The remodeling of the central nervous system (CNS) during metamorphosis in Drosophila melanogaster is a prime model system in which to study the genetic control of the sexual dimorphisms in the abdominal ganglion of the CNS. I have been using a P[tau-lacZ] enhancer trap line, 4.078, to label a segmentally repeated subset of abdominal motorneurons in order to assess the function of the sex determination hierarchy in controlling sex-specific development of the adult nervous system. In both the male and female larva there are 8 sets of these labeled abdominal motorneurons but only six sets in males and five sets in females survive in the adult. When this P[tau-lacZ] reporter construct is placed into a doublesex (dsx) mutant background, all 8 sets of these labeled abdominal motorneurons survive in both male and female adults. These results strongly suggest that dsx plays a role in the sex-specific survival of larval neurons that have functions in the adult. During the construction of mutant strains containing the sex determining genes transformer (tra) and transformer-2 (tra2), a genetic interactor was discovered in the P[tau-lacZ] 4.078 line. Female flies heterozygous for either tra or tra-2 alleles and the P[tau-lacZ] 4.078 developed with masculinized external and internal sex-specific structures. The external sex-specific structures, such as the genitalia, and ventral muscles are dependent on dsx gene function and a dorsal sex-specific muscle is dependent on fruitless (fru) gene function. From standard genetic crosses, I have characterized and demonstrated that the genetic interaction is linked to the P-element insertion site, which maps to the 85-87 region on the right arm of the third chromosome. By genetic analysis, this new genetic interactor appears to interfere with the tra and tra2 regulated female specific functions of both dsx and fru, potentially by reducing the female-specific splicing of the primary transcripts of the genes dsx and fru. To test the possibility that this newly described genetic interactor was allelic to a known gene, B52, that maps to the same region of the chromosome and alters dsx splicing, complementation tests were conducted which showed that the P[tau-lacZ] is not allelic B52. Additional phenotypes were observed in the crosses that first detected the interaction, suggesting that this newly described locus may affect other gene functions as well. Among the phenotypes observed were XX intersexes, male-female gynandromorphs (XX//XO mosaics), and non-disjunction events evident as XO males and XXY females. This new locus may represent a new member of the family of genes that influence regulated splicing events. / Graduation date: 1999

In most mammals, sexual fate is determining genetically by the presence of the SRY gene which encoded the testis-determining factors on the Y chromosome. Likewise, avian sex is determined genetically. At day 3.5 (stage 22; HH) in chicken embryogenesis, the gonadal primordium begins forming. Thus, to identify the novel sex-determinating genes in early chicken embryos, subtractive cDNA libraries from male-minus-female (M-F) and female-minus-male (F-M) of 3 Dpc. embryos were established. Both collected male and female chicken total mRNAs were purified using Dynabeads. After a blund-end restriction endonuclease Rsa I digestion of cDNA, adaptor ligation for tester cDNA was performed. When first and second cDNA hybridization was finished, those nonredundant cDNA between tester and driver will be amplified by two rounds of PCR. Subsequently, TA-cloning was performed and the cDNA fragments were PCR-amplified using M13 primers. PCR products of Clones were first screened by differential screening hybridization to decrease false positive inserts. Then, gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA). Finally, 40 known and 71 novel transcripts of M-F cDNA library, 88 known and 128 novel transcripts of F-M cDNA library were identified. In M-F subtracted library, 4 identified known genes were located on Z sex chromosome such as WD repeat domain 36 (WDR36), PC4 and SFRS1 interacting protein 1 (PSIP1), serum response factor binding protein 1 (SRFBP1) and glycine dehydrogenase (decarboxylating) (GLDC). Another two identified known genes, laminin alpha 1 (LAMA1) and leukocyte cell derived chemotaxin 1 (LECT1) were reported be relate to cell differentiation and development. In F-M subtracted library, only Wpkic-8 was located on W sex chromosome. Other identified genes like slowmo homolog 2 (Drosophila) (SLMO2), collagen, type IV, alpha 1 (COL4A1), anterior gradient 2 homolog (Xenopus laevis), transcript variant 2 (AGR2), solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 6 (SLC25A6) and prolyl endopeptidase (PREP) were also found expressed higer in human ovary then testis. PREP was proposed that it may play a role in mediating sperm death by regulating the levels of thyrotropin-releasing hormone analogs and in mediating sperm death associated with necrozoospermia. These transcripts located on W or Z sex chromosome identified from subtracted libraries may play an important role in sex determination mechanism.

