Uterine and vaginal insemination optimised in brushtail possums (Trichosurus vulpecula) superovulated with pregnant mare serum gonadotrophin and porcine luteinising hormone.

Artificial insemination of brushtail possums (Trichosurus vulpecula) is being developed as an assisted breeding model for endangered marsupials, as well as a bioassay for testing fertility control vaccines to manage overabundant populations. Procedures were optimised in animals superovulated with pregnant mare serum gonadotrophin (PMSG) and porcine luteinising hormone (pLH). Of three intervals examined, yields were maximal following uterine insemination at 27-29.5 h after pLH treatment (four eggs, two to three embryos per female). Compared with no insemination, uterine-inseminated animals ovulated 30-36 h rather than 28-34 h after pLH treatment. For the vaginal route, yields were maximal following insemination at 10-13 h after pLH treatment (six to seven eggs, four embryos per female) than at five other intervals, and when using acclimatised females during the autumn breeding season. This protocol was suitable for testing fertility control vaccines in April-June and was influenced by the housing location of animals, the presence of an active corpus luteum and PMSG batch, but not other factors (year of trial, Freund's adjuvant treatment, changes in bodyweight, dose of PMSG kg(-1)). Embryos developed to the eight- to 16-cell or unilaminar blastocyst stage after uterine or vaginal insemination, respectively. With the timing of artificial insemination optimised, new methods to synchronise or induce oestrus and ovulation are required to achieve year-round testing of fertility control vaccines or birth of offspring.

Development of a porcine follicle-stimulating hormone and porcine luteinizing hormone induced ovulation protocol in the seasonally anoestrus brushtail possum (Trichosurus vulpecula).

Monovulatory brushtail possums (Trichosurus vulpecula) were stimulated with exogenous hormones during seasonal anoestrus to overcome ovarian insensitivity and induce ovulation. Seasonal ovarian insensitivity to pregnant mare serum gonadotrophin (PMSG) was overcome by a new porcine follicle-stimulating hormone/porcine luteinizing hormone (pFSH/pLH) protocol. This protocol was refined because the original treatment produced oocytes with abnormal morphology. Possums (n = 12 per group) received eight injections of pFSH of 1.5, 3.0 or 6.0 mg per injection (at 12-h intervals for 4 consecutive days). Ovulation was induced 12 h after the final pFSH injection with a 4-mg injection of pLH. Control animals were treated with the established protocol of a single injection of 15 IU of PMSG, followed 48 h later with an injection of 4 mg of pLH. All females responded to pFSH/pLH treatment, although optimal stimulation occurred in those receiving 8 x 3 mg pFSH, with 13-14 ovulations and recovery of 11-12 oocytes per female (8 x 1.5 mg pFSH: 13 ovulations, 8-9 oocytes; 8 x 6 mg pFSH: 7-8 ovulations, 4-5 oocytes). In contrast, only seven of 12 females responded to PMSG/pLH and, of those responding, only 2-3 ovulations occurred and only 1-2 oocytes per female were recovered. However, around 80% of oocytes recovered after PMSG/pLH treatment had undergone nuclear maturation (metaphase II/1st polar body) compared with around 60% of oocytes from pFSH/pLH-treated animals. In possums killed from 27 to 39 h after pLH treatment, ovulation onset was first observed from 30 h and by 31.5 h, all animals had completed ovulation. Laparoscopic artificial insemination (LAI) was performed on pFSH/pLH-treated animals to determine whether the oocytes produced were capable of fertilization. Uterine LAI performed 27.5-28.5 h after pLH treatment yielded 11/26 fertilized oocytes (up to 4-cell stage), whereas vaginal LAI performed 13-14 h after pLH treatment yielded 21/53 fertilized oocytes. A proportion of oocytes generated from the refined pFSH/pLH protocol are thus properly mature and capable of fertilization. Further refinement of the protocol is now needed to improve the yield of fully matured oocytes.

Transgenic rescue of defective Cd36 ameliorates insulin resistance in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) display several features of the human insulin-resistance syndromes. Cd36 deficiency is genetically linked to insulin resistance in SHR. We show that transgenic expression of Cd36 in SHR ameliorates insulin resistance and lowers serum fatty acids. Our results provide direct evidence that Cd36 deficiency can promote defective insulin action and disordered fatty-acid metabolism in spontaneous hypertension.

Cd36 and molecular mechanisms of insulin resistance in the stroke-prone spontaneously hypertensive rat.

