Ultrastructural studies of the morphological variations of the egg surface and envelopes of the African catfish Clarias gariepinus ( Burchell , 1822 ) before and after fertilisation , with a discussion of the fertilisation mechanism

Much of the existing knowledge of the mechanisms involved in teleost fertilisation is based on a few small model species that have no commercial value. Research is therefore urgently required to address mechanisms involved in fertilisation in species of great commercial value. In this study, the ultrastructural morphological variations in the surface of the egg of Clarias gariepinus were recorded before and after fertilisation by using electron microscopy. The outer surface of the unfertilised egg was smooth, whereas the fertilised egg acquired a network of projections on the vegetal hemisphere. Moreover, different patterns of ornamentation on the egg surface were evident. This pattern of ornamentation varied with the progress of embryonic development. The micropyle of the C. gariepinus egg consisted of a funnel-shaped vestibule, from the bottom of which a cylindrical micropylar canal extended. The micropylar canal decreased in diameter after completion of fertilisation, forming a micropylar disc. The sperm behaviour on the egg surface was oriented towards any depression on the chorion surface. The chorion of ovulated eggs consisted of one layer. After fertilisation the chorion was differentiated into three layers: the double-layered coat, the zona radiata externa and the zona radiata interna. Four protein subunits of the chorion of C. gariepinus were identified by SDS-PAGE. IR-spectra obtained from C. gariepinus chorion revealed that the vibration of chorion proteins exhibited different weak activities in the IR-spectra with minor difference between preand post-fertilisation chorion proteins.


INTRODUCTION
Teleosts include more than half the vertebrate species (Baldacci et al., 2001).A key feature of teleost evolutionary success is their reproductive system, which must be functional in all aquatic environmental conditions.Much of the existing knowledge of the mechanisms involved in teleost fertilisation is based on a few small model species such as zebrafish, medaka and bitterling, which have no real commercial value (Coward et al., 2002).Research is required to address mechanisms involved in fertilisation in species of commercial value, which represent a cornerstone of the field of aquaculture and the associated biotechnology, genetic engineering and biodiversity fields.
Interspecific differences in the microstructure of the chorion (egg envelope) of teleost fishes have been recognised for almost 30 years (Merrett and Barnes, 1996).Such differences have been used not only to identify eggs in plankton samples (Merrett and Barnes, 1996), but also as potentially useful taxonomic characters (Gill and Mooi, 1993;Johnson and Brothers, 1993;Britz et al., 1995;Chen et al., 1999;Britz and Breining, 2000).Chen et al. (1999) concluded that the ultrastructural features of the egg envelope were found to be helpful in species identification of the distantly related species, but not of closely related ones.Merrett and Barnes (1996) clarified Marshall's (1973) suggestion that egg envelope ornamentation is a family characteristic.
The chorion structure and its chemical constituents is the end product of different evolutionary trends, adaptation processes and environmental factors (Yamagami et al., 1992;Celius and Walther, 1998;Quagio-Grassiotto and Guimuraes, 2003).Hyllner et al. (2001) concluded that the chorion proteins from the vertebrate groups of fishes and their amphibian, avian and mammalian counterparts share a common ancestry and form a unique group of structural proteins.Such proteins exhibit dramatic changes during chorion hardening.
Several important questions arise with regard to fertilisation.Where do the instructions for the process of teleost fertilisation lie?What is the language of these instructions?Are these instructions and their language influenced by genetic programs of both parents?To answer these questions, the fertilisation process of a fish must be described in the light of reorganisation of the chorion at fertilisation, its physiological roles, the dynamic changes of the egg cortex, the secretory functions of cortical granules, mechanisms of sperm-egg interactions, and mechanical blocking of polyspermy (Lönning and Hagstrom, 1975;Hart, 1990;Yamagami et al., 1992;Griffin et al., 1996;Merrett and Barnes, 1996;Linhart and Kudo, 1997;Chen et al., 1999;Britz and Breining, 2000;Coward et al., 2002).
Literature dealing with Clarias gariepinus (Burchell, 1822) chorion and fertilisation and the associated mechanisms is very rare.Only two articles (Riehl and Appelbaum, 1991;Wenbiao et al., 1991) are at hand.The present study aimed to elucidate the morphological variations of the chorion of C. gariepinus eggs before and after fertilisation.It also aimed to emphasize the process of chorion hardening and its biochemical composition, in addition to the structure and behaviour of spermatozoa on the chorion surface, using transmission and scanning electron microscopy.The micropyle shape and its closure, the micropyle-like depressions, the folded chorion and the polyspermy prevention and fertilisation mechanisms were also considered and discussed in the light of the available information.

