Transdifferentiation, Metaplasia and Tissue Regeneration

Transdifferentiation is defined as the conversion of one cell type to another. It belongs to a wider class of cell type transformations called metaplasias which also includes cases in which stem cells of one tissue type switch to a completely different stem cell. Numerous examples of transdifferentiation exist within the literature. For example, isolated striated muscle of the invertebrate jellyfish (Anthomedusae) has enormous transdifferentiation potential and even functional organs (e.g., tentacles and the feeding organ (manubrium)) can be generated in vitro. In contrast, the potential for transdifferentiation in vertebrates is much reduced, at least under normal (nonpathological) conditions. But despite these limitations, there are some well-documented cases of transdifferentiation occurring in vertebrates. For example, in the newt, the lens of the eye can be formed from the epithelial cells of the iris. Other examples of transdifferentiation include the appearance of hepatic foci in the pancreas, the development of intestinal tissue at the lower end of the oesophagus and the formation of muscle, chondrocytes and neurons from neural precursor cells. Although controversial, recent results also suggest the ability of adult stem cells from different embryological germlayers to produce differentiated cells e.g., mesodermal stem cells forming ecto- or endodermally-derived cell types. This phenomenon may constitute an example of metaplasia. The current review examines in detail some well-documented examples of transdifferentiation, speculates on the potential molecular and cellular mechanisms that underlie the switches in phenotype, together with their significance to organogenesis and regenerative medicine.Key Words: transdifferentiation, metaplasia, tissue regeneration, stem cells, plasticity, reprogramming, regenerative medicine     

Sexing fish by palpation: a simple method for gonadal assessment of fluvial salmonids

This paper presents a non‐destructive sexing method that can be employed in fluvial salmonids and presumably some other taxa of fish. The sex of a fish is identified and assessed by indirectly palpating the gonad.     

Regulation of Sex Determination in Mice by a Non-coding Genomic Region

To identify novel genomic regions that regulate sex determination, we utilized the powerful C57BL/6J-Y^POS (B6-Y^POS) model of XY sex reversal where mice with autosomes from the B6 strain and a Y chromosome from a wild-derived strain, Mus domesticus poschiavinus (Y^POS), show complete sex reversal. In B6-Y^POS, the presence of a 55-Mb congenic region on chromosome 11 protects from sex reversal in a dose-dependent manner. Using mouse genetic backcross designs and high-density SNP arrays, we narrowed the congenic region to a 1.62-Mb genomic region on chromosome 11 that confers 80% protection from B6-Y^POS sex reversal when one copy is present and complete protection when two copies are present. It was previously believed that the protective congenic region originated from the 129S1/SviMJ (129) strain. However, genomic analysis revealed that this region is not derived from 129 and most likely is derived from the semi-inbred strain POSA. We show that the small 1.62-Mb congenic region that protects against B6-Y^POS sex reversal is located within the Sox9 promoter and promotes the expression of Sox9, thereby driving testis development within the B6-Y^POS background. Through 30 years of backcrossing, this congenic region was maintained, as it promoted male sex determination and fertility despite the female-promoting B6-Y^POS genetic background. Our findings demonstrate that long-range enhancer regions are critical to developmental processes and can be used to identify the complex interplay between genome variants, epigenetics, and developmental gene regulation.     

Why boys will be boys and girls will be girls: Human sex development and its defects

Among the most defining events of an individual's life, is the development of a human embryo into male or a female. The phenotypic sex of an individual depends on the type of gonad that develops in the embryo, a process which itself is determined by the genetic setting of the individual. The development of the gonads is different from any other organ, as they possess the potential to differentiate into two functionally distinct organs, testes, or ovaries. Sex development can be divided into two distinctive processes, “sex determination,” which is the commitment of the undifferentiated gonad into either a testis or an ovary, a process that is genetically programmed in a critically timed manner and “sex differentiation,” which takes place through hormones produced by the gonads, once the developmental sex determination decision has been made. Disruption of any of the genes involved in either the testicular or ovarian development pathway could lead to disorders of sex development. In this review, we provide an insight into the factors important for sex determination, their antagonistic actions and whenever possible, references on the “prismatic” clinical cases are given. Birth Defects Research (Part C) 108:365–379, 2016. © 2016 Wiley Periodicals, Inc.     

