Ovarian follicular superstimulation and oocyte maturation in the anoestrous southern hairy-nosed wombat, Lasiorhinus latifrons

Animal Reproduction Science 99 (2007) 363–376

Ovarian follicular superstimulation and oocyte maturation in the anoestrous southern hairy-nosed wombat, Lasiorhinus latifrons G.V. Druery a,∗ , G.A. Shimmin b,c , D.A. Taggart b,e , P.D. Temple-Smith d , W.G. Breed e , C.H. McDonald e , G.R. Finlayson f,g , M.C.J. Paris f,h a

School of Biological and Environmental Sciences, Central Queensland University, Rockhampton, Queensland 4702, Australia b School of Earth and Environmental Science, The University of Adelaide, Adelaide, South Australia 5005, Australia c Department of Environment & Heritage, GPO Box 1047, Adelaide, South Australia 5001, Australia d The University of Melbourne, Zoology Department, Parkville, Victoria 3056, Australia e Department of Anatomical Sciences, The University of Adelaide, South Australia 5005, Australia f Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia g Institute of Wildlife Research, School of Biological Sciences, University of Sydney, Sydney, New South Wales 2006, Australia h Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Received 24 November 2005; received in revised form 10 May 2006; accepted 12 June 2006 Available online 1 August 2006

Abstract This study investigates the effect of three exogenous gonadotrophin regimens on ovarian follicular development in southern hairy-nosed wombats during the non-breeding season. Females were given either porcine follicle stimulating hormone (pFSH; total of 200 mg at 12 h intervals over 7 (Group 1), or 4 days (Group 2)), or pregnant mares’ serum gonadotrophin (PMSG; single dose of 150 I.U. (Group 3)). In all treatment groups 25 mg of porcine luteinising hormone (pLH) was used to trigger maturation; Groups 1 and 2 received pLH 12 h after the final pFSH injection and Group 3 received pLH 72 h after PMSG. The results showed Group 1 produced significantly more follicles per ovary (5.91 ± 1.28) than Group 2 (1.67 ± 0.62), or Group 3 (2.17 ± 1.16) at p < 0.05. Control females received saline injections concurrently with the three treatment groups (n = 6; 2 control animals for each treatment group). No follicular development occurred in any control female. Analysis of oocyte nuclear status revealed that while oocytes from all three treatment groups had

∗

Corresponding author. Tel.: +61 749 310400; fax: +61 749 222085. E-mail address: [email protected] (G.V. Druery).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.06.011

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resumed meiosis, only those in Group 1 (7-day pFSH/pLH treatment) progressed to metaphase II. These results have implications for the development of assisted breeding strategies in this species. © 2006 Elsevier B.V. All rights reserved. Keywords: Marsupial; Wombat; Assisted breeding; Exogenous gonadotrophin