Gamete collection
Mature African catfish, C. gariepinus (weight of 900-1500 g) were collected from the River Nile at Assuit, Egypt and transported to the Fish Lab, Zoology Department, Assuit University.The catfish were kept in 100 l glass tanks to be acclimatised for a two-week period at 24-26ºC.The photoperiod was a 12 hour light to 12 hour dark cycle and the catfish were fed on a commercial pellet diet (3% of the body weight/day).
For collection of semen, male fish were anaesthetised with 200 mg/l MS-222 and one of the testes was removed surgically.Alternatively, the fish were killed and whole gonads were removed.Testes were cleaned from the blood by surgical towels.Sperm from the testes was pressed through a mesh fabric into a sterile dry petri dish and used directly for dry fertilisation.For collection of eggs, ovulation was artificially induced.Females were injected intraperitoneally with pellets (gonadotropin-releasing hormone analogue, GnRHa, D-Ala 6 , Pro 9 Net) containing 2.5-3.0 mg of water-soluble dopamine antagonist metoclopramide (Interfish Ltd, Hungary) dis-solved in 0.65% NaCl.One pellet was used per kg body weight.Eleven to 12 h after injection, the fish were stripped and the eggs were collected in clean dry plastic containers.

Light microscope
Pieces of the ripe ovary of C. gariepinus were fixed in 10% neutral buffered formalin (pH 7.5), dehydrated in ascending series of ethyl alcohol, and cleared in methyl benzoate.Embedded tissues were sectioned at 3 µm and stained with hematoxylin and eosin (H&E) (Bancroft and Stevens, 1982).

Transmission electron microscope (TEM)
Pieces of the eggs and the testis were immediately fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 24 h at 4ºC.The specimens were washed in 0.1 M cacodylate buffer (pH 7.2) for 1-3 h and then post-fixed in 1% osmium teraoxide for 2 h.The tissue pieces were placed in propylene oxide for 60 min, then in pure Epon 812.
Tissues were sectioned at 1 µm and stained with toluidine blue.Sections were examined by light microscope to identify different representative regions to be sectioned.Ultrathin sections were mounted in copper grids, stained with uranyl acetate and lead citrate (Bancroft and Stevens 1982), and examined with a TEM (JEOL 100, CXII) operated at 80 kv.

Scanning electron microscope (SEM)
Eggs before insemination and after fertilisation were fixed with 5% glutaraldehyde in 100 mM phosphate buffer (pH 7.4, 4ºC) for 24 h.They were post fixed with 1.5 osmium tetraoxide for 2 h and washed four times with 100 mM phosphate buffer (pH 7.4).Some eggs were cut into halves with a fine razor.After slow dehydration with an ethanol series, the eggs were dried at 30-40ºC, glued to stubs coated with 20 nm of gold, and viewed with SEM (GAOL, GSMS 400 LV) at 15 kv.

Gel electrophoresis of chorion proteins
The isolated chorion of the fertilised and unfertilised eggs of C. gariepinus were dissolved in buffer containing 150 mM NaCl, 20 mM Tris, 10 mM EDTA, and 1% SDS with boiling at 100ºC for 5 min and centrifugation to remove undissolved remnants.The chorion was also dissolved in the same buffer with 8 M urea for another run because one-dimensional SDS-PAGE using 10% acrylamide was performed according to the procedure of Laemmli (1970) and according to Swank and Munkres (1971) for SDS-Urea-PAGE.Gradient SDS-PAGE (5-20%) was prepared according to Hames (1981).The low molecular weight standards (Pierce, USA) were run concurrently and the protein molecular mass was determined using Gel-Pro Analyser package (Media Cybernetics, 1998).