To produce male Muscovy only for fatty liver and meat-type production is an important economic goal in animal husbandry, although the sex-determining mechanism in poultry remains to be elucidated. Manipulation of sex-determining gene(s) in poultry provides enormous opportunities on the development of sex pre-selection reproductive systems. DSX and MAB-3 genes in Drosophila and C. elegans are conserved across the human, mice, chickens, fish, turtles, and reptiles revealing an ancient sex-determining locus DMRT1. Thus the Z-linked, DMRT1 in chicken is an excellent candidate regulatory gene controlling similar aspects of sexual development in poultry. This dissertation is aimed to clone and characterize Muscovy DMRT1 gene for further application in sex pre-selection. Partial cDNA sequences of Muscovy DMRT1 was determined and revealed 95% identity and 83% with chicken and red-eared slider turtle DMRT1 cDNA sequences. DMRT1 orthologs among various species were analyzed by Phylip program and phylogenetic tree was constructed by MEGA2 programs. Results indicated that Muscovy, chicken and red-eared slider turtle DMRT1 revealing 95%, and 83% identity at cDNA and 61%, 54% identity at amino acid level.

Two hypotheses currently exist regarding to the determining factors for sexual development and differentiation in birds. One is based on the unbalanced sex chromosome, meaning that avian sex determination is dominated by ¡§Z-chromosome dosage¡¨. The other brings up (reconsider this) the key factor of ¡§W chromosome¡¨ which is a particular sex chromosome in female birds (ZW). In the previous studies, we constructed a female-subtract-male cDNA library before morphological gonad differentiation. After sequencing and annotation, a total of 279 expression sequence taqs (ESTs) were identified, with potentially higher expression levels in females. By utilizing quantitative RT-PCR, 16 potential ESTs and three marker transcripts (HINT1, FET1 and WDR36), which identified to be involved in sexual development at 3, 5, 7, 9 days post-coitum (dpc) was analyzed in chicken embryos. Results indicated that AGR2, CPT2, DUSP19, HINTW, LOC771368 and EY53070791 had higher expression levels in female than in male embryos at 3 and 5 dpc; FET1 expression level in female embryos gradually increased from 3 to 9 dpc. Moreover, both HINT1 and WDR36 were higher expressed in male than in female embryos across 3 to 9 dpc. However, HINT1 exhibited higher expression levels starting at early stage, whereas WDR36 at later stage. Next, we constructed HINT1-GFP fusion protein and overexpressed this protein in chicken B-cell line (DT40), resulting in upregulation of WDR36 expression. On the contrary, overexpressed HINTW-GFP fusion protein in DT40 cells had decreased WDR36 expression level. Moreover, we designed a small hairpin RNA by utilizing RNA interference technique to knockdown expression of HINTW, which resulted in WDR36 upregulation. Finally, we then estimated the regulation of WDR36 promoter activity through analyzing HINT1-GFP overexpression. Results had shown that HINT1-GFP can improve WDR36 promoter activity. Therefore, we suppose that HINT1 can regulate WDR36 transcription via WDR36 proximal promoter region. Ongoing HINT1 responsive element(s) must be identified to characterize whether HINT1 or HINTW regulates WDR36.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 153-164). Available also in a digital version from Dissertation Abstracts.

The leopard gecko (Eublepharis macularius) is a reptile species in which embryonic temperature contributes both to sex determination and within- sex polymorphisms. Its life history makes the leopard gecko a model system for seeking ontogenic and proximate explanations for within-sex variation in sexually dimorphic behavior and neurophysiology, necessary attributes for reproductive success. For my dissertation I have incorporated the role of androgens that potentially modulate incubation temperature effects on behavioral and brain variation, which I approached using embryo and adult leopard geckos. First, I found that that the bias of same-sex clutch siblings is primarily incubation temperature- dependent and any maternal or genetic effects on same-sex clutch siblings are secondary. Second, I found that testosterone concentrations in the yolk-albumen were higher in eggs of late development than early development at 26 °C, a female-producing incubation temperature, but did not differ from eggs incubated at another female-biased temperature. This increase in testosterone concentrations during the temperature sensitive period in putative females is a finding opposite of reported trends in most other reptiles studied to date. Further, I found that the embryonic environment influences male sociosexual investigation in the absence of gonadal hormones. Lastly, in adult males of 32.5 °C, a male-biased incubation temperature, I found that the phosphoprotein DARPP-32 that is activated by the D1 dopamine receptor in limbic brain regions is correlated to this sociosexual investigatory behavior. Neurons immunopositive for phosphorylated DARPP-32 were not only less dense in the nucleus accumbens of males who spent more time with other males, but also more dense in the preoptic area of males who spent more time with females. The use of phosphorylated DARPP-32 as marker for sociosexual exposure is novel in a lizard species. Taken together, in support of previous studies, these results show that differences in embryonic environment stem primarily from incubation temperature, can explain behavioral differences in adulthood in the absence of hormones, and, in concert with hormonal manipulation, can influence neuronal marker sensitivity to sociosexual exposure. / text

