M. C. T. Penedo

Spermatogonial Stem Cell Transplantation into Rhesus Testes Regenerates Spermatogenesis Producing Functional Sperm

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout a mans life and may have application for treating some cases of male infertility, including those caused by chemotherapy before puberty. We performed autologous and allogeneic SSC transplantations into the testes of 18 adult and 5 prepubertal recipient macaques that were rendered infertile with alkylating chemotherapy. After autologous transplant, the donor genotype from lentivirus-marked SSCs was evident in the ejaculated sperm of 9/12 adult and 3/5 prepubertal recipients after they reached maturity. Allogeneic transplant led to donor-recipient chimerism in sperm from 2/6 adult recipients. Ejaculated sperm from one recipient transplanted with allogeneic donor SSCs were injected into 85 rhesus oocytes via intracytoplasmic sperm injection. Eighty-one oocytes were fertilized, producing embryos ranging from four-cell to blastocyst with donor paternal origin confirmed in 7/81 embryos. This demonstration of functional donor spermatogenesis following SSC transplantation in primates is an important milestone for informed clinical translation.

Generation of Chimeric Rhesus Monkeys

Totipotent cells in early embryos are progenitors of all stem cells and are capable of developing into a whole organism, including extraembryonic tissues such as placenta. Pluripotent cells in the inner cell mass (ICM) are the descendants of totipotent cells and can differentiate into any cell type of a body except extraembryonic tissues. The ability to contribute to chimeric animals upon reintroduction into host embryos is the key feature of murine totipotent and pluripotent cells. Here, we demonstrate that rhesus monkey embryonic stem cells (ESCs) and isolated ICMs fail to incorporate into host embryos and develop into chimeras. However, chimeric offspring were produced following aggregation of totipotent cells of the four-cell embryos. These results provide insights into the species-specific nature of primate embryos and suggest that a chimera assay using pluripotent cells may not be feasible.

Origins and genetic diversity of New World Creole cattle: inferences from mitochondrial and Y chromosome polymorphisms

The ancestry of New World cattle was investigated through the analysis of mitochondrial and Y chromosome variation in Creoles from Argentina, Brazil, Mexico, Paraguay and the United States of America. Breeds that influenced the Creoles, such as Iberian native, British and Zebu, were also studied. Creoles showed high mtDNA diversity (H = 0.984 +/- 0.003) with a total of 78 haplotypes, and the European T3 matriline was the most common (72.1%). The African T1a haplogroup was detected (14.6%), as well as the ancestral African-derived AA matriline (11.9%), which was absent in the Iberian breeds. Genetic proximity among Creoles, Iberian and Atlantic Islands breeds was inferred through their sharing of mtDNA haplotypes. Y-haplotype diversity in Creoles was high (H = 0.779 +/- 0.019), with several Y1, Y2 and Y3 haplotypes represented. Iberian patrilines in Creoles were more difficult to infer and were reflected by the presence of H3Y1 and H6Y2. Y-haplotypes confirmed crossbreeding with British cattle, mainly of Hereford with Pampa Chaqueño and Texas Longhorn. Male-mediated Bos indicus introgression into Creoles was found in all populations, except Argentino1 (herd book registered) and Pampa Chaqueño. The detection of the distinct H22Y3 patriline with the INRA189-90 allele in Caracú suggests introduction of bulls directly from West Africa. Further studies of Spanish and African breeds are necessary to elucidate the origins of Creole cattle, and determine the exact source of their African lineages.

An MHC-defined primate model reveals significant rejection of bone marrow after mixed-chimerism induction despite full MHC matching

In murine models, mixed hematopoietic chimerism induction leads to robust immune tolerance. However, translation to primates and to patients has been difficult. In this study, we used a novel MHC‐defined rhesus macaque model to examine the impact of MHC matching on the stability of costimulation blockade‐/sirolimus‐mediated chimerism, and to probe possible mechanisms of bone marrow rejection after nonmyeloablative transplant. Using busulfan‐based pretransplant preparation and maintenance immunosuppression with sirolimus, as well as CD28 and CD154 blockade, all recipients demonstrated donor engraftment after transplant. However, the mixed chimerism that resulted was compartmentalized, with recipients demonstrating significantly higher whole blood chimerism compared to T cell chimerism. Thus, the vast majority of T cells presenting posttransplant were recipient—rather than donor‐derived. Surprisingly, even in MHC‐matched transplants, rejection of donor hematopoiesis predominated after immunosuppression withdrawal. Weaning of immunosuppression was associated with a surge of antigen‐experienced T cells, and transplant rejection was associated with the acquisition of donor‐directed T cell alloreactivity. These results suggest that a reservoir of alloreactive cells was present despite prior costimulation blockade and sirolimus, and that the postimmunosuppression lymphocytic rebound may have lead to a phenotypic shift in these recipient T cells towards an activated, antigen‐experienced phenotype, and ultimately, to transplant rejection.

