Spermatozoal Morphology and Ultrastructure of Channel Catfish, Ictalurus punctatus

Ë d m o n d e J. J a s p e r s , J a m e s W. A v a u l t , J r ., a n d J o s e p h D. R o u s s e l

Made in the United States of America Reprinted from T ransactions o f t h e A m e r ic a n F i s h e r ie s S o c ie t y Vol. 105, No. 3, May 1976 pp. 475-480 © Copyright by the American Fisheries Society, 1976

Spermatozoal Morphology and Ultrastructure of Channel Catfish, Ictalurus punctatus1,2

E d m o n d e J . J a s p e r s 3 Department of Marine Sciences

J a m e s W . A v a u l t , J r . Fisheries Division School of Forestry and Wildlife Management

J o s e p h D. R o u s s e l Department of Dairy Science Louisiana State University Baton Rouge, Louisiana 70803

ABSTRACT The morphology, dimensions, aberrations, and ultrastructure of channel catfish (Ictalurus punctatus) spermatozoa were studied. Spermatozoa of a wild catfish stock from Louisiana were compared to those of a domestic stock from Mississippi. The spermatozoon of channel catfish consists of a rounded dark head, a collar-like mid­ piece, and a long flagellum. The average dimensions were: head length, 2.3 pm; midpiece length, 1.6 /an; midpiece width, 3.1 Aim; and flagellum length, 94.9 pm. The flagellum had the classical (2.9 + 2) axoneme. On the average, 4.7% of the spermatozoa in the fish studied were biflagellar. Each flagellum arose from an individual centriolar complex. Domestic fish stock had a higher percentage of normal gametes compared to wild stock.

