In vitro fertilization of lobster oocytes

Abstract

The feasibility of fertilizing preovulatory lobster oocytes was examined in vitro under various experimental conditions. Large (1.6 mm diameter) and small (0.6 mm diameter) oocytes were compared in fertilization trials. Small oocytes were surrounded by a thin envelope thought to correspond to envelope 1A of mature oocytes. Large oocytes were surrounded by a fully formed vitelline envelope comprised of two distinct sublayers, 1A and 1B. Although sperm bound very effectively to the coat surrounding small oocytes, none penetrated the coat or fertilized the oocytes. Large diameter oocytes that were removed from follicles by dissection were fertilized by sperm from the proximal vas deferens of males and by sperm from the seminal receptacle of females. Fertilized oocytes showed a high degree of polyspermy. Higher numbers of sperm bound to, penetrated, and fertilized large diameter oocytes when inseminations were done in a saline solution (2.5LSH) than when done in artificial sea water (ASW). Sperm failed to bind to large diameter oocytes that were induced to ovulate in vitro by collagenase treatment. Contaminating enzymes may have destroyed the sperm receptor in envelope 1A of collagenase treated oocytes. Our in vitro fertilization method will allow the process of fertilization to be studied experimentally in lobsters, and it may be applicable to other decapods.Many aspects of lobster (Homarus) reproductive biology have been well characterized in numerous studies done during the past 100 years (reviewed by Aiken and Waddy, '80; Talbot, '90). The structure of the gametes has been examined in considerable detail at both the light and electron microscopic level (Herrick '09; Kessel, '68; Talbot, '81a,b; Pochon Mason, '68; Talbot and Chanmanon, 80a; Schade and Schivers, '80). Morphological changes occurring in sperm during the acrosome reaction have also been described (Pochon-Mason, '68; Talbot and Chanmanon, '80b), and the role of the cortical reaction in formation of the fertilization envelope has recently been analyzed (Talbot and Goudeau, '88).In spite of much interest in this topic, relatively little is known about the mechanism of fertilization in Homarus americanus. This is due to the biennial ovarian cycle characteristic of this species and the difficulty in obtaining ovulated unfertilized oocytes or freshly spawned oocytes. Studies of gamete interaction in the lobster would be facilitated by an in vitro technique for fertilizing eggs. Goudeau and Goudeau ('86) have successfully fertilized lobster eggs in vitro using sperm from the seminal receptacle of a female that was spawning. To catch a female in spawning, however, requires constant monitoring of mature females and is not practical for most experimental applications.The main purpose of this study was to examine the feasibility of fertilizing preovulatory lobster oocytes in vitro. Oocytes were collected from ovaries by dissecting them free of follicle cells and inseminating them in vitro with sperm from either the proximal vas deferens (PVD) of males...

The morphology of the male reproductive system, spermatogenesis and the spermatozoon of Daphnia magna (Crustacea: Branchiopoda)

“…The role and fate of radial arms in spermatozoa of decapod crustaceans are not known. However, it is known that after 2 hrs of egg‐spermatozoon binding in the lobster Homarus americanus , radial arms depolymerize (Talbot, Poolsanguan, Poolsanguan, & Al‐Hajj, ). Some similarities in the structural and molecular levels have been detected in the radial arms of immotile crustacean spermatozoa and flagellum of the highly evolved spermatozoa of mammalian species that revealed an evolutionary link between these two distant groups (Niksirat et al, ).…”

Evaluating the impact of moulting and chilled storage of spermatophores on the integrity of plasma membrane, acrosome and DNA of black tiger prawn ( Penaeus monodon ) spermatozoa

“…Three types of sperm extender were used in this study: (a) artificial crustacean haemolymph (AH) (NaCl 27.058 g/L, KCl 1.118 g/L, CaCl 2 0.368 g/L, MgCl 2 ·6H 2 0 0.813 g/L, MgSO 4 ·7H 2 0 0.912 g/L, HEPES Na salt 5.206 g/L, pH 7.5) (Talbot et al, ); (b) calcium free saline (CS) (NaCl 21.63 g/L, KCl 1.12 g/L, H 3 BO 3 0.53 g/L, NaOH 0.19 g/L, MgSO 4 ·7H 2 0 4.93 g/L, pH 7.5) (Vuthiphandchai et al, ); and (c) filtered natural seawater (FS) (salinity 35 ± 1.0 ppt, pH 8.0) by 10 μm pore diameter filter paper (Whatman ® , Maidstone, UK). Five animals (5 replicates) from each grouped moult stage (B‐C, D0‐1 and D2‐3) were assigned to each type of the sperm extender (AH, CS and FS), so that the total number of animals in the experiment was 45 (P01–P45, see Figure ).…”

“…Calcium free saline is the most commonly used prawn sperm extender for sperm cryopreservation (Bart, Choosuk, & Thakur, ; Vuthiphandchai, Nimrat, Kotcharat, & Bart, ) or chilled sperm storage (Bray & Lawrence, ; Morales‐Ueno, Paniagua‐Chávez, Martínez‐Ortega, Castillo‐Juárez, & Alfaro‐Montoya, ) as the depletion of calcium typically prevents sperm AR in penaeid species. Alternative sperm extenders that have been reported include artificial crustacean haemolymph (Nimrat, Sangnawakij, & Vuthiphandchai, ; Talbot, Poolsanguan, Poolsanguan, & Al‐Hajj, ), natural seawater (Bray & Lawrence, ), phosphate buffer (Nimrat et al, ) and mineral oil (Nimrat et al, ). Within the spermatophore, there is a large volume of glutinous fluid covering the sperm mass, possibly providing extra protection for the embedded spermatozoa (Sasikala & Subramoniam, ); consequently, it may be prudent to examine whether sperm quality is affected by breaching the spermatophore wall, in which case the spermatozoa are either chilled preserved as a free sperm suspension or retained within the intact spermatophore.…”

Video microscopic analysis of ionophore induced acrosome reactions of lobster (Homarus americanus) sperm

“…All solutions were made using nanopure water collected from a Barnstead D2798 four holder ion exchanger. Sperm were handled in a physiological saline solution, 2.5LSH, which supports in vitro fertilization of lobster oocytes (Talbot et al, 1991a) and in vitro contraction of ovarian muscle strips (Howard and Talbot, 1992). 2.5LSH was made as described previously (Ro et al, 1990), adjusted to pH 7.4, and stored at 4°C.…”