Worldwide frequency distribution of the ‘Gait keeper’ mutation in the DMRT3 gene

For centuries, domestic horses have represented an important means of transport and served as working and companion animals. Although their role in transportation is less important today, many horse breeds are still subject to intense selection based on their pattern of locomotion. A striking example of such a selected trait is the ability of a horse to perform additional gaits other than the common walk, trot and gallop. Those could be four-beat ambling gaits, which are particularly smooth and comfortable for the rider, or pace, used mainly in racing. Gaited horse breeds occur around the globe, suggesting that gaitedness is an old trait, selected for in many breeds. A recent study discovered that a nonsense mutation in DMRT3 has a major impact on gaitedness in horses and is present at a high frequency in gaited breeds and in horses bred for harness racing. Here, we report a study of the worldwide distribution of this mutation. We genotyped 4396 horses representing 141 horse breeds for the DMRT3 stop mutation. More than half (2749) of these horses also were genotyped for a SNP situated 32 kb upstream of the DMRT3 nonsense mutation because these two SNPs are in very strong linkage disequilibrium. We show that the DMRT3 mutation is present in 68 of the 141 genotyped horse breeds at a frequency ranging from 1% to 100%. We also show that the mutation is not limited to a geographical area, but is found worldwide. The breeds with a high frequency of the stop mutation (>50%) are either classified as gaited or bred for harness racing.

Genetic characterization of Latin-American Creole cattle using microsatellite markers

Genetic diversity in and relationships among 26 Creole cattle breeds from 10 American countries were assessed using 19 microsatellites. Heterozygosities, F-statistics estimates, genetic distances, multivariate analyses and assignment tests were performed. The levels of within-breed diversity detected in Creole cattle were considerable and higher than those previously reported for European breeds, but similar to those found in other Latin American breeds. Differences among breeds accounted for 8.4% of the total genetic variability. Most breeds clustered separately when the number of pre-defined populations was 21 (the most probable K value), with the exception of some closely related breeds that shared the same cluster and others that were admixed. Despite the high genetic diversity detected, significant inbreeding was also observed within some breeds, and heterozygote excess was detected in others. These results indicate that Creoles represent important reservoirs of cattle genetic diversity and that appropriate conservation measures should be implemented for these native breeds in order to minimize inbreeding and uncontrolled crossbreeding.

Genetic diversity, structure, and breed relationships in Iberian cattle1

In Iberia there are 51 officially recognized cattle breeds of which 15 are found in Portugal and 38 in Spain. We present here a comprehensive analysis of the genetic diversity and structure of Iberian cattle. Forty of these breeds were genotyped with 19 highly polymorphic microsatellite markers. Asturiana de los Valles displayed the greatest allelic diversity and Mallorquina the least. Unbiased heterozygosity values ranged from 0.596 to 0.787. The network based on Reynolds distances was star-shaped with few pairs of interrelated breeds and a clear cluster of 4 breeds (Alistana/Arouquesa/Marinhoa/Mirandesa). The analysis of the genetic structure of Iberian cattle indicated that the most probable number of population clusters included in the study would be 36. Distance results were supported by the STRUCTURE software indicating a relatively recent origin or possible crossbreeding or both between pairs or small groups of breeds. Five clusters included 2 different breeds (Betizu/Pirenaica, Morucha/Avileña, Parda de Montaña/Bruna de los Pirineos, Barrosã/Cachena, and Toro de Lidia/Brava de Lide), 3 breeds (Berrenda en Negro, Negra Andaluza, and Mertolenga) were divided in 2 independent clusters each, and 2 breeds were considered admixed (Asturiana de los Valles and Berrenda en Colorado). Individual assignation to breeds was not possible in the 2 admixed breeds and the pair Parda de Montaña/Bruna de los Pirineos. The relationship between Iberian cattle reflects their geographical origin rather than their morphotypes. Exceptions to this geographic clustering are most probably a consequence of crossbreeding with foreign breeds. The relative genetic isolation within their geographical origin, the consequent genetic drift, the adaptation to specific environment and production systems, and the influence of African and European cattle have contributed to the current genetic status of Iberian cattle, which are grouped according to their geographical origin. The greater degree of admixture observed in some breeds should be taken into account before using molecular markers for genetic assignment of individuals to breeds.

