As mentioned above (page 11) there is a relationship between somatic and gonadal growth in decapod crustaceans. Reproduction takes place during the intermoult period, and, although in synchrony with the moult cycle, there may be several moult cycles during a single reproductive period (Adiyodi, 1985; Chamiaux-Cotton, 1985; Chamiaux-Cotton, and Payen, 1988; Meusy and Payen,
1988).
Vitellogenesis is the process by which yolk is deposited within the developing oocyte. There is no clear stage at which this process starts and so the term early vitellogenic phase or primary vitellogenesis is used to refer to the period preparatory to vitellogenesis (Adiyodi and Subramoniam, 1983). Vitellogenesis can be divided into two phases. Primary vitellogenesis is a period of slow oocyte
growth while secondary vitellogenesis, in contrast, is a period of rapid ovarian growth immediately preceding oviposition, during which, there are large increases in gonad weight, with yolk coming both from the gonad itself (endogenous source) and from the hepatopancreas (exogenous source) (Chamiax-Cotton, 1985; Blanchet-Toumier, 1982). Female reproduction is regulated mainly at the time of secondary vitellogenesis.
Reproduction and its regulation in the Decapoda is under the inhibitory control of an eyestalk neuropeptide. Panouse (1943) demonstrated that eyestalk ablation during genital quiescence removed this inhibition, stimulated the rapid development of the ovary in the shrimp Leander serratus, and resulted in precocious egg deposition. This effect was not observed in juvenile females (Panouse, 1946). The presence, therefore, of an ovarian inhibiting hormone in the eyestalk had been demonstrated. This hormone was to be known as gonad inhibiting hormone (GIH) as it retards gonad maturation and secondary vitellogenesis. The MTGXO was later identified as the site of neurohormonal synthesis and release (for review see Kleinholz and Keller, 1979). The role of GIH has been confirmed in a number of decapod species (for reviews see Chamiaux- Cotton and Kleinholz, 1964; Adiyodi and Adiyodi, 1970; Demeusy, 1970; Adiyodi and Subramoniam, 1983; Charniaux-Cotton, 1985; Chamiaux and Payen, 1988). Until recently, however, the term vitellogenesis inhibiting hormone or VIH was considered to be more accurate due to the peptides inhibitory activity on the secondary vitellogenic phase. To complicate the nomenclature of this peptide still further. Van Herp et al (1992 conference abstract) noted the presence of GIH/VIH in the X-OSG complex of the male lobster, and suggests the peptides role in male reproductive strategies. The name GIH (gonad inhibiting hormone) instead of VIH (vitellogenesis inhibiting hormone) is suggested by the authors as this encompasses the general function of the neuropeptide. As this is likely to be a particular area of debate in the future and as the presence of GIH has not been demonstrated as an
Chapter 1
active peptide within the male reproductive system, GIH will be used to describe this hormone throughout this thesis.
By the use of a heterologous in vivo bioassay, which measured the retardation of oocyte growth in the prawn Palaemonetes varians, the purification and characterisation of sinus gland peptides representing GIH was possible from
Homarus americanus (Soyez et al, 1987; Tensen et al, 1989; Van Deijnen, 1986). Characterisation of GIH from the crayfish, Procambarus bouvieri by Aguilar et al
(1992) indicated a single peptide of 8388Da. However, Soyez et al (1990), isolated two related peptides of approximately 8kDa from Homarus americanus
and a complete amino acid sequence for these neuropeptides with a Fast Atom Bombardment Mass Spectrometry (FAB/MS) determined molecular weight of 9135Da, has now been established (Soyez et al, 1991). Each isoform is a neutral (PI 6.8) 77 amino acid long peptide, with a free N-terminus, six cysteine residues
and is devoid of CHH activity (Tensen, 1991). The structural significance of these isoforms of GIH will be discussed in section 1.3.4.2 under the structural aspects of CHH. Alignment of sequences of Homarus GIH and Carcinus MIH show a closer relationship between these two peptides than that with CHH from the same species and it is clear that these are two separate types of peptide that have diverged considerably from CHH (Keller, 1992). There is no homology between the sequence of GIH or indeed CHH and MIH with any other known peptide, thus suggesting that this group is unique to the Crustacea.
Polyclonal mouse antisera raised against high performance liquid chromatography (HPLC) purified GIH have been tested against extracts of sinus glands from several crustacean species, including sinus gland peptides from
Homarus americanus. A preliminary immunocytochemical detection of GIH terminals in the sinus glands of Homarus americanus and the wood louse,
Porcellio dilatatus was demonstrated (Meusy et at, 1987). Using both GIH and CHH antibodies, partial co-localisation of both peptides with CHH perikarya in the Page 28
MTGXO was identified, resulting in the suggestions that both GIH and CHH are synthesised in the same cells, that they may be encoded by the same RNA transcript or that both may originate from the same precursor. This last hypothesis had been supported by similar reports (Stuenkel, 1986; Eipper and Mains, 1988; Thomas et al, 1988). However, recent work by Van Herp et al, (1992) demonstrates the detection of the Homarus CHH and GIH mRNAs in combination with immunocytochemical techniques, demonstrating a degree of cellular colocalisation for both peptides within the eyestalk. The suggestion that both peptides may be synthesised within one cell group of the X-organ may be premature due to the possibility of three cell groups; one for each peptide and one with the capability of dual synthesis. As the expression appears not to be simultaneous, it is suggested that there is no common precursor and that both peptides originate from different primary transcripts.
Finally, initial studies of the mode of action of GIH suggests the inhibition of receptor mediated endocytotic uptake of yolk by oocytes in vitro (Jugan and Soyez, 1985; Jugan and Van Herp, 1989).