Optimisation of an oviposition protocol employing human chorionic and pregnant mare serum gonadotropins in the Barred Frog Mixophyes fasciolatus (Myobatrachidae)

Pituitary extracts have been widely used for many decades as an effective and practical tool for obtaining viable oocytes from gravid amphibians [1‐6] since the first bioassays demonstrated the effects of pituitary extracts on amphibian ovulation. Pituitary extracts from the same (homologous) species are the most effective at inducing ovulation [2]. Homologous pituitaries are presumably the most suitable and potent source of LH activity [2] for the induction of ovulation because of sequence variation in the active β unit of the gonadotropins [7] that impacts on hormone-receptor specificity, an effect that is likely to increase with phylogenetic distance. Heterologous pituitary extracts from different amphibian species are a “second best” source of LH activity [5] but still (with exceptions such as Xenopus, Table 1) much more effective than phylogenetically distant mammalian gonadotropins [2, 8]. However, disadvantages of using heterologous or homologous pituitary extracts as a routine tool for inducing ovulation and oviposition in gravid females include individual, gender and seasonal variability in FSH and LH activity [6], availability of a source of pituitaries, and the cost and inconvenience of the collection process. In the case of endangered species, the potential for transfer of pathogens in raw extracts [9] as well as the inappropriateness of using rare or endangered animals to obtain pituitary extracts argues for alternative induction protocols.

One great advantage of working with Xenopus spp (X. laevis and X. (Silurana) tropicalis) in reproductive and developmental biology studies involving amphibians has been the ease with which ovulation of fertile eggs can be induced with mammalian gonadotropic hormones, especially hCG as a source of LH activity [8] (Table 1). Xenopus gained widespread attention in the 1930s [10] providing the first effective, large scale pregnancy test for women because of the reliability of its ovulatory response to hCG administration (an application that persisted for many years). This turned out to be an aspect of Xenopus biology shared with few other amphibians (Table 2). The poor response to mammalian gonadotropins in most species (Table 2) and the desire to move away from pituitary extracts for reasons outlined above has led to the development of protocols based on other parts of the hypothalamic-pituitary-gonadal axis. These include gonadotropic releasing hormones (GnRH) to stimulate release of endogenous gonadotropins [30‐32], progesterone to directly induce oocyte maturation and germinal vesicle breakdown [33, 34], and dopamine antagonists [35, 36], as well as various combinations of these with or without gonadotropins (for recent reviews and discussions see [9, 36, 37]). Nevertheless, as a first step to inducing ovulation in a novel target species, it is reasonable to test the use of synthetic mammalian gonadotropins, to determine whether these are an effective and inexpensive means of obtaining viable eggs, as is the case in Xenopus.

Global declines since the 1960s have resulted in amphibians experiencing the highest rate of decline and extinction of any vertebrate class over that period [44‐46]. This loss of amphibian biodiversity is primarily a function of a global pandemic of chytridiomycosis. Amongst Australian frogs, the genus Mixophyes (commonly known as the Barred Frogs) is amongst the most threatened genera. Currently, >40% (3 out of 7) of extant Mixophyes species are listed as vulnerable or endangered under Australian federal and state legislation, as well as the IUCN Red List. In the state of New South Wales, this figure is as high as 75% (3 out of 4) species of Mixophyes listed as endangered or vulnerable. Consequently, protocols for assisted reproduction, such as hormonal induction of ovulation have the potential to contribute to management and conservation of species across the genus. Currently, there is no data available for induced ovulation in gravid females of Mixophyes species. Adding to this, the group (Family: Myobatrachidae) is phylogenetically distant from many other amphibian taxa, and may consequently be quite different in its response to protocols that work well for other amphibian taxa, including its response to the injection of pituitary homogenates from unrelated species. Collectively the myobatrachids constitute about 50% of Australian amphibian species; 22% (n = 23) are considered vulnerable or endangered with 3 (2.8%) considered extinct; and they also constitute 48% of all threatened Australian frogs [47].