Σκοπός της έρευνας ήταν να μελετήσουμε την επίδραση της θερμοκρασίας στο ολικό μεταγράφωμα νυμφών zebrafish (Danio rerio, Hamilton 1822), θέλοντας να εστιάσουμε το ενδιαφέρον μας στη διερεύνηση μηχανισμών και μορίων που εμπλέκονται στο φυλοκαθορισμό του συγκεκριμένου ψαριού. Kατορθώσαμε να επιδράσουμε με τρεις διαφορετικές θερμοκρασίες σε μία θερμοκρασιακά ευαίσθητη οντογενετική περίοδο, η οποία οριοθετείται από την 10 έως την 20dpf. Η επιλογή των θερμοκρασιών έγινε με σημείο αναφοράς τους 28 ºC, η οποία είναι η πρότυπη θερμοκρασία ανάπτυξης των zebrafish. Η μελέτη της επίδρασης της θερμοκρασίας επιτεύχθη με διατήρηση των πειραματικών μας πληθυσμών σε θερμοκρασίες που αποκλίνουν κατά 4-6 ºC από την πρότυπη, δηλαδή στους 32 ºC και 22 ºC αντίστοιχα. Ως εκ τούτου, ολόκληρος ο πειραματικός πληθυσμός αναπτύχθηκε στη πρότυπη θερμοκρασία των 28 ºC μέχρι και την 10 dpf, όπου μετά την πάροδο της χωρίστηκε σε τρεις υπoπληθυσμούς καθένας από τους οποίους εκτέθηκε στους 22 ºC, 28 ºC ή 32 ºC αντίστοιχα για 280 θερμοημέρες (χρονική περίοδος :10-20dpf). Ακολούθησε δειγματοληψία (100 περίπου ατόμων) από κάθε υποπληθυσμό και εν συνεχεία πραγματοποιήθηκε απομόνωση ολικού RNA. Τα δείγματα μας υβριδοποιήθηκαν σε microarrays, τα οποία έφεραν αντιπροσωπευτικές αλληλουχίες για 15.500 γονίδια του zebrafish. Τα δεδομένα επεξεργάστηκαν με τη χρήση δυο εξειδικευμένων βιοπληροφορικών προγραμμάτων, τα Biosystanse και Dchip, με τη βοήθεια των οποίων συγκρίθηκαν οι εξής καταστάσεις: 10dpf _28 ºC με 20dpf _28 ºC, η οποία αποτελεί την αναπτυξιακή σύγκριση ώστε να ελέγξουμε γονίδια των οποίων η έκφραση αυξάνεται ή ελαττώνεται στα δυο αυτά στάδια αλλά και 20dpf_22 ºC με 20dpf_32 ºC, 20dpf_22 ºC με 20dpf_28 ºC και 20dpf_28 ºC με 20dpf_32 ºC, ώστε να παρατηρηθεί η επίδραση του περιβαλλοντικού παράγοντα ″θερμοκρασία″ στο μεταγράφωμα των ιχθυδίων. / The purpsose of this research was to analyse how temperature effects the total trascriptome at juvenile zebrash (Danio rerio, Hamilton 1822). So, we focus our investigation in the inspection of mechanisms and molecules which probably associated with the sex determination at this specific type of fish. We militated in the thermosensitive ontogenetic period ( from 10 to 20 days post fertilization) with three different temperatures. These three temperatures were 22, 28 and 32οC. Exemplary temperature for zebrafish is 28οC (environmental temperature). Initially, experimental population grew up to 28οC until the 10th day post fertilization. After this temporal period, population separated to three smaller experimental populations which of them grew up to 22, 28 and 32οC respectively for 280 thermodays ( chronic period: 10-20dpf). The next step was sampling (about 100 fish from each experimental population) and then isolating total RNA from each one of the samples. Total RNA samples hybridized on microarrays. Microarrays contained complementary sequences for about 15.500 genes of zebrafish genome. The data analyzed with two specific bioinformatics programs, Biosystance and Dchip. The use of these bioinformatics programs helped us comparing the following situations: 10dpf _28 ºC to 20dpf _28 ºC (developmental comparison) over and above 20dpf_22 ºC to 20dpf_32 ºC, 20dpf_22 ºC to 20dpf_28 ºC and 20dpf_28 ºC to 20dpf_32 ºC (temperature comparison).