Even though research is in progress to ge­ from Yazoo City, Mississippi. From each netically improve the commercial value of stock, twelve 2-year-old and twelve 3-year-old channel catfish, Ictalurus punctatus (Rafin­ male specimens were used to study morphol­ esque), through selective breeding, various ogy, dimensions, aberrations, and ultrastruc­ aspects of its reproductive biology remain ture of their spermatozoa. The fish were rather obscure. Since selective breeding will stunned by electric shocking and the testes sooner or later involve cryogenic preserva­ dissected to expose the male gametes. tion of semen, obtaining an insight in the morphology of male gametes is a prerequisite Spermatozoal Morphology and Dimensions to the study of their physiology. This paper Gross morphology of spermatozoa from reports on various morphological properties the anterior, medial, and posterior areas of of channel catfish spermatozoa. the spermatogenic part of the testes was studied separately. A tissue sample from the METHODS appropriate region was teased apart in a drop Male gametes of a wild stock of channel of 0.65% NaCl on a glass slide and covered catfish from Lake des Allemands, Louisiana, with a number 1 cover slip. The sample was were compared with those of a domestic stock examined under oil immersion with a phase contrast microscope at a magnification of 1 Research supported by NOAA Department of 1,569 X . Spermatozoal dimensions were de­ Commerce through the Sea Grant Program, by the termined to the nearest 0.8 /im with an ocular LSU Agricultural Experiment Station, by the Lou­ isiana Catfish Farmers Association, and by a private micrometer. grant from Mrs. Addie R. Cormie. During the first 3 months of the study 2 Based on a section of a dissertation by E. J. (November 1971-December 1972), 20 sper­ Jaspers submitted in partial fulfillment of the re­ quirements for the Ph.D. degree at Louisiana State matozoa were measured in each testicular University, December 1972. area per fish. Statistical analysis of these 3 Present address: I.Z.W.O. (Institute for Marine data revealed that, due to the small variation Scientific Research), 69, Prinses Elisabethlaan, 8401 Bredene, Belgium. in the measurements, the number of obser- 475 476 TRANS. AM. FISH. SOC., 1976, NO. 3 valions could be reduced to five for the fol­ Beem capsules4 (Spurr 1969). After harden­ lowing 3 months (February 1972—April ing overnight at 70 C, thin sections were cut 1972), without losing accuracy. with an LKB ultramicrotome4 with thermal The following spermatozoal measurements advance. Thickness of the sections was de­ were taken : total length of the head and mid­ termined by their interference colors, when piece, length of the head, length of the mid­ viewed under a dissecting microscope, ac­ piece (these three measurements were made cording to the technique described by Peachy along the longitudinal axis), width of the (1958). Silver to silver-gray thin sections head, width of the midpiece (both along the (60-90 nm thick) were then mounted on axis perpendicular to the longitudinal one), unsupported 300 mesh copper grids. Staining and length of the flagellum. was done with 0.5% uranyl acetate in 90% ethanol and Reynold’s lead hydroxide che­ Spermatozoal Aberrations lated with citrate (Reynolds 1963; Dawes After determining their dimensions, a total 1971). of 200 spermatozoa were examined for each Thin sections were examined with an RCA of the three testicular areas in every speci­ EMU-3G electron microscope at an electron men. A differential count of the morphologi­ acceleration of 50 kV, and magnifications cally normal spermatozoa and those with ranging from 5,800 to 16,000 X . Micro­ aberrations was carried out. Only morpho­ graphs of 5 cm X 5 cm were taken on Kodak logical aberrations pertaining to the flagel­ Electron Microscopy Film (Estar thick base) lum were observed. Distinction was made with extremely fine grain. between biflagellar gametes and gametes with a flagellum that appeared double or split at Statistical Procedures some regions, and single in other areas of Morphological data were analysed by least the tail. square analysis of variance and corrected for The number of spermatozoa recorded in unequal numbers within classes, which oc­ the differential count as either normal, bi­ curred in a few cases. flagellar or with a split flagellum was then The means of the different variables were divided by two to give the respective per­ calculated within and among each source of centiles. variation (month of examination, stock, age, and testes area). Standard error of the mean, Spermatozoal Ultrastructure standard deviation from the mean, and co­ Ultrastructure of channel catfish sperma­ efficient of variation were computed. tozoa was investigated by electron micros­ copy. RESULTS AND DISCUSSION Tissue from the three areas of the sperma- Spermatozoal Morphology and Dimensions togenic part of the testes was macerated separately and fixated in a 3% gluteralde- The spermatozoon of channel catfish com­ hyde, CHO(CH2) 3CHO solution at 4 C for prises a rounded head, a collar-like midpiece, 2 hours and left overnight in Sorensen’s and a long flagellum (Fig. 1), which are phosphate buffer with sucrose (McLean and characteristic for the primitive type of meta- Cook 1952). The material was then postfixed zoan spermatozoon (Franzén 1969). The in a 1% osmium tetroxide (OsCL) solution head appears dark due to the dense nuclear for 1.5 hours, followed by a cold water rinse. chromatin, while the midpiece, containing The sample was then carried through a de­ the mitochondria is lighter in color. In gross hydration series of ethanol: 25%, 50%, 75% morphology the channel catfish spermatozoon each for 5 minutes, 90% until room tempera­ resembles the schematic drawing of the male ture was reached, and 100% with three gamete of Clarias senegalis, belonging to the changes, 10 minutes each. Embedding was done in Spurr’s low vis­ * The use of trade names does not imply endorse­ cosity epoxy resin medium, in polyethylene ment of the commercial products. JASPERS ET AL.—STRUCTURE OF CATFISH SPERM 477

T able 1.—Overall mean, standard error of the mean, and coefficient of variation of spermatozoal di­ mensions of 35 male channel catfish all months (December 1971-April 1972), stocks, ages, and testes areas combined.

