Reproductive Biology of Chlorophthalmus agassizi Bonaparte, 1840 (Teleostei: Aulopiformes: Chlorophthalmidae) as Revealed through Histology of Archival Museum Specimens
Teleost fishes exhibit a range of spawning modes that differ among the recruitment and timing of release of gametes. Oocyte staging is critical for understanding when females will spawn and in what spawning mode. Most of the data on reproductive biology of marine fishes is generated by fisheries biologists using specimens that are not archived in museum collections. We turned to these collections to investigate reproductive biology, including spawning mode and frequency, in the deep-sea hermaphrodite, the Shortnose Green Eye, Chlorophthalmus agassizi. Histological examination of specimens preserved over 50 years ago revealed both ovarian and testicular tissues in an ovotestis. Simultaneous activity of the female germinal epithelium and ongoing oocyte maturation and ovulation in these fishes which were preserved while spawning constitutes the basic mechanism of the asynchronous spawning mode in fishes: the germinal epithelium produces new follicles during or between spawning events. Well-preserved, archival museum specimens can provide information on reproductive biology for many taxa, especially rare species, which may never be the subject of detailed fisheries biology studies.
Chlorophthalmus agassizi, USNM 185473, 121 mm SL. Collected off Florida, on 12 June 1958. Bar = 1 cm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) Ovary of an ovulating individual (1F) to illustrate an oocyte in the Eccentric Germinal Vesicle Step of maturation (OMegv) with coalescing oil globules (cog). A late secondary growth oocyte (SGl), a cell nest (CN) with pachytene oocytes, and a postovulatory follicle complex (POC) are present. The POC is composed of the postovulatory follicle (POF) and the postovulatory theca (POT). Normally separated by a basement membrane, this is not visible in H&E sections, but a small space (*) separates the POF and the POT. Primary growth oocytes in the Perinucleolar Step (PGpn) are common. The germinal epithelium (GE) borders the ovarian lumen (OL). Yolk globules (y) and oil droplets (od) are observed in the ooplasm. Coalescing oil droplets are labeled oil globules (cog) to distinguish growth from maturation. Germinal vesicle (gv); Zona pellucida (ZP). Bar = 50 µm. (B) An enlargement of the cell nest (CN) in A illustrates the pachytene oocytes that extend from the germinal epithelium (GE) into the stroma. Bar = 10 µm. (C) The POF opening through which the egg emerged from the follicle is illustrated (large arrow). Bar = 10 µm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) A cell nest features a prophase oogonium (pOG), an anaphase oogonium (aOG), and a metaphase oogonium (mOG) within the germinal epithelium (GE) of individual 3F. Ovarian lumen (OL). Bar = 10 μm. (B) In the same individual, a single oocyte, within the germinal epithelium still undergoing folliculogenesis, has a nucleus with a single nucleolus and stained ooplasm, being in the One Nucleolus Step of Primary Growth. Cell nuclei lateral to the oocyte are prefollicle cells (PF) differentiated from epithelial cells (E) of the germinal epithelium. Early diplotene oocyte in the chromatin nucleolus stage (CNed). Bar = 10 μm. (C) Early in the Perinucleolar Step of Primary Growth (PG), the germinal vesicle (gv) nucleoli (nu) are arranged around the periphery and are spherical. The germinal vesicle in this individual (1F) has a smooth contour. Atretic oocyte (aOC); follicle cell (FC); Primary Growth perinuclear oocyte (PGpn). Bar = 10 μm. (D) As primary oocytes grow, the germinal vesicle (gv) contour starts to become irregular and increasingly so (asterisk) as growth continues in individual 2F. Bar = 10 μm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) Oocytes with basophilic ooplasm and one nucleolus mark the first step of primary growth (PGon) that is followed by oocytes with multiple nucleoli and then the perinucleolar oocytes (PGpn). Cortical alveoli (ca) become arranged around the oocyte periphery, the Cortical Alveolar Step of primary growth (PGca), and the germinal vesicle (gv) contour is highly irregular. Atretic oocytes are common (aOC). Individual 1F. Bar = 20 µm. (B) Small yolk globules (y) appear during the Early Secondary Growth Step (SGe). Oil droplets (od) begin to appear along the irregular periphery of the germinal vesicle (gv). Cortical alveoli (ca) are along the oocyte periphery. With H&E, both oil droplets and cortical alveoli remain unstained and appear similar. Individual 