scholarly journals The knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs

2019 ◽  
Author(s):  
Stefanie Kurtz ◽  
Andrea Lucas-Hahn ◽  
Brigitte Schlegelberger ◽  
Gudrun Göhring ◽  
Heiner Niemann ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Mammalian Species ◽  
High Mobility ◽  
Digital Pcr ◽  
Sex Reversal ◽  
Female Genitalia ◽  
Large Animal Model ◽  
Large Animal ◽  
Sry Gene ◽  
Male Sex

1AbstractThe sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development. Mutations within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species such as mice and rabbits. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular and morphological level. In our study, we successfully generated a knockout of the porcine SRY gene by microinjection of two clustered regularly interspaced short palindromic repeats (CRISPR) – associated protein - 9 nuclease (Cas9) ribonucleoprotein (RNP) complexes targeting the centrally located “high mobility group” (HMG) domain of the SRY gene. Mutations within this region resulted in the development of complete external and internal female genitalia in genetically male pigs. The internal female genitalia including uteri, ovaries, and oviducts, revealed substantial size differences in 9-months old SRY-knockout pigs compared to age-matched female wild type controls. In contrast, a deletion within the 5’ flanking region of the HMG domain was not associated with sex reversal. Results of this study demonstrates for the first time the central role of the HMG domain of the SRY gene in male sex determination in pigs. Moreover, quantitative analysis by digital PCR revealed evidence for a duplication of the porcine SRY locus. Our results pave the way towards the generation of boars exclusively producing phenotypically female offspring to avoid surgical castration without anesthesia in piglets. Moreover, the study establishes a large animal model that is much more similar to humans in regard of physiology and anatomy and pivotal for longitudinal studies.

scholarly journals Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs

2020 ◽  
Vol 118 (2) ◽  
pp. e2008743118
Author(s):  
Stefanie Kurtz ◽  
Andrea Lucas-Hahn ◽  
Brigitte Schlegelberger ◽  
Gudrun Göhring ◽  
Heiner Niemann ◽  
...  
Keyword(s):  
Sex Determination ◽  
Mutational Analysis ◽  
Mammalian Species ◽  
Digital Pcr ◽  
Sex Reversal ◽  
Large Animal Model ◽  
Pcr Analysis ◽  
Large Animal ◽  
Sry Gene ◽  
Sex Development

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located “high mobility group” (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.

scholarly journals Investigation of intersexual polled goats

2015 ◽  
pp. 11-15
Author(s):  
Judit Bordán ◽  
András Kovács ◽  
Szilárd Bodó
Keyword(s):  
Sex Ratio ◽  
Mammalian Species ◽  
Sex Reversal ◽  
Ovary Development ◽  
X Chromosomes ◽  
Ovary Cells ◽  
Male Sex ◽  
Sox9 Gene ◽  
Foxl2 Gene

The sex reversal may occur in all mammalian species, but is connected to a favourable trait – the polledness – only in the goat. Later abnormal sex ratio was noticed in these goat populations, in which a part of the phenotypically male individuals was sterile. These males have two X chromosomes. In goats the PIS (Polled Intersex Syndrome) mutation is responsible for the absence of horns in homozygous and heterozygous individuals. This same mutation causes a female-to-male sex reversal, but only in the homozygous polled genetic female goats. The PIS mutation inhibits the expression of the FOXL2 gene which is responsible for ovary development, and a protein encoded by this gene inhibits the activity of the Sox9 gene. The Sox9 gene stimulates the development of the cells of the testis. When the FOXL2 gene is inhibited, the Sox9 gene is activated and transforms the ovary cells into testis cells. In our article we briefly introduce the morphological and chromosome investigations of three intersex individuals we found.

5 DISRUPTION OF THE HIGH MOBILITY GROUP AT-HOOK 2 (HMGA2) GENE IN SWINE REDUCES POSTNATAL GROWTH

2014 ◽  
Vol 26 (1) ◽  
pp. 117 ◽  
Author(s):  
J. Chung ◽  
X. Zhang ◽  
B. Colins ◽  
K. Howard ◽  
S. Simpson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mutant Cell ◽  
High Mobility ◽  
Postnatal Growth ◽  
High Mobility Group ◽  
Large Animal Model ◽  
Large Animal ◽  
Wild Type ◽  
Adult Growth ◽  
Fetal Fibroblasts

