333
INTEGR. COMP. BIOL., 44:333?348 (2004)
Evolution and Phylogeny of Gonad Morphology in Bony Fishes1
LYNNE R. PARENTI2,* AND HARRY J. GRIER?*
*Division of Fishes, Department of Zoology, National Museum of Natural History, MRC 159, Smithsonian Institution,
P.O. Box 37012, Washington, D.C. 20013-7012
?Fish and Wildlife Research Institute, 100 8th Avenue, SE, St. Petersburg, Florida 33701-5095
SYNOPSIS. Gonad morphology at the gross anatomical or histological levels has long been studied by
fisheries biologists to identify annual reproductive cycles and length of breeding season, among other goals.
Comparative surveys across vertebrate taxa have not been detailed enough, however, to describe fully the
differences and similarities among gonads of bony fishes and other vertebrates, and to use gonad morphology
in phylogenetic systematic analyses. An emerging constant among vertebrates is the presence of a germinal
epithelium composed of somatic and germ cells in both males and females. In females, the germinal epithe-
lium lines the ovarian lamellae. In males, arrangement of the germinal epithelium into compartments varies
among osteichthyans: basal taxa have an anastomosing tubular testis, whereas derived taxa have a lobular
testis. The lobular testis is proposed as a synapomorphy of the Neoteleostei. The annual reproductive cycle
is hypothesized to be the source of morphological variation among testis types. Elongation of germinal
compartments during early maturation may result in a transition from anastomosing tubular to lobular
testes. In all male atherinomorphs surveyed, spermatogonia are restricted to the distal termini of lobules
rather than being distributed along the lobule; there is an epithelioid arrangement of Sertoli and germ cells
rather than a germinal epithelium. Arrest of the maturation-regression phases is hypothesized to lead to
formation of the atherinomorph testis. Atherinomorphs also have a distinctive egg with fluid, rather than
granular, yolk. Variation among germinal epithelia is interpreted in a developing phylogenetic framework
to understand evolution of gonad morphology and to propose gonad characters for phylogenetic analyses.
INTRODUCTION
Gonad morphology at the gross anatomical or his-
tological levels has long been studied by fisheries bi-
ologists to identify annual reproductive cycles, length
of breeding season, onset of reproductive maturity,
spawning rhythms, fecundity and various other aspects
of reproductive biology that can be applied to fisheries
questions and concerns. Of necessity, these studies
have focused on a restricted set of commercial or rec-
reational fishing species, such as common snook, Cen-
tropomus undecimalis (e.g., Grier and Taylor, 1998;
Taylor et al., 1998; Neidig et al., 2000), redfish or red
drum, Sciaenops ocellatus (e.g., Murphy and Taylor,
1990), bonefish, Albula vulpes (e.g., Crabtree et al.,
1997), and trout, Oncorhynchus species (Billard,
1987), among others. A second field of investigation
is the reproduction of marine fishes, many of which
have long been known to switch sex and are her-
maphroditic (e.g., Warner and Robertson, 1978; Has-
tings, 1981; Cole, 1988, 1990). A third, well-studied
area of fish reproduction is the modes of viviparity in
the atherinomorph orders Cyprinodontiformes (viz.,
Parenti, 1981), including the poeciliids (e.g., Rosen
and Bailey, 1963; Hoar, 1969), the four-eyed fishes,
genus Anableps (e.g., Turner, 1950), and the Mexican
goodeids (e.g., Miller and Fitzsimons, 1971), and Be-
loniformes, including the viviparous halfbeaks (e.g.,
Downing and Burns, 1995; Meisner and Burns, 1997;
1 From the Symposium Patterns and Processes in the Evolution
of Fishes presented at the Annual Meeting of the Society for Inte-
grative and Comparative Biology, 4?8 January 2003, at Toronto,
Canada.
2 E-mail: parentil@si.edu
Meisner, 2001). Despite a few efforts aimed at using
reproductive characters in comprehensive classifica-
tions of bony fishes (e.g., Breder and Rosen, 1966),
these areas of research remain relatively independent.
Comparative surveys across vertebrate taxa have not
been broad or detailed enough to describe fully the
differences and similarities among gonads of bony
fishes and other vertebrates, and to use gonad mor-
phology routinely in phylogenetic systematic analyses.
These are our aims.
An emerging constant among vertebrates is the pres-
ence of a germinal epithelium (Grier, 2000, 2002;
Grier and Lo Nostro, 2000). Almost all osteichthyans
have a germinal epithelium composed of somatic and
germ cells in male and female gonads. We classify the
atherinomorph testis as epithelioid based on refinement
of definitions of an epithelium as applied to gonad
morphology. In teleosts, the germinal epithelium is ac-
tive throughout the life of the organism and is corre-
lated with indeterminate reproduction of females.
Among the Perciformes, five reproductive classes have
been described in males of common snook, Centro-
pomus undecimalis (see Taylor et al., 1998), spotted
sea trout, Cynoscion nebulosus (see Brown-Peterson,
2003), cobia, Rachycentron canadum (see Brown-Pe-
terson et al., 2002), and the freshwater goby, Pado-
gobius bonelli (as Padogobius martensi, see Cinquetti
and Dramis, 2003):?regressed, early maturation, mid
maturation, late maturation, and regression?based on
the alternation of the germinal epithelium between
continuous and discontinuous types and the stages of
germ cells present (Grier and Taylor, 1998; Taylor et
al., 1998; Grier, 2002). These changes in the germinal
epithelium have also been used to describe annual
334 L. R. PARENTI AND H. J. GRIER
TABLE 1. Survey of testis types of osteichthyans.*
Species Testis Type
Reference and/or
Material
CLASS SARCOPTERYGII
Order Coelacanthiformes
Latimeria chalumnae anastomosing tubular Millot et al., 1978
CLASS ACTINOPTERYGII
SUBCLASS CHONDROSTEI
Order Acipenseriformes
Polyodontidae
Polyodon spathula anastomosing tubular USNM/FMRI
SUBCLASS NEOPTERYGII
Order Lepisosteiformes
Lepisosteidae
Lepisosteus platyrhinchus anastomosing tubular Grier, 1993
DIVISION TELEOSTEI
Order Elopiformes
Elopidae
Megalops atlanticus anastomosing tubular USNM/FMRI
CLUPEOCEPHALA
Order Clupeiformes
Clupeidae
Dorosoma petenense
Opisthonema oglinum
anastomosing tubular
anastomosing tubular
Grier, 1993
Grier, 1993
Ostariophyi
Order Cypriniformes
Cyprinidae
Abbottina rivularis
Barbus kahajanii
Danio rerio
Notemigonus crysoleucas
Notropis hypselopterus
anastomosing tubular
anastomosing tubular
anastomosing tubular
anastomosing tubular
anastomosing tubular
USNM 336887
Grier et al., 1980
USNM/FMRI; Maack and Segner, 2003
Grier et al., 1980
Grier et al., 1980
Order Characiformes
Characidae
Gymnocorymbus ternetzi anastomosing tubular Grier et al., 1980
Order Siluriformes
Pimelodidae
Conorhynchus conirostris anastomosing tubular Lopes et al., 2004
Ictaluridae
Ictalurus natalis anastomosing tubular Grier, 1993
SUBDIVISION EUTELEOSTEI
Protacanthopterygii
Order Salmoniformes
Salmonidae
Oncorhynchus mykiss
Oncorhynchus kisutch
anastomosing tubular
anastomosing tubular
USNM/FMRI
Grier et al., 1980
NEOGNATHI
Esociformes
Esox lucius
Esox niger
anastomosing tubular
anastomosing tubular
Grier et al., 1980; Grier, 1993
Grier et al., 1980; Grier, 1993
NEOTELEOSTEI
Paracanthopterygii
Order Percopsiformes
Amblyopsidae
Amblyopsis spelaea unrestricted lobular USNM 127055
Percopsidae
Percopsis omiscomaycus unrestricted lobular USNM 308217
Order Ophidiiformes
Bythitidae
Dinematichthys sp. unrestricted lobular USNM 338466
335GONAD MORPHOLOGY IN BONY FISHES
TABLE 1. (Continued)
Species Testis Type
Reference and/or
Material
Order Lophiiformes
Lophiidae
Lophiodes mutilus unrestricted lobular USNM 322221
Order Polymixiiformes
Polymixiidae
Polymixia lowei unrestricted lobular USNM 157839
SERIES ATHERINOMORPHA
Order Atheriniformes
Atherinidae
Labidesthes sicculus
Leuresthes sardina
Menidia beryllina
restricted lobular
restricted lobular
restricted lobular
Grier et al., 1980, 1990; USNM 108573
USNM 177811
Grier et al., 1980
Melanotaeniidae
Melanotaenia nigrans restricted lobular Grier et al., 1980
Phallostethidae
Gulaphallus bikolanus
Gulaphallus mirabilis
Neostethus bicornis
Neostethus borneensis
Neostethus lankesteri
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier and Parenti, 1994
Grier et al., 1980; Grier and Parenti, 1994
Grier and Parenti, 1994
Grier and Parenti, 1994
Grier and Parenti, 1994
Phenacostethus smithi
Phenacostethus posthon
restricted lobular
restricted lobular
Munro and Mok, 1990; Grier and Parenti, 1994
Grier and Parenti, 1994
Order Cyprinodontiformes
Aplocheilidae
Aphyosemion gardneri restricted lobular USNM 339706
Rivulidae
Pterolebias hoignei restricted lobular USNM 245947
Profundulidae
Profundulus guatemalensis restricted lobular USNM 134600
Fundulidae
Adinia xenica
Fundulus chrysotus
Fundulus grandis
Fundulus seminolis
Lucania goodei
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980; USNM/FMRI
Grier et al., 1980
Grier et al., 1980
Goodeidae
Ameca splendens
Ataeniobius toweri
Characodon lateralis
Xenotoca eiseni
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980; USNM 374494
Anablepidae
Anableps anableps
Anableps dowi
Jenynsia lineata
Jenynsia multidentata
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980
Mart??nez and Monasterio de Gonzo, 2002
Poeciliidae
Cnesterodon decemmaculatus
Gambusia affinis
Heterandria formosa
Poecilia latipinna
Poecilia reticulata
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
USNM 360480
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980
Poeciliopsis gracilis
Tomeurus gracilis
Xiphophorus helleri
Xiphophorus maculatus
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier et al., 1980
Grier et al., 1980; USNM 225463
Grier et al., 1980
Grier et al., 1980
Cyprinodontidae
Cyprinodon variegatus
Jordanella floridae
restricted lobular
restricted lobular
Grier et al., 1980
Grier et al., 1980
336 L. R. PARENTI AND H. J. GRIER
TABLE 1. (Continued)
Species Testis Type
Reference and/or
Material
Order Beloniformes
Adrianichthyidae
Horaichthys setnai
Oryzias latipes
Oryzias matanensis
restricted lobular
restricted lobular
restricted lobular
Grier, 1984
Grier, 1976
USNM 340428
Exocoetidae
Cypselurus heterurus
Hirundichthys speculiger
Oxyporhamphus micropterus
Prognichthys gibbifrons
restricted lobular
restricted lobular
restricted lobular
restricted lobular
USNM 294785
USNM 299274
USNM 216327
USNM 185882, 185883
Hemiramphidae
Dermogenys bispinna
Dermogenys burmanica
Dermogenys orientalis
Dermogenys pusilla
Dermogenys siamensis
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Downing and Burns, 1995
Downing and Burns, 1995; Meisner, 2001
Downing and Burns, 1995
Grieret al., 1980; Downing and Burns, 1995
Downing and Burns 1995; Meisner, 2001
Euleptorhamphus velox
Hemiramphus brasiliensis
Hemirhamphodon chryopunctatus
Hemirhamphodon kapuasensis
Hemirhamphodon kuekenthali
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier et al., 1980
Grier et al., 1980; USNM/FMRI
Downing and Burns, 1995
Downing and Burns, 1995
Downing and Burns, 1995
Hemirhamphodon phaisoma
Hemirhamphodon pogonognathus
Hemirhamphodon tengah
Hyporhamphus quoyi
Hyporhamphus regularis
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Downing and Burns, 1995
Downing and Burns, 1995
Downing and Burns, 1995
Grier et al., 1980
Grier et al., 1980
Nomorhamphus brembachi
Nomorhamphus celebensis
Nomorhamphus ebrardtii
Nomorhamphus liemi
Nomorhamphus rossi
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Downing and Burns, 1995; Meisner, 2001
Downing and Burns, 1995
Downing and Burns, 1995; Meisner, 2001
Downing and Burns, 1995
Downing and Burns, 1995; Meisner, 2001
Nomorhamphus towoetii
Nomorhamphus vivipara
Nomorhamphus weberi
Zenarchopterus buffonis
Zenarchopterus caudovittatus
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Downing and Burns, 1995
Downing and Burns, 1995; Meisner, 2001
Downing and Burns, 1995; Meisner, 2001
Grier and Collette, 1987
Grier and Collette, 1987
Zenarchopterus dispar
Zenarchopterus dunckeri
Zenarchopterus ectuntio
Zenarchopterus gilli
Zenarchopterus kampeni
restricted lobular
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier and Collette, 1987
Grier and Collette, 1987
Grier and Collette, 1987
Grier and Collette, 1987
Grier and Collette, 1987
Zenarchopterus novaeguineae
Zenarchopterus ornithocephala
Zenarchopterus rasori
Zenarchopterus robertsi
restricted lobular
restricted lobular
restricted lobular
restricted lobular
Grier and Collette, 1987
Grier and Collette, 1987
Grier and Collette, 1987
Grier and Collette, 1987
SERIES MUGILOMORPHA
Mugilidae
Agnostomus monticola
Mugil cephalus
unrestricted lobular
unrestricted lobular
USNM 318360
USNM 101188; 111387; Grier et al., 1980
SERIES PERCOMORPHA
Order Synbranchiformes
Synbranchidae
Synbranchus marmoratus unrestricted lobular LoNostro et al., 2003
Mastacembelidae
Mastacembelus armatus unrestricted lobular USNM 319481
Order Gasterosteiformes
Gasterosteidae
Pungitius sinensis unrestricted lobular USNM 336886
Order Perciformes
Elassomatoidei
Elassomatidae
Elassoma evergladei unrestricted lobular USNM 357366
337GONAD MORPHOLOGY IN BONY FISHES
TABLE 1. (Continued)
Species Testis Type
Reference and/or
Material
Percoidei
Centrarchidae
Enneacanthus gloriosus
Lepomis macrochirus
Micropterus salmoides
Pomoxis nigromaculatus
unrestricted lobular
unrestricted lobular
unrestricted lobular
unrestricted lobular
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980
Grier et al., 1980
Centropomidae
Centropomus undecimalis unrestricted lobular Taylor et al., 1998
Percidae
Perca flavescens unrestricted lobular Grier et al., 1980
Rachycentridae
Rachycentron canadum unrestricted lobular Brown-Peterson et al., 2002
Gerreidae
Diapterus plumieri unrestricted lobular Grier et al., 1980
Serranidae
Centropristis striata unrestricted lobular USNM/FMRI
Sparidae
Archosargus probatocephalus unrestricted lobular Grier et al., 1980
Sciaenidae
Cynoscion nebulosus
Sciaenops ocellatus
unrestricted lobular
unrestricted lobular
Brown-Peterson, 2003
Grier, 1993
Labroidei
Labridae
Thalassoma bifasciatum unrestricted lobular Koulish et al., 2002
Cichlidae
Cichlasoma nigrofasciatum
Labeotropheus trewavasae
Oreochromis sp.
Pterophyllum scalare
Sarotherodon aurea
unrestricted lobular
unrestricted lobular
unrestricted lobular
unrestricted lobular
unrestricted lobular
Grier et al., 1980
Grier et al., 1980
Grier, 1993
Grier et al., 1980
Grier et al., 1980
Blennioidei
Blenniidae
Ophioblennius steindachneri unrestricted lobular USNM 292427
Gobioidei
Odontobutidae
Micropercops swinhonis unrestricted lobular USNM 336883
Microdesmidae
Microdesmus bahianus
Microdesmus dorsipunctatus
unrestricted lobular
unrestricted lobular
Thacker and Grier, 2004
Thacker and Grier, 2004
Gobiidae
Bathygobius lineatus
Neogobius fluviatilis
Padogobius bonelli
Pandaka pygmaea
Tridentiger bifasciatus
unrestricted lobular
unrestricted lobular
unrestricted lobular
unrestricted lobular
unrestricted lobular
Thacker and Grier, 2004
Thacker and Grier, 2004
Cinquetti and Dramis, 2003
Thacker and Grier, 2004
Thacker and Grier, 2004
Schindleriidae
Schindleria praematura restricted lobular Thacker and Grier, 2004
* Classification follows Nelson (1994), Johnson and Patterson (1996), and Parenti (2004), in part. See text for further explanation.
male reproductive classes in the synbranchiform
swamp eel, Synbranchus marmoratus (see Lo Nostro
et al., 2003). Further, arrangement of the male ger-
minal epithelium into compartments is a fixed char-
acteristic among taxa that may be used to define testis
types (Grier, 1993).
One diagnostic character of the atherinomorph fish-
es was described by Rosen and Parenti (1981, p. 11)
as ??. . . spermatogonia . . . restricted to the distal end
of the tubule immediately beneath the tunica albuginea
whereas other groups of teleosts have the spermato-
gonia distributed along the length of the tubule.?? The
338 L. R. PARENTI AND H. J. GRIER
previous year, Grier et al., (1980) identified this dis-
tinctive testis type and reported it in 31 atherinomorph
species representing each of the three currently rec-
ognized orders (viz., Parenti, 2004). The more taxo-
nomically widespread condition was reported in 19 te-
leost species, including Esox and Oncorhynchus spe-
cies, as well as an array of ostariophysans and perco-
morphs (Table 1). Description and definition of testis
types in osteichthyans was reconsidered by Grier
(1993) who concluded that primitive osteichthyans
have an anastomosing tubular testis, whereas derived
teleosts, including atherinomorphs, have a lobular tes-
tis, and that the lobular testis could be divided into
two types based on distribution and arrangement of
spermatogonia. Germinal compartments that extend to
the periphery of the testis and terminate blindly are
termed lobules, not tubules (Figs. 1A?D; 2A?D; see
Grier, 1993, and Discussion, below). Hence, the ath-
erinomorph testis has a restricted distribution of sper-
matogonia at the distal ends of lobules (Fig. 1A,C,D),
in contrast to the ??perciform testis type,?? so-called
because of its initial description in fishes at one time
classified in the order Perciformes, such as the striped
mullet, Mugil cephalus (Fig. 1B), in which spermato-
gonia are distributed along the lengths of testis lobules.
Surveys of gonad morphology during the past two de-
cades have confirmed the presence of the unique, re-
stricted testis type in atherinomorphs (viz., Grier and
Collette, 1987; Grier and Parenti, 1994; Downing and
Burns, 1995; Figs. 1, 2; Table 1). There have been no
proposals, however, regarding the hierarchical level at
which we can recognize the lobular testis as a syna-
pomorphy.
Our collaboration began as an attempt to clarify
both the definition of testis types and their distribu-
tion among bony fish taxa. It has expanded necessar-
ily to include ovarian and egg characters as they also
vary phylogenetically (Figs. 3, 4). Variation among
germinal epithelia is interpreted in a developing phy-
logenetic framework to understand the evolution of
gonad morphology and also to begin to propose go-
nad characters that may be used in phylogenetic anal-
yses.
MATERIALS AND METHODS
Gonad material was obtained from either freshly
fixed specimens or from specimens maintained in the
fish collection of the National Museum of Natural His-
tory (USNM), Smithsonian Institution. The museum
material is identified by the prefix USNM (United Stat-
ed National Museum), followed by a catalogue num-
ber. Material that was collected and processed at the
Florida Marine Research Institute (now the Fish and
Wildlife Research Institute) is identified by the abbre-
viation USNM/FMRI. Voucher material will be de-
posited in the permanent collections of the USNM.
Paddlefish, Polyodon spathula, gonads were obtained
from Kentucky State University. Rainbow trout, On-
corhynchus mykiss, gonads were obtained from the El
Zarco trout hatchery outside of Mexico City, Mexico.
Testis type was recorded in 136 osteichthyan species
from our own observations, the literature or both (Ta-
ble 1). Egg type was observed in a more limited set
of taxa and is cited in the text.
The museum material is likely to have been fixed
in formalin, a common fixative starting in the late
1800s. It is currently stored in 75% ethanol. Gonads
from fresh material were fixed in Bouin?s solution
or buffered 10% formalin. Formalin-fixed, alcohol-
preserved whole fish specimens stored in museum
collections for decades proved as useful and reliable
for examination of histological structure of gonads
as recently fixed material. Museum collections are
the only source of gonads of certain taxa. When mu-
seum specimens did not prove to be of high histo-
logical quality, our opinion was that the initial fix-
ation was at fault, not the prolonged storage in eth-
anol.
Whole or sectioned gonads were embedded in plas-
tic (glycolmethacrylate [Polysciences]) or paraffin.
Tissue sections were cut at 6?8 
m (paraffin) or 3.5
and 4 
m (plastic). Paraffin sections were stained with
hematoxylin and eosin; plastic sections were stained
with thionin or metanil yellow-periodic acid/
Schiff?s (PAS) hematoxylin (Quintero-Hunter et al.,
1991).
RESULTS
Testes
Anastomosing tubular testes are found throughout
primitive teleost taxa, ranging from the tarpon, Me-
galops atlanticus (Fig. 2E), the cypriniform, Abbottina
rivularis (Fig. 2C), to the rainbow trout, Oncorhynchus
mykiss and the pikes and pickerels, genus Esox (see
Table 1 for references and material). In some histolog-
ical preparations of anastomosing tubular testes, the
germinal compartments may appear somewhat lobular,
as in Figure 2E, probably owing to the plane of section
through a three-dimensional tissue. Our descriptions
are based upon two-dimensional histological represen-
tations of the three-dimensional germinal compart-
ments. A tubular testis, which appears to be anasto-
mosing, characterizes the primitive sarcopterygian, the
coelacanth, Latimeria chalumnae, and the primitive
non-teleost actinopterygians, the paddlefish, Polyodon
spathula, and the gar, Lepisosteus platyrhinchus (Table
1).
Lobular testes characterize all fishes of the Neote-
leostei that we have surveyed or for which we found
citations (Figs. 1, 2A,B,D; Table 1). Lobular testes
may be further divided into restricted and unrestricted
types (Grier et al., 1980; Grier, 1993). In all male ath-
erinomorph fishes surveyed to date, 79 species repre-
senting all three orders, including taxa with a range of
reproductive modes, spermatogonia are restricted to
the distal termini of lobules rather than being distrib-
uted along the lobule (Fig. 1A,C,D; Table 1). Further-
more, there is an epithelioid arrangement of Sertoli and
germ cells; that is, the germ cells and Sertoli cells
339GONAD MORPHOLOGY IN BONY FISHES
FIG. 1. A. Cross section of the testis from the Gulf killifish, Fundulus grandis. The lobules terminate at the periphery of the testis, where
spermatogonia (SG) are located. Proceeding proximally, meiotic germ cells are arranged almost in rows between juxtaposed lobules, and
primary spermatocytes (1SC), spermatids (ST), and sperm (SP) in spermatocysts or within the lumina of efferent ducts (ED) are observed.
Bar  50 
m. B. A lobule from the striped mullet, Mugil cephalus, collected in late October, approximately one month before spawning
condition. Sperm (SP) accumulate within the lobule lumen. Spermatogonia (SG) are observed both at the distal terminus of the lobule and
also along the lateral walls as are spermatocysts containing primary spermatocytes (1SC), secondary spermatocytes (2SC), and spermatids
(ST). The germinal epithelium has become discontinuous as extensive regions of the lobule lack spermatocysts. Bar  50 
m. C. Testis lobules
from the ballyhoo, Hemiramphus brasiliensis, have spermatogonia (SG) restricted to their distal termini. Later stage developing sperm within
spermatocysts, primary spermatocytes (1SC) and spermatids (ST) are located progressively closer to the efferent ducts (ED) which are filled
with sperm (SP). Bar  50 
m. D. The distal termini of lobules from the testis of Fundulus grandis. Spermatogonia (SG), some dividing and
in metaphase (M), are restricted to the lobule distal termini. The borders of spermatocysts with primary spermatocytes (1SC) are delineated
by lightly-staining Sertoli cells (SE). Bar  10 
m. E. The efferent duct (ED) cells in the testis of Fundulus grandis contain eosinophilic
secretory material (bright pink). At the arrow, spermiogenesis, or release of sperm into the efferent ducts is occurring as Sertoli cell processes
separate, the lumen of the spermatocyst then becomes continuous with that of the efferent duct. Bar  10 
m.
within the lobules do not border directly onto a lumen.
By definition, epithelia border a body surface, lumen,
or tube (viz., Grier, 2000; Grier and Lo Nostro, 2000).
In atherinomorphs, the Sertoli cells extend processes
across the widths of the lobules; spermatocysts, there-
fore, extend across the lobules, and there is no lumen
within the lobule (Fig. 1A,C,D). At spermiation, sperm
are voided from the spermatocyst the lumen of which
340 L. R. PARENTI AND H. J. GRIER
FIG. 2. A. A lobule from the Everglades pygmy sunfish, Elassoma evergladei, illustrating primary spermatogonia (SG) with different-sized
nuclei. Some spermatogonia are in metaphase (M) or anaphase (A) along the wall of the lobule. Secondary spermatogonia (2SG) are within
spermatocysts. Bar  10 
m. B. A testicular lobule from the trout-perch, Percopsis omiscomaycus, illustrating spermatogonia (SG) at the
distal terminus of and along the lobule wall. The lobule is occluded, and no lumen is observed, with spermatocysts containing synchronously-
developing primary spermatocytes (1SC). Bar  10 
m. C. Testicular lobules from the Chinese false gudgeon, Abbottina rivularis. Sperma-
tocysts (CY) containing spermatocytes are numerous, and a few scattered spermatogonia (SG) are located at the distal termini and along the
341GONAD MORPHOLOGY IN BONY FISHES
?
FIG. 2. (Continued) lobule (L) walls. Bar  10 
m. D. Testis lobules of the beardfish, Polymixia lowei, have spermatogonia (SG) located
along their walls and at the distal termini. Spermatocytes (SC) and one testis ovum (TO) are observed. Bar  50 
m. E. The anastomosing
tubular testis of the tarpon, Megalops atlanticus, is illustrated. Anastomosing tubules do not terminate at the testis periphery, but rather form
continuous, interconnected loops as observed in the lobules shaded in turquoise. Lobules to the right of those shaded appear to terminate at
the periphery of the testis owing to plane of section. The lobules in this reproductive fish are filled with stored sperm, and developing sperm
in spermatocysts are not observed, a characteristic of reproductive, synchronous spawning fish. Bar  100 
m.
becomes continuous with that of the efferent duct (Fig.