1. Introduction There are three species of wombats in Australia. These are the Common Wombat, Vombatus ursinus, abundant throughout most of southeastern Australia, the southern hairy-nosed (SHN) wombat, Lasiorhinus latifrons, confined to four populations in southern Australia (St John, 1998), and the northern hairy-nosed (NHN) wombat, Lasiorhinus krefftii, which has declined significantly and exists as a single population estimated at 83 individuals in central Queensland (Taylor et al., 2004). The NHN wombat is more closely related to the SHN than the common wombat. The local abundance of the SHN wombat in South Australia and its relationship to the NHN wombat makes it a valuable model species for the development of assisted breeding techniques and their potential application to recovering the NHN wombat. The female SHN wombat is monovular, polyoestrus, and usually produces only one young per year (Gaughwin and Wells, 1978; Tyrell, 2001; Paris et al., 2002b; Finlayson et al., 2004; Finlayson et al., 2006). The breeding season occurs mainly from July to December, and the majority of births usually occur in October (Gaughwin and Wells, 1978; Gaughwin et al., 1998; Tyrell, 2001). Peak breeding activity, defined as the maximum number of females with new pouch young, changes from year to year and may be dependant on appropriate environmental conditions like seasonal rainfall followed by fresh pasture growth (Gaughwin et al., 1998). Assisted reproductive technologies have been developed and implemented for the conservation of several mammalian species including marsupials. The development of these techniques in marsupials has the potential to benefit endangered species or captive populations of marsupials by increasing their productivity (Rodger, 1990; Mate et al., 1998). Techniques such as superovulation, artificial insemination, in vitro fertilisation and embryo transfer have resulted in pregnancies and live births in a number of non-domesticated eutherian species, e.g. blackbuck (Holt et al., 1988); Siberian tiger, Panthera tigris (Donoghue et al., 1990); black-footed ferret, Mustela putorius furo (Howard et al., 1991); puma, Felis concolor (Moore et al., 1981; Barone et al., 1994). More recently, the use of artificial insemination has successfully resulted in live births in marsupial species, e.g. koala (Johnston et al., 2003); tammar wallaby (Paris et al., 2002a). Superovulation provides a further approach to manipulating the reproductive capacity of a monovular group like the hairy-nosed wombats. Using this technique, the seasonal nature of this species reproductive strategy can potentially be overcome, and if achieved then could be used to increase reproductive output when carried out with other assisted breeding techniques such as artificial insemination or cross-fostering (Taggart et al., 1997). A number of superovulation protocols have been investigated in monovular marsupial species, e.g. brushtail possum, Trichosurus vulpecula (Rodger and Mate, 1988; Glazier, 1998; Glazier and Molinia, 1998, 2002; Molinia et al., 1998a; McLeod et al., 1999); tammar wallaby, Macropus eugenii (Renfree et al., 1988; Rodger et al., 1992b, 1993; Molinia et al., 1998a,b); brushtail bettong, Bettongia penicillata (Rodger et al., 1992b); fat-tailed dunnart, Sminthopsis crassicaudata (Rodger et al., 1992a); common wombat (West et al., 2002). These studies have concentrated on stimulating follicular development using either pregnant mare serum gonadotrophin (PMSG) or

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porcine FSH (pFSH). With the exception of the fat-tailed dunnart (Rodger et al., 1992a), ovulation in marsupials stimulated with exogenous gonadotrophins appears to require exogenous induction. The ovulatory stimulus has been treatment with either single or multiple doses of gonadotrophin releasing hormone (GnRH), single doses of pLH, or human chorionic gonadotrophin (hCG) (Smith and Godfrey, 1970; Rodger and Mate, 1988; Rodger et al., 1992a,b; Glazier and Molinia, 1998; Jungnickel et al., 2000; Glazier and Molinia, 2002). The present study examined three exogenous gonadotrophin treatment regimens aimed at inducing ovarian follicular development outside of the breeding season of the SHN wombat. The hormone regimens used either PMSG or pFSH in combination with pLH. 2. Materials and Methods 2.1. Animals All females used in this study were wild-caught in June 2002 on Kooloola Station near Swan Reach in the Murraylands District of South Australia. Animals were netted and examined for pouch condition, body condition and weight. Adult females were included in the study if they were adults with pouch conditions typical of the anoestrous condition, which meant they were pink, dirty and dry or only very slightly moist (Tyrell, 2001). Further, included females weighed over 20 kg, and had a head width greater than 124 mm to ensure they were adults (Tyrell, 2001). Suitable females were held in a purpose-built temporary holding facility and maintained on a diet of Complete ‘O’ (Magill Grain Store, Magill, S.A., Australia), lucerne and oaten hay. Water was provided ad libitum. Efforts were made to exclude light from the holding facility. Animals were randomly assigned to treatment groups and hormone administration was commenced the day following capture. Experimentation was approved by the University of Adelaide’s Animal Ethics Committee on Certificate No. S-12-2002. A Permit (No. A24016-5) to undertake scientific research on SHN wombats was granted by The Department for Environment and Heritage, South Australia. 2.2. Hormonal stimulation There were three gonadotrophin treatment groups. Group 1 animals (n = 6) received 200 mg porcine follicle stimulating hormone (pFSH, Folltropin-V; Bioniche, Canada) via i.m. injections over 7 days at 12-h intervals. Group 2 animals (n = 6) received the same total dose as Group 1, but over 4 days at the same 12-h intervals and also administered i.m. Group 3 received a single i.m. injection of 150 I.U. PMSG (Folligon; Bioniche, Canada). Porcine luteinising hormone (pLH, Lutropin-V; Bioniche, Canada) was given at the end of either pFSH or PMSG administration to induce oocyte maturation. Both the 7-day and 4-day pFSH treatment groups received 25 mg pLH 12 h after the final pFSH injection. PMSG-treated animals also received 25 mg pLH, but 72 h after the single dose of PMSG. Saline was concurrently administered to the three control groups (n = 2 per group). 2.3. Reproductive tract collection and processing Females were anaesthetised with a combination of Zolazepam and Tiletamine (Zoletil® ; Virbac Australia Pty Ltd.) (3 mg kg−1 ) 24 h after the pLH or final saline injection. Once anaesthetised, animals were euthanased with a .22 calibre shot to the occiputal region and reproductive tracts