Insulin resistance is of pathogenic importance in several common human disorders including type 2 diabetes, hypertension, obesity and hyperlipidemia, but the underlying mechanisms are unknown. The spontaneously hypertensive rat (SHR) is a model of these human insulin resistance syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridemia, and hypertension map to a single region on rat chromosome 4. Genetic analysis of an SHR derived from a National Institutes of Health colony led to the identification of a causative mutation in the SHR Cd36. We have investigated glucose and fatty acid metabolism in the stroke-prone SHR (SHRSP). We demonstrate defects in insulin action on 2-deoxy-D-glucose transport (SHRSP 3.3 +/- 1.5 vs. 21.0 +/- 7.4 pmol x min(-1) x [20 microl packed cells](-1), SHRSP vs. WKY, respectively, P = 0.01) and inhibition of catecholamine-stimulated lipolysis (P < 0.05 at all concentrations of insulin) in adipocytes isolated from SHRSP. In contrast, basal levels of catecholamine-stimulated nonesterified free fatty acid (NEFA) release and plasma levels of NEFA are similar in SHRSP and WKY. These results are in agreement with the data on the SHR.4 congenic strain, which suggested that the QTL containing Cd36 mutations accounted for the entire defect in basal catecholamine action but only for approximately 40% of the SHR defect in insulin action. In the SHR, both abnormalities appear consequent of defective Cd36 expression. Because Cd36 sequence and expression are apparently normal in SHRSP, it is likely that the molecular mechanism for defective insulin action in this strain is caused by a gene(s) different than Cd36.

Sperm binding and penetration of the zona pellucida in vitro but not sperm-egg fusion in an Australian marsupial, the brushtail possum (Trichosurus vulpecula).

Sperm capacitation and in vitro fertilisation (IVF) have been achieved in most eutherian mammals and American marsupials under relatively simple culture conditions. In contrast sperm capacitation in Australian marsupials has not been achieved in vitro and attempts at IVF have previously been characterised by a complete lack of sperm-zona pellucida (ZP) binding. Recently, co-culture of sperm with oviduct epithelial cell monolayers or with oviductal explant conditioned media has been shown to prolong the viability and motility of brushtail possum spermatozoa, as well as to induce capacitation-associated changes such as transformation of sperm to the T-shape orientation. In this study we report that these in vitro produced T-shaped sperm, and in vivo derived T-shaped sperm flushed from the oviduct of artificially inseminated possums as a control, are able to bind to and penetrate the ZP of approximately 25% of eggs recovered from PMSG/LH-superovulated possums in vitro. Development of ZP receptivity and penetrability towards sperm was also identified as a major factor affecting the outcome of IVF. Neither in vivo nor in vitro derived T-shaped sperm were able to bind to or penetrate the ZP if eggs were obtained from animals that were treated with pLH less than 76 h after PMSG. Thus this study provides preliminary evidence for the necessity of sperm-oviduct epithelial cell interactions for capacitation in Australian species and lends further support to the suggestion that the T-shape head orientation is indicative of sperm capacitation. Despite the occurrence of sperm-ZP binding and penetration, sperm-egg membrane fusion and egg activation were not observed. Although the factor(s) responsible for the lack of sperm-egg membrane fusion in the possum have not been identified it is possible that egg capacity for membrane fusion develops independently of zona receptivity and is defective in these eggs, or alternatively that membrane fusion requires strictly defined ionic conditions which are not provided by the IVF media used in this study.

A high-resolution radiation hybrid map of the proximal region of rat chromosome 4.

Radiation hybrid (RH) mapping has been used to produce genome maps in the human and mouse, but as yet the technique has been applied little to other species. We describe the use of RH mapping in the rat, using a newly available rat/hamster RH panel, to construct an RH map of the proximal part of rat Chromosome (Chr) 4. This region is of interest because quantitative trait loci (QTLs) for defective insulin and catecholamine action, hypertension, and dyslipidemia map to this region. The RH map includes 23 rat genes or microsatellites previously mapped to this part of Chr 4, one rat gene not previously mapped in the rat, and markers for four new genes, homologs of which map to the syntenic region of the mouse genome. The RH map integrates genetic markers previously mapped on several rat crosses, increases the resolution of existing maps, and may provide a suitable basis for physical map construction and gene identification in this chromosomal region. Our results demonstrate the utility of RH mapping in the rat genome and show that RH mapping can be used to localize, in the rat genome, the homologs of genes from other species such as the mouse. This will facilitate identification of candidate genes underlying QTLs on this chromosomal segment.

Time of ovulation in the brushtail possum (Trichosurus vulpecula) following PMSG/LH induced ovulation.