This paper reports the outcome of analyses of data on multiple ovulation and oviposition induction protocols employed on Mixophyes fasciolatus (the great barred frog). This species is the only non-threatened species of the Mixophyes in New South Wales and one of the few non-threatened species in Australia, and as such is an appropriate model for establishing protocols for assisted reproductive techniques (ART) for this genus. The data set does not contain all feasible protocols for the induction of ovulation (e.g. no data is available for effects of GnRHs or dopamine antagonists), but has focussed on the use of amphibian pituitary homogenates and mammalian FSH and LH preparations.

This study adds M. fasciolatus to the small group of amphibian species (including Xenopus laevis, Silurana tropicalis, and some bufonids) that will ovulate and oviposit in response to protocols based only on mammalian gonadotropins. The range of hCG doses associated with successful oviposition in M. fasciolatus are similar to the range of doses reported in various Xenopus/Silurana protocols (this study up to 1400 IU, but optimised to 200 IU with PMSG priming; compared to Xenopus/Silurana 90 to 1000 IU without FSH/PMSG priming, 250 to 500 IU with priming; Table 1). The absolute dose for M. fasciolatus (not adjusted for body weight differences) is similar or less than that required to induce B. americanus and B. fowleri (Table 2). It is assumed that oviposition in this study also indicates ovulation occurring as a result of the induction process (as would be the case with most studies investigating the induction of oviposition in amphibians), although this was not tested directly; there are no published data on anurans known to the authors where ovulation and oviposition have been shown not to be linked, sequential events.

In M. fasciolatus there is a distinct benefit of priming with a gonadotropin with FSH activity. It was demonstrated in this study that the rate of ovulation can be increased from around 30% to more than 50% by priming females with PMSG. Interestingly, the benefits of priming were increased by extending the interval between the first PMSG priming dose and the first hCG dose. Based on the data from this study, the optimal protocol for inducing oviposition in M. fasciolatus involves injection of two priming doses of PMSG (50 IU and 25 IU) administered 6 and 4 days, respectively, prior to the injection of two ovulatory doses of 100 IU hCG, administered 24 hours apart.

Published induction protocols utilising hCG (Tables 12) identify Xenopus and Silurana species from the family Pipidae as those that respond most strongly to hCG, administered alone or in combination with a mammalian gonadotropin with FSH priming activity, or with other components of the hypothalamo-pituitary-gonadal pathway (Table 1). The data in Table 1 indicates a wide range of hCG concentrations (from 90 IU to 1000 IU in X. laevis as single hCG doses, a range of an order of magnitude) have been used to induce Xenopus laevis and Silurana tropicalis. An apparent higher sensitivity to hCG by S. tropicalis may reflect its smaller body size [48]. Various protocols in Table 1 have also reported benefits of priming the ovaries with either lower, anovulatory doses of hCG, or with PMSG to prime for maturation. Nevertheless, there do not appear to be reports of data on the generation of dose response curves for induction (hence the wide variation in protocols), even in Xenopus, which might indicate optimal doses that achieve maximum egg generation with the minimum hCG dose, nor which indicate the quantitative impacts of priming with hCG or PMSG. Most published Xenopus/Silurana protocols do not incorporate FSH priming (although a number of protocols have an hCG “priming” dose - see Table 1).

Although some Xenopus/Silurana protocols report priming doses of PMSG (Table 1), the authors of this study are unaware of a direct comparison in any study of protocols with and without priming that would prove a benefit of priming with FSH. This study thus provides the only published data that demonstrates in any amphibian species an increase in ovulation rate as a result of such priming, and that the maturational effect of priming increases with time since administration. FSH is common in mammalian protocols, recognising the role that FSH plays in recruiting follicles and priming the maturing follicle for ovulation by cumulus expansion during the LH induced resumption of meiosis and germinal vesicle breakdown [49, 50]. LH but not FSH [51] causes a rise in progesterone in Xenopus follicles, indicating a separate, non-progesterone effect of FSH on follicle maturation.