Infrared spectroscopy analysis
The chorion of fertilised and unfertilised eggs of C. gariepinus were isolated under dissecting microscope and washed thoroughly several times with distilled water in a sonicator to remove the newly formed embryo and other remnants.The chorion was air-dried.Infrared spectra were recorded on the infrared spectroscopy SHIMADZU (IR-470) according to Iconomidou et al. (2000) with modification.

Terminology of the chorion
There is considerable variation in the nomenclature used to describe the external membrane of teleost eggs.Commonly used terms for this outer covering include zona radiata, zona pellucida, chorion, radiate membrane, egg membrane, primary membrane, vitelline membrane, vitelline envelope, egg envelope, egg shell and egg capsule.In the present work, the term chorion is used according to Yamagami et al. (1992).

The ripe testes and sperm structure
The testes of C. gariepinus in a ripe stage displayed various stages of active spermatogenesis.The spermatozoa of C. gariepinus were tightly packed in the lumen of the testes lobules (Fig. 1A).Spermatozoa consisted of a head, midpiece and very long tail (Fig. 1B).The head region contained the nucleus, which consisted of variable electron dense granular chromatin materials.The head region was relatively triangular or rectangular.The midpiece had an inverted conical shape forming an oval shaped structure with the head.The head-midpiece surface showed irregularity (surface with irregular folding, Fig. 1D).The irregular inverted conical shape of the midpiece reflected an increased number of mitochondria.The midpiece had a reticular structure around the flagellum (Fig. 1C), in which the mitochondria were distributed separately.These findings were in contrast with those reported by Mansour et al. (2002), who stated that several single mitochondria were fused with each other and formed a complex chondriosome.It was difficult to postulate such a chondriosome structure with the reticulate structure of the midpiece of C. gariepinus.Figure 1 (E, F) showed other related structures, especially the flagellum structure in cross and longitudinal sections.The topography of spermatozoa of C. gariepinus seemed to be adjusted to the diameter of the inner aperture of the micropylar canal.This condition was indicated by Linhart and Kudo (1997) as contributing to the prevention of polyspermy.Also, the topography of the head-midpiece region provided binding facets for the attachment of the sperm on the chorion surface by a ligand-receptor mechanism.

The ripe ovary and the oocyte
The ripe period of C. gariepinus gonad was characterised chiefly by migration of the nucleus toward the animal pole (Zaki et al., 1986).The ovarian ripe oocyte of C. gariepinus had four distinct layers: an outermost follicular layer (outer theca + inner granulosa layer), a median zona radiata (the chorion), and an inner oolemma or oocyte plasma membrane (Fig. 2A, B).Zaki et al. (1986) confused the terminology of the layers surrounding the oocyte since they referred to the outer layer (zona granulose) as chorion and the inner layer as zona radiata (with clearly discernible pores).Rizkalla (1960)  to 5 layers encircling the ovarian ripe oocyte of C. gariepinus: theca folliculi, membrane proporia folliculi, the follicular epithelium, the definitive membrane proper (zona radiata with faint striation) and the zonoid layer (peripheral cytoplasm).The incomplete second layer of Rizkalla (1960) was not observed in the present work.The chorion of the ovarian ripe oocyte was a single layer (sections shown in Fig. 2C, D, and E).Al-Absawy ( 2004) reported a single-layered chorion of ovarian oocyte of Trachinotus ovatus observed by light microscope.
A chorion with more than one layer has been recorded in the ovarian oocyte of some other teleost fishes.The ovarian mature oocyte of Carassius auratus had a three-layered chorion (Cotelli et al., 1988).
The egg envelope of the full-grown oocyte before fertilisation in viviparous species of Goodeidae was composed of one to three layers: filamentous-zona radiata interna and an electron dense zona radiata externa covered by an additional flocculent layer (Riehl and Greven, 1993).Other oviparous teleosts exhibited a division of the zona radiata into two or more layers (Schoots et al., 1982).The chorion thickness of viviparious fishes was considerably reduced in comparison with that of related oviparious species (Riehl and Greven, 1993).