This study aimed to determine the timing of ovulation in response to a new induced ovulation regime for the monovulatory brushtail possum (Trichosurus vulpecula). Ovarian stimulation was achieved by a single subcutaneous injection of 15 i.u. pregnant mare serum gonadotrophin (PMSG). This treatment resulted in promotion of a large number (9-16) of Graafian follicles > 2 mm diameter on the ovaries. Seventy-two hours later a single intramuscular injection of 4 mg porcine luteinizing hormone (LH) was administered to induce ovulation. Groups of possums were killed 24 hr post-LH injection and subsequent groups were killed at 6-hr periods up to 48 hr later. Ovulation occurred from 30 hr to 42 hr post-LH. The ovulatory success increased from 3% at 30 hr post-LH to 83% at 48 hr post-LH. Oocytes were recovered primarily from the oviducts at 36 hr post-LH. Thereafter oocytes were recovered increasingly from the uteri and by 48 hr post-LH, were only found there. The implications of these observations for artificial breeding in possums are discussed.

Identification of Cd36 (Fat) as an insulin-resistance gene causing defective fatty acid and glucose metabolism in hypertensive rats.

The human insulin-resistance syndromes, type 2 diabetes, obesity, combined hyperlipidaemia and essential hypertension, are complex disorders whose genetic basis is unknown. The spontaneously hypertensive rat (SHR) is insulin resistant and a model of these human syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridaemia and hypertension map to a single locus on rat chromosome 4. Here we combine use of cDNA microarrays, congenic mapping and radiation hybrid (RH) mapping to identify a defective SHR gene, Cd36 (also known as Fat, as it encodes fatty acid translocase), at the peak of linkage to these QTLs. SHR Cd36 cDNA contains multiple sequence variants, caused by unequal genomic recombination of a duplicated ancestral gene. The encoded protein product is undetectable in SHR adipocyte plasma membrane. Transgenic mice overexpressing Cd36 have reduced blood lipids. We conclude that Cd36 deficiency underlies insulin resistance, defective fatty acid metabolism and hypertriglyceridaemia in SHR and may be important in the pathogenesis of human insulin-resistance syndromes.

Secretory proteins from the female reproductive tract of the brushtail possum (Trichosurus vulpecula): binding to sperm and effects on sperm survival in vitro.

Previous studies have demonstrated that co-culture of brushtail possum epididymal spermatozoa with oviduct epithelial cell monolayers prolongs sperm survival and results in the re-orientation of the sperm head and tail to the T-shape (thumbtack) configuration. Transformation of sperm to thumbtack orientation is believed to be associated with marsupial sperm capacitation. Here we report that incubation in oviduct-conditioned media also significantly prolongs sperm survival and results in the transformation of sperm to the thumbtack orientation. The major objective of the current study was to examine the proteins present in the conditioned media, to determine whether any of these proteins specifically bound to sperm and the relationship between these proteins and sperm survival and thumbtack orientation. Co-culturing brushtail possum sperm with biotin-labeled proteins in conditioned media (CM) from ampulla, isthmus and uterine explants demonstrated strong binding of two proteins of molecular mass 230 and 61 kD and weak binding of nine proteins of molecular mass 200, 180, 120, 140, 55, 52, 48, 34, 30 kD to sperm within 30 min. The binding of the 61-kD protein from the conditioned media appeared specific as increasing concentrations of non-labeled oviduct proteins, but not serum proteins, inhibited the binding of labeled proteins. The binding of oviduct and uterine proteins in the conditioned media significantly prolonged sperm survival and percentage motility and also transformed a large number of sperm to a thumbtack orientation. The implication of binding of these proteins is discussed in the context of sperm survival and capacitation in this species.

Improved method of superovulation in monovulatory brushtail possums (Trichosurus vulpecula) using pregnant mares' serum gonadotrophin-luteinizing hormone.

A new superovulation regimen for the monovulatory brushtail possum (Trichosurus vulpecula) has been devised. It reduces the number of hormone treatments required and elicits a better rate of ovulation than the established pregnant mares' serum gonadotrophin (PMSG)-GnRH method. Ovarian stimulation was achieved by a single intramuscular injection of 15 iu PMSG. This treatment resulted in the recruitment and development of a large number (9-12) of Graafian follicles on the ovaries. Induction of ovulation was achieved by a single intramuscular injection of 2-10 mg pig LH or multiple intramuscular injections of GnRH, (4 x 50 micrograms, 90 min apart) given 72 h after PMSG treatment. Superovulation occurred in all animals (n = 48) examined 48 h after treatment irrespective of dose of LH or type of ovulatory stimulus used. The highest ovulatory success (83%), the maximum number of ovulation sites (9.5 +/- 2.8) and the highest number of oocytes recovered (9.0 +/- 2.5) were achieved after treatment with PMSG and 4 mg LH. These results were significantly greater than the ovulatory success (43%), numbers of ovulation sites (3.9 +/- 1.1) and number of oocytes recovered (2.1 +/- 0.9) after PMSG-GnRH treatment (P < 0.05). This simpler and more effective superovulation protocol should assist with more effective manipulation of possum reproduction in captivity.

