California Sea Grant College Program

The overall aim of this project was to understand the mode of sex determination mechanism operates in chondrostean species. The following hypotheses were tested: i) white sturgeon has a genetic sex determination system, ii) one of the sexes in sturgeon is heterogametic, and iii) sