Within the GE are oocyte cell nests and two chromatin nucleolus, early diplotene oocytes. Long arrow in upper left points to extension of the germinal epithelium into the stroma. PAS/MY, Bar = 20 μm. (C) The edge of an ovarian lamella illustrates pachytene oocytes in the Chromatin Nucleolus Stage of development. A one nucleolus, primary growth oocyte is within the germinal epithelium that is separated from stroma by a black basement membrane and from the ovarian lumen by epithelial cells that become prefollicle cells when associated with oocytes. A blood vessel underlies the germinal epithelium. RET, Bar = 10 μm. bb, Balbiani body; BMGE, germinal epithelium basement membrane; BV, blood vessels; ca, cortical alveoli; CN, cells nest; CNed, Chromatin Nucleolus Stage, early diplotene step oocyte; CNp, Chromatin Nucleolus Stage, pachytene step oocyte; E, epithelial cells; LA, lamella; MO, mesovarium; nu, single nucleolus; OL, ovarian lumen; PF, prefollicle cell; PGca, Primary Growth Stage, cortical alveolar step oocyte; PGon, Primary Growth Stage, one nucleolus step oocyte.
Micropterus floridanus. (A) The germinal epithelium is the border between ovarian lamellae and the ovarian lumen. Two oogonia are observed within the germinal epithelium, and dark-staining cell nuclei on either side are either epithelial cells or prefollicle cells. PAS/MY, Bar = 10 μm. (B) A cell nest containing numerous pachytene oocytes in the Chromatin Nucleolus Stage is separated from the ovarian lumen by prefollicle cells. The basement membrane subtending the cell nest is PAS-positive, but hardly resolved. A primary growth oocyte and an oocyte in the cortical alveolar step of primary growth, with PAS-positive cortical alveoli, are visible. PAS/MY, Bar = 10 μm. (C) After pachytene, oocytes enter the early diplotene step in the Chromatin Nucleolus Stage (CNed). A single CNed oocyte is observed associated with a prefollicle cell. Numerous fibrils, reticular fibers, and two primary growth oocytes with basophilic ooplasm are observed. PAS/MY, Bar = 10 μm. (D) Folliculogenesis is the process by which developing, primary growth oocytes are separated from the germinal epithelium by a basement membrane. The developing follicle maintains contact with the germinal epithelium containing epithelial cells and those that have associated with other primary growth oocytes to become prefollicle cells. The fully-formed, primary growth oocyte in a follicle is surrounded by a basement membrane, and the germinal epithelium is subtended by a basement membrane. Ooplasm of two primary growth oocytes in the germinal epithelium (each *) are sectioned tangentially. When the basement membrane completely encloses the developing follicle (between pair of arrows), a follicle is formed and its prefollicle cells become follicle cells. RET, Bar = 10 μm. (E) A Primary Growth Stage, one nucleolus step oocyte has a spherical germinal vesicle that is rimmed with ribosomal ribonucleic acid (rRNA). The oocyte is surrounded by a basement membrane that appears to be attached to the germinal epithelium by reticular fibers that emanate from the basement membrane of the germinal epithelium. RET, Bar = 10 μm. BM, basement membrane; BMF, follicle basement membrane; BMGE, germinal epithelium basement membrane; ca, cortical alveoli; CNed, Chromatin Nucleolus Stage, early diplotene step oocyte; CNp, Chromatin Nucleolus Stage, pachytene step oocytes; E, epithelial cells of the germinal epithelium; EVS, extravascular space; GE, germinal epithelium; gv, germinal vesicle; nu, nucleolus; OG, oogonia; OL, Ovarian lumen; PF, prefollicle cells; PG, Primary Growth oocyte; PGca, Primary Growth Stage, cortical alveolar step oocytes; PGon, Primary Growth oocyte with one nucleolus; R, reticular fibers; ST, stroma.