Egg surface
The outer surface of the unfertilised egg of C. gariepinus exhibited no prominent elongated projections (Fig. 3A), whereas the fertilised egg acquired a network of projections 1 h after fertilisation on the vegetal hemisphere (Fig. 3C-F).On the vegetal pole, such projections formed a base-like circle (Fig. 3F), which might represent an adhesive apparatus for contacting substratum.At high magnification (×7500), some of these elongated projections had a dentate lateral process (Fig. 3D).The network projections seemed to be inserted in the chorion.White spots were recorded within the network projections (Fig. 3C, E).Such network projections and their related white spots were unique for C. gariepinus fertilised egg, since no Clarias-related studies referred to such variability.Moreover, no chorion-related studies on other teleost fishes referred to such projections.Different patterns of ornamentation on the egg surface of C. gariepinus were evident (Fig. 4).Such ornamentation has been represented as tubercle and/or reticular (Fig. 4A), debris-like dots and batches (Fig. 4B,D), irregularly lobulated ornamentations (Fig. 4C) and partially reticulated debris (Fig. 4F).These patterns were recorded in different regions and at different postfertilisation times.Irregularly distributed pore bulges were recorded 1/2 h after postfertilisation (Fig. 4E).The debris-like dots and the partially reticulated debris might represent the poorly preserved remains of the diffuse mucus layer (Lönning and Hagstrom, 1975;Johnson and Werner, 1986).
The ornamentation varied from the germinal disc region to the vegetal hemisphere (Fig. 5).Moreover, the pattern of ornamentation varied with the progress in embryonic development since hairs and depressions appeared on the animal hemisphere but not on the vegetal one (Fig. 5D).Further changes were recorded at the 30 h stage (Fig. 5E).Similar ornamentation patterns have been recorded by many authors working on different teleost species belonging to different taxonomic groups (Johnson and Werner, 1986;Cotelli et al., 1988;Merrett and Barnes, 1996;Chen et al., 1999;Rizzo et al., 2002;Chiou et al., 2004).Some of these authors considered the egg surface ornamentation as taxonomic characters at the specific level.The variability in ornamentation of C. gariepinus makes their use in species identification difficult owing to their association with the fertilisation and development process.Chen et al. (1999) referred to the importance of the outer surface of the chorion in egg identification and phylogenetic study.However, they added that the outer surface of the chorion did not show remarkable differences in microstructure among species in a genus or a family.

