Gamete maturation and interaction in the brushtail possum (Trichosurus vulpecula)

Abstract

Maturation of both male and female gametes of the brushtail possum (Trichosurus vulpecula) following in vivo and in vitro culture was investigated in these studies and suitably mature gametes were co-cultured to effect, for the first time in an Australian marsupial, successful in vitro fertilization (IVF).

It was shown that induced ovulation of the possum using pregnant mares' serum gonadotrophin (PMSG) is subject to seasonal variation in ovarian response. Maximal ovarian response was observed during the peaks in breeding of the natural seasonal cycle, whereas during summer (December - February, Southern Hemisphere) response to PMSG was minimal.

Prior to these studies, induced ovulation of the possum was effected by the use PMSG followed by GnRH. When this protocol was applied to possums in New Zealand, very few oocytes were recovered. Subsequently a higher dose of PMSG was used (15 IU) and ovulation was induced by the use of porcine luteinizing hormone (pLH) in place of GnRH. This led to the recovery of 9-13 oocytes per female but was dependent on time of the year when stimulation was attempted. Possums treated in this manner ovulated from 30 h post LH injection with a biphasic response. To overcome seasonal effects on ovarian sensitivity to PMSG, porcine follicle stimulating hormone (pFSH) was used. Treatment of animals with 8 X 6 mg pFSH followed by 4 mg pLH led to the recovery of oocytes displaying abnormal morphology. Adaptation of the protocol to reduce the dose of pFSH to 8 X 3 mg pFSH overcame hyperstimulation and led to the recovery of 11.8 ± 2.3 oocytes per female that demonstrated their ability to fertilize following laparoscopic artificial insemination. The time of ovulation was similar to when PMSG is utilised however oocytes were liberated synchronously.

Following induced ovulation using PMSG/LH, possum oocytes could be matured in vitro to metaphase II stage (MII) after 48 h in culture in EMEM media + 10 % fetal calf serum (FCS). Oocytes recovered from follicles < 2 mm in diameter had a much reduced capability to complete meiotic maturation, with oocytes from follicles < 1.5 mm failing to progress. The rate of maturation of oocytes recovered from follicles > 2 mm was unaffected by the presence of either exogenous hormones or granulosa cells. Possum oocytes matured in vivo following PMSG/LH treatment completed maturation to MII within 1 h prior to ovulation.

When possum sperm were co-cultured in the presence of oviduct epithelial cell (OEC) monolayers or their products, they underwent both conformational and functional changes that are consistent with capacitation events observed for in vivo recovered sperm. This change was obtained following 2 h in co-culture. There did not appear to any specific interaction with the monolayer cells and sperm undergo no detectable modification of the acrosome.

The cumulative development of techniques to mature both male and female possum gametes led to the penetration of oocyte zona pellucidae by sperm following IVF. This is the first recorded observation of early fertilization events in any Australian marsupial species.