Ž .
in fish, but may also suggest additional roles for gonadotropin GtH and 17P, indicating that spermiation may not be simply 17,20bP dependent. q 2001 Elsevier Science B.V. All rights
reserved.
Keywords: Atlantic salmon; GnRHa; Spermiation; Plasma steroids
1. Introduction
Ž .
Ž .
Gonadotropin releasing hormone analog GnRHa and gonadotropins GtHs have Ž
. been used to stimulate milt production in a variety of teleosts see Pankhurst, 1994 . In
salmonids, increased milt volume or accelerated production of milt has been observed in Ž
response to GnRHa and GtH treatments e.g. Crim et al., 1983; Weil and Crim, 1983; .
Ueda et al., 1985 where the resulting increase in circulating levels of those compounds Ž
. is believed to imitate endogenous changes in hormone levels Pankhurst, 1994 . In-
creases in, or advancements of, milt production are unlikely to have occurred as a result of any direct effect of GtH but rather, they are elicited in response to the GtH-stimulated
Ž .
synthesis of gonadal steroids Pankhurst, 1994 . In salmonids, 17a ,20b-dihydroxy-4- Ž
. pregnen-3-one 17,20bP has been used to stimulate milt production in amago salmon,
Ž .
Oncorhynchus rhodurus Ueda et al., 1985 , and in brook trout, SalÕelinus fontinalis Ž
. Marshall et al., 1989 , while GnRH and GtH-stimulated increases in 17,20bP have
been observed in other teleosts, thereby lending support to the hypothesis that milt Ž
. production is stimulated by GtH-dependent steroid production Pankhurst, 1994 .
Enhanced 17,20bP production often coincides with the decline in androgen produc- tion that occurs during the transition from spermatogenesis to spermiation in a number
Ž of male teleosts Ueda et al., 1983, 1985; Baynes and Scott, 1985; Sakai et al., 1989;
. Miura et al., 1991; Swanson, 1991; Schulz et al., 1991, 1992 . Spermiation, the release
Ž .
of hydrated semen i.e. with lower spermatocrit than during intra-testicular storage from the genital pore, appears to be dependent on the production of 17,20bP. 17,20bP also
acts to increase sperm duct pH, which in turn, increases sperm cyclic adenosine Ž
. monophosphate cAMP, Morisawa et al., 1992, 1993; Miura et al., 1992 . cAMP is
known to trigger the initiation of sperm motility in salmonids through the protein Ž
. phosphorylation cascade Morisawa et al., 1992 .
Prior to commencement of the present study, we had attempted to control milt Ž
. production in male Atlantic salmon Salmo salar in order to ensure that spermiation
occurred in synchrony with the ovulation of naturally ripening females. These attempts generated anecdotal evidence which tended to suggest that use of a single injection of a
commercial salmon GnRHa preparation resulted in a marked increase in milt production at the height of the spawning season, whereas no such increase in milt production was
elicited prior to, or during, the very early stages of the recognised spawning season Ž
. King, unpublished observations . The lack of effect of GnRHa injection on milt
production immediately prior to spawning could be due to several factors, including failure of GnRHa to elicit adequate GtH secretion andror lack of substrate for 17,20bP
Ž production. Kawauchi et al. have confirmed that two chemically distinct GtHs GtH-I
. and GtH-II with structural homology to mammalian FSH and LH exist in salmonids
Ž .
reviewed by Swanson, 1991 . GtH-I and GtH-II are produced by different gonadotropin Ž
. cell-types Nozaki et al., 1990a; Naito et al., 1993 . During vitellogenesis and spermato-
genesis, large numbers of GtH-I cells were observed relative to low numbers of GtH-II cells and blood GtH-I levels increased and peaked prior to spawning while GtH-II levels
remained around or below assay detection limits. In contrast, the numbers of GtH-II cells exceeded the number of GtH-I cells during spawning, a time when circulating
Ž GtH-I declined and GtH-II displayed a rapid increase Suzuki et al., 1988; Nozaki et al.,
. 1990b; Swanson, 1991; Naito et al., 1993 . GtH-I and GtH-II are equipotent in
Ž .
stimulating 11-ketotestosterone 11KT and 17,20bP production during spermatogenesis Ž
. although 11KT production exceeded 17,20bP production Planas et al., 1991 . Later,
however, GtH-II was more potent than GtH-I in stimulating 17,20bP synthesis. Thus, early GnRHa treatment may have had a relatively greater impact on GtH-I cell activity
and GtH-I production while the effectiveness of GnRHa injection at the height of the spawning season is consistent with increased impact on GtH-II production and 17,20bP
synthesis at that stage of the reproductive cycle.
One of the primary factors limiting the early synthesis of 17,20bP may also be the Ž
lack in availability of its precursor 17a-hydroxyprogesterone 17P; Sakai et al., 1989; .
Schulz et al., 1991, 1992 due to the dominance of the androgen biosynthetic pathway. A number of authors have demonstrated increased 17,20bP production and increased
Ž spermiation in response to exogenous 17P both in vitro and in vivo Ueda et al., 1983,
. 1984, 1985; Sakai et al., 1989; Miura et al., 1991; Pankhurst, 1994 . Thus, the variation
in the efficacy of GnRHa treatment as a stimulant of spermiation during different stages of reproductive development may also be related to 17P availability. However, the roles
of GtH-I and GtH-II in the production of 17P, relative to androgens during spermatogen-
Ž .
esis, remain to be assessed Swanson, 1991 . We hypothesised that providing substrate for 17,20bP synthesis would enhance the
effectiveness of the GnRHa treatment. Three experiments were conducted immediately Ž
prior to the commencement of the 1993 and 1994 spawning seasons late April in .
Tasmania in order to investigate the effect of exogenous 17,20bP, 17P and GnRHa, alone or in combination, on the spermiation response of mature non-spermiating male
Atlantic salmon.
2. Materials and methods