Novel variants in the KIT and PAX3 genes in horses with white-spotted coat colour phenotypes.

Variants in the EDNRB, KIT, MITF, PAX3 and TRPM1 genes are known to cause white spotting phenotypes in horses, which can range from the common white markings up to completely white horses. In this study, we investigated these candidate genes in 169 horses with white spotting phenotypes not explained by the previously described variants. We identified a novel missense variant, PAX3:p.Pro32Arg, in Appaloosa horses with a splashed white phenotype in addition to their leopard complex spotting patterns. We also found three novel variants in the KIT gene. The splice site variant c.1346+1G>A occurred in a Swiss Warmblood horse with a pronounced depigmentation phenotype. The missense variant p.Tyr441Cys was present in several part-bred Arabians with sabino-like depigmentation phenotypes. Finally, we provide evidence suggesting that the common and widely distributed KIT:p.Arg682His variant has a very subtle white-increasing effect, which is much less pronounced than the effect of the other described KIT variants. We termed the new KIT variants W18-W20 to provide a simple and unambiguous nomenclature for future genetic testing applications.

Genetic characterization of specific pathogen‐free rhesus macaque (Macaca mulatta) populations at the California National Primate Research Center (CNPRC)

A study based on 14 STRs was conducted to understand intergenerational genetic changes that have occurred within the California National Primate Research Centers (CNPRC) regular specific pathogen‐free (SPF) and super‐SPF captive rhesus macaque populations relative to their conventional founders. Intergenerational genetic drift has caused age cohorts of each study population, especially within the conventional population, to become increasingly differentiated from each other and from their founders. Although there is still only minimal stratification between the conventional population and either of the two SPF populations, separate derivation of the regular and super‐SPF animals from their conventional founders has caused the two SPF populations to remain marginally different from each other. The regular SPF and, especially, the super‐SPF populations have been influenced by the effects of differential ancestry, sampling, and lost rare alleles, causing a substantial degree of genetic divergence between these subpopulations. The country of origin of founders is the principal determinant of the MHC haplotype composition of the SPF stocks at the CNPRC. Selection of SPF colony breeders bearing desired genotypes of Mamu‐A*01 or ‐B*01 has not affected the overall genetic heterogeneity of the conventional and the SPF research stocks.

Estimated prevalence of the Type 1 Polysaccharide Storage Myopathy mutation in selected North American and European breeds.

The GYS1 gene mutation that is causative of Type 1 Polysaccharide Storage Myopathy (PSSM) has been identified in more than 20 breeds of horses. However, the GYS1 mutation frequency or Type 1 PSSM prevalence within any given breed is unknown. The purpose of this study was to determine the frequency of the GYS1 mutation and prevalence of genetic susceptibility to Type 1 PSSM in selected breeds from Europe and North America. The GYS1 mutation was detected in 11 breeds, including, in order of increasing allele frequency, Shires, Morgans, Appaloosas, Quarter Horses, Paints, Exmoor Ponies, Saxon-Thuringian Coldbloods, South German Coldbloods, Belgians, Rhenish German Coldbloods and Percherons. The prevalence of genetic susceptibility to Type 1 PSSM in these breeds varied from 0.5% to 62.4%. The GYS1 mutation was not found in the sampled Thoroughbreds, Akhal-Tekes, Connemaras, Clydesdales, Norwegian Fjords, Welsh Ponies, Icelandics, Schleswig Coldbloods or Hanoverians, but failure to detect the mutation does not guarantee its absence. This knowledge will help breed associations determine whether they should screen for the GYS1 mutation and will alert veterinarians to a possible differential diagnosis for muscle pain, rhabdomyolysis or gait abnormalities.