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

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.

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DNA Methylation Reprogramming during Sex Determination and Transition in Zebrafish

10.1101/2020.09.17.301580 ◽

2020 ◽

Author(s):

Xinxin Wang ◽

Xin Ma ◽

Gaobo Wei ◽

Weirui Ma ◽

Zhen Zhang ◽

...

Keyword(s):

Dna Methylation ◽

Sex Determination ◽

Sexual Development ◽

Epigenetic Modification ◽

Sex Differentiation ◽

Primordial Germ Cells ◽

Regulation Of Gene Expression ◽

Adult Zebrafish ◽

Germline Development ◽

Male Sex

AbstractIt is a mystery about sex determination and sexual plasticity in species without sex chromosomes. DNA methylation is a prevalent epigenetic modification in vertebrates, which has been shown to involve in the regulation of gene expression and embryo development. However, it remains unclear about how DNA methylation regulates sexual development. To elucidate it, we used zebrafish to investigate DNA methylation reprogramming during juvenile germ cell development and adult female-to-male sex transition. We revealed that primordial germ cells (PGCs) undergo significant DNA methylation reprogramming during germline development and set to an oocyte/ovary-like pattern at 9 days post fertilization (9 dpf). When blocking DNMTs activity in juveniles after 9 dpf, the zebrafish preferably develops into females. We also show that Tet3 involves in PGC development. Notably, we find that DNA methylome reprogramming during adult zebrafish sex transition is similar to the reprogramming during the sex differentiation from 9 dpf PGCs to sperm. Furthermore, inhibiting DNMTs activity can prevent the female-to-male sex transition, suggesting that methylation reprogramming is required for zebrafish sex transition. In summary, DNA methylation plays important roles in zebrafish germline development and sexual plasticity.

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MALE SEX DETERMINATION: Current Concepts of Male Sexual Differentiation

Annual Review of Medicine ◽

10.1146/annurev.med.45.1.505 ◽

1994 ◽

Vol 45 (1) ◽

pp. 505-524 ◽

Cited By ~ 32

Author(s):

Michael L. Gustafson, M.D ◽

Patricia K. Donahoe, M.D

Keyword(s):

Sex Determination ◽

Sexual Differentiation ◽

Male Sex

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

10.1101/617589 ◽

2019 ◽

Cited By ~ 1

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.

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Putative aromatase inhibitor induces male sex determination in a female unisexual lizard and in a turtle with temperature-dependent sex determination

Journal of Endocrinology ◽

10.1677/joe.0.1410295 ◽

1994 ◽

Vol 141 (2) ◽

pp. 295-299 ◽

Cited By ~ 120

Author(s):

T Wibbels ◽

D Crews

Keyword(s):

Sex Determination ◽

Aromatase Inhibitor ◽

Sexual Differentiation ◽

Trachemys Scripta ◽

Aromatase Activity ◽

Temperature Dependent ◽

Endogenous Production ◽

Diverse Species ◽

Male Sex ◽

Cnemidophorus Uniparens

Abstract Treatment of developing embryos of two diverse species of reptiles with fadrozole (a potent and specific nonsteroidal inhibitor of aromatase activity in mammals) resulted in the induction of male sex determination. In the first experiment, males were produced in an all-female parthenogenic species of lizard (Cnemidophorus uniparens). In the second experiment, male sex determination was induced in a turtle (Trachemys scripta) with temperature-dependent sex determination. The results support the hypothesis that the endogenous production of oestrogen may represent a pivotal step in the sex determination cascade of reptiles. Further, the production of male C uniparens indicates that the genes required for male sexual differentiation have not been lost in this parthenogenic lizard. Journal of Endocrinology (1994) 141, 295–299

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Genetics of sex determination: what can we learn from Drosophila?

Development ◽

10.1242/dev.101.supplement.17 ◽

1987 ◽

Vol 101 (Supplement) ◽

pp. 17-24

Author(s):

Rolf Nöthiger ◽

Monica Steinmann-Zwicky

Keyword(s):

Molecular Biology ◽

Sex Determination ◽

Genetic Control ◽

Sexual Development ◽

Sexual Differentiation ◽

Regulatory Elements ◽

Negative Control ◽

Homeotic Genes ◽

Specific Expression ◽

X Chromosomes

The combined efforts of genetics, developmental and molecular biology have revealed the principles of genetic control of sexual differentiation in Drosophila. In combination with maternal components, a quantitative chromosomal signal, provided by the ratio of X chromosomes to sets of autosomes (X: A), regulates a key gene (Sxl). The functional state, ON or OFF, of Sxl, via a few subordinate regulatory genes, controls a switch gene (dsx) that can express two mutually exclusive functions, M or F. These serve to repress either the female or the male set of differentiation genes, thus directing the cells either into the male or into the female sexual pathway. Investigations of control genes and their regulation show that they have properties of homeotic genes. Their role is to select one of two alternative developmental programs. Their function, or lack of function, is required throughout development to maintain the cells in their respective sexual pathway. Differentiation genes are under negative control by dsx. We discuss the cis- and tams-regulatory elements that are needed for sex-, tissue- and stage-specific expression of the differentiation genes. A comparison of Drosophila to other organisms such as Caenorhabditis, mammals and other insects indicates similarities that we interpret as evidence for a basically invariant genetic strategy used by various organisms to regulate sexual development.

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Mab-3 is a direct tra-1 target gene regulating diverse aspects of C. elegans male sexual development and behavior

Development ◽

10.1242/dev.127.20.4469 ◽

2000 ◽

Vol 127 (20) ◽

pp. 4469-4480 ◽

Cited By ~ 3

Author(s):

W. Yi ◽

J.M. Ross ◽

D. Zarkower

Keyword(s):

Transcription Factor ◽

Sex Determination ◽

Sexual Development ◽

Sexual Differentiation ◽

Sense Organ ◽

Yolk Protein ◽

Bhlh Transcription Factor ◽

Organ Differentiation ◽

And Behavior ◽

Protein Transcription

Sex determination is controlled by global regulatory genes, such as tra-1 in Caenorhabditis elegans, Sex lethal in Drosophila, or Sry in mammals. How these genes coordinate sexual differentiation throughout the body is a key unanswered question. tra-1 encodes a zinc finger transcription factor, TRA-1A, that regulates, directly or indirectly, all genes required for sexual development. mab-3 (male abnormal 3), acts downstream of tra-1 and is known to be required for sexual differentiation of at least two tissues. mab-3 directly regulates yolk protein transcription in the intestine and specifies male sense organ differentiation in the nervous system. It encodes a transcription factor related to the products of the Drosophila sexual regulator doublesex (dsx), which also regulates yolk protein transcription and male sense-organ differentiation. The similarities between mab-3 and dsx led us to suggest that some aspects of sex determination may be evolutionarily conserved. Here we find that mab-3 is also required for expression of male-specific genes in sensory neurons of the head and tail and for male interaction with hermaphrodites. These roles in male development and behavior suggest further functional similarity to dsx. In male sensory ray differentiation we find that MAB-3 acts synergistically with LIN-32, a neurogenic bHLH transcription factor. Expression of LIN-32 is spatially restricted by the combined action of the Hox gene mab-5 and the hairy homolog lin-22, while MAB-3 is expressed throughout the lateral hypodermis. Finally, we find that mab-3 transcription is directly regulated in the intestine by TRA-1A, providing a molecular link between the global regulatory pathway and terminal sexual differentiation.

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Ontogeny and cellular localization of SRY transcripts in the human testes and its detection in spermatozoa

Reproduction ◽

10.1530/rep.1.00413 ◽

2005 ◽

Vol 130 (5) ◽

pp. 603-613 ◽

Cited By ~ 32

Author(s):

D Modi ◽

C Shah ◽

G Sachdeva ◽

S Gadkar ◽

D Bhartiya ◽

...

Keyword(s):

Sex Determination ◽

Sertoli Cells ◽

Cellular Localization ◽

Reproductive Physiology ◽

Spermatogenic Cells ◽

Rt Pcr ◽

Sry Gene ◽

Single Transcript ◽

Determining Factor

The sex-determining region on the Y (SRY) gene is unequivocally designated as the testis-determining factor in mammals; however, its roles beyond sex determination, if any, have been hitherto unknown. To determine whether SRY has any roles beyond sex determination, herein the expression of SRY mRNA was investigated in the midtrimester human fetal, infantile and adult testes as well as in ejaculated spermatozoa. High levels of SRY transcripts werein situlocalized to the Sertoli cells of the developing testis at 9 weeks of gestation, and the expression persisted at comparable levels throughout the midtrimester (until 22 weeks) and also in the testis of an infant at 3 months of age. The germ cells and other somatic cells in the testes of fetuses and the infant were negative for SRY expression. The mRNA for SRY was detected in the spermatogenic cells, particularly the spermatogonia and the round spermatids; the expression was negligible in the meiotic stages. A single transcript of ~1.2 kb was detected in the adult testes and isolated spermatogonial cells. In the adult testis,in situhybridization (ISH) studies revealed a switch in the cellular localization of SRY transcripts. SRY transcripts were also demonstrable by RT-PCR of RNA from ejaculated human spermatozoa. ISH revealed the presence of SRY transcripts in the midpiece of 50% of ejaculated sperm. These results suggest that SRY may have extensive roles in male reproductive physiology, such as maturation of fetal testis, spermatogenesis, sperm maturation and early embryonic development.

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