Many anuran taxa, including various species of ranids, hylids and some bufonids (listed in Table 1) do not respond well (do not ovulate or ovulate at a very low rate) to induction with mammalian homologues of the amphibian gonadotropic hormones (a fact recognised many years ago: see [2, 6]). The data in Table 2 shows that, in particular, ranids as a group are the least responsive to mammalian homologues of any higher amphibian taxon that has been studied in detail (resulting in the continued use of pituitary extracts for induction in this group, see below); bufonids are highly variable in their responses – many species have not been recorded as responding to mammalian homologues, others respond in conjunction with other effectors of the hypothalamic-pituitary-gonadal axis such as LHRH and progesterone, while at least two species (B. americanus and B. fowleri) respond reasonably well to hCG only inductions. Some less studied groups such as the Australasian hylids and the eleutherodactylids may or may not be poor responders, with results obtained on a small sample of species. In contrast, a number of urodeles appear to respond well to hCG.

Nevertheless, effective hCG doses for Xenopus and Mixophyes fasciolatus (allowing for differences in body weight) are high compared to doses reported to be effective in inducing ovulation in mammals (mouse 5 IU [52, 53], dog 500 IU [54], rhesus monkey 4000 IU [55], cheetah 100–250 IU [56], cows 1500 to 2500 IU [57, 58], mares 2,000 – 3,300 IU [59, 60]; for example, the optimised dose of hCG used to induce oviposition in M. fasciolatus in this study (200 IU with PMSG priming) would have been sufficient to induce ovulation in 2 cheetahs [56]. This large differential in effective dose between mammals and responsive amphibians is also noted by Kouba and Vance [37] who observed that the ovulatory dose of hCG for Xenopus is 2000 times for that for the tiger. hCG is a convenient source of mammalian LH activity, but other mammalian sources are not necessarily more potent [8].

Given the challenges of inducing ovulation with mammalian homologues, pituitary extracts have continued to be used in many species to induce ovulation including bufonids [61], ranids [3, 62, 63] and at least one other myobatrachid [4]. No data were generated in this study on the capacity of amphibian pituitary gland extracts alone to induce ovulation and oviposition in M. fasciolatus, and there was no evidence (although the approach was not exhaustively investigated) of pituitary extracts potentiating the effects of the mammalian gonadotropins. The source of amphibian pituitary extracts in this study was Rhinella marina, which is not closely related to the Mixophyes[64]. The effect of homologous pituitary extracts remains untested in this species.

The results of this study do not preclude further work on this or other myobatrachid species to improve the rate and control of ovulation in assisted reproduction approaches. The use of gonadotropin releasing hormone analogues to induce ovulation has been reported in other myobatrachid species, Pseudophryne guentheri[65] and P. corroboree[66]. Investigations focussing on combining gonadotropin releasing hormones and dopamine antagonists [39] might also improve success rates and the control of timing of ovulation and oviposition in M. fasciolatus and other Mixophyes species, as might various combinations of hCG or other gonadotropins with progesterone [35, 37].

Mixophyes frogs are one of the most threatened Australian myobatrachid genera (with only Taudactylus containing a higher proportion of threatened species). There is one instance of captive breeding reported in a Mixophyes species (M. fasciolatus at Melbourne Zoo; [67]). In the future, captive breeding approaches in this and other Mixophyes species may be augmented by assisted reproduction using techniques including induced ovulation for in vitro fertilisation that allow specific individuals to be paired for optimal genetic management, and improve efficiency and reduce costs in resource limited captive programs, and play a role in breeding programs selecting for disease resistance, such as against chytridiomycosis.

This study found that M. fasciolatus is amongst the few amphibian species (including Xenopus (Silurana) and some bufonids) that respond well to the induction of ovulation utilising mammalian gonadotropins (hCG). The optimal protocol for M. fasciolatus involved two priming doses of PMSG (50 IU and 25 IU) administered at 6 and 4 days respectively, prior to two doses of hCG (100 IU), 24 hours apart. This study is also the first to demonstrate in an amphibian species that responds to mammalian gonadotropins that an increase in the ovulation rate occurs after priming with a gonadotropin (PMSG) with FSH activity.