D.A.F. Villagómez

Derivation and Characterization of Canine Embryonic Stem Cell Lines with In Vitro and In Vivo Differentiation Potential

Embryonic stem cells (ESCs) represent permanent cell lines that can be maintained in an undifferentiated state. In an environment that induces differentiation, they form derivatives of the three embryonic germ layers: mesoderm, ectoderm, and endoderm. These characteristics give ESCs great potential for both basic research and clinical applications in the areas of regenerative medicine and tissue engineering. The establishment of ESCs from large animals that model human diseases is of significant importance. We describe the derivation of permanent canine cell lines from preimplantation‐stage embryos. Similar to human ESCs, canine ESCs expressed OCT3/4, NANOG, SOX2, SSEA‐3, SSEA‐4, TRA‐1–60, TRA‐1–81, and alkaline phosphatase, whereas they expressed very low levels of SSEA‐1. They maintained a normal karyotype and morphology typical of undifferentiated ESCs after multiple in vitro passages and rounds of cryopreservation. Plating cells in the absence of a feeder layer, either in attachment or suspension culture, resulted in the formation of embryoid bodies and their differentiation to multiple cell types. In vivo, canine ESCs gave rise to teratomas comprising cell types of all three embryonic germ layers. These cells represent the first pluripotent canine ESC lines with both in vitro and in vivo differentiation potential and offer the exciting possibility of testing the efficacy and safety of ESC‐based therapies in large animal models of human disease. STEM CELLS 2009;27:329–340

Classical and Molecular Cytogenetics of Disorders of Sex Development in Domestic Animals

The association of abnormal chromosome constitutions and disorders of sex development in domestic animals has been recorded since the beginnings of conventional cytogenetic analysis. Deviated karyotypes consisting of abnormal sex chromosome sets (e.g. aneuploidy) and/or the coexistence of cells with different sex chromosome constitutions (e.g. mosaicism or chimerism) in an individual seem to be the main causes of anomalies of sex determination and sex differentiation. Molecular cytogenetics and genetics have increased our understanding of these pathologies, where human and mouse models have provided a substantial amount of knowledge, leading to the discovery of a number of genes implicated in mammalian sex determination and differentiation. Additionally, other genes, which appeared to be involved in ovary differentiation, have been found by investigations in domestic species such as the goat. In this paper, we present an overview of the biology of mammalian sex development as a scientific background for better understanding the body of knowledge of the clinical cytogenetics of disorders of sex development in domestic animals. An attempt to summarize of what has been described in that particular subject of veterinary medicine for each of the main mammalian domestic species is presented here.

Characterization of canine embryonic stem cell lines derived from different niche microenvironments.

Embryo-derived stem cells hold enormous potential for producing cell-based transplantation therapies, allowing high-throughput drug screening and delineating early embryonic development. However, potential clinical applications must first be tested for safety and efficacy in preclinical animal models. Due to physiological and genetic parity to humans, the domestic dog is widely used as a clinically relevant animal model for cardiovascular, neurodegenerative, orthopedic, and oncologic diseases. Therefore, we established numerous putative canine embryonic stem cell (cESC) lines by immunodissection of the inner cell mass (ICM), which we termed OVC.ID.1-23, and by explant outgrowths from whole canine blastocysts, named OVC.EX.1-16. All characterized lines were immunopositive for OCT4, SOX2, NANOG, SSEA-3, and SSEA-4; displayed high telomerase and alkaline phosphatase (ALP) activities; and were maintained in this state up to 37 passages ( approximately 160 days). Colonies from OVC.EX lines showed classic domed hESC-like morphology surrounded by a ring of fibroblast-like cells, whereas all OVC.ID lines exhibited a mixed cell colony of tightly packed cESCs surrounded by a GATA6+/CDX2- hypoblast-derived support layer. Spontaneous serum-only differentiation without feeder layers demonstrated a strong lineage selection associated with the colony niche type, and not the isolation method. Upon differentiation, cESC lines formed embryoid bodies (EB) comprised of cells representative of all germinal layers, and differentiated into cell types of each layer. Canine ESC lines such as these have the potential to identify differences between embryonic stem cell line derivations, and to develop or to test cell-based transplantation therapies in the dog before attempting human clinical trials.