Micropterus floridanus. (A) As primary growth proceeds, nucleoli within the germinal vesicle become numerous; they originate from division (arrow) of the larger nucleoli. RET, Bar= 10 μm. (B) Initially, oocytes in the perinucleolar step of primary growth have a germinal vesicle with a smooth contour. A smaller nucleolus is separating (arrow) from another nucleolus. Clearer areas in the ooplasm (*) are probably due to breakup of the Balbiani body. RET, Bar = 10 μm. (C) By December, full-grown, cortical alveolar oocytes represent a major component in the ovarian lamellae and have large, PAS-positive cortical alveoli. Oil droplets surround the germinal vesicle. Stroma contains numerous cells and the extravascular space. PAS/MY, Bar = 100 μm. ca, cortical alveoli; EVS, extravascular space; gv, germinal vesicle; nu, nucleoli; od, oil droplets; OL, ovarian lumen; PGpn, Primary Growth Stage, perinucleolar step oocyte; ST, stroma.
Micropterus floridanus. A cortical alveolar oocyte from a Florida Bass collected in December. Cortical alveoli are peripheral. The germinal vesicle is surrounded by oil droplets. The oocyte is surrounded by a layer of follicle cells, an acellular zona pellucida and a theca, barely resolved at the magnification of the micrograph. Capillaries are within the theca. The arrow points to the site where ovulation will occur: the only cells present are the follicle cells and those of the germinal epithelium. Epithelial cells separate the germinal epithelium from the ovarian lumen. PAS/MY, Bar = 50 μm. BM, basement membrane; c, capillaries; ca, cortical alveoli; E, epithelial cells; EVS, extravascular space; FC, follicle cells; GE, germinal epithelium; gv, germinal vesicle; od, oil droplets; OL, ovarian lumen; PGca, Primary Growth Stage, cortical alveolar step oocyte; T, theca; ZP, zona pellucida.
Micropterus floridanus. (A) A single Primary Growth Stage, one nucleolus step oocyte is attached to the germinal epithelium. Within the ovarian lamella is an extensive extravascular space and stromal cells are next to a Primary Growth Stage, perinucleolar step oocyte. An early secondary growth oocyte, with small yolk globules, is next to a late secondary growth oocyte with maximum-sized yolk globules. Oil droplets surround the germinal vesicles which have irregular outlines. With H&E staining, cortical alveoli around the oocyte periphery are primarily clear vesicles, some with eosin staining. H&E, Bar = 100 μm. (B inset) Thecal cells associate with stroma (arrow) from an oocyte with a well-defined zona pellucida and cortical alveoli. H&E, Bar = 10 μm. ca, cortical alveoli; EVS, extravascular space; FC, follicle cells; GE, germinal epithelium; gv, germinal vesicle; od, oil droplets; PGon, Primary Growth Stage, one nucleolus step oocyte; PGpn, Primary Growth Stage, perinucleolar step oocyte; SGe, Secondary Growth Stage oocyte, early step; SGl, Secondary Growth Stage oocyte, late step; ST, stromal cells; T, theca; y, yolk globules; ZP, zona pellucida.
Micropterus floridanus. Portions of juxtaposed oocytes. The oocyte on the left is in maturation, with oil droplets fusing (small arrows) to become larger oil globules; the oocyte on the right is preovulatory, with a single, large oil globule. The oocytes are attached by stromal cells (large arrow between oocytes). Each oocyte is encompassed by a layer of follicle cells and a zona pellucida. Cortical alveoli rim the periphery of each oocyte. The ooplasm of the preovulatory oocytes is intensely PAS-positive (*). PAS/MY, Bar = 50 μm. ca, cortical alveoli; EVS, extravascular space; FC, follicle cells; od, oil droplet; og, oil globule; OL, ovarian lumen; y, yolk; yg, yolk globules; ZP, zona pellucida.
Micropterus floridanus. (A) At the initiation of oocyte maturation, small oil droplets surrounding the germinal vesicle begin to fuse, becoming oil globules. Cortical alveoli are around the periphery of the oocyte, beneath the zona pellucida. A postovulatory follicle complex is next to the oocyte beginning maturation. H&E, Bar = 100 μm. (B) As oil globules fuse, they are more restricted to the region around the germinal vesicle and are surrounded by yolk globules, exterior to which is a layer of cortical alveoli beneath the zona pellucida. A single nucleolus, primary growth oocyte is attached to the germinal epithelium. H&E, Bar = 100 μm. (C) As oil globules continue to fuse, one becomes larger than others. As above, the oocyte has a peripheral layer of cortical alveoli beneath the zona pellucida. H&E, Bar = 100 μm. (D) A preovulatory oocyte with but one oil globule, numerous fluid yolk globules, peripheral cortical alveoli and surrounded by a zone pellucida. The fluid yolk globules are separated (*). PAS/MY, Bar = 100 μm. ca, cortical alveoli; GE, germinal epithelium; gv, germinal vesicle; og, one or more oil globules; PGon, Primary Growth Stage, one nucleolus step oocyte; POC, postovulatory follicle complex; y, yolk globules; ZP, zona pellucida.
Micropterus floridanus. (A) Ovulation is the emergence of the oocyte from the follicle, in which it developed, to become an egg or ovum. Small, unlabeled arrows point to the “break” in the germinal epithelium and follicle cells indicating the beginning of ovulation. The zona pellucida is exposed (*) to the ovarian lumen between the arrows as the follicle cells and the epithelial cells in the germinal epithelium part. A single, large oil globule is semi-surrounded by smaller oil globules, still fusing. Fluid yolk globules fill the deeply PAS-positive ooplasm. Cortical alveoli are located around the periphery of the oocyte. PAS/MY, Bar = 100 μm. (B) Enlargement of the ovulating oocyte where epithelial cells of the germinal epithelium form a continuum with follicle cells (unlabeled arrow) of the ovulating oocyte. The follicle cells are vacuolated, and thecal cells are miniscule. At the zona pellucida label, the zona pellucida contacts the ovarian lumen. PAS/MY, Bar = 20 μm. BV, blood vessels; ca, cortical alveoli; FC, follicle cells; GE, germinal epithelium; og, oil globule; OL, ovarian lumen; T, thecal cell; vFC, vacuolated follicle cells; ZP, zona pellucida.
Micropterus floridanus. A preovulatory oocyte below a postovulatory follicle complex, in which the ooplasm has peripheral cortical alveoli and yolk globules. The zona pellucida is bi-layered. The vacuolated follicle cells are similar to the postovulatory follicle cells (large arrows). Outside of the follicle cell layer is a squamous thecal cell layer. A basement membrane (arrows) separates the follicle cells from the theca. A postovulatory follicle complex is composed of a postovulatory follicle, having vacuolated cells (arrows) and the postovulatory theca. The basement membrane, which separates the postovulatory theca from the stroma, is resolved. PAS/MY, Bar = 10 μm. BM, basement membrane; BV, blood vessels; ca, cortical alveoli; GE, germinal epithelium; OL, ovarian lumen; POC, postovulatory follicle complex; POF, postovulatory follicle; POT, postovulatory theca; T, thecal cells; vFC, vacuolated follicle cells; y, yolk globules; ZP, zona pellucida.
Micropterus floridanus. A postovulatory follicle complex is composed of a postovulatory follicle and a postovulatory theca separated by a basement membrane. Blood vessels are in the postovulatory theca, and some granulomas are in the stroma. Large arrow marks the opening through which the oocyte entered the ovarian lumen to become an egg at ovulation. Small arrow indicates where the postovulatory follicle joins the germinal epithelium. PAS/MY, Bar = 50 μm. Inset: an enlargement from POF2 to illustrate vacuolated postovulatory follicle cells. PAS/MY, Bar = 10 μm. BM, basement membrane; BV, blood vessels; F, tangential section of a follicle; FC, follicle cells; GE, germinal epithelium; GR, granuloma; n, nucleolus; OL, ovarian lumen; POF, postovulatory follicle; POF2, tangential portion of POF; POT, postovulatory theca; v, PAS-positive vacuole in POF cells.