The high mobility group AT-hook 2 (HMGA2) protein has been shown to be a crucial gene for cell growth, proliferation, and apoptosis; HMGA2 is also a strong biological candidate for growth, because mutations in this gene alter body size in mice and humans. Compared with wild-type controls, adult mice lacking HMGA2 are 60% smaller, and adult heterozygous mutants are 20% smaller. In humans, HMGA2 has been associated with adult and childhood height without any other deleterious effect. Additionally, a microdeletion in the HMGA2 gene in a human patient resulted in short stature, with no dysmorphologies and normal puberty. In order to determine the effect of HMGA2 on fetal and adult growth in pigs, a transgenic pig line deficient in HMGA2 expression was generated by gene targeting in fetal fibroblasts (FF). Using a targeting vector carrying a reporter gene, and homology arms specific to HMGA2, heterozygous mutant cell lines were generated. The cell lines were then used to generate 6 heterozygous females by somatic cell nuclear transfer (SCNT). Bodyweights and lengths from snout to base of tail were measured every 2 weeks for a year for mutant (n = 6) and wild-type farm gilts (n = 6). Data were analysed by one-way ANOVA. As in mice, disruption of one allele of the HMGA2 gene resulted in 25% reduction in weight (P < 0.0001) and 14% reduction in length (P < 0.0001). Early in postnatal growth (2 months), weights of mutants were not different than wild-type. However, mutants were 20 to 35% lighter (P < 0.05) during mid stages (6 months) and 25 to 30% (P < 0.0001) in late stages (3 months). The same insertional mutation generated 8 heterozygous male clones by SCNT. In addition, 7 nontransgenic males from the same FF line were generated as SCNT controls. Bodyweights and lengths were measured every 2 weeks for 30 weeks for HMGA2 heterozygous mutants (n = 8), control SCNT (n = 7) and wild-type farm boars (n = 5). The weight curve of boars showed similar pattern as for mutant gilts. At 30-week postnatal stage, mutants were 17% (P < 0.05) and 16% (P < 0.05) lighter in weight compared with littermate and wild-type animals, respectively. We are presently developing homozygous HMGA2 mutant lines. Currently, 3 of 6 heterozygous gilts have been bred with heterozygous boars, with 1 confirmed pregnancy. The expectation is that the homozygous animals will, like mice, be 60% smaller than the wild-type animals. The approach described here will result not only in a valuable large-animal model of dwarfism, but also in a tool to reduce the size of existing transgenic and nontransgenic swine lines. This, in turn, will increase the receptivity of valuable transgenic lines by the biomedical community. Funding for this work was provided by NIH grant R21-OD010553 to JP.

282 IDENTIFICATION OF SRY AND STEROIDOGENIC FACTOR-1 (SF1) GENES IN CANINE XY MALE-TO-FEMALE SEX DEVELOPMENTAL DISORDER

2011 ◽  
Vol 23 (1) ◽  
pp. 239
Author(s):  
G. H. Jang ◽  
Y. H. Jeong ◽  
I. S. Hwang ◽  
Y. W. Jeong ◽  
S. H. Hyun ◽  
...  
Keyword(s):  
Sex Determination ◽  
Cultured Cells ◽  
Mammalian Species ◽  
Gene Mutations ◽  
Sex Reversal ◽  
Normal Donor ◽  
Sry Gene ◽  
German Shepherd ◽  
Steroidogenic Factor 1 ◽  
Wide Range

Cloning by the process of somatic cell nuclear transfer (SCNT) has been achieved in a variety of mammalian species and has many promising applications. In this process, however, due to reasons beyond current scientific understanding, many results contrary to expectation have also been produced. For instance, abnormal sex development such as demasculinization has been observed in 1 of 6 healthy German shepherd offspring produced with SCNT (1 normal donor (not cloned), 5 cloned but normally developed progenies, 1 cloned sex reversed progeny, and 1 recloned sex reversed progeny from 1 cloned sex reversed progeny). Sex-determining region Y (SRY) is one of the most basic and crucial genes that initiate male sex determination in many mammals. Steroidogenic factor-1 (SF1, NR5A1), which is closely related to SRY, also regulates several genes involved in sex determination. Numerous studies have reported that reduced or deleted SRY gene expression as well as SF1 gene mutations can produced XY sex reversal. To verify the hypothetical association between phenotypic disorder of sex determination and genetic modification by SCNT, we extracted genomic DNA from tissues of normal progeny (not cloned), primary cultured cells of cloned but normally developed progeny, cloned sex reversed progeny, and recloned sex reversed progeny at the age of 1 year and carried out PCR with produced primers based on available SRY and SF1 gene information (SRY gene from AF107021 in GenBank; SF1 gene from ENSCAFG00000023086 in Ensembl). The cloned PCR products were subcloned into T-vector for sequence analysis, which showed no mutation in genetic sequences of SRY and SF1. Taken together, in our case of abnormal sex determination, there was no apparent association between phenotypic sex determination disorder and SRY/SF1 gene mutation. Other sex reversal and related mutation studies have pointed to a wide range of signal networks that include Sox9 and so forth. Further studies should be focused on these other genes in the signal network.