The micropyle
The micropyle of C. gariepinus egg consisted of a funnel-shaped vestibule from the bottom of which a cylindrical micropylar canal extended (Fig. 6A B).Such a micropyle was similar to Type III of Riehl and Schulte (1977), inasmuch as no micropyle pit was recorded in C. gariepinus eggs.No micropylar pit was found in Epinephelus malabaricus, E. coioides, Sciaenops ocellatus and Mugil cephalus (Li et al., 2000).The micropylar canal of C. gariepinus eggs apparently decreased in diameter after completion of fertilisation-related ooplasmic changes.The narrowing of the micropyle was involved in the polyspermy-preventing reaction (Hart, 1990, Linhart andKudo, 1997).Riehl and Schulte (1977) described two other types of micropyles: micropyles with a deep pit and short micropylar canal (Type I) and micropyles with a flat pit and a corresponding longer canal (Type II).In addition, a cylindrical, a conical and a funnel shaped micropyle have been described in Gadus morhuu marisabli, Mugil cephalus and Gleginus navaga (Mikodina, 1987).Deung et al., (1997Deung et al., ( , 1999Deung et al., ( , 2000)), Kim (1998) and Kim et al. (1993Kim et al. ( , 1996Kim et al. ( , 1998Kim et al. ( a,b, 1999Kim et al. ( , 2001) ) described the micropyle of different species belonging to four families, Cichlidae, Characidae, Cyprinidae and Belontiidae, referring to taxonomic validity of the micropyle (the egg built-in advantage, Brummett and Dumont, 1979).The shape of the chorion around it appeared to facilitate the movement of spermatozoa toward the micropyle.The micropyle represents the initial isolating mechanism for preventing interspecific hybridisation (Chen et al., 1999) at least in teleost species, and it is considered to be species-specific (Kobayashi and Yamamoto, 1981).
With development progress, the micropyle of C. gariepinus egg continued to narrow with the formation of the micropylar disc (Fig. 6C, E).Riehl and Appelbaum (1991) and Wenbiao et al. (1991) referred to this micropylar disc.The development of such a micropylar disc made C. gariepinus eggs have a unique characteristic shape (a fur cap) that differs from that of other catfish such as Silurus glanis (Kobayakawa, 1985;Riehl and Appelbaum, 1991).The micropylar disc of C. gariepinus resembled that of P. mattereri, S. spiloplema, R. aspera, Cichlasoma nigrofasciatum and Polypeteus spp (Wirz-Hlavacek and Riehl, 1990 Appelbaum, 1991; Bartsch and Britz, 1997;Rizzo et al., 2002).In the absence of data that elucidate the mechanism by which eggs of these species adhere to substratum, Riehl and Appelbaum (1991) concluded that the micropylar disc may play a role in their adhesiveness.Similarly, Wenbiao et al. (1991) termed the micropylar disc as the attachment disc.
As such, the eggs should be fertilised before their attachment to the substratum (Wirz-Hlavacek and Riehl, 1990).The network of projections on the vegetal hemisphere founding C. gariepinus leads to the conclusion that these projections might represent another attachment mechanism.In fertilisation experiments of C. gariepinus, the animal pole was usually directed upward.Most catfish, including C. gariepinus, possess demersal eggs, which become sticky after encountering water.Catfish eggs adhere to substratum with several other methods (Riehl and Appelbaum, 1991).In Silurns glanis and two Japanese Silurus species (S. asotus and S. biwaensis), the eggs adhered with a voluminous layer of jelly (Kobayakawa, 1985;Hilge et al., 1987;Riehl and Appelbaum, 1991), whereas the eggs of Japanese Silurus lithophilus were not adhesive.The jelly layer coat was also present in the adhesive eggs of other siluriformes, including Ictalurus spp and Chrysichthys spp, in addition to Silurus spp.(Sato, 1999;Rizzo et al., 2002) and in non-adhesive eggs of siluriformes including Paulicea luetkeni, Pimelodus maculatus and Conorhynchus conirostris (Sato, 1999;Rizzo et al., 2002).The adhesive apparatus of C. gariepinus was formed of a large number of tiny attaching filaments, which were embedded in a certain cement substance (Riehl and Appelbaum, 1991).Such tiny structures were observed in the present work as microvilli extending from the outer surface of the chorion in embryonic stages before hatching (Fig. 5E).
The spermatozoa of C. gariepinus move along the surface of the chorion and enter the micropyle or any micropyle-like depression in a directed fashion (Fig. 7).Receptors of various sources or motility stimulating factors of C. gariepinus must be involved in aggregating spermatazoa.Kudo (1997) reported that the multiplicity of micropyles recorded in some fishes, such as Acipenserid species, was less favourable to the prevention of polyspermy than that of other fish eggs that possess only one micropyle.They also referred to the uncertainty of how the multi-micropylar eggs responded to stimulus of fertilisation by forming a cytoplasmic process underneath several micropyles of the same egg by polyspermy or other mechanisms.Accordingly, the multiplicity of micropylelike depression of C. gariepinus eggs was more beneficial for trapping sperm without the formation of cytoplasmic processes.Brummett and Dumont (1979) stated that the main block to polyspermy in the teleost was inherent in the morphology of the chorion with its single point of entry.Cosson et al. (2002) reported that for spermatozoa, the interface trapping mechanism was a very efficient means of increasing their concentration on a surface of the egg instead of their being dispersed in a threedimensional volume.They also added that this mechanism was highly efficient and crucial for species in which spermatozoa had a very short peri-od of motility to reach the micropyle.Our findings were also corroborated by the observations of experimentally induced polyspermic eggs by Iwamatsu and Ohta (1978) and Ohta (1985), who reported that spermatozoa can bend and enter the teleost egg at locations other than the site of sperm entry.Moreover, in their studies of polyspermic and monospermic fertilised eggs of Oryzias, Iwamatsu andOhta (1978, 1981) described folds of the egg surface that rapidly engulf the sperm in a "Cave-like pit" before the fusion of egg and sperm plasma lemmae, which occurred some 20 s later.The sperm behaviour of C. gariepinus on the chorion surface, micropylar area, micropyle-like depression and folded chorion (Fig. 7) indicate that spermatozoa are oriented inherently towards any depression on the chorion surface in addition to their trapping mechanism on the chorion fibres (Fig. 8A, B).