Micropterus floridanus. (A) An unfertilized egg is encompassed by an egg envelope, has basophilic ooplasm and eosinophilic, peripheral cortical alveoli. The animal pole has slightly more ooplasm than does the vegetal pole. There is a single, large oil globule and a few smaller ones (*). Vegetal pole yolk is more homogeneous than animal pole yolk. H&E, Bar = 200 μm. (B) An infertile egg to illustrate basophilic ooplasm and distinctive, eosinophilic yolk globules and cortical alveoli at the animal pole. The egg envelope is bi-layered and has a single micropyle at the animal pole. (C) 15 to 20 seconds after fertilization, cortical alveoli begin to be released at the animal pole and result in its having a scalloped ooplasm surface in sections. Release of cortical alveoli is not yet observed at the equator or vegetal pole. Ooplasm is PAS-positive. A distinctive difference exists between globule animal pole yolk and more homogeneous vegetal pole yolk. The oocyte has a single oil globule. PAS/MY, Bar = 10 μm. (D) A fertilized egg after all cortical alveoli have been released. Remnants of cortical alveoli are against the egg envelope (arrow heads), and there is a single oil globule. Cortical alveoli contents apparently break down and form a homogeneous, granular substance between the egg and egg envelope (*). (E) Animal pole egg surface 1.5 minutes after activated sperm were poured onto swirled eggs. Cortical alveoli are still being released from the fertilized egg at the animal pole; the released contents have accumulated beneath an egg envelope. The micropyle contains three sperm which cannot pass through the lower micropyle orifice because of PAS-positive cortical alveoli release (arrow, asterisk). PAS/MY, Bar = 20 μm. (F) Enlargement of the egg envelope to illustrate the micropyle with three sperm that are blocked from passing through the micropyle by PAS-positive cortical alveoli release (arrow, asterisk). Sperm flagella are visible. PAS/MY, Bar = 10 μm. AP, animal pole; ca, cortical alveoli; car, cortical alveoli being released; car*, cortical alveoli contents against egg envelope; EE, egg envelope; f, sperm flagella; mp, micropyle; og, oil globule; oop, animal pole ooplasm; sp, sperm; VP, vegetal pole; y, vegetal pole yolk; y*, globular animal pole yolk.
Micropterus floridanus. (A) Atretic, primary growth oocyte. Basophilic ooplasm is fragmented. The atretic oocyte extends into the extravascular space from the germinal epithelium that borders the ovarian lumen. H&E, Bar = 20 μm. (B) Atresia of a secondary growth oocyte shows disorganized yolk globules and numerous lipid droplets. The atretic oocyte is bordered by primary growth oocytes and a late secondary growth oocyte with yolk globules. H&E, Bar = 50 μm. (C) Atresia of a secondary growth oocyte, still with yolk globules, but follicle cell nuclei have invaded the ooplasm. A primary growth, perinucleolar oocyte and one with cortical alveoli are present. H&E, Bar = 20 μm. (D) An atretic oocyte in late atresia resides in the extravascular space and is composed of only lipid vacuoles and follicle cell nuclei, at the level of resolution. A germinal epithelium separates the extra vascular space from the ovarian lumen. PAS/MY, Bar = 10 μm. aPG, atretic, Primary Growth oocyte; ATl, late atresia; ca, cortical alveoli; EVS, extravascular space; FC, follicle cells; FCn, follicle cell nuclei; GE, germinal epithelium; l, lipid vacuoles; OL, ovarian lumen; PG, Primary Growth oocyte; PGca, Primary Growth Stage, cortical alveolar step oocyte; PGpn, Primary Growth Stage, perinucleolar step oocyte; SGl, late Secondary Growth oocyte; ST, stromal cells; y, yolk globules.
Micropterus floridanus. (A) Atretic preovulatory oocyte. The zona pellucida is still intact, but slightly internalized, separated from the follicle cell layer that appears “granular.” There are two oil globules, and cortical alveoli have largely disappeared. Those that remain are internalized. Yolk has become fluid aside from a few globules that still remain. An early secondary growth oocyte has numerous cortical alveoli around its periphery, and small yolk globules and oil droplets internal to them are seen along with a postovulatory follicle complex. PAS/MY, Bar = 100 μm. (B) Surface of the atretic, preovulatory oocyte. The follicle cells are phagocytizing yolk; their nuclei are located between ingested yolk particles. Within the ooplasm, the internalized zona pellucida, fluid yolk, cortical alveoli remnants, and the oil globule are seen. PAS/MY, Bar = 20 μm. (C) Late atresia wherein a couple of yolk globules remain in the ooplasm that is being phagocytized by follicle cells whose nuclei are scattered throughout the atretic oocyte. An intact basement membrane surrounds the atretic oocyte. Reticulin Stain, Bar = 10 μm. BM, basement membrane; ca, cortical alveoli; FC, follicle cell layer; FCn, follicle cell nuclei; fy, fluid yolk; og, oil globule(s); OL, ovarian lumen; POC, postovulatory follicle complex; SGe, early secondary growth oocyte; ST, stromal cells; y, yolk globule; ZP, zona pellucida.
Contributor Notes
Associate Editor: M. P. Davis.