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were immediately recovered and dissected of mesenteric tissue and fat. Ovaries were removed from the tract before gross measurements and weights were recorded. The size and number of antral follicles >1.5 mm were then recorded and oocytes were immediately recovered. Oocyte recovery involved piercing each visible follicle wall with two 25G needles and gently expelling the contents into phosphate buffered saline (PBS). A ‘positive response’ was defined as an animal in which five or more follicles >1.5 mm in diameter had formed. Uteri were dissected, measured and weighed. Recovered oocytes were fixed in 2.5% glutaraldehyde made up in PBS for 24 h at 4 ◦ C. Oocytes were then washed in PBS and stored in PBS supplemented with 4% foetal calf serum at 4 ◦ C to facilitate gamete handling. 2.4. Oocyte analysis Oocytes collected from the three groups were stained with the DNA-specific dye DAPI (4,6-diamidino-2-phenylindole dihydrochloride; Sigma) diluted 1 in 104 and examined using fluorescence at ultraviolet wavelength using an Olympus BH2-RFL-T2 fluorescence microscope at ×40 magnification. Phase contrast and Nomarski differential optics microscopy were also used to confirm nuclear status. 2.5. Blood progesterone analysis Progesterone levels in blood were analysed at the Reproductive Endocrine Unit, Queen Elizabeth Hospital in Adelaide. The competitive immunoassay was performed using an automated chemiluminescence system (Bayer ACS:180), and was validated for measuring SHN wombat progesterone levels by performing tests for linearity, spiking recoveries, and intra- and inter-assay variation. Intra-assay coefficients of variation (CV) were 5.7% at 4.8 nmol/L, and <5% at 22.9 and 66.5 nmol/L. Inter-assay CV were 11.1% at 4.8 nmol/L, 5.6% at 23.3 nmol/L and 6.7% at 70 nmol/L. The ACS:180 shows high specificity for progesterone and has a cross reactivity of less than 0.95% with other compounds. The assay measures progesterone concentrations within the range of 0.11–60 ng/mL. The sensitivity of the assay was considered to be appropriate for the expected progesterone levels in SHN wombats (Paris et al., 2002b). 2.6. Data analysis Follicular development, ovarian and uterine weights are presented as means ± S.E.M. One way ANOVA was used for comparison of treatment means. Where ANOVA detected a statistically significant difference among treatment means (p < 0.05), a Tukey test was used for pairwise comparisons to determine which treatment groups were significantly different from each other. 3. Results 3.1. Ovarian follicular response Only animals administered exogenous gonadotrophins had ovaries with antral follicles. Among the treated females 4/6 of Group 1, 3/6 of Group 2 and 2/6 of Group 3 had ovaries with antral follicles >1.5 mm (Fig. 1). In treated animals all antral follicles >1.5 mm appeared transparent, were turgid and fluid filled, and appeared to be normal morphologically (Fig. 2). Group 1 had significantly more follicles than the other groups (p < 0.05) (Table 1), but the mean follicle size (of

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Fig. 1. Variation in ovarian follicular response in each treatment group displayed as the total number of follicles >1.5 mm (n = 6 per group). Only four and three data points are visible for Group 2 and Group 3 respectively, as three and four females respectively in these groups failed to respond to treatment. The dotted line signifies the cut off point for an ‘ovarian response’.

Fig. 2. Variation in ovarian response to treatment: (a and b) ovaries recovered from control animals. Ovaries look largely homogeneous with no obvious ovarian activity and the exception of old corpora albicantia (b). The ovaries of two females from the 7-day pFSH treatment group are shown in (c and d), where numerous antral follicles are present (bars = 5 mm).