The chorion structure and fertilisation
The microscope and TEM (Fig. 2C, D).The single-layered chorion of the unfertilised egg of C. gariepinus had faint striations (Fig. 2E).Directly after fertilisation, the single zona radiata was differentiated into a zona radiata externa (ZRE) and a zona radiata interna (ZRI) (Fig. 8C, D).At 1/2 h and 1 h after fertilisation, the chorion of C. gariepinus was differentiated into three layers (Figs. 9, 10), the double-layered coat (DLC, Figure 10C, E), which might have been involved in the first steps of fertilisation, the middle layer, the zona radiata externa (ZRE) and the innermost layer, the zona radiata interna (ZRI), which was half the thickness of the ZRE.The pore canals were expressed in both ZRE and ZRI.These pore canals and striations were more prominent in ZRI (Fig. 9A-C).Following ZRI, there was an electron opaque layer (Fig. 10).This latter unknown layer invaded the perivitelline space (PVS) and came into contact with the oolemma.The three layers of the chorion identified by TEM were detected by SEM with elucidation of their pore canals (Fig. 11).The ZRE showed variability in its thickness in comparison with the ZRI on the same egg, especially in the region of the mycropylar disc (Fig. 11D).The ZRE disappeared completely in the micropyle, in the micropylar-like depression and in the vegetable pole (Fig. 11E, F).The lamellar and tubular nature of the ZRI was evident (Fig. 11A, B).The disappearance of the ZRE and the increased tubular nature of the ZRI in the vegetal pole facilitated the embedding of spermatozoa on this region.Griffin et al. (1996) reported that for C. pallasi eggs the chorion at the animal pole was distinct functionally, structurally and biochemically from the remainder of the surface.The postfertilisation changes of the chorion of C. gariepinus reflected the morphological aspect of chorion hardening and fertilisation processes.
The current postfertilisation morphological changes of the chorion of C. gariepinus were associated with cortical reactions and cortical alveoli discharge in the perivitelline space (Fig. 12 echinoderms (Vacquier, 1981), the cortical layer of the egg was considered to be a gel with specialised viscoelastic mechanical properties (Hart, 1990).Moreover, the egg cortex became increasingly contractile after sperm-egg union, causing a twist-ing movement of oil droplets toward the animal pole; a meshwork of polymerised actin appeared in the egg cytoplasm and microfilaments became highly organised in the microvilli (Vacquier, 1981;Mabuchi, 1983).66, 61 and 55 kDa Celius and Walther (1998) Actin and actin-containing filaments have been described in the cortical layer of Brachydanio (Wolenski and Hart, 1988) and Oncorhynchus (Kobayashi, 1985) eggs.These lectins, as major components in vertebrate cortical granules (Krajhanzl, 1990), are involved in the formation of the egg envelope and in turn in its polyspermyblocking functions (Quill and Hedrick, 1996).Dong et al. (2004) identified a C-type lectin from oocyte of a freshwater fish species Carassius auratus gibelio.TEM showed that the cortical cytoplasm of the eggs of C. gariepinus contained membrane-limited cortical granules with an internal matrix of varying electron density (Fig. 12).The determination of the chemical and molecular composition of cortical granules is essential to understanding the role of these organelles in fertilisation and early development (Hart, 1990).Cortical reaction seems to be a prerequisite for the chorion hardening process, which has been considered by some authors to be independent of fertilisation (Lönning et al., 1984;Davenport et al., 1986).There was a precise relationship between sperm behaviour, chorion hardening and cortical reaction of C. gariepinus.