1E).
Ovaries and oocytes
The ovarian germinal epithelium is the origin of fol-
licles in the fish ovary (Grier, 2000), actively produc-
ing follicles throughout the annual reproductive cycle
in the perciform Centropomus undecimalis. Morphol-
ogy of the germinal epithelium is identical in C. un-
decimalis and the synbranchiform Synbranchus mar-
moratus (see Ravaglia and Maggese, 2003). In the ath-
erinomorph Gulf killifish, Fundulus grandis (Fig. 3A),
the ovarian germinal epithelium has essentially the
same features as the above two species, and separates
the ovarian lumen from the stroma. A consistent fea-
ture of the ovarian stroma in teleosts is a conspicuous
extravascular space of varying size, and sometimes
seemingly nonexistent. If they do not dissolve in his-
tological preparation, the fixed ??fluids?? within the ex-
travascular space stain positively, if only lightly, for
glycoproteins as does blood plasma. As capillaries are
??leaky,?? that is, their endothelial cells are not joined
by tight junctions, we infer that fluids within the ex-
travascular space are derived from the circulatory sys-
tem. Furthermore, their demonstration may be depen-
dent upon type of fixative, rate of penetration of fix-
ative, and stain (Grier, unreported).
The germinal epithelium is composed of epithelial
cells that become prefollicle cells when associated
with oogonia, as in Fundulus grandis (Fig. 3A). It is
subtended by prethecal cells that are identical to cells
in the ovarian stroma (Grier, unreported). Once an oo-
gonium enters meiosis, it becomes an oocyte that pro-
gresses through the initial phases of the first meiotic
prophase until the diplotene stage when division is ar-
rested, and the other events of oocyte maturation com-
mence: primary oocyte growth (previtellogenesis [Pa-
tin?o and Sullivan, 2002], Fig. 3A,B), secondary oocyte
growth (vitellogenesis [Patin?o and Sullivan, 2002],
Fig. 3B,C), and final oocyte maturation (Figs. 3D, 4A).
During primary growth, the yolk nucleus appears as
do cortical alveoli (Fig. 3B).
From the onset of formation of oocyte cytoplasmic
yolk, osteichthyans have yolk that is distinctly granu-
lar, i.e., during vitellogenesis, the process of yolk for-
mation, protein yolk is formed into spherical globules
(Fig. 4B). Atherinomorphs have what we describe as
fluid yolk; it is relatively uniform in contrast to that
of other osteichthyans (Figs. 3C,D, 4A). The differ-
ence between fluid and granular yolk is discerned read-
ily histologically. Granular yolk becomes fluid during
the final, pre-ovulatory events of oocyte maturation
(Wallace and Selman, 1981; Selman and Wallace,
1989; Neidig et al., 2000), to which the term ??final
oocyte maturation?? (FOM) has been applied (Jalabert
et al., 1977). In atherinomorphs, protein yolk appears
globular at the oocyte surface, but these globules con-
tinuously fuse (Fig. 3C) to form a yolky mass that
stains positively with the periodic acid Schiff reaction
for glycoproteins.
The events of final oocyte maturation have not been
documented in atherinomorphs, but in Hemiramphus
brasiliensis (see McBride and Thurman, 2003; Fig.
4A) there is both an increase in oocyte diameter and
a reduction of the periodic acid Schiff staining of yolk.
In contrast to the fluid yolk of atherinomorphs, yolk
is organized into globules in other taxa. In Mugil ce-
phalus (Fig. 4B), yolk is distinctively globular and eo-
sinophilic, not staining with periodic acid Schiff. In
Elassoma evergladei (Fig. 4C), the yolk is periodic
acid Schiff-positive, but globular.
DISCUSSION AND CONCLUSIONS
Our survey confirms the initial observation of Grier
(1993) that an anastomosing tubular testis character-
izes basal osteichthyans, including basal teleosts,
whereas a lobular testis characterizes higher teleosts
(Table 1). The lobular testis type is proposed as a di-
agnostic or synapomorphic character of the Neoteleos-
tei, as discussed below. Further, we confirm the results
of Grier et al. (1980) that the restricted lobular type
of testis is diagnostic of atherinomorph fishes.
Testis types in fishes have been defined poorly,
based on examination of too few species or indiscrim-
inate application of terms such that ??lobule?? and ??tu-
bule?? were used interchangeably. For example, Grier
et al. (1980) noted that the literature does not ??clearly
distinguish structural differences between ??lobular??
and ??tubular?? testicular types,?? and that use of either
term varied by author. The testes of fishes, both the
basal osteichthyans and the neoteleosts, were termed
??tubular?? (Grier et al., 1980; Grier, 1981) deliberately
to avoid confusion with the erroneous concept that
some fishes have ??lobule?? boundary cells as Leydig
cell homologs. It was demonstrated subsequently that
one teleost species, Esox lucius, which was reported
to have lobule boundary cells as Leydig cell homo-
logs, had a typical distribution of interstitial, hormone-
secreting Leydig cells (Grier et al., 1989). The so-
called lobule boundary cells were Sertoli cells within
the germinal compartment. Further comparative inves-
tigations of testis morphology in teleosts led to a new
interpretation: the basal osteichthyans have anasto-
mosing tubular testes in which the germinal compart-
342 L. R. PARENTI AND H. J. GRIER
FIG. 3. A. The germinal epithelium of Fundulus grandis borders the ovarian lumen (OL). In it are observed an oogonium (OG) associated
with a prefollicle cell (PFC), distal to which is an epithelial cell (E). Epithelial cells that are associated with oogonia are prefollicle cells.
Beneath the germinal epithelium is a prethecal cell (PT). An extravascular space (EVS) is prominent, and primary growth oocytes (PGOC),
with dense-staining cytoplasm, are located beneath the germinal epithelium. Bar 
 10 m. B. In Fundulus grandis the germinal epithelium
separates the ovarian lumen (OL) from an extensive extravascular space (EVS) in which a primary growth oocyte, with cortical alveoli (ca),
is observed. Its nucleoli (nu) are located around the periphery of the nucleus (n). A smaller primary growth oocyte has a prominent yolk
nucleus (yn) within its cytoplasm, and the nucleoli are scattered within the nucleus (n). Cortical alveoli are also observed within a vitellogenic
343GONAD MORPHOLOGY IN BONY FISHES
?