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Table 1 Number of follicles >1.5 mm, and weights (g) of uteri and ovaries in saline-, pFSH- or PMSG-treated female SHN wombats

Follicles/ovary Uteri weight Ovarian weight

Group 1 (n = 6)

Group 2 (n = 6)

Group 3 (n = 6)

Control (n = 6)

5.91 ± 1.28b

1.67 ± 0.62a

2.17 ± 1.16a

0.08 ± 0.08a 0.92 ± 0.08a 0.44 ± 0.03a

1.51 ± 0.13b 0.95 ± 0.12b

1.23 ± 0.25a,b 0.56 ± 0.10a,b

1.01 ± 0.12a 0.65 ± 0.11a,b

Tissues were collected 24 h after pLH administration or final saline injection; Group 1: 7-day pFSH; Group 2: 4-day pFSH; Group 3: PMSG. Means with different superscripts (a and b) are significantly different from each other at p < 0.05.

Fig. 3. Mean ± S.E.M. follicle size in the three treatment groups. The mean number of follicles in Group 1 is higher than the other groups, though not significantly. Only follicles >1.5 mm were considered. Group 4 (control animals) is absent due to a lack of response. Group 1: 7-day pFSH; Group 2: 4-day pFSH; Group 3: PMSG.

Fig. 4. Nuclear status of oocytes recovered from PMSG, 4-day and 7-day FSH treatment groups (n = 6 per group). Meiosis had resumed in all treatments, as evidenced by the presence of the breakdown of the germinal vesicle membrane or the progression to anaphase I or metaphase I. Treatment with pFSH over 7 days was the only protocol that produced MII oocytes.

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Fig. 5. Various stages of meiosis in recovered oocytes (Nomarski optics microscopy and fluorescence microscopy): (a) MII oocyte from 7-day pFSH-treated female showing a thickened zona (ZP) and polar body (red arrow). (b) Resumption of meiosis showing metaphase I stage. Note the chromosomal alignment (arrow). (c and d) Extruded polar bodies are clearly visible (red arrows) in oocytes recovered from females treated with pFSH for 7 days. (e) An immature oocyte, evident by the intact nuclear membrane of the germinal vesicle stage (arrows); bars = 40 ␮m.

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follicles >1.5 mm) was not statistically significant to Group 2 or 3 (Fig. 3). Compared to control animals, Group 1 also had significantly greater ovarian and uterine weights. No ovulations had occurred. 3.2. Nuclear status of recovered oocytes Fluorescence microscopy revealed that some of the oocytes from the three stimulated groups had resumed meiosis, as indicated by the breakdown of the nuclear membrane surrounding the germinal vesicle (GVBD) or progression to either anaphase I/metaphase I, or metaphase II (Fig. 4). In the 7-day pFSH group approximately 47% (14/30) had progressed to either anaphase I or metaphase I, and 37% (11/30) of oocytes had progressed to metaphase II. Oocytes displaying a polar body were considered to have progressed to metaphase II (Fig. 5). Although 50% (4/8) and 23% (3/13) of the 4-day pFSH and PMSG treatment groups respectively had resumed meiosis, no oocytes from either of these treatments were found to have progressed to the metaphase II stage. 3.3. Qualitative observations of reproductive tracts Observations of reproductive tracts suggest that animals administered gonadotrophin responded not only at the ovarian level, but also in growth and development of the entire reproductive tract. There was an obvious difference in the size of the urogential sinus, vaginal complex and urogenital sinus glands compared to control animals. Fig. 6 clearly demonstrates differences in tracts from controls and females from the 7-day pFSH treatment group. 3.4. Progesterone analysis Plasma progesterone levels were low in all groups including controls before commencing treatments. A repeated measures ANOVA on the three times that progesterone levels were measured in all four treatment groups showed a significant effect of time (F1,20 = 14.83, p < 0.01), no interaction between time and treatment (F3,20 = 2.29, p = 0.25) and no effect of treatment (F3,20 = 0.64, p = 0.54). Mid cycle data were excluded because they were incomplete. The analysis showed that progesterone levels changed over time, but there was no difference among treatments and the changes were similar in all four treatment groups. 4. Discussion This is the first report to detail the ovarian follicular response of anoestrous SHN wombats to exogenous gonadotrophin administration. Compared to the other treatments, pFSH administered over a 7-day period was most effective for producing antral follicles >1.5 mm in diameter. In another marsupial, the tammar wallaby, pFSH also has a greater stimulatory effect on the total number of follicles compared to treatment with PMSG (Molinia et al., 1998a,b). Early studies in tammars used low doses of PMSG and multiple doses of GnRH late in the oestrous cycle, and found that the active corpus luteum (CL) from the previous cycle had a significant effect on the ovarian response of the CL-bearing ovary (Renfree et al., 1988; Rodger et al., 1993). As our study was performed prior to the start of the breeding season (Gaughwin et al., 1998; Tyrell, 2001), females had no active CL’s at the time of gonadotrophin administration. Glazier and Molinia (2002) have also reported that pFSH results in a higher percentage of anoestrous