Chorion hardening is a process of initiated chain polymerisation of substances within the membrane itself (Hart, 1990) to form insoluble proteins of higher molecular weight (Yamagami et al., 1992).The solubility of chorion of unfertilised egg is a requirement for the hardening system or machinery   Grassiotto and Guimaraes, 2003).The electrophoretic profile showed interesting similarities among some but not all.The variability reported for teleost chorion (Table 1) might be explained by species difference (Griffin et al., 1996).Also, variability might be due to the difficulty of extracting certain polypeptides from the teleost chorion (Oppen-Berntsen et al., 1990).Four protein subunits of the chorion of C. gariepinus were identified by SDS-PAGE: three proteins of low molecular weights (MW) and one protein of relatively higher MW that had the highest percentages, 29.8-47.89%(Tables 2-6).Hardening of ovulated eggs resulted in no or minor variation in the MW.Fertilisation influenced the chorion proteins of the first three categories .According to SDS-UREA-PAGE, more chorion protein subunits were identified, especially those of higher MW (Tables 3, 4).The chorion protein of higher MW detected by normal SDS-PAGE was 86.8-100.3kDa and represented the highest percentage, 28.3-52.9%.Two other categories of protein subunits were identified: one with relatively low MW (5.2-80.3kDa) and another with higher MW after hardening and fertilisation (133.87-248.46kDa).More protein subunits were also recorded by gradient SDS-PAGE (Tables 5, 6); each was represented by a low percentage.Different analyses resulted in different patterns of protein subunits.
Chemical analysis of chorion protein subunits of C. gariepinus from eggs 2 min to 3 h after fertilisation by partial hydrolysis indicated the raw material of the polymerisation process.Polymerisation in influenced by the length in time of chorion hardening.Zotin (1958) indicated that the chorion began to harden from between <1 min of insemination to 2 to 4 h in salmonid and coregonid eggs, whereas Hart (1990) found that maximum hardening of the chorion was reached within 3-7 days in the trout and 1-2 days in the whitefish.For lump sucker (Cyclopteurs) and cod (Gadus), chorion hardening started shortly after exposure to sea water and reached a maximum resistance by about 24 h (Lönning et al., 1984).
Hardening in these two salt water species did not require fertilisation (Hart, 1990).Iconomidou et al. (2000) and Hamodrakas et al. (1987) indicated that the β-pleated sheet was the molecular conformation of protein macromolecules that constitute the fibrils and of fibrils of the egg shell with a helicoidal architecture.These studies did not deal with the interaction between chorion hardening, fertilisation and progress in development.Further spectroscopic studies must be done to demonstrate a relationship between chorion hardening and the time of development based on different amide ranges.
FIG. 1. -Spermatozoa in the lobules of the rip testis of Clarias gariepinus (A: ×10000).The structures of the spermatozoa as revealed by SEM (B, C) in semen (×3500) and TEM (×20000).The irregularity of sperm head and midpiece were evident with a very long tail (B) and a midpiece process (D: ×20000).E, F represent transverse and longitudinal sections of the flagellum (×20000).
FIG. 7. -Different spermatozoa trapped on the surface of Clarias gariepinus eggs in increased number (A) with different white secretion on their heads and tails (B, C).Different spermatozoa of Clarias gariepinus swarmed and trapped or embedded in the outer surface of the micropyle-like depressions at the vegetal pole (D-F).Note the pore outer surface of the chorion in this region.The micropyle-like depressions and the folded chorion at the vegetal pole of Clarias gariepinus eggs (G-I) with spermatozoa trapped and directed in lines (arrow).(A-I) 30 seconds postfertilisation (A,I: ×2000; B,C: ×15000; D,E: ×7500; F: ×5000; G: ×3500; H: ×1500).