Peter Temple-Smith

Human Pregnancy by in Vitro Fertilization (IVF) Using Sperm Aspirated from the Epididymis

Spermatozoa were collected by microaspiration from the corpus epididymidis of a 42-year-old man with secondary obstructive azoospermia and used for in vitro fertilization. At insemination 61% of the spermatozoa were motile, with a motility index of 157. One of five eggs was fertilized and this was subsequently transferred to the patients wife at the two-cell stage. Ultrasound examination and changing hormone levels confirmed an ongoing pregnancy, which is currently at 30 weeks of gestation. This technique will provide a useful alternative for the management of some infertile men with obstructive azoospermia.

Defensins and the convergent evolution of platypus and reptile venom genes

When the platypus (Ornithorhynchus anatinus) was first discovered, it was thought to be a taxidermists hoax, as it has a blend of mammalian and reptilian features. It is a most remarkable mammal, not only because it lays eggs but also because it is venomous. Rather than delivering venom through a bite, as do snakes and shrews, male platypuses have venomous spurs on each hind leg. The platypus genome sequence provides a unique opportunity to unravel the evolutionary history of many of these interesting features. While searching the platypus genome for the sequences of antimicrobial defensin genes, we identified three Ornithorhynchus venom defensin-like peptide (OvDLP) genes, which produce the major components of platypus venom. We show that gene duplication and subsequent functional diversification of beta-defensins gave rise to these platypus OvDLPs. The OvDLP genes are located adjacent to the beta-defensins and share similar gene organization and peptide structures. Intriguingly, some species of snakes and lizards also produce venoms containing similar molecules called crotamines and crotamine-like peptides. This led us to trace the evolutionary origins of other components of platypus and reptile venom. Here we show that several venom components have evolved separately in the platypus and reptiles. Convergent evolution has repeatedly selected genes coding for proteins containing specific structural motifs as templates for venom molecules.

Factors influencing paternity success in Antechinus agilis: last‐male sperm precedence, timing of mating and genetic compatibility

We describe the patterns of paternity success from laboratory mating experiments conducted in Antechinus agilis, a small size dimorphic carnivorous marsupial (males are larger than females). A previous study found last‐male sperm precedence in this species, but they were unable to sample complete litters, and did not take male size and relatedness into account. We tested whether last‐male sperm precedence regardless of male size still holds for complete litters. We explored the relationship between male mating order, male size, timing of mating and relatedness on paternity success. Females were mated with two males of different size with either the large or the small male first, with 1 day rest between the matings. Matings continued for 6 h. In these controlled conditions male size did not have a strong effect on paternity success, but mating order did. Males mating second sired 69.5% of the offspring. Within first mated males, males that mated closer to ovulation sired more offspring. To a lesser degree, variation appeared also to be caused by differences in genetic compatibility of the female and the male, where high levels of allele‐sharing resulted in lower paternity success.

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

Cryopreservation of ovarian tissue is an important option for preserving the fertility of cancer patients undergoing chemotherapy and radiotherapy. In this study, we examined the viability and function of oocytes derived in vitro from pre-antral follicles as an alternative method for restoring fertility. Pre-antral follicles (specified as secondary follicle with a diameter around 100-130 μm) were mechanically isolated from vitrified-warmed and fresh adult mouse ovarian tissues and cultured for 12 days followed by an ovulation induction protocol at the end of this period to initiate oocyte maturation. Oocytes were then released from these follicles, fertilized in vitro, and cultured to the blastocyst stage and vitrified. After storage in liquid nitrogen for 2 weeks, groups of vitrified blastocysts were warmed and transferred into pseudo-pregnant recipient females. Although most of the isolated mouse pre-antral follicles from fresh (79.4%) and vitrified (75.0%) ovarian tissues survived the 12-day in vitro culture period, significantly fewer mature oocytes developed from vitrified-warmed pre-antral follicles than from the fresh controls (62.2 vs 86.4%, P<0.05). No difference was observed in embryo cleavage rates between these two groups, but the proportion of embryos that developed into blastocysts in the vitrification group was only half that of the controls (24.2 vs 47.2%, P<0.05). Nevertheless, live births of healthy normal pups were achieved after transfer of vitrified blastocysts derived from both experimental groups. This study shows that successful production of healthy offspring using an in vitro follicle culture system is feasible, and suggests that this procedure could be used in cancer patients who wish to preserve their fertility using ovarian tissue cryopreservation.

Paternity success and the direction of sexual selection in a field population of a semelparous marsupial, Antechinus agilis

Antechinus agilis is a small sexually size dimorphic marsupial with a brief annual mating period of 2–3 weeks. All males die after this period, and females give birth to up to 10 young. Mating is thought to be promiscuous, however, there is no field data to confirm this. Using microsatellites, we investigated paternity patterns over two seasons in a wild population. Male weight was significantly positively related to the number of females fertilized and with the number of offspring sired, in both years. Furthermore, selection gradients indicated selection for larger males. Both results suggest that size dimorphism in A. agilis can be explained by sexual selection for larger males. The proportion of offspring sired within litters, did not relate to male size. Therefore, larger males are more successful through higher mating access, not through their sperm outcompeting that of smaller males. As expected from their known ranging behaviour, the number of offspring within litters left unassigned to a father did not depend on the grid location of the mother. Female size did not differ between successful reproducing and unsuccessful females. However, females that weaned offspring had larger heads than females that did not wean offspring. Males did not ‘prefer’ mating with larger females, nor did assortative mating occur. From our results, the mating system of A. agilis is clearly promiscuous. Selection for larger males occurred in both years, even though in one year the operational sex ratio was highly female biased, suggesting that the potential reproductive rate is a better predictor of the direction of sexual selection in A. agilis.

