Damien B.B.P. Paris

Clinical and genetic study of Friedreich ataxia in an Australian population

Friedreich ataxia is an autosomal recessive disorder caused by mutations in the FRDA gene that encodes a 210-amino acid protein called frataxin. An expansion of a GAA trinucleotide repeat in intron 1 of the gene is present in more than 95% of mutant alleles. Of the 83 people we studied who have mutations in FRDA, 78 are homozygous for an expanded GAA repeat; the other five patients have an expansion in one allele and a point mutation in the other. Here we present a detailed clinical and genetic study of a subset of 51 patients homozygous for an expansion of the GAA repeat. We found a correlation between the size of the smaller of the two expanded alleles and age at onset, age into wheelchair, scoliosis, impaired vibration sense, and the presence of foot deformity. There was no significant correlation between the size of the smaller allele and cardiomyopathy, diabetes mellitus, loss of proprioception, or bladder symptoms. The larger allele size correlated with bladder symptoms and the presence of foot deformity. The duration of disease is correlated with wheelchair use and the presence of diabetes, scoliosis, bladder symptoms and impaired proprioception, and vibration sense but no other complications studied.

The correlation of clinical phenotype in Friedreich ataxia with the site of point mutations in the FRDA gene

ABSTRACTMost cases of Friedreich ataxia (FRDA) are due to expansions of a GAA trinucleotide repeat sequence in the FRDA gene coding for frataxin, a protein of poorly understood function which may regulate mitochondrial iron transport. However, between 1% and 5% of mutations are single base changes in the sequence of the FRDA gene, causing missense, nonsense, or splicing mutations. We describe three new mutations, IVS4nt2 (T to G), R165C, and L182F, which occur in patients in association with GAA expansions. These cases, and a further five reported cases of point mutations causing FRDA, demonstrate that splicing, nonsense, or initiation codon mutations (which cause a complete absence of functional frataxin) are associated with a severe phenotype. Missense mutations, even in highly evolutionally conserved amino acids, may cause a mild or severe phenotype.

Equine embryos and embryonic stem cells: Defining reliable markers of pluripotency

Cartilage and tendon injuries are a significant source of animal wastage and financial loss within the horse-racing industry. Moreover, both cartilage and tendon have limited intrinsic capacity for self-repair, and the functionally inferior tissue produced within a lesion may reduce performance and increase the risk of reinjury. Stem cells offer tremendous potential for accelerating and improving tissue healing, and adult mesenchymal stem cells (MSCs) are already used to treat cartilage and tendon injuries in horses. However, MSCs are scarce in the bone marrow isolates used, have limited potential for proliferation and differentiation in vitro, and do not appear to noticeably improve long-term functional repair. Embryonic stem cells (ESCs) or induced pluripotent stem (iPS) cells could overcome many of the limitations and be used to generate tissues of value for equine regenerative medicine. To date, six lines of putative ESCs have been described in the horse. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit pluripotency in vivo. Moreover, it is becoming clear that the markers used to characterize the putative ESCs were inadequate, primarily because studies in domestic species have revealed that they are not specific to ESCs or the pluripotent inner cell mass, but also because the function of most in the maintenance of pluripotency is not known. Future derivation and validation of equine embryonic or other pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing horse embryo.

Sperm DNA analysis in a Friedreich ataxia premutation carrier suggests both meiotic and mitotic expansion in the FRDA gene.

Friedreich ataxia is usually caused by an expansion of a GAA trinucleotide repeat in intron 1 of the FRDA gene. Occasionally, a fully expanded allele has been found to arise from a premutation of 100 or less triplet repeats. We have examined the sperm DNA of a premutation carrier. This mans leucocyte DNA showed one normal allele and one allele of approximately 100 repeats. His sperm showed an expanded allele in a tight range centering on a size of approximately 320 trinucleotide repeats. His affected son has repeat sizes of 1040 and 540. These data suggest that expansion occurs in two stages, the first during meiosis followed by a second mitotic expansion. We also show that in all informative carrier father to affected child transmissions, with the notable exception of the premutation carrier, the expansion size decreases.

Birth of Pouch Young after Artificial Insemination in the Tammar Wallaby (Macropus eugenii)

Abstract Timing of artificial insemination (AI) in marsupials is critical because fertilization must occur before mucin coats the oocyte during passage through the oviduct. In this study, timing and the site of insemination were examined to develop AI in the tammar wallaby (Macropus eugenii). Birth and postpartum (p.p.) estrus was synchronized in 46 females. Epididymal spermatozoa (n = 4) or semen collected by electroejaculation (n = 42) were inseminated early (4–21 h p.p.) into the urogenital sinus (n = 7), the anterior vaginal culs de sac (n = 7), the uterus by transcervical catheter (n = 5), or the uterus by injection (intrauterine artificial insemination, IUAI) (n = 5). A further 16 females were inseminated late (19–48 h p.p.) by IUAI. All females were monitored for birth. A third group of six females was inseminated late (21–54 h p.p.) by IUAI and 0.4–6.6 h later, sperm had reached the oviduct in all animals. In total, an oocyte to which spermatozoa were attached was recovered and two young were born after IUAI using epididymal (n = 1) or electroejaculated (n = 2) spermatozoa, but no young resulted from insemination at other sites. Two females were successfully inseminated at 43 and 47 h p.p., later than most other animals, and the third was inseminated much earlier (18 h p.p.) but with highly motile spermatozoa. These young represent the first macropodids born by AI and the first marsupials conceived using epididymal spermatozoa.