Stan­ dard Number Mean error Coeffi­ of obser­ cient of Variable vations fim variation

Total length of head and midpiece 525 3.9 0.01 7.64 Head length 525 2.3 0.01 10.29 Midpiece length 525 1.6 0.01 12.94 Head width 525 2.4 0.01 8.21 Midpiece width 525 3.1 0.01 8.09 Flagellum length 525 94.9 0.26 6.38

lucius Linnaeus; 2.5 pm and 1.5 to 2 pm for rainbow trout, Salmo gairdneri Richard­ son and brown trout, Salmo trutta fario Linnaeus; 4 pm and 1 /xm for guppy, Poecilia reticulata Peters. Very little variation occurred in the mean dimensions for observations grouped by month, by stock, by age, and area of the testes. For some dimensions a significant difference (P < 0.05) existed among months. However, since these relate only to a fraction of a micrometer, they can for all practical F igure 1.— Photograph of a normal spermatozoon purposes of be disregarded because of human channel catfish, magnified 5,020 X (only % of error and limited accuracy of the ocular the flagellum is shown). micrometer. same order of Siluriformes as the Ictaluridae Spermatozoal A berrations (Mattei 1969). For all the fish examined, an average of As in all teleost species, except for Anguilla 14.1% ± 0.6% of the spermatozoa showed anguilla Cuvier (Tuzet and Fontaine 1937), morphological aberrations of the flagellum. the channel catfish spermatozoon lacks an The existence of biflagellar spermatozoa at acrosome. Pasteels (1965a, 1965b) sug­ an average rate of 4.7% ± 0.6% in each gested that the absence of an acrosome might specimen was confirmed by electron micro­ be associated with the presence of a micro- scopical examination. The split flagellum, phyle in the eggs of the teleost fishes. which may have actually consisted of two Dimensions of channel catfish spermatozoa separate tails overlapping each other at some are shown in Table 1. Measurements were points, occurred with a mean frequency of of the same magnitude as was reported by 9.4% ± 0.6% in each fish examined. Billard (1969) for four teleost fishes. The The number of flagella seems to be less length of the spermatozoal head and its consistent in fishes than in other animal maximal width reported by Billard (1969) groups. Billard and Fléchon (1969) ob­ were, respectively: 3.3 pm and 2.5 /j,m for served two flagella on some spermatids of common carp, Cyprinus carpio Linnaeus; 2 guppy. Biflagellar spermatids occurred in pm and 1.8 pm for the northern pike, Esox plainfin midshipman, Porichthys notatus 478 TRANS. AM. FISH. SOC., 1976, NO. 3

gametes (87.3% ± 1.4%) compared to the wild fish (84.4% ± 1.5%).

Spermatozoal Ultrastructure Although spermatozoal ultrastructure of some fish species have been studied (Ginz­ burg 1972), those of Ictaluridae have never been examined, and a first attempt was made in this study. No apparent differences in spermatozoal ultrastructure existed between the wild and domestic stock of channel cat­ fish, neither between the age group, nor the different areas of the spermatogenic part of the testes. A normal spermatozoon of channel catfish is shown in Fig. 2. Most of the rounded head consists of dense nuclear chromatin, contain­ ing DNA during interphase (De Robertis et al. 1965). In channel catfish the chromatin is slightly granular, as in common carp. In F ig ü r e 2 .—Electron micrograph of a longitudinal section of the head region of a normal spermato­rainbow trout, brown trout, and northern zoon of channel catfish, magnified 10,360 X- Sym­ pike chromatin occurs as large granules, bols: cm, cytoplasmic membrane; eye, cytoplas­while in guppies it appears as an almost mic canal; de, distal centriole; f, flagellum; mp, midpiece; nm, nuclear membrane (karyotheca); homogenous mass (Billard 1969). The nu­ nu, nucleus. clear membrane is only slightly undulated and an acrosome is lacking (Fig. 2). A midpiece with collar-like appearance, Girard (Stanley 1965), and in an African such as in channel catfish, is usually referred bichir, Protopterus annectens (Owen) (Bois­ to as cytoplasmic or mitochondrial collar. In son et al. 1968). The African teleost Gym­ channel catfish this collar is low (mean narchus niloticus (Cuvier) is to date the only length 1.6 pm) , but broad (mean width 3.1 vertebrate known to have tailless sperma­ pm, Table 1). tozoa (Mattei et al. 1967a). Morphology of the cytoplasmic collar in Occasionally spermatozoa with wide trans­ teleosts might be associated with fertilization. lucent round heads were encountered, but Low collars, as in channel catfish, occur if this was probably the result of water ab­ fertilization is external and high ones in sorption and swelling and not a morphologi­ viviparous species (Porte and Follenius cal abnormality (Ginzburg 1972). 1960; Dadone and Narbaitz 1967; Franzén Among the anterior, medial, and posterior 1969; Stanley 1969). part of the testes little variation in the rela­ The flagellum of the channel catfish sper­ tive percentages of normal and abnormal matozoon is separated from the mito­ spermatozoa existed. A highly significant chondrial collar by an invagination of the difference (P < 0.01) in the mean percent cytoplasmic membrane, forming a cytoplas­ normal spermatozoa was apparent during mic canal, comparable to that of mammalian March and April (respectively, 91.4% ± 2.3% spermatids (Meander 1968; Billard 1969, and 94.5% ± 2.2% versus a range from 1970) (Figs. 2, 3, and 4). 70.0% ± 2.5% to 87.1% ± 1.4% for the other The midpiece contains mitochondria and months). This was coupled with a decrease vesicles, irregularly distributed in the cyto­ in the number of spermatozoa with a split plasm (Fig. 3). flagellum. Domestic fish stock showed a The centriolar complex of spermatozoa in significantly higher percentage of normal vertebrates consists usually of two structures, JASPERS ET AL.—STRUCTURE OF CATFISH SPERM 479