The sex-determining gene on the Y chromosome, named Sry, initiates differentiation of gonadal somatic cells into testes, which in turn regulate the development of male phenotype. / The B6.YTIR sex-reversed mouse provides a good model for studying sex-determining mechanisms. We proposed a hypothesis that the testis-determining pathway is impaired downstream of Sry transcription in the B6.YTIR fetus. / The current study aimed to determine the hierarchical order of Sry, Sox9, Pn1, and Mis by examining their expression in B6.YTIR gonads as compared to normal B6.XY gonads by RT-PCR. / Results. Sry expression was comparable between B6.Y TIR and B6.XY gonads, with its onset between 10.5 and 11.5 dpc, a peak at 11.5 dpc, and downregulation thereafter. Sox9 expression was detectable in both B6.XX and B6.XY gonads at 11.5 dpc at comparable levels, but was then downregulated in B6.XX gonads at 12.5 dpc, by which stage testicular cord formation had began in B6.XY gonads. Pn1 was expressed in both B6.XX and B6.XY gonads at comparable levels at 11.5 dpc and was upregulated in B6.XY gonads at 12.5dpc. Mis expression in B6.Y TIR gonads was low at 10.5 and 11.5 dpc with a peak at 12.5dpc and higher levels only in ovotestes at 14.5dpc. / These results indicate that all Sox9, Pn1, and MIS genes follow a sexually dimorphic pattern of expression associated with development of testicular cords. Therefore, these genes are placed downstream of Sry in the fetal mouse gonad. Furthermore, we conclude that the testis-determining pathway is impaired upstream of Sox9 and Pn1 and Mis in the B6.YTIR gonad. (Abstract shortened by UMI.)

A plethora of anthropological studies have been undertaken on the skull, including many analyses of sexual dimorphism. Sexual dimorphism reflected in the eye orbits has not always demonstrated consistent or reliable results. However, recent studies (Pretorius, Steyn, & Scholtz, 2006; Ji et al., 2010) suggest some positive results utilizing geometric morphometrics to predict sex. Utilizing 97 post-mortem CT (computed tomography) scans, established morphological and metric techniques for sex determination were assessed from 3D rendered models of the crania. In addition, landmark data were collected on the orbital margin to evaluate the accuracy of sex determination using geometric morphometric techniques. Traditional methods demonstrated poor levels of accuracy for prediction of sex, however, utilizing generalised procrustes analysis and discriminant function analysis on 3D landmark data resulted in 94.95% overall accuracy. Application of recent methodological advances, including geometric morphometrics, should continue to be developed as it increases the ability to assess sexual dimorphism which will allow for greater identification of unknown remains.

A plethora of anthropological studies have been undertaken on the skull, including many analyses of sexual dimorphism. Sexual dimorphism reflected in the eye orbits has not always demonstrated consistent or reliable results. However, recent studies (Pretorius, Steyn, & Scholtz, 2006; Ji et al., 2010) suggest some positive results utilizing geometric morphometrics to predict sex. Utilizing 97 post-mortem CT (computed tomography) scans, established morphological and metric techniques for sex determination were assessed from 3D rendered models of the crania. In addition, landmark data were collected on the orbital margin to evaluate the accuracy of sex determination using geometric morphometric techniques. Traditional methods demonstrated poor levels of accuracy for prediction of sex, however, utilizing generalised procrustes analysis and discriminant function analysis on 3D landmark data resulted in 94.95% overall accuracy. Application of recent methodological advances, including geometric morphometrics, should continue to be developed as it increases the ability to assess sexual dimorphism which will allow for greater identification of unknown remains.