Artificial insemination in marsupials

Assisted breeding technology (ART), including artificial insemination (AI), has the potential to advance the conservation and welfare of marsupials. Many of the challenges facing AI and ART for marsupials are shared with other wild species. However, the marsupial mode of reproduction and development also poses unique challenges and opportunities. For the vast majority of marsupials, there is a dearth of knowledge regarding basic reproductive biology to guide an AI strategy. For threatened or endangered species, only the most basic reproductive information is available in most cases, if at all. Artificial insemination has been used to produce viable young in two marsupial species, the koala and tammar wallaby. However, in these species the timing of ovulation can be predicted with considerably more confidence than in any other marsupial. In a limited number of other marsupials, such precise timing of ovulation has only been achieved using hormonal treatment leading to conception but not live young. A unique marsupial ART strategy which has been shown to have promise is cross-fostering; the transfer of pouch young of a threatened species to the pouches of foster mothers of a common related species as a means to increase productivity. For the foreseeable future, except for a few highly iconic or well studied species, there is unlikely to be sufficient reproductive information on which to base AI. However, if more generic approaches can be developed; such as ICSI (to generate embryos) and female synchronization (to provide oocyte donors or embryo recipients), then the prospects for broader application of AI/ART to marsupials are promising.

Friedreich's ataxia presenting as adult-onset spastic paraparesis.

ABSTRACTWe have studied a man with an atypical form of Friedreichs ataxia (FRDA), who presented at age 26 years with a 2-year history of unsteadiness and clumsiness. The predominant feature of his initial neurological examination was a spastic paraparesis, along with a mild distal weakness and hyperreflexia of the upper limbs. He also displayed limb ataxia. Frataxin GAA repeat sizes were 1040/690. This unusual FRDA presentation is not dissimilar to that of Acadian spastic ataxia.

Maternal age and in vitro culture affect mitochondrial number and function in equine oocytes and embryos

Advanced maternal age and in vitro embryo production (IVP) predispose to pregnancy loss in horses. We investigated whether mare age and IVP were associated with alterations in mitochondrial (mt) DNA copy number or function that could compromise oocyte and embryo development. Effects of mare age (<12 vs ≥12 years) on mtDNA copy number, ATP content and expression of genes involved in mitochondrial replication (mitochondrial transcription factor (TFAM), mtDNA polymerase γ subunit B (mtPOLB) and mitochondrial single-stranded DNA-binding protein (SSB)), energy production (ATP synthase-coupling factor 6, mitochondrial-like (ATP-synth_F6)) and oxygen free radical scavenging (glutathione peroxidase 3 (GPX3)) were investigated in oocytes before and after in vitro maturation (IVM), and in early embryos. Expression of TFAM, mtPOLB and ATP-synth-F6 declined after IVM (P<0.05). However, maternal age did not affect oocyte ATP content or expression of genes involved in mitochondrial replication or function. Day 7 embryos from mares ≥12 years had fewer mtDNA copies (P=0.01) and lower mtDNA:total DNA ratios (P<0.01) than embryos from younger mares, indicating an effect not simply due to lower cell number. Day 8 IVP embryos had similar mtDNA copy numbers to Day 7 in vivo embryos, but higher mtPOLB (P=0.013) and a tendency to reduced GPX3 expression (P=0.09). The lower mtDNA number in embryos from older mares may compromise development, but could be an effect rather than cause of developmental retardation. The general down-regulation of genes involved in mitochondrial replication and function after IVM may compromise resulting embryos.

Sperm transport, size of the seminal plug and the timing of ovulation after natural mating in the female tammar wallaby Macropus eugenii

The distribution of spermatozoa and seminal plug in the reproductive tract and the timing of ovulation were examined at various times in a naturally mated monovular macropodid marsupial, namely the tammar wallaby (Macropus eugenii). After the first post partum (p.p.) mating, 28 females were isolated and their reproductive tracts dissected at 0.5, 6, 18, 36 and 40 h post coitum (p.c.). Each tract was ligated into 13 major anatomical sections and spermatozoa and eggs were recovered by flushing. Mating was possibly delayed by handling and occurred 21.7 +/- 2.5 h p.p. in these animals. Copulation lasted 7.8 +/- 0.7 min. Within 0.5 h after a single mating, the tract contained 25.8 +/- 10.2 x 10(6) spermatozoa and 21.6 +/- 8.8 g of seminal plug, 96% and 70% of which was lost within 6 h p.c. respectively. Spermatozoa reached the uterus, isthmus and ampulla of the oviduct on the side of the developing follicle within 0.5, 6 and 18 h p.c., respectively, and a uterine population of 26.1 +/- 12.10(3) spermatozoa was maintained for over 40 h. Sperm numbers were reduced at the cervix (up to 57-fold) and uterotubule junction (eight-fold) and only one in approximately 7500 ejaculated spermatozoa (3.4 +/- 0.9 x 10(3)) reached the oviduct on the follicle side. Differential transport of spermatozoa was not observed. Although the numbers of spermatozoa were reduced in the parturient uterus, they were highly variable and were not significantly different to those in the non-parturient uterus. Ovulation and recovery of sperm-covered eggs from the isthmus occurred 36-41 h p.c. (49-72 h p.p.). In contrast with the polyovular dasyurid and didelphid marsupials, the tammar wallaby ejaculates large numbers of spermatozoa, but transport is relatively inefficient and sperm storage in the tract before ovulation is limited.

Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.