Rapid DNA extraction and PCR-sexing of mouse embryos

We have devised a PCR-based sexing method that is quick, simple, and highly reproducible. DNA is first extracted from embryonic mouse yolk sac via a 15 min, two-step incubation procedure utilizing PCR-compatible proteinase K buffer. Without any further manipulation the lysate is subjected to 30 cycles of PCR, optimized to run in less than 1 hr. The reaction includes multiplexed primer pairs for Sry and Myog (myogenin) that generate a male specific band of 441 bp and an internal control band of 245 bp, respectively. This robust method is used routinely in our laboratory and gives rapid genotyping results with 98% reliability and 100% accuracy.     

Morphology and immunolocalization of intertubular steroidogenic cell in mesonephros of Podocnemis expansa during gonadal differentiation

Sex steroid hormones are critical in gonadal differentiation in turtles. The gonads are not the only organs responsible for producing these hormones during this phase. Mesonephros play an important role in steroidogenesis. The present study aimed to investigate the presence of steroidogenic cells in mesonephros of Podocnemis expansa during gonadal differentiation and to evaluate their morphology and ultrastructure. Ten embryos of P. expansa were collected from 5 nests on day 36 of incubation, during spawning period on an artificial beach. Embryos were extracted from eggs by slicing the shell and euthanized. They were dissected under a stereoscopic microscope to collect the gonad-mesonephro complex, in which were fixed and subsequently processed for light microscopy, immunohistochemistry and transmission electron microscopy analysis. During histological analysis was observed mesonephros has typical morphological structure. Immunohistochemistry showed immunoreaction to aromatase in cells of intertubular space. Confirming these findings, it was possible to observe a type of intertubular cell in several regions of mesonephro, being more predominant in region close to blood vessels, distal and proximal tubules. In ultrastructural analysis these cells were characterized by having a clear, large, and rounded nucleus with evident nucleolus and cytoplasm rich in electron-dense droplets. This study demonstrated for the first time the presence of cells with morphological, immunohistochemical and ultrastructural characteristics similar to steroid-producing cells in P. expansa mesonephrons, suggesting that this organ may contribute to gonadal differentiation in this species.     

哺乳动物性别决定和性反转

目前已知SRY仅是涉及性别决定过程的基因之一.近年来又发现和克隆了许多可能参与性腺分化与发育的基因,如副中肾抑制基因MIS,也称抗副中肾激素基因AMH;SRY相关基因SOX9;编码甾类因子的基因SFI;X-连锁的DAX基因;Wilm′s肿瘤抑制基因WTI;以及X-连锁的剂量敏感基因DSS等,并新建立了性别决定的Z-基因模型,DSS-基因模型和Jimenez等的模型,较合理地解释了哺乳动物性别决定的分子机理和以前难以解释的各种奇特的性反转现象,使性别决定的研究取得了长足的进展,但仍有一些悬而未决的问题有待于进一步探索.     

Prolonged use of letrozole causes morphological changes on gonads in Galea spixii

Letrozole is used as a therapeutic agent in reproductive disorders caused by high estrogen levels. Letrozole inhibits cytochrome P450 aromatase and reduces estrogen levels. However, the effects of long-term use on reproductive traits are unknown. The aim of this study was to evaluate the prolonged use of letrozole in the gonads of rodents (Spix''s yellow-toothed cavy; Galea spixii). Forty-eight rodents (24 males and 24 females) were randomly divided into the treated and control groups. Letrozole administration started at 15 days of age and continued weekly until 30, 45, 90, and 120 days of age. The body, testis, and ovary weights were analyzed, as well as the morphological progression of spermatogenesis and folliculogenesis. Macroscopically, body weight gain and gonads weight were increased in the letrozole group. Microscopically, the ovaries of treated females showed stratified epithelium and a cellular disorder of the tunica albuginea. In the testes of treated males, the development of seminiferous tubules was delayed and sperm was absent. The collective findings indicate that the prolonged use of letrozole alters secondary sexual characteristics, and causes weight gain, reproductive changes, and male infertility.     