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F i g u r e 3.—Electron micrograph of a longitudinal F igure 4.—Electron micrograph of a cross section of section of the head region of a biflagellar sper­the midpiece region of a biflagellar spermatozoon matozoon of channel catfish, magnified 19,690 X- of channel catfish, showing the two flagella with Symbols: ch, chromatin; cm, cytoplasmic mem­ outer and central fibrils and the cytoplasmic canal, brane; eye, cytoplasmic canal; de, distal cen-magnified 64,130 X - Symbols: cf, central fibril; triole; f, flagellum; mi, mitochondrion; nm, nu­ eye, cytoplasmic canal; f, flagellum; of, outer clear membrane (karyotheca); nu, nucleus; pc, fibril. possibly proximal centriole; v, vesicle.

northern pike, rainbow and brown trout a proximal and a distal centriole, instead of (Billard 1969) ; tidepool sculpin, Oligocottus the single basal body as in cilia. Structural maculosus Girard (Stanley 1969). modifications may occur in either or both On the average 4.7% ± 0.2% of the sper­ parts. In channel catfish spermatozoa, a matozoa examined were biflagellar and found distal centriole could be identified (Figs. 2 in every fish, regardless of age, stock, or and 3) and possibly a proximal one (Fig. 3). time. Each flagellum arises from an indi­ To determine its structural properties, a vidual centriolar complex (Fig. 3) and is more detailed study of the centriolar complex separated from the cytoplasm of the midpiece would be necessary. and from one another by a cytoplasmic canai The spermatozoal flagellum of channel cat­ (Figs. 3 and 4 ). Both flagella have the fish possesses the classical (2.9 + 2) axo- (2.9 + 2) axoneme with the central fibrils neme of nine double outer fibrils and two showing identical orientation. Stanley (1965) smaller single central fibrils (Fig. 4 ). The reported a similar structure of the biflagellar flagellum is bisymmetrical because of the spermatozoa of plainfin midshipman. nine double peripheral microtubules. A plane perpendicular to the one through the two ACKNOWLEDGMENTS central fibrils always cuts through one of the Dr. M. D. Socolofsky, Professor of Micro­ outer fibrils. The flagellum of the channel catfish spermatozoon lacks a peripheral sheet biology, and Mrs. Gale LoPicollo of LSU are or ridge as is found among others in the fol­ thanked for their valuable help with the elec­ lowing fish species: silver salmon, Oncorhyn­ tron microscopy work. Appreciation is ex­ chus kisutch (Walbaum) (Lowman 1953) ; pressed to Dr. Luther E. Franklin, Depart­ guppy (Mattei et al. 1967b) ; common carp, ment of Biology, University of Houston, for 480 TRANS. AM. FISH. SOC., 1976, NO. 3 his assistance with the interpretation of the of silver salmon spermatozoa (Oncorhynchus electron micrographs. The authors are in­ kitsuch (Walbaum)). Exp. Cell Res. 5:335- 360. debted to Dr. Kenneth L. Koonce, Associate M a t t e i, X. 1969. Spermiogenèse comparée des Professor of Experimental Statistics at LSU, poissons. Pages 57-69 in Baccio Bacetti, ed. Comparative spermatology. Academic Press, for assistance in statistical analyses. New York, London. 573 pp. The Thompson-Anderson Enterprises, Yazoo , C. B o is s o n, C. M a t t e i, a n d C. R e iz e r . City, Mississippi, are thanked for providing 1967a. Spermatozoïdes aflagellés chez un pois­ son: Gymnarchus niloticus (Téléostéen, Gym­ the domestic fish used in this study. narchidae). C. R. Acad. Sei., Sér. D 265:2010- 2012. LITERATURE CITED , C. M a t t e i, a n d C. B o is s o n. 1967b. L’ex­ trémité flagellaire du spermatozoïde de Lebistes B il l a r d, R. 1969. Ultrastructure comparée de reticulatus (Poecilidae). C. R. Soc. Biol. 161: spermatozoïdes de quelques poissons téléostéens. 884-887. Pages 71-79in Baccio Bacetti, ed. Comparative M c L e a n , R. C., a n d W. R. I. C o o k. 1952. Plant spermatology. Academic Press, New York, Lon­ science formulae. MacMillan and Co. Ltd., Lon­ don. 573 pp. don, England. 205 pp. . 1970. La spermatogenèse de Poecilia reti­ N ic a n d e r, L. 1968. Gametogenesis and the ul­ culata. IV La spermiogenèse. Etude ultrastruc- trastructure of germ cells in vertebrates. VIeme turale. Ann. Biol. Anim. Biochim. Biophys. Congrès International Reproduction Animale 10(3) : 493-510. et Insémination ArtificieUe, Volume 1:89-107. — ■, a n d J. E. F l e c h o n . 1969. Spermatogonies P a s t e e l s, J. J. 1965a. La fécondation étudiée au et spermatocystes flagellés chez Poecilia reti­ microscope électronique. Etude comparative. culata (Téléostéens Cyprinodontiformes). Ann. Bull. Soc. Zool. Fr. 90: 195-224. Biol. Anim. Biochim. Biophys. 9(2) : 281-286. •. 1965b. Aspects structuraux de la fécon­ B o is s o n, C., X. M a t t e i, a n d C. M a t t e i. 1968. Le dation vus au microscope électronique. Arch. spermatozoïde de Dactylopterus volitans, Linné Biol. Belges 76:463-509. (Poisson Céphalacanthidae), étudié au micro­ P e a c h y, L. D. 1958. Thin sections I. A study of scope électronique. C. R. Soc. Biol. 162: 820- section thickness and physical distortion pro­ 823. duced during microtomy. J. Biophys. Biochem. D a d o n e, L., a n d R. N a r b a it z. 1967. Submicro- Cytol. 4: 233-242. scopic structure of spermatozoa of a cyprino- P o r t e , A., a n d E. F o l l e n iu s. 1960. La spermio­ dontiform teleost, Jenynsia lineata. Z. Zell- genèse chez Lebistes reticulatus, étude au mi­ forsch. Mikrosk. Anat. 80(21:214-219. croscope électronique. Bull. Soc. Zool. 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S. 1972. Fertilization in fishes and spermiogenesis in the teleost fish Oligocottus the problem of polyspermy. Israel Program maculosus. J. Ultrastruct. Res. 27:230-243. Scientific Translations, Jerusalem. 366 pp. T u z e t , O ., a n d M. F o n t a in e. 1937. Sur la sper­ (Academy of Sciences of the USSR, Institute of matogenèse de l’anguille argentée (Anguilla Development Biology.) vulgaris Cuv.). Arch. Zool. Exp. Gén. Notes L o w m a n, F. G. 1953. Electron microscope studies Rev. 4: 199-215.