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future.

Olfactory cues, genetic relatedness and female mate choice in the agile antechinus (Antechinus agilis)

Females show mate preferences for males that are genetically dissimilar to themselves in a variety of taxa, but how females choose these males is not clearly understood. In this study, we examined the effects of olfactory stimuli and genetic relatedness on female mate choice in a small carnivorous marsupial, the agile antechinus (Antechinus agilis), during two breeding seasons. Captive female antechinus in oestrus were provided with a combination of male urine and body scent from two novel males, one more genetically similar and one more dissimilar to the females, in a Y-maze olfactometer. Genetic relatedness between females and pairs of males was determined using highly polymorphic, species-specific, microsatellite markers. Females consistently chose to visit the scents of males that were genetically dissimilar to themselves first, spent significantly more time near the source of those scents and showed more sexual and non-exploratory behaviours near those scents. These data demonstrate that chemosensory cues are important in mate choice in the agile antechinus and that females prefer males that are genetically dissimilar to themselves.

Frozen-thawed epididymal spermatozoa for intracytoplasmic sperm injection

OBJECTIVE To report the fertilization rate and pregnancy results for intracytoplasmic sperm injection (ICSI) cycles using frozen-thawed epididymal spermatozoa. DESIGN Retrospective analysis of consecutive ICSI cycles. SETTING Tertiary referral center for infertility. PATIENT(S) Infertile couples in whom 39 patients (59 ICSI cycles) required the use of frozen-thawed epididymal spermatozoa. As no cycles were performed using fresh epididymal spermatozoa, outcomes were compared with those of 130 couples (170 ICSI cycles) using fresh ejaculated spermatozoa for severe oligoasthenozoospermia-teratozoospermia, in which < 200,000 motile spermatozoa were retrieved after Percoll density gradient centrifugation. INTERVENTION(S) Epididymal sperm aspiration during microsurgery, followed by ICSI. MAIN OUTCOME MEASURE(S) Fertilization, embryo cleavage, ET, and clinical pregnancy rates (PRs). RESULT(S) A total of 484 metaphase II oocytes were injected with frozen-thawed epididymal spermatozoa, resulting in a two-pronuclear fertilization rate per injected metaphase II oocyte of 47%, significantly lower than in cycles using fresh ejaculated spermatozoa (73%). Embryo implantation rates (12.0% versus 13.3%) and clinical PRs per transfer (18.4% versus 27.0%) were not different. When the prefreeze vitality was > 20%, compared with lower vitality, both the fertilization (56% versus 22%) and embryo cleavage (91% versus 57%) rates were significantly greater. CONCLUSION(S) The routine collection and cryopreservation of epididymal spermatozoa at microsurgery allows multiple ICSI treatment cycles with success rates similar to those of ejaculated spermatozoa. However, when the vitality of aspirated spermatozoa is < 20%, the poor fertilization rates indicate the need to consider an alternative source of viable spermatozoa.

Burrow use and ranging behaviour of the southern hairy-nosed wombat ( Lasiorhinus latifrons ) in the Murraylands, South Australia

This study investigated burrow use and ranging behaviour in the southern hairy-nosed wombat Lasiorhinus latifrons in semi-arid South Australia. Sixteen adult wombats were fitted with radio transmitters and monitored monthly from July 2001 to February 2002. Wombats generally used between one and five warrens, preferred large warrens with a greater number of entrances and showed a preference for one or two warrens. Across the study period there was no apparent change in burrows used within warrens. Radio-tracking indicated that animals spent very little time above ground (26% of 1115 night-time fixes), centred their activity around their preferred warrens, and moved, on average, 99 m/h and 221 m/night. Mean home-range size, estimated using minimum convex polygons and the harmonic mean method from location data, obtained through triangulation, and daytime warren fixes, ranged from 1.3 to 4.8 ha. Home-range size was similar between males and females and home ranges overlapped substantially. The data highlight the importance of burrows to southern hairy-nosed wombats in shaping their home ranges. It seems likely that the use of burrows and a specialized diet are important energy saving strategies for this species in such unpredictable regions of South Australia.

Effect of cooling and cryopreservation on sperm motility and morphology of several species of marsupial

The effects of long-term cooling and freezing on sperm motility are described for six marsupial species: the fat-tailed dunnart, koala, brushtail possum, long-footed potoroo, northern brown bandicoot and ring-tailed possum. The effects of up to eight days of cooling at 4 degrees C on the motility of dunnart spermatozoa and the effect of cryopreservation on spermatozoa of the other species were determined. The cryoprotectant used was a Tris-citrate-fructose-egg yolk-glycerol diluent. The percentage and rating of sperm motility, and sperm structure, as determined by light microscopy, were investigated. Sperm motility in the fat-tailed dunnart was retained for up to six days when cooled to 4 degrees C, suggesting that sperm from this species have some degree of tolerance to cold shock. After this time, however, the percentage of motile spermatozoa and their motility rating declined. In all species except the fat-tailed dunnart, reinitiation of motility following cryopreservation occurred across a range of glycerol concentrations (4-17%). Cryoprotectant containing 6% and/or 8% glycerol resulted in little change of motility rating or of the percentage of live sperm after thawing, although there was some decline in the percentage of motile sperm. The unusual structural and motility characteristics of dunnart spermatozoa may account for the lack of success of sperm cryopreservation in this species.