3F. Zona pellucida (ZP); follicle cells (F). Bar = 20 µm. (C) Many yolk globules (y) in maximum-sized oocytes in the Full-grown Step of secondary growth (SGfg) have attained their maximum size. Oil droplets surround the germinal vesicle (gv). Primary growth, perinucleolar oocytes (PGon), and early secondary growth oocytes (SGe) are present. Individual 1F. Bar = 50 µm. (D) Oocyte maturation begins when oil droplets begin to coalesce around the germinal vesicle (gv), becoming oil globules (og); the germinal vesicle loses its irregular contour, becoming polymorphic due to oil globules coalescing in the center of the oocyte. Individual 1F. Eccentric Germinal Vesicle Step of maturation (OMegv); germinal epithelium (GE); ovarian lumen (OL). Bar = 50 µm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) A section through an ovarian lamella from an ovulating individual (1F) also illustrates oocyte maturation in the Eccentric Germinal Vesicle Step of Maturation when oocytes have coalescing oil globules (isolated arrows). One oocyte has progressed to having a single oil globule (og), its germinal vesicle (gv) has become ovoid, and it is about to start migration. The lamella is bounded by a germinal epithelium (GE). An egg (E), with an irregular contour due to tissue processing, is within the ovarian lumen (OL). Within the lamellae, there are numerous primary growth oocytes (PG), early secondary growth oocytes (SGe), late secondary growth oocytes (SGl), and multiple postovulatory follicle complexes (POC). Bar = 200 µm. (B) Two oocytes, one in early and one in the late Eccentric Germinal Vesicle Step of oocyte maturation are observed in the same individual. Initially, oil globules coalesce in the center of the oocyte and deform the germinal vesicle (gv). As they coalesce, the germinal vesicle becomes capped over the oil globule (og), the end of the Eccentric Germinal Vesicle Step. Bar = 50 µm. (C) As oocyte maturation proceeds, the germinal vesicle becomes oval to spherical, and ooplasm is evident between it and the oil globule (arrow) marking the beginning of the Germinal Vesicle Migration Step in the same individual. Yolk globules (y) are larger than in the previous step of oocyte maturation. Bar = 50 µm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) The Germinal Vesicle Breakdown Step in maturation is marked by disappearance of the germinal vesicle membrane (gvb) to leave nucleoli (nu) surrounding the metaphase chromosomes (mch) free in the ooplasm in the region of the animal pole. Yolk is clearing, particularly at the vegetal pole of the oocyte (to the right) where it is no longer globular, but is a fluid mass. Cortical alveoli (ca) are clear vesicles around the oocyte periphery beneath the zona pellucida (ZP). Perinucleolar oocyte (PGpn); atretic oocyte (aOC). Individual 5F. Bar = 20 µm. (B) In preovulatory oocytes, the yolk is entirely fluid (fy) and the ooplasm (op) is restricted to the periphery within which are cortical alveoli. The preovulatory oocyte is surrounded by a zona pellucida and a layer of follicle cells (FC) and is separated from the theca (T) by a basement membrane that is not apparent in H&E stained material. Late secondary growth oocyte (SGl). Individual 1F. Bar = 20 µm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) Germinal vesicle migration might not cover a great intercellular distance. The oil globule (og) and germinal vesicle (gv) are close to the animal pole where a micropyle is observed (arrow) in the zona pellucida (ZP). Cortical alveoli (ca); yolk (y). Individual 3F. Bar = 20 µm. (B) At higher magnification, a micropylar cell (MPC) is observed to extend a process into the micropyle (mp), through the zona pellucida (ZP). Nucleoli (nu) are no longer located around the germinal vesicle periphery. Individual 3F. Bar = 10 µm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) In ovarian tissue with ovulating oocytes, some oocytes become atretic (aOC), the zona pellucida (ZP) of which is undulating and follicle cells (FC) are irregular. Both new (nPOC) and old (oPOC) postovulatory follicle complexes are also observed, the latter being smaller. A second difference is that nPOCs have postovulatory follicles (POF), the cells of which have spherical nuclei, whereas oPOCs possess follicle cells with pycnotic nuclei (n) and numerous dense bodies (arrow), evidence of karyolysis and apoptosis. Postovulatory theca (POT). Individual 1F. Bar = 20 µm. (B) Enlargement of a section of the old postovulatory follicle complex (oPOC) in the rectangle in A illustrates dense bodies and small nuclei. Individual 1F. Bar = 10 µm.