Analysis of Oocyte-Like Cells Differentiated from Porcine Fetal Skin-Derived Stem Cells

We previously reported the differentiation of cells derived from porcine female fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cells expressed germ cell markers and formed aggregates resembling cumulus-oocyte complexes. Some of these aggregates extruded large oocyte-like cells (OLCs) that expressed markers consistent with those of oocytes. The objective of the current study was to further characterize OLCs differentiated from porcine skin-derived stem cells. Reverse transcriptase (RT)-polymerase chain reaction and Western blot revealed the expression of connexin37 and connexin43, both of which are characteristic of ovarian follicles. The expression of meiosis markers DMC1 and synaptonemal complex protein, but not STRA8 and REC8, was detected in the OLC cultures. Immunofluorescence with an antibody against synaptonemal complex protein on chromosome spreads revealed a very small subpopulation of stained OLCs that had a similar pattern to leptotene, zytotene, or pachytene nuclei during prophase I of meiosis. Sodium bisulfite sequencing of the differentially methylated region of H19 indicated that this region is almost completely demethylated in OLCs, similar to in vivo-derived oocytes. We also investigated the differentiation potential of male skin-derived stem cells in the same differentiation medium. Large cells with oocyte morphology were generated in the male stem cell differentiation cultures. These OLCs expressed oocyte genes such as octamer-binding transcription factor 4 (OCT4), growth differentiation factor-9b (GDF9B), deleted in azoospermia-like (DAZL), VASA, zona pellucida B (ZPB), and zona pellucida C (ZPC). It was concluded that skin-derived stem cells from both male and female porcine fetuses are capable of entering an oocyte differentiation pathway, but the culture system currently in place is inadequate to support the complete development of competent oocytes.

Chromosomal abnormalities, meiotic behavior and fertility in domestic animals

Since the advent of the surface microspreading technique for synaptonemal complex analysis, increasing interest in describing the synapsis patterns of chromosome abnormalities associated with fertility of domestic animals has been noticed during the past three decades. In spite of the number of scientific reports describing the occurrence of structural chromosome abnormalities, their meiotic behavior and gametic products, little is known in domestic animal species about the functional effects of such chromosome aberrations in the germ cell line of carriers. However, some interesting facts gained from recent and previous studies on the meiotic behavior of chromosome abnormalities of domestic animals permit us to discuss, in the frame of recent knowledge emerging from mouse and human investigations, the possible mechanism implicated in the well known association between meiotic disruption and chromosome pairing failure. New cytogenetic techniques, based on molecular and immunofluorescent analyses, are allowing a better description of meiotic processes, including gamete production. The present communication reviews the knowledge of the meiotic consequences of chromosome abnormalities in domestic animals.

Equine Disorders of Sexual Development in 17 Mares Including XX, SRY-Negative, XY, SRY-Negative and XY, SRY-Positive Genotypes

We described the clinical, cytogenetic and molecular findings of 17 clinical equine cases presented for abnormal sexual development and infertility. Six horses with an enlarged clitoris had an XX, SRY-negative genotype, which displayed male-like behavior (adult individuals). Bilateral ovotestes were noted in 2 of those cases, while another case showed increased levels of circulating testosterone. Six horses with a female phenotype, including normal external genitalia, had an XY, SRY-negative genotype. These individuals had small gonads and an underdeveloped internal reproductive tract. Four horses with normal appearing external genitalia had an XY, SRY-positive genotype, 3 of them had hypoplastic testes and male-like behavior. In addition, one young filly with enlarged clitoris and hypoplastic testes had the same genotype but did not show male-like behavior due to her age. Three of these horses were related with 2 being siblings. These findings demonstrate the diversity of disorders of sexual development seen in the horse. Furthermore, they emphasize the need for further research to identify genes involved in abnormal sex determination and differentiation in the horse.

Synaptonemal complex analysis of a three-breakpoint translocation in a subfertile bull

Somatic chromosome analysis of a subfertile Brown Swiss bull demonstrated a three-breakpoint translocation involving chromosomes 1, 8, and 9 in G- and R-banded karyotypes. Based on standard bovine chromosome nomenclature, the translocation was defined as t(1;8;9)(q43;q13;q26). Synaptonemal complex analysis of the chromosome aberration by electron microscopy revealed a hexavalent configuration in 52 of 53 pachytene cells. Twenty-seven cells (51%) had a completely paired hexavalent configuration showing distinctly nonhomologous pairings between normal and/or translocated chromosomes involved in the exchanges. Thirteen cells showed a hexavalent configuration with centrally unpaired chromosome segments but with completely paired terminal arms. In 13 cells (including one at zygotene) the translocation chromosomes formed an open hexavalent, and in one cell there were two completely paired trivalents. Thirty-two cells at diakinesis-MI demonstrated 28 configurations, including one large hexavalent. Testicular histology, testis size, and seminal characteristics were normal.