FIG. 3. (Continued) oocyte (V). Bar 
 50 m. C. In vitellogenic oocytes of Fundulus grandis the yolk globules (y) fuse, being quite separate
and small at the oocyte periphery and larger centrally. Because of staining characteristics, it is difficult to distinguish between fat globules
and cortical alveoli which become progressively lighter-staining as oocyte development proceeds. The oocyte is surrounded by a zona pellucida
(ZP), often called a chorion. Exterior to this is a follicle cell (FC) layer, and theca (T). The oocyte nucleus (n) is dwarfed by the increasing
size of the oocyte. Germinal epithelium (GE). Extravascular space, (EVS). Bar 
 100 m. D. Portion of a mature oocyte from Fundulus
grandis wherein the cytoplasm is filled with periodic acid Schiff-positive fluid, protein yolk. Cortical alveoli (ca) are restricted to the periphery
of the oocyte, as is the nucleus (n), indicating that this oocyte is preovulatory. The oocyte is surrounded by the zona pellucida (zp). Bar 

100 m.
ments do not terminate at the testis periphery, but form
highly branched, anastomosing loops or tubules. The
term ??tubule?? was retained as for mammals in which
testicular tubules do not terminate at the testis periph-
ery, but also form loops (viz., Grier, 1993). In neote-
leosts, the germinal compartments may form anasto-
mosing networks proximally, but distally they extend
to the periphery of the testis and terminate blindly.
This character defines the lobular testis, which is either
unrestricted (neoteleosts) or restricted (atherino-
morphs) with regard to the distribution of spermato-
gonia within the lobules (Grier, 1993). Furthermore,
the testes in Atherinomorpha do not form anastomos-
ing networks, but the lobules may branch as they ex-
tend from the efferent ducts to the periphery of the
testis. This may be another atherinomorph synapo-
morphy, but support for more than this simple proposal
is lacking. The description of the germinal compart-
ments in atherinomorphs as ??lobular?? (Grier, 1993)
replaces use of the term ??tubular?? (Grier et al., 1980;
Grier, 1981; Nagahama, 1983; Rosen and Parenti,
1981) and emphasizes the distinctive differences in
testis structure between basal and higher teleosts as
subsequently defined by Grier (1993). The testis of the
Everglades pygmy sunfish, Elassoma evergladei, a
species examined here, is lobular, not anastomosing
tubular as described for the banded pygmy sunfish
Elassoma zonatum by Walsh and Burr (1984). We in-
terpret the testis of E. zonatum as unrestricted lobular
and suggest that description of the testis as anasto-
mosing tubular was due to the inconsistent way in
which these terms have been applied.
The unrestricted lobular testis is found throughout
the Neoteleostei (Table 1), including the paracanthop-
terygian Percopsis (Fig. 2B), the beardfish Polymixia
(Fig. 2D), and all other neoteleosts, except for the ath-
erinomorphs and the diminutive gobioid Schindleria,
discussed below. Basal, deep-sea neoteleosts in the or-
ders Myctophiformes, Stomiiformes, and Aulopifor-
mes (following the classification of Johnson and Pat-
terson, 1996) have not been surveyed; that they have
an unrestricted lobular testis is a prediction open to
test.
Atherinomorphs have been proposed by morpholo-
gists to be closely related to the percomorphs (Rosen
and Parenti, 1981), to mullets (Stiassny, 1993), to a
group of taxa in a higher-level teleost category, the
Smegmamorpha (Johnson and Patterson, 1993), or
with unresolved relationships to the percomorphs and
paracanthopterygians (Parenti, 1993). Smegmamorphs
were considered by Johnson and Patterson (1993) to
include, in addition to the atherinomorphs, the mullets,
Mugilidae, the swamp and spiny eels, Synbranchifor-
mes, the sticklebacks and relatives, Gasterosteiformes,
and the pygmy sunfishes, Elassoma. We examined rep-
resentatives of each of the non-atherinomorph smeg-
mamorphs and found all to have an unrestricted lob-
ular testis (Table 1). Similarly, all paracanthopterygi-
ans examined have an unrestricted lobular testis (Table
1).
That the restricted lobular testis of atherinomorph
fishes is not found in any of the other proposed smeg-
mamorphs does not test smegmamorph monophyly,
but does refute a recent molecular hypothesis in which
atherinomorph monophyly was challenged (e.g., Chen
et al., 2003). Atherinomorph monophyly has not been
questioned by morphologists since Gosline (1971), and
has been corroborated in a recent, broad-scale molec-
ular analysis (Miya et al., 2003). The list of diagnostic
morphological characters for atherinomorphs is exten-
sive and includes characters of the egg (viz., Parenti,
2004) to which we add fluid, rather than granular, yolk.
All other so-called smegmamorphs have globular pro-
tein yolk within vitellogenic and mature oocytes (Fig.
4B,C). Only during final oocyte maturation, preceding
ovulation, does the yolk become fluid?as observed
throughout vitellogenesis in atherinomorphs (Fig. 4A).
We also note that atherinomorphs have periodic acid
Schiff-positive yolk, but the phylogenetic significance
of this observation remains to be determined with fur-
ther surveys.
Ultrastructural investigation has shown that oogonia
are scattered in the simple epithelium lining the ovar-
ian cavity or lumen in common snook, Centropomus
undecimalis (see Grier, 2000) and the swamp eel, Syn-
branchus marmoratus (see Ravaglia and Maggese,
2003). It is a discontinuous germinal epithelium, fol-
lowing definitions of germinal epithelia (continuous
and discontinuous) developed for common snook
(Grier and Taylor, 1998). Our examination of the ovary
of Fundulus grandis, using plastic embedded tissue,
revealed the presence of oogonia that are scattered
within a germinal epithelium, similarly associated with
epithelial and prefollicle cells as in the snook and the
swamp eel. Contrary to Brummett et al. (1982), who
indicated that ??oocytes appear to be derived from oo-
gonia located immediately beneath the simple epithe-
lium lining the ovarian cavity,?? we conclude that the
luminal epithelium in the ovary of Fundulus is the
germinal epithelium. Furthermore, it is established that
344 L. R. PARENTI AND H. J. GRIER
FIG. 4. A. Periphery of the ovary in Hemiramphus brasiliensis with a preovulatory oocyte (POV) whose periodic acid Schiff-positive (PAS),
protein yolk is lighter-staining than the protein yolk in an oocyte that is not preovulatory. In both oocytes, the protein yolk (y) is fluid. The
germinal epithelium separates these two oocytes, and primary growth oocytes (PG), from the ovarian lumen (OL). Bar 
 50 m. B. In the
ovary of reproductive Mugil cephalus, the germinal epithelium (GE) separates the ovarian lumen (OL) from the stroma in which a prominent
extravascular space (EVS) is observed. Within the germinal epithelium, two small primary growth oocytes (arrows) are observed. Larger
primary growth oocytes (PG) and mature oocytes (MOC) are surrounded by the EVS. Mature oocytes have globular protein yolk granules
(yg) rather than fluid yolk. These become fluid during final oocyte maturation, just before ovulation. Lipids (1) are circumnuclear in position,
and cortical alveoli (ca) are peripheral. Bar 
 50 m. C. Portion of a mature oocyte from the Everglades pygmy sunfish, Elassoma evergladei.
The content of cortical alveoli is periodic acid Schiff-positive (purple). Globular yolk (y) is also periodic acid Schiff-positive, particularly the
intensely-staining, small yolk globules at the oocyte periphery. The germinal epithelium, separating the oocyte from the ovarian lumen (OL),
is composed of epithelial cells with flattened nuclei (E) and an oogonium (OG). Bar 
 10 m.
345GONAD MORPHOLOGY IN BONY FISHES
the ovarian germinal epithelium in the protogynous
swamp eel, Synbranchus marmoratus, produces folli-
cles during the female phase of the reproductive cycle.
During sex reversal, however, the same germinal epi-
thelium produces the initial male germ cells before
lobules are formed (Lo Nostro and Grier, 2002). This
observation introduces a new approach to study of the
mechanism of sex reversal in other protogynous spe-
cies, for example in the perciform families Serranidae,
Labridae, and Gobiidae.
Our investigation reveals that there is constant ori-
gin of ovarian follicles from the germinal epithelium
among taxa. Terminology should reflect this proposal
of homology. The histology text book definition of a
follicle (see Grier and Lo Nostro, 2000) precisely re-
flects its origin from a germinal epithelium. The fol-
licle is composed of the germ cell, the oocyte, and
surrounding follicle (granulosa) cells that originate
from the epithelial (somatic) cells of the germinal ep-
ithelium. The follicle is surrounded by a theca, derived
from the stromal compartment of the ovary (Grier,
2000) and is always separated from this compartment,
throughout development and final oocyte maturation,
by a basement membrane. As a basement membrane
separates an epithelium from the underlying lamina
propria or supportive tissue, so it also separates the
follicle from the theca. The follicle basement mem-
brane is derived from that underlying the germinal ep-
ithelium (Grier, 2000). The term ??follicle complex??
(Grier and Lo Nostro, 2000) has been proposed to in-
clude the follicle, basement membrane, and the theca,
including its blood vessels. The various definitions of
a follicle within the fish literature are based primarily
on function rather than form, however. These defini-
tions of a follicle include the surrounding theca, with
(Redding and Patin?o, 1993; Sullivan et al., 1997) or
without (Tyler and Sumpter, 1996) the basement mem-
brane.
Terminology may be confusing due to our ignorance
of comparative morphology, the use of synonyms, the
still-emerging physiological and molecular events
causing oocyte growth, the now documented role of a
germinal epithelium in follicle formation and in sex
reversal (vide supra), and an array of egg types in fish-
es that is only dealt with superficially herein. The term
??final oocyte maturation?? has not become ??irrelevant
and misleading,?? as argued by Patin?o and Sullivan
(2002). Final oocyte maturation is used commonly to
describe changes in oocytes leading to ovulation ob-
served in common snook (Neidig et al., 2000) and
other marine fishes (viz., Brown-Peterson et al., 1988,
2002) and is embedded in the fisheries literature. Oo-
cyte changes leading to ovulation include cytoplasmic
and nuclear events (as in Patin?o and Sullivan, 2002),
and, in our opinion, these events mark final oocyte
maturation prior to ovulation. All of the changes in
follicles as they mature can be viewed as maturation
or growth.
Definitions based on homology of form can aid in
understanding reproduction across a broad array of
taxa. For example, mammals have an advanced follic-
ular morphology not found in teleosts. As above, the
teleost follicle consists merely of the oocyte and its
encompassing, monolayer of follicle (granulosa) cells,
defined as a ??primordial follicle?? (viz., Van Blerkom
and Motta, 1979). In mammals, however, the follicle
cells divide, become many cells deep, surround the
oocyte to produce a primary follicle. Then, a fluid-
filled space develops, to form the antrum (Gray et al.,
1995) of a tertiary follicle. From the standpoint of
comparative anatomy, the fish ovarian follicle corre-
sponds to the mammalian ??primordial follicle,?? and
all oocyte maturation occurs within the homologue of
the mammalian ??primordial follicle.?? Furthermore, as
in fish (Grier, 2000), mammalian follicles originate
from a germinal epithelium (Zamboni, 1972), i.e., they
are homologues. Recent studies of mammalian repro-
duction confirm that follicle cells in sheep develop
from cells of the germinal epithelium (Heywood et al.,
2002), and that a possibly active germinal epithelium
is present in mice (Johnson et al., 2004: fig. 2). We
anticipate that additional homologous characteristics
within reproductive systems across a broad array of
taxa are yet to be revealed.
The annual reproductive cycle is hypothesized to be
the source of morphological variation among testis
types. Five reproductive classes have been described
in males of the common snook, Centropomus unde-
cimalis (see Taylor et al., 1998), seatrout, Cynoscion
nebulosus (see Brown-Peterson, 2003), the cobia, Ra-
chycentron canadum (see Brown-Peterson et al.,
2002), the swamp eel, Synbranchus marmoratus (see
Lo Nostro et al., 2003), and the freshwater goby, Pa-
dogobius bonelli (see Cinquetti and Dramis, 2003): re-
gressed, early maturation, mid maturation, late matu-
ration, and regression. These are identified by the al-
ternation of the germinal epithelium between contin-
uous and discontinuous types and the stages of germ
cells present (Grier, 2000).
The transition between basal and neoteleosts is
marked by numerous physiological and morphological
modifications, such as type 4 tooth attachment (Fink,
1981) and acellular bone (Parenti, 1986), characters
that Esox shares with neoteleosts (Parenti, 1986; John-
son and Patterson, 1996). Teleost evolution was de-
scribed as paedomorphic by Fink (1981) because some
characters observed in adults of more advanced tele-
osts, such as tooth attachment mode, approximate the
early developmental stages of primitive teleost fishes.
The evolutionary transition from an anastomosing tu-
bular to a lobular testis could have resulted from the
elongation of the testis germinal compartments during
the early maturation class when the testis enlarges pri-
or to the breeding season. The process could have
evolved as a simple change in formation of the sup-
porting basement membrane of the germinal epitheli-
um. Similarly, restriction of spermatogonia to the dis-
tal ends of lobules in the atherinomorphs is mirrored
in perciforms by the establishment of distal epithelioid
cords of Sertoli cells and spermatogonia in cobia testes
346 L. R. PARENTI AND H. J. GRIER
during the regression and regressed classes of the an-
nual reproductive cycle (Brown-Peterson et al., 2002).
Clusters of spermatogonia become established during
the same reproductive classes in common snook (Grier
and Taylor, 1998). During regression in these perci-
forms, but not in atherinomorphs, spermatogonia also
become established along the walls of lobules. There
is a marked change in the arrangement of Sertoli cells
in atherinomorphs compared to other fishes. Sertoli
cells extend processes across the lobules, and a lobule
lumen is absent. We hypothesize that the difference in
the way in which Sertoli cell processes bridge the
widths of the lobules, as in Fundulus grandis (Fig. 1A)
and Hemiramphus brasiliensis (Fig. 1C), prevents the
colonization of the lateral lobule walls by spermato-
gonia?they are only observed at the distal termini of
the lobules. Evolution of the atherinomorph testis type,
the strongest evidence supporting atherinomorph
monophyly (viz., Parenti, 2004), is hypothesized to en-
tail mechanisms that prevent the repopulation of sper-
matogonia along lobule walls during regression and
when regressed. Atherinomorphs may be said to have
a ??regressed?? testis that undergoes a functional mat-
uration.
There is scant information on the mechanism of lob-
ule elongation during gonad maturation between
spawning seasons, and practically nothing is known
about the process of regression. It has recently been
proposed that fish testes shift between meiosis-domi-
nated to mitosis-dominated cell divisions during their
annual reproductive cycles (Grier, 2002). The supply
of spermatogonia, from which meiotic germ cells are
derived, becomes progressively exhausted between
early maturation, mid maturation, and late maturation.
It has not been appreciated that in the latter part of the
annual reproductive cycle, especially regression, the
testes are actively preparing for the next reproductive
cycle, i.e., the lobules become repopulated by sper-
matogonia. The same is true in the teleost ovary, at
least in Centropomus undecimalis, in which the pro-
cess of folliculogenesis was interpreted using ovaries
from regressed fish (Grier, 2000). There is significant
cell division during the regressed class in Synbranchus
marmoratus that involves both Sertoli cells and sper-
matogonia (Lo Nostro and Grier, 2003). Sertoli cells
have been demonstrated to divide in the atherinomorph
Poecilia latipinna (see Grier, 1993: fig. 25), and the
wrasse, Thalassoma bifasciatum (see Koulish et al.,
2002). Mitotic cell division in fish testes during the
regression and regressed classes, as defined by Taylor
et al. (1998), has hardly been investigated. In light of
growth processes during these classes, however,
Brown-Peterson et al. (2002) suggested that the term
??resting stage?? no longer be used. Active cell division
likely takes place in fish gonads throughout the annual
reproductive cycle, even when not spawning.
There appear to be two locations within the testes
of perciforms from which spermatogonia are derived:
in common snook (Grier and Taylor, 1998) and cobia
(Brown-Peterson et al., 2002), clusters or elongation
of the lobules composed of spermatogonia and Sertoli
cells become established at the distal ends of lobules.
Divisions of these cells result in lobule elongation
(Grier, 1993). Lobule growth was inferred to occur via
a branching process; in common snook, anastomosing
testis morphology results from fusion of lateral lobule
walls (Grier and Taylor, 1998). We have not observed
anastomosing of the lobules in any atherinomorph.
Spermatogonia also repopulate the lateral walls of
testicular lobules. By the end of the breeding season,
the population of spermatogonia is exhausted, these
cells having formed sperm. There are always scattered
spermatogonia along the lobule walls that apparently
do not divide, however. They compose a ??discontin-
uous germinal epithelium,?? one that spans reproduc-
tive seasons and are the prima facie evidence for a
permanent germinal epithelium in fishes (Grier, 1993;
Lo Nostro et al., 2003). During the regression and re-
gressed classes?the mitosis dominated classes?sper-
matogonia within the discontinuous germinal epithe-
lium become mitotically active, repopulate the lateral
lobule walls and form a continuous germinal epithe-
lium, again composed of spermatogonia and Sertoli
cells. The term ??epithelioid?? was first applied to a
description of the testes in the cobia in reference to
cords of spermatogonia and Sertoli cells that grow
from the distal termini of the lobules during the re-
gressed class in the annual reproductive cycle (Brown-
Peterson et al., 2002). Because they lack a lumen,
these cells do not fulfill the criteria that define an ep-
ithelium (Grier, 2000; Grier and Lo Nostro, 2000). The
epithelioid arrangement of cells at the distal termini of
lobules is transitory. When a lumen develops, these
cells compose a germinal epithelium.
Changes in the male germinal epithelium, used to
define annual reproductive classes first in common
snook (Taylor et al., 1998), cannot be used to define
annual reproductive classes in females. The basic dif-
ference between the sexes that prevents this simple
characterization is the early initiation of meiosis in fe-
males. In common snook, the germinal epithelium is
active throughout the year, producing follicles where
the oocyte is in arrested meiosis, diplotene of the first
meiotic prophase, and the germinal epithelium is al-
ways discontinuous. In males, only diploid spermato-
gonia persist in the regressed class and the germinal
epithelium is continuous, becoming discontinuous dur-
ing mid maturation and late maturation. The annual
alternation between continuous and discontinuous ger-
minal epithelia in the male teleost germinal epithelium
permits the use of changes to be used for defining
annual reproductive classes.
These proposals emphasize that morphological
change within the testis during the annual reproductive
cycle can lead to the formation of distinct testis types.
Interestingly, Schindleria, a gobioid fish that has been
characterized as ?? . . . the most radically ontogeneti-
cally truncated fish?? (Johnson and Brothers, 1993, p.
469), reportedly has a restricted lobular testis (Thacker
and Grier, 2004). This corroborates our hypothesis that
347GONAD MORPHOLOGY IN BONY FISHES
arrest of the late maturation-regression phases leads to
formation of the atherinomorph testis type; that is, it
is like the paedomorphic testis of mature adult Schin-
dleria.
Testis type provides another way to distinguish
among model fish organisms: the zebrafish, Danio re-
rio, has an anastomosing tubular testis, whereas the
atherinomorph medaka, Oryzias latipes, has a restrict-
ed lobular testis (Table 1). We predict that the fugu,
Takifugu rubripes, a derived percomorph, has an un-
restricted lobular testis. Our proposal is that simple
changes in the annual reproductive cycle have resulted
in these different testis types. The genetic basis of
these differences is unknown. Identifying morpholog-
ical variation, and proposing the source of that mor-
phological variation, is a first step in understanding
underlying genetic mechanisms.
ACKNOWLEDGMENTS
We are grateful to Francesco Santini and Gustavo
Ybazeta, University of Toronto, for organizing the
symposium on teleost fishes and for inviting us to par-
ticipate. Helen F. Wimer, USNM, skillfully prepared
the bulk of the histological sections. She was granted
access to plastic embedding and sectioning facilities
by William D. Swaim, Director, Cellular Imaging Core
Facility, National Institute of Dental and Craniofacial
Research, NIH. Steve Mims, Kentucky State Univer-
sity, Rich McBride, FMRI, and Maria del Carmen Uri-
be, UNAM, Mexico City, helped obtain gonad mate-
rial. Llyn French, FMRI, graciously prepared the fig-
ures and helped edit the manuscript. Jeffrey M. Clay-
ton, USNM, and Yvonne Waters, FMRI, provided
invaluable technical assistance.
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