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Fig. 6. Comparison between reproductive tracts from control animals (a and b), and animals treated with pFSH over 7 days (c and d). The differences in size of the vaginal complex, urogenital sinus (UGS) and urogenital sinus glands are clearly evident.

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brushtail possums responding, with a higher number of follicles and ovulations compared to PMSG. The present study used higher hormone doses than have previously been reported for either the common wombat or tammar wallaby (Molinia et al., 1998a,b; Jungnickel and Hinds, 2000). In the common wombat 48 mg of pFSH over 4 days followed by 4 mg of pLH 12 h after the final pFSH injection lead to the production of multiple follicles and the recovery of MII oocytes when treatment was started early in the follicular phase of a natural oestrous cycle (West et al., 2002). Evidence for the importance of adjusting the stimulatory dose to the body mass is scarce in marsupials although large brushtail possums form fewer follicles than small brushtail possums in response to the same PMSG dose (Molinia et al., 1998a; Glazier and Molinia, 1998; Rodger and Mate, 1988). This study chose a higher dose to compensate for the SHN wombats large body size and their reproductive status (seasonal anoestrus). The range in the number of developing follicles in the 7-day pFSH group (2–24 follicles) is comparable to that observed in tammar wallabies treated with pFSH (3–47; Molinia et al., 1998b), but wider than that reported for brushtail possums following treatment with PMSG (8–17 medium to large Graafian follicles (Rodger and Mate, 1988); 9–12 follicles > 2 mm (Glazier and Molinia, 1998); 9–16 Graafian follicles > 2 mm (Glazier, 1999)). The range in the number of developing follicles in our 4-day pFSH and PMSG treatment groups was also very large (0–11 and 0–21 respectively), which suggests that unknown factors may be affecting the response. Although statistically there was no significant difference in the weight of females in this experiment and females appeared to be in a similar body condition, it is difficult to exclude the possibility of environmental influences on the response of treated wombats. The study site was experiencing the early stages of a drought at the time of experimentation, and pasture quality and quantity were both low. It is well documented that nutrition has a profound influence on the reproductive capacity of mammals, and improved or increased dietary intake can have a significant effect on ovarian follicular recruitment, fertilisation and embryo development in animals including cattle (Gong, 2002; Gong et al., 2002; Diskin et al., 2003), and sheep (Munoz-Gutierrez et al., 2002; Lozano et al., 2003). Drought is known to have an effect on seasonal reproduction in southern hairy-nosed wombats, and the incidence of breeding in drought years appears to drop significantly (Gaughwin et al., 1998; Taggart et al., unpublished data). Another possibility for the varied response at least in the PMSG-group is a seasonal variation in response to this particular gonadotrophin. PMSG-primed brushtail possums exhibit variation in ovarian response that closely follows the natural breeding season (Glazier, 1998). The lowered response in the anoestrous brushtail possum can be overcome by administering pFSH instead of PMSG and contrasts the response of seasonally breeding eutherian species such as sheep and deer to PMSG where no seasonal variation to treatment is observed (Ryan et al., 1991; Argo et al., 1994; McLeod et al., 2001). It must be considered that SHN wombats administered PMSG in June may also have a lowered ovarian response due to timing in relation to breeding season. The gross appearance of control SHN wombat’s ovaries was largely homogeneous with all follicles >1.5 mm and only visible under a dissecting microscope. Tyndale-Biscoe and Renfree (1987) have described this ovarian appearance in marsupials as being indicative of anoestrus. This experiment would need to be repeated at other times of the year to determine if there are seasonal changes in responsiveness to stimulation. There is little information about the normal size range of antral follicles in SHN wombats. Tyrell (2001) provides only sparse information on antral follicle size in naturally cycling SHN wombat females. Graafian follicles range from 4.9 to 9.9 mm with an average diameter of 6.7 ± 1.6 mm (Tyrell, 2001). Moritz et al. (1998) reported the mean diameter of Type 8 Graafian follicles