Successful fertilization after superovulation and laparoscopic intrauterine insemination of the brushtail possum, Trichosurus vulpecula, and tammar wallaby, Macropus eugenii.

Fertilization has been achieved in superovulated brushtail possums and tammar wallabies after laparoscopic intrauterine artificial insemination. Various superovulation protocols and insemination times were examined but a maximum of 2-5 eggs including 1-2 embryos per possum were recovered. The female possums were superovulated by treatment with 15 iu pregnant mares' serum gonadotrophin and then either GnRH (4 x 50 micrograms, at intervals of 90 min) or 4 mg LH, 3 days later. Inseminations were performed within 6 h before or 4-10 h after (pregnant mares' serum gonadotrophin-GnRH group only) the expected onset of ovulation using epididymal spermatozoa. Superovulation in wallabies was achieved by treatment with FSH (8 x 6 mg, at intervals of 12 h for 4 days) followed by 4 mg LH on day 5. Inseminations were performed 4-6 h before the expected onset of ovulation using ejaculated spermatozoa, which resulted in the recovery of 7-8 eggs including 3-4 embryos per female. All embryos recovered were from possums and wallabies examined 1-2 days after insemination and included fertilized eggs, two-cell and four-cell embryos. Motile spermatozoa were recovered from the oviducts and uteri but only immotile spermatozoa were found in the vaginal complex. Five to thirty per cent of spermatozoa recovered from the oviducts of possums examined 2-6 h after insemination had thumbtack morphology, which is thought to be correlated with capacitation. Although embryo yields per female were low, this study has established that intrauterine artificial insemination after superovulation is a feasible assisted breeding strategy for marsupials with implications for species conservation and population control.

Seasonal variation in ovarian response to pregnant mares serum gonadotrophin in the brushtail possum (Trichosurus vulpecula).

This study examined the seasonal variation in ovarian response to the exogenous hormone pregnant mares serum gonadotrophin (PMSG) in the brushtail possum (Trichosurus vulpecula). Ovarian stimulation was achieved by administration of a single intramuscular injection of 15 IU PMSG. Animals (n = 165) responded to this treatment in a variable manner depending on the month of the year. The maximum response (measured by the total number of large (>2 mm) follicles) was 15.8 +/- 1.5 follicles per animal (n = 12) in May and the minimum response was 4.9 +/- 1.6 follicles per animal (n = 7) in January. The response throughout the rest of the year closely paralleled the birth seasonal distribution observed in wild possums in published reports. In the summer months (December, January and February) when few females carried pouch young, 55%, 42% and 17% of PMSG-primed animals, respectively, exhibited a nil response to PMSG (i.e. no follicles >2 mm were found on the ovaries of treated animals). In addition, when wild possums were in lactational anoestrus after the autumnal breeding peak, possums with a nil response to PMSG accounted for 17% (July) and 6% (August) of those treated, but the other animals gave near maximal numbers of large follicles (July, 5-21; August, 2-25). Whether the ovaries of non-responders are downregulated in some manner outside the breeding season or they become insensitive to gonadotrophin remains to be investigated. These observations pinpoint times of the year when the highest productivity can be expected following PMSG treatment, and together with information on the timing of ovulation provide critical data for the development of artificial breeding strategies for the possum.

A retrospective analysis of the in-vitro development of 'spare' human in-vitro fertilization preimplantation embryos using 'in-house' prepared medium and 'Medi-Cult' commercial medium.