scholarly journals A rare case of male sex reversal syndrome (46, XX) with negative SRY gene: a disorder of sexual differentiation (DSD)

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Mohamed Ahmed Abd El Salam ◽  
Noha Hassan Ibrahim ◽  
Nehad Nabil Eskarous
Keyword(s):  
Rare Case ◽  
Clinical Presentation ◽  
Ambiguous Genitalia ◽  
Sex Reversal ◽  
Normal Male ◽  
Testosterone Replacement Therapy ◽  
Obstructive Azoospermia ◽  
Sry Gene ◽  
Case Presentation ◽  
Male Sex

Abstract Background Male sex reversal syndrome is a rare genetic cause of male infertility with an overall incidence of 1/20,000–1/100,000 males. There is mismatching between the genetic make-up and the apparent clinical features. The clinical presentation of such cases is variable ranging from ambiguous genitalia at birth, failed puberty, up to normal male phenotype with infertility and hypogonadism. The exact molecular and genetic bases of this syndrome are still unclear. Most of the recorded cases were SRY positive (i.e. representing 80–90% of all cases), and they showed translocated SRY gene on the Y chromosome. Moreover, fewer cases of male sex reversal (46, XX) were SRY negative. Case presentation Herby, we report a rare case of a 35-year-old infertile male patient who presented with azoospermia, hypergonadotropic hypogonadism, and abnormal classical (46, XX) karyotype, as well as negative FISH for SRY gene. He had a previous negative biopsy and was asking for redoing micro-TESE, whoever he was discouraged as chances to find sperm is eventually nil, and instead, he was prescribed testosterone replacement therapy to correct hypogonadism. Conclusion Therefore, any case of non-obstructive azoospermia should be offered genetic testing trying to exclude non-treatable cases and for genetic counseling.

scholarly journals CRISPR/Cas9-mediated mosaic mutation of SRY gene induces hermaphroditism in rabbits

2018 ◽  
Vol 38 (2) ◽  
Author(s):  
Yuning Song ◽  
Yuanyuan Xu ◽  
Mingming Liang ◽  
Yuxin Zhang ◽  
Mao Chen ◽  
...  
Keyword(s):  
Sex Determination ◽  
Sexual Development ◽  
Sexual Differentiation ◽  
Rabbit Model ◽  
Gonad Development ◽  
High Mobility ◽  
Testicular Tissue ◽  
Spermatogenic Cells ◽  
Sry Gene ◽  
Male Sex

Hermaphroditism is a rare disorder that affects sexual development, resulting in individuals with both male and female sexual organs. Hermaphroditism is caused by anomalies in genes regulating sex determination, gonad development, or expression of hormones and their receptors during embryonic development during sexual differentiation. SRY is a sex-determination gene on the Y chromosome that is responsible for initiating male sex determination in mammals. In this study, we introduced CRISPR/Cas9-mediated mutations in the high-mobility-group (HMG) region of the rabbit SRY. As expected, SRY-mutant chimeric rabbits were diagnosed with hermaphroditism, characterized by possessing ovotestis, testis, ovary and uterus simultaneously. Histopathology analysis revealed that the testicular tissue was immature and lacked spermatogenic cells, while the ovarian portion appeared normal and displayed follicles at different stages. This is the first report of a rabbit hermaphroditism model generated by the CRISPR/Cas9 system. This novel rabbit model could advance our understanding of the pathogenesis of hermaphroditism, and identify novel therapies for human clinical treatment of hermaphroditism.

DEVELOPMENT OF A LARGE ANIMAL MODEL OF OPIATE ADDICTION TO EVALUATE CARDIOVASCULAR FUNCTION.

Analgesia ◽  
1995 ◽  
Vol 1 (4) ◽  
pp. 598-602 ◽  
Author(s):  
L.D. Napier ◽  
Z. Mateo ◽  
D.A. Yoshishige ◽  
B.A. Barron ◽  
J.L. Caffrey
Keyword(s):  
Animal Model ◽  
Cardiovascular Function ◽  
Large Animal Model ◽  
Opiate Addiction ◽  
Large Animal
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