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observed on the ovaries of common wombats (n = 3) as 4.2 ± 0.2 mm. It is not known how large SHN wombat or common wombat follicles are at the point of ovulation. The average size of the antral follicles that were measured in this study (<1.5 mm) was not significantly different between treatment groups. Only 10 follicles greater than 6.7 mm were found, 8 of these were in two females in the 7-day pFSH treatment group, and the other two occurred in one female in the PMSG treatment group. No follicles greater than 6.7 mm were observed on the ovaries of any female that received pFSH over 4 days. No ovulations had occurred by 24 h post-pLH in the present study despite a few follicles measuring 10 mm in diameter. Follicles may need to be >10 mm before ovulation commences in SHN wombats stimulated with pFSH (Druery et al., unpublished data). A protocol that stimulates the largest number of harvestable follicles may not be the same as those which give rise to most MII oocytes. In the case of the anoestrous brushtail possum, protocols using pFSH and PMSG both have advantages and disadvantages in relation to the number of follicles recruited and the percentage of oocytes that mature. Other studies suggest that this is also the case in the tammar wallaby as pFSH overrides the inhibiting effect of the luteal ovary on follicle development. Glazier and Molinia (2002) suggest that the pFSH dose in the brushtail possum is not optimised, and that the protocol needs refining to improve the number of mature oocytes retrieved. Only 39% of SHN wombat oocytes recovered from the 7-day pFSH group in this study were found to be at the metaphase II stage indicating that the hormone doses and/or recovery times were not optimal. Glazier et al. (2002) using pLH to trigger ovulation in possums stimulated with PMSG found that oocytes recovered from Graafian follicles >2 mm did not resume meiosis until after 27 h post-pLH. The collection of MII oocytes from Graafian follicles in our study is consistent with other marsupials in which the completion of the first meiotic maturation division occurs before ovulation (Hinds et al., 1996). Cleary et al. (2003) showed that it was possible to produce a similar percentage (34%) of common wombat MII oocytes retrieved from antral follicles in vitro after culturing for 60 h. The length of the oestrous cycle in captive SHN wombats was initially determined noninvasively from faecal pregnanes as 41.1 ± 12.8 days, with a follicular phase of 27.9 ± 12.3 days (Paris et al., 2002b). A similar length oestrous cycle has also been reported for the common wombat in Victoria (West et al., 2001). More recently the oestrous cycle of captive SHN wombats during the breeding season has been reported as 36.33 ± 0.67, determined from circulating levels of progesterone and oestradiol (Finlayson et al., 2006). The 7-day pFSH stimulation protocol may have been more effective at eliciting an ovarian response, as the SHN wombat appears to have a lengthy follicular phase during the oestrous cycle (Paris et al., 2002b). Follicles and their associated oocytes may require a longer maturational period than from a 4-day FSH stimulation period, or administration of a single PMSG dose. Lawrence et al. (1997) determined the level of homology between brushtail possum FSH ␤-subunit gene-expressing cells isolated from anterior pituitary tissue and the same in six eutherian species. They showed that porcine FSH had the closest amino acid sequence (79.8% identical full-length precursor protein) compared to the same precursor protein in the six eutherian species. Although the amino acid sequence has not been determined for wombat gonadotrophins, porcine FSH may also show a closer affinity with wombat FSH. In conclusion, the 7-day pFSH treatment protocol reported for the SHN wombat results in the production of multiple mature oocytes within morphologically normal follicles. Thus the primary aim of this study has been achieved. The next step is to determine the timing of ovulation in relation to the pLH trigger injection, so this knowledge may provide the opportunity to attempt

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