Synaptonemal complex analysis of a reciprocal translocation, rcp(20;24) (q17;q25), in a subfertile bull

A reciprocal translocation was identified in a subfertile artificial insemination bull. Somatic chromosome investigation of G-banded metaphases demonstrated a 60,XY,rcp(20;24)(q17;q25) karyotype for the carrier. Synaptonemal complex analysis of the translocation by electron microscopy revealed an irregular pairing behavior of the chromosome axes involved, which resulted in a variety of configurations at pachytene. Not only was the expected quadrivalent configuration present, but also a trivalent plus univalent and two heteromorphic bivalents. Most common was an incompletely or completely paired quadrivalent configuration, which was non-homologously paired. XY association with the multivalent was seen only rarely. Histological analysis of testicular tissue showed meiotic arrest in some tubules. However, the semen picture was normal.

GTG Mutation in the Start Codon of the Androgen Receptor Gene in a Family of Horses with 64,XY Disorder of Sex Development

Genetic sex in mammals is determined by the sex chromosomal composition of the zygote. The X and Y chromosomes are responsible for numerous factors that must work in close concert for the proper development of a healthy sexual phenotype. The role of androgens in case of XY chromosomal constitution is crucial for normal male sex differentiation. The intracellular androgenic action is mediated by the androgen receptor (AR), and its impaired function leads to a myriad of syndromes with severe clinical consequences, most notably androgen insensitivity syndrome and prostate cancer. In this paper, we investigated the possibility that an alteration of the equine AR gene explains a recently described familial XY, SRY + disorder of sex development. We uncovered a transition in the first nucleotide of the AR start codon (c.1A>G). To our knowledge, this represents the first causative AR mutation described in domestic animals. It is also a rarely observed mutation in eukaryotes and is unique among the >750 entries of the human androgen receptor mutation database. In addition, we found another quiet missense mutation in exon 1 (c.322C>T). Transcription of AR was confirmed by RT-PCR amplification of several exons. Translation of the full-length AR protein from the initiating GTG start codon was confirmed by Western blot using N- and C-terminal-specific antibodies. Two smaller peptides (25 and 14 amino acids long) were identified from the middle of exon 1 and across exons 5 and 6 by mass spectrometry. Based upon our experimental data and the supporting literature, it appears that the AR is expressed as a full-length protein and in a functional form, and the observed phenotype is the result of reduced AR protein expression levels.

Reproductive consequences of a reciprocal chromosomal translocation in two Duroc boars used to provide semen for artificial insemination.

Cytogenetic analysis of 58 boars at an artificial insemination (AI) centre revealed the presence of a reciprocal chromosome translocation, rcp(1;11)(q-;p+), in two Duroc boars. Pedigree analysis of these two boars suggested familial transmission of the chromosome rearrangement. The reproductive consequences of this translocation were determined in a herd of sows that had received semen doses from these and other boars. All sows underwent multiple AI, with different groups established retrospectively depending on the percentage of semen doses provided by the carrier boars ([number of carrier boar doses/total number doses provided] x 100): 0%, 25%, 50%, 75%, 100%. The fertility rates (percentage of successful multiple AIs/total multiple AIs) recorded for multiple AI including semen doses from the carrier boars were not significantly different from those recorded when all semen doses were supplied by normal-karyotype boars. A reduction in litter size of 29.38% was observed, however, in litters sired by one of the carrier boars when its participation in multiple AI was 100%. The number of live-born piglets per litter gradually decreased (P<0.05) as the percentage participation in multiple AI (25, 50, or 75%) of the carrier boar increased. In addition, both carrier boars sired some piglets with signs of cleft palate and complex malformations of the front legs; these died soon after birth. In conclusion, the boars carrying the translocation rcp(1;11)(q-;p+) showed reduced reproductive performance.