Representative micrographs of the ovarian components of Chlorophthalmus agassizi. (A) A new postovulatory follicle complex (POC) composed of a postovulatory follicle (POF) that is separated from the postovulatory theca (POT) by a basement membrane (BM). The POF extends from the POC lumen (L) to the basement membrane outside of which is the postovulatory theca. New POF cells have spherical nuclei (n). Morphological differences in germinal epithelium (GE) cell nuclei and those of the postovulatory theca are obvious. Ovarian lumen (OL). Individual 3F. Bar = 10 µm. (B) A new postovulatory follicle complex (POC), again illustrating the postovulatory follicle (POF) being separated from the postovulatory theca (POT) by a periodic acid Schiff-positive (purple) basement membrane (BM). While POF nuclei are still spherical, dense bodies in the POF cell layer characterize apoptosis of POF cells (arrows). Individual 3F. Bar = 10 µm.
Representative micrographs of the hermaphroditic gonadal components of Chlorophthalmus agassizi. (A) A section of dorsal ovary from an ovulating individual reveals an array of oocyte stages: primary growth (PG), late secondary growth (SGl), oocyte maturation at the Eccentric Germinal Vesicle Step (OMegv), and preovulatory (OMpov). Testis tissue (T) in this oocyte spawning individual is also observed. Postovulatory follicle complexes (POC) are also present. Individual 1F. Bar = 100 µm. (B) Cross section of the largely ovarian gonad with extensive testicular tissue and presence of sperm (SP) in the lumen of the male tissue. The gonad is composed of lamellae (L) in which follicles are observed, the latest stage oocyte being in the Cortical Alveolar Step (PGca), most oocytes being prior to this step. Arrow points to mesentery. Individual 4F. Bar = 200 µm. (C) A fully developed individual (1M) gonad with extensive testis (T) and ovary with only primary growth oocytes (PG). The germinal epithelium (arrows) forms the surfaces of ovarian lamellae that border an ovarian lumen (OL). An active testis has numerous spermatocysts (CY) forming the male germinal epithelium, and sperm (SP) fill the testis lumen. Bar = 200 µm.
Representative micrograph of the gonadal components of Chlorophthalmus agassizi. (A) The peripheries of ovarian lamellae of individual 1F have numerous oogonia (asterisks) and primary growth oocytes (PG) and an active germinal epithelium with cell nests (CN). Epithelial cells in the germinal epithelium (EGE); ovarian lumen (OL). Bar = 10 µm. (B) Cross section through the testis tissue of individual 2M to illustrate spermatocytes (SC) and sperm in the lumen (SP) and about to be released from spermatocysts. Primary growth oocytes with one (PGon) and multiple (PGmn) nucleoli are in the testicular germinal epithelium. Red blood cells (RBC); Sertoli cell processes (SE). Bar = 10 µm.
Contributor Notes
Associate Editor: G. Arratia.