Sry on the Y-chromosome triggers the fetal gonad to begin differentiation into testis in mammals. Mutation or absence of Sry results in development of ovaries and the female phenotype. However, XY sex reversal in the presence of wild-type Sry exists in mice and man. One such example is the B6-YTIR mouse, whose autosomes and X-chromosome are of the C57BL6/J mouse (Mus musculus molossinus) whereas the Y-chromosome is from a mouse originating in Tirano, Italy (Mus musculus domesticus). B6-YTIR mice develop only ovaries or ovotestes in fetal life. The objective of my thesis was to identify the mechanism of sex reversal in the B6-YTIR mouse. The results indicate that onset of Sry transcription in B6-YTIR gonads is comparable to control B6 XY gonads. On the other hand, onset of Mis, 17alpha-HA, 3beta-HSD (testicular cell products), p450arom as well as inactivation of Sry transcription are delayed or absent in the sex reversed gonads. It has been suggested that low levels of Sry transcription may account for aberrant testis differentiation in B6-YTIR mice. We observed relatively low levels of Sry transcripts not only in B6-YTIR but also in B6 mice. However, levels in normal B6-YSJL mice were significantly greater. On the SJLB6F1 background, where no sex reversal occurs, Sry transcript levels of the TIR allele increased while those of B6 and SJL alleles remained the same as in the B6 background. Thus, low levels of Sry transcript from the B6 allele are sufficient whereas the levels from TIR and SJL alleles (both DOM type) appear to be critical for testis determination. We then compared the levels of endogenous Sry proteins. A combination of immunoprecipitation and immunoblotting succeeded in detecting a protein band whose expression profile and molecular size are consistent with those of the predicted Sry. Sry protein levels in B6-Y TIR gonads were roughly two fold greater than in B6 XY gonads. We hypothesize that the Sry protein of the TIR/SJL alleles is less efficient

To date, the majority of studies into sex determination and sexual development have focused on the mammalian system due to the mouse model being an excellent tool for developmental biology as well as obvious implications to human health and development. However, the focus on the mammalian system has caused a large fraction of other vertebrate groups to be overlooked. The last decade has seen an array of studies into various non-mammalian organisms like fishes, birds, reptiles, and amphibians. These studies have unveiled a remarkably conserved molecular background utilized for sexual differentiation amongst all vertebrates, ranging from the alligator which employs temperature dependent sex determination to the mouse, which employs genotypic sex determination In this project, I implemented molecular methods traditionally used to study model organisms, to investigate an amphibian species, the cane toad (Bufo marinus). The cane toad was chosen due to its invasive status in Australia, as well as being a representative of one of the most successful and specious families of vertebrates, Bufonidae. Since, its introduction, this species has rapidly spread across the continent, adversely effecting native species throughout its introduced range. Recent studies have identified the disruption of the sex-determination pathway as a realistic goal in an otherwise, seemingly futile, effort to curtail their expansion. We decided to approach the study of cane toad sex determination and development on three levels, in order to have a broader understanding of the similarities between toads and other groups, and additionally, to better assess the feasibility of utilizing molecular means to control cane toads in non-native habitats. Firstly, I used a candidate gene approach to clone and characterize five genes which were previously known to be involved in both mammalian as well as amphibian sex determination: Sox9, Dmrt1, p450arom, Sf1, and Dax1. I chose Sox9 and Dmrt1 due to their known involvement in the male-specific pathway of mammals. Dmrt1 was also known to be male specific in other groups, including amphibians. Conversely, I also decided to investigate p450arom and Dax1, due to their affiliation with the female pathway, while Sf1 has a role in both sexes. All of these genes were expressed in the gonads of both sexes of cane toads. However, Sox9 exhibited strong transcriptional up-regulation in testes at the time of sexual differentiation, similar to mammals. Thus, using the candidate gene approach, I was successful in identifying a sex-specific marker which could be utilized in the manipulation of the sex determination pathway (e.g. female to male sex reversal). VIIThe second approach involved the study of sex determination on a cytogenetic level. Traditionally, Bufonids have been assumed to utilize a ZZ/ZW (male homogametic/female heterogametic) sex chromosome system, yet only one recent study has been successful in the identification of sex chromosomes in a toad species. Moreover, the authors identified the sex chromosomes in only one population (of 18 studied), highlighting the scarcity of discernible sex chromosomes in toads. When we began to investigate the karyotype of cane toads, we were able to identify a female-specific length polymorphism in the nucleolus organizer region (NOR) of chromosome 7, making this chromosome pair a strong candidates for the Z and W sex chromosomes. In order to verify our hypothesis, we also performed chromomycin A3 staining to reveal a differential signal between the chromosome pair, indicative of heterochromatin accumulation on the brighter NOR. This study lent strong support to the observed difference between the chromosomes, yet we required further investigation in order to identify the W and the Z chromosomes, specifically. By utilizing comparative genomic hybridization (CGH), I was able to identify a female specific region on the chromosome with the larger NOR, identifying it as the W chromosome. This study was successful in identifying the sex chromosomes in the cane toad. Moreover, it allows us to make a more accurate prediction of the possible sex determination method utilized: either a dosage dependent male determination system, or a female-specific gene in a female determination system. The third approach focused on the Bidder’s organ and its role in sexual development of cane toads. The Bidder’s organ is a gonadal structure comprised of ovary-like tissue, found in both males and females, with no known function. Since our study was focused on female to male sex-reversal of cane toads, we decided to investigate the potential of the Bidder’s organ to interrupt this process. By utilizing the candidate genes, which I had previously cloned, I was able to identify the Bidder’s organ as having an entirely distinct transcriptional pattern in comparison to the gonads of either sex. Additionally, the Bidder’s organ showed significantly higher levels of p450arom expression than the gonads, identifying it as a possible key player in the production of aromatase enzyme for oestrogen production. Moreover, gene expression patterns in the Bidder’s organ strongly correlated with the significant developmental time points in the sexual development of the toads, implying a possible function in the development process of toads. In conclusion, I have performed the first study of the molecular, cytogenetic and anatomical aspects of sexual development in a toad. I was able to verify that cane toads utilize a ZZ/ZW chromosome system and furthermore, likely utilize the mammalian male-linked gene, Sox9, in male development. I was also able to show that the Bidder’s organ is transcriptionally active at key time points, likely indicative of a functional role during development.

Abstract Biotechnological strategies aimed at producing male-only offspring have the potential to improve the yield of the Australian beef industry. As a proof-of-concept project, I aimed to target the primary male sex-determining gene Sry to the X chromosome in mice, to produce a transgenic XY male that would transmit Sry – and hence maleness – to both XX and XY offspring. In this project I aimed to target a 14.5 kb DNA fragment containing Sry to an X-chromosome locus that escapes X-inactivation. After considering many potential loci, a targeting strategy and construct were designed for the SMCX locus, which is well conserved between mouse, human and bovine. A targeting vector with 5kb and 3kb arms of homology was also constructed without Sry, to target the locus. Attempts to introduce the 14.5 kb Sry fragment into the construct were unsuccessful, and a smaller construct, containing only the coding sequence of the Sry gene driven by a strong promoter, is currently being made. In order to translate this transgenic approach into cattle, other facets of bovine sex determination required investigation. First, it was important to identify the necessary regulatory regions upstream of bovine SRY needed for the gene to be functional, and secondly to investigate the timing of testis development in male bovine embryos. To enable sequence comparison, I sequenced upstream of the bovine and goat SRY gene and through bioinformatic analysis identified regulatory regions common to several mammals. I identified four regions of high homology upstream of bovine SRY conserved between human, goat, and pig, but not mouse. These regions are likely to be important for the regulation of the gene in these species, as they share unique transcription factor binding sites. From this research I concluded that 9 kb upstream of bovine SRY were likely to be useful in transgenic strategies to produce sex-reversed cattle. Although I attempted to use a 15 kb bovine genomic fragment containing SRY to sex reverse XX mice, this project was unsuccessful. I also investigated the expression pattern of genes known to have a role in sex determination, including SRY, in early bovine embryos. I identified the major time points important for male sex determination, including the first appearance of the gonadal ridge from the mesonephros at day 31, the onset of SRY expression and its peak at day 39, and the appearance of testis cords at day 42, along with the pattern of expression of many other genes downstream of SRY. This information will allow future researchers to check that transgenic SRY expression is occurring at the correct time and place for it to be able to cause XX sex reversal in cattle. I also identified some of the major time points important for female sex determination, including that ovigerous cords form between CRL 37-91 in female bovine embryos. In addition I show the cellular differentiation of the cortex and medulla at this time. I have also predicted the female germ cell entry into meiosis around CRL 40 in bovine embryos through the use of qRT-PCR for STRA8 and SYCP3. This is the first detailed account of gene expression profiles in early female bovine embryos, unfortunately the data is incomplete due to an uneven distribution of embryo ages due to the difficulty of obtaining embryos from timed matings. Hopefully in the future obtaining more female embryos of the missing stages can complete the female data. This project has provided additional basic knowledge about bovine sex-determination events to ensure the possibility of making single-sex livestock a real possibility in the future. The similarity between human and bovine developmental time frames also points to cattle being a good alternative model for human development, and emphasises the need for further research in species other than mouse, with the aim of ultimately understanding our own biology.

Thesis (Ph. D. biochemistry)--Washington State University, May 2008. / Includes bibliographical references.

With a large number of unidentified skeletal remains found in South Africa, the development of osteometric standards to determine sex from various bones is crucial. It is imperative that a forensic anthropologist have access to a variety of techniques both morphological and metric, which can be used to establish accurate demographic profiles from complete, fragmentary and/or commingled remains. Standards for South African populations are available for the cranium, humerus, pelvis, femur, tibia, calcaneus, and talus. No research has been done on bones of the forearm, even though they are known through international studies to exhibit sexual dimorphism. The purpose of this research was to develop discriminant function formulae to determine sex from the radius and ulna for a South African population. The sample consisted of 200 male and 200 female skeletons from the Pretoria Bone (University of Pretoria) and Raymond A. Dart (Witwatersrand University) collections. Sixteen standard anthropometric measurements were taken from the radius (9) and ulna (7) and subjected to stepwise and direct discriminant function analysis. Distal breadth, minimum midshaft diameter and maximum head diameter were the best discriminators of sex in the radius, while minimum midshaft diameter and olecranon breadth were selected for the ulna. Classification accuracy for the forearm ranged from 76% to 86%. In summary, the radius and ulna can be considered moderate discriminators for determining sex in a South African group. However, it is advised that whenever possible these formulae are used in conjunction with visual methods to determine sex from skeletal remains. Copyright 2007, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Barrier, ILO 2007, Sex determination from the bones of the forearm in a modern South African sample, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-08052008-090115 / > E961/ag / Dissertation (MSc)--University of Pretoria, 2008. / Anatomy / unrestricted

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22781号 / 農博第2424号 / 新制||農||1081(附属図書館) / 学位論文||R2||N5301(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 近藤 直, 准教授 小川 雄一, 教授 飯田 訓久 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Northern Pike (Esox lucius) is an economically and ecologically valuable species with a circumpolar distribution across the Northern Hemisphere. Northern Pike have been shown to have low levels of genetic variation despite their great capacity to colonize new environments. Here, high-resolution resequencing data from 47 Northern Pike from across North America was used for SNP discovery and population analysis. Our analysis reveals an extraordinary lack of genetic variation among Northern Pike with observed heterozygosity (Ho) of just 0.0835. Our analyses suggest that two major groups of Northern Pike exist in North America that are separated by the North American Continental Divide. Genetic variation associated with the stratification of these two groups resides across the genome particularly in gene regions with multiple copy number variants and functions related to immunity, tissue permeability, and development. Northern Pike from Alaska and the Yukon River harbour about two times more heterozygosity than Northern Pike east of the Continental Divide with an average of one heterozygous SNP every 6,250 bases. Populations east of the Continental Divide possess a remarkable level of genetic homogenization with an average of just one heterozygous SNP every 16,500 bases. For comparison, an average of one heterozygous SNP per 309 bases was reported in herring (Martinez Barrio et al., 2016), one per 500 in Atlantic cod (Star et al., 2011), and one per 750 bases in Coho and chinook salmon (Koop, 2018). This is at least 5 – 10 fold less variation than is seen in humans (the 1000 Genomes Project Consortium, 2015). We observed a recently described master sex-determining gene, amhby, in three western North American populations but not in populations east of the Continental Divide. We could not resolve any signals indicating a genetic sex determination system was present in populations from southern Manitoba or the St. Lawrence River. This may indicate that environmental sex determination is at play in these populations. We found evidence of a possible female-heterozygous, male homozygous ZW-ZZ genetic sex-determination system in New Jersey Northern Pike. With the highest average of 181,268 heterozygous SNPs genome wide and the greatest Ho (0.3228) of all populations, as well as the presence of the sex-determining gene amhby indicate that Northern Pike from our Alaskan population are the oldest in North America. Fewer numbers of heterozygous SNPs (61,073), low Ho (0.0922), and the absence of amhby in Northern Pike east of the Continental Divide suggests that these are relatively young populations and are descended from a small founding population. These results imply that Northern Pike first came to North America through Beringia and colonized its North American range from there, possibly via pro-glacial lake formation and drainage. However, from the data herein it was not possible to trace how re-colonization occurred after the final retreat of glaciers at the end of the last ice age. This thesis provides a genetically high-resolution snapshot of Northern Pike population structure in North America. It demonstrates that organisms with largely homogenous genomes can be incredibly successful and resilient. Finally, it adds to the complex subject of sex determination in fish and provides insight into a sex determination system in transition. / Graduate / 2020-10-15

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