In-house prepared medium was used routinely in our in-vitro fertilization (IVF) facility prior to the introduction of the commercial 'Medi-Cult' products. A comparative study of the in-vitro development of embryos cultured in two [T6 and Earle's balanced salt solution (EBSS)] human-inactivated serum (HIS)-supplemented media from days 0 to 5 showed that 44.7% (46/103) of the embryos developed to the blastocyst stage in the T6 medium compared with 22.3% (23/103) in EBSS. Following the introduction of the commercial Medi-Cult IVF M2 medium, which is used routinely to culture fertilized eggs from days 0 to 2, new baseline data were required for the in-vitro development of 'spare' embryos from days 2 to 5. When Medi-Cult M3 medium was used, 35.6% (37/104) of the 'spare' day 2 embryos achieved the blastocyst stage. However, if morphologically similar (four normal nucleated blastomeres with no fragmentation) day 2 embryos were selected, an increase in the blastocyst rate to 50.0% (33/66) was achieved. This compared favourably with the 45.0% blastocyst rate (published in the Medi-Cult literature) for M2/M3 medium cultured human embryos. A small series of experiments with T6 + HIS medium and human serum albumin (HSA)-supplemented Ham's F-10, MCDB 302 and M3 media was undertaken to identify a suitable medium which could be used for the culture of M2 medium day 2 embryos. Results show that M2 medium cultured embryos placed in Ham's F-10 medium supplemented with 10 mg/ml HSA gave an acceptable 37.8% (14/45) blastocyst rate. Therefore, this medium could be substituted for M3 medium in an emergency. A total of 483 IVF embryos donated by patients, which were surplus to the therapeutic IVF programme, were used for these studies over a period of 30 months. Late day 2 IVF spare embryos were assigned an embryo score based on a high-power phase-contrast microscopic examination prior to being placed in culture. The embryo score provides an effective in-vitro parameter with which embryos from different patients can be compared. The cleavage and development of individual embryos were monitored on days 2 to 5. In some cases, the continuing normal development and viability of the day 5 cultured embryo were assessed by monitoring the hatching, attachment and outgrowth of the cavitated blastocyst.

Genetic diagnosis using polymerase chain reaction and fluorescent in-situ hybridization analysis of biopsied cells from both the cleavage and blastocyst stages of individual cultured human preimplantation embryos.

Cultured human preimplantation embryos have been used to develop methods which allow preimplantation genetic diagnosis (PGD) analyses by polymerase chain reaction (PCR) and fluorescent in-situ hybridization (FISH) on biopsied blastomeres and trophectoderm cells from the same embryo. An experimental design is described and experiments undertaken, which demonstrate the feasibility of extending biopsy and PGD procedures currently in use. We have shown that dual-stage biopsies are possible, and that the PCR and FISH analyses of the biopsied cell samples are effective. One to two blastomeres were biopsied from an 8- to 10-cell embryo and processed for the simultaneous PCR amplification of a beta-globin and a cytosine adenine (CA) repeat sequence, or a Y chromosome sequence. FISH procedures were also used to detect the presence of Y chromosome markers. The biopsied cleavage-stage embryo can be cultured to the blastocyst stage, where the serial biopsy of three to five mural trophectoderm cells provides two further cell samples. These can be used to repeat and/or undertake additional PGD analyses. The biopsied blastocyst is either used to confirm earlier diagnoses, or placed in culture for a further 4-24 h. Maintenance of a blastocoele cavity, hatching and formation of an outgrowth demonstrates continuing viability following the dual-stage biopsy procedures. The PCR DNA amplification procedures are effective at the cellular level for both biopsied blastomeres and mural trophectoderm cells. The FISH techniques have shown a definitive Y signal in 50% (one out of two) and 100% (two out of two) of the biopsied blastomeres and 72% (two out of three, four out of five and 7 out of 10) for the trophectoderm cell nuclei. Preliminary experiments have demonstrated that the FISH preparations can be re-amplified to improve the signal, and dual fluorescent procedures using the X and Y probes are effective. A retrospective PCR analysis has also been undertaken on preparations of biopsied cells which were previously used for PGD analysis by FISH.

Biopsy of the human blastocyst and polymerase chain reaction (PCR) amplification of the beta-globin gene and a dinucleotide repeat motif from 2-6 trophectoderm cells.

Cultured human blastocysts have been biopsied on day 5-6 post insemination and 2-6 extra-embryonic cells from the trophectoderm were removed and their DNA subjected to specific amplification by the polymerase chain reaction (PCR). Simultaneous amplification of part of the beta-globin gene and a dinucleotide repeat sequence has been achieved in a high percentage of cases when using the DNA from both trophectoderm cell biopsies and biopsied blastocysts as template for the PCR. A similar success rate was achieved when serial biopsies were taken from the same blastocyst, thus allowing one cell sample to be held in reserve for use should equivocal results be obtained. Over the entire experimental period (5 months), no contamination was experienced with biopsy or PCR procedures. Following biopsy of the trophectoderm cells all blastocysts had reformed a blastocoele cavity within 3-4 h of the biopsy procedure. Those blastocysts remaining in culture after this time showed a high incidence (78-83%) of hatching and outgrowth.