Temperature-dependent sex determination in reptiles: Proximate mechanisms,ultimate outcomes,and practical applications

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). In TSD sex determination is an “all or none” process and intersexes are rarely formed. How is the external signal of temperature transduced into a genetic signal that determines gonadal sex and channels sexual development? Studies with the red-eared slider turtle have focused on the physiological, biochemical, and molecular cascades initiated by the temperature signal. Both male and female development are active processes—rather than the crganized/default system characteristic of vertebrates with genotypic sex determination—that require simultaneous activation and suppression of testis- and ovary-determining cascades for normal sex determination. It appears that temperature accomplishes this end by acting on genes encoaing for steroidogenic enzymes and steroid hormone receptors and modifying the endocrine microenvironment in the embryo. The temperature experienced in development also has long-term functional outcomes in addition to sex determination. Research with the leopard gecko indicates that incubation temperature as well as steroid hormones serve as organizers in shaping the adult phenotype, with temperature modulating sex hormone action in sexual differentiation. Finally, practical applications of this research have emerged for the conservation and restoration of endangered egg-laying reptiles as well as the embryonic development of reptiles as biomarkers to monitor the estrogenic effects of common environmental contaminants. © 1994 Wiley-Liss, Inc.     

Characterization of Mesonephric Cells That Migrate into the XY Gonad during Testis Differentiation

In mouse fetal gonads, sex differentiation begins at 10.5-11.5 days postcoitum (dpc). With XY gonads of 12.5 dpc, cord-like structures are visible and stromal cells migrate from adjacent mesonephros, unlike in XX gonads. However, the migrated mesonephric cells, except for the endothelial cells, have not been specifically identified because they have not expressed differentiation markers over the course of organ coculture in previous experiments. In this study, we have for the first time succeeded in isolating only the mesonephric cells that migrate into the XY gonad from the mesonephros with alive and then cultured these cells in vitro through the use of an organ coculture system using EGFP-transgenic mice and a FACS Vantage. The migrated and isolated cells were used for morphological and molecular characterization. The migrated mesonephric cells contained three cell forms; a sharp cell form, a round cell form, and a cluster-forming cell. The sharp cells have the characters of peritubular myoid cells. The round cells and cluster-forming cells have the potential to differentiate into Leydig cells, as some of them are 3beta-HSD-positive. In in vitro culture of migrated mesonephric cells, the cluster-forming cells proliferated well and then differentiated into round cells, suggesting that the cluster-forming cells may be stem or precursor cells for the round cells. Thus, our findings provide important information related to the migration and differentiation of migrated mesonephric cells in the XY gonad.     

孕牛外周血中胎儿Y-特异性序列的检测

根据公牛Sry特异性序列设计两对寡核苷酸引物。并对20份妊娠晚期母牛外周血DNA样品进行PCR扩增,结果有9份样品检测出有Sry片断(阳性),其余11份为阴性。经分娩牛犊性别验证,在20份被检测的样品中,有16份PCR检测结果与犊牛实际性别相符,其余4例不符。我们的实验结果说明,胎儿细胞可以进入到孕牛的外周血中去。     

In search of determinants: gene expression during gonadal sex differentiation

The diversity of inputs that guide sexual fate during development is both intriguing and daunting. In the field of fish biology, the study of sex determination is of great importance. For example, in aquaculture, sexually dimorphic growth rates and overall size leads to one sex being more marketable than the other. Moreover, for breeding purposes it is important to maintain balanced sex ratios. Furthermore, sex determination is sensitive to environmental factors, such as temperature and contaminants, which can lead to skewed sex ratios, intersexes and sterility in wild or farmed fish. The gonad is typically the first organ to exhibit morphological signs of sexual dimorphism and therefore is likely to be the primary organ system whose fate is controlled by the sex determination cues in many fish species. Additionally, the sexual fate of the gonad has been shown to fully or partially control organismal sex differentiation. Thus, understanding the genetic regulation of gonadal sex differentiation is critical in studies of fish sex determination. This review summarizes recent knowledge of genes expressed during gonadal sex differentiation in gonochoristic teleost fish. Three species are discussed, which serve as excellent model systems for probing teleost sex differentiation: the Oreochromis niloticus, Oryzias latipes and Danio rerio. The similarities and differences between gonadal gene expression in these three species and in comparison to mammals suggest conserved roles during vertebrate gonadal sex differentiation. In the future, it will be essential to develop tools to assay the function of genes expressed during gonadal sex differentiation in fish.     

Asymmetry in sexual development of gonads in intersex rainbow trout

Large‐scale sampling of spontaneous rainbow trout Oncorhynchus mykiss intersexes indicated a strong asymmetry of gonad differentiation in XX females; the right gonad was more sensitive to the mutation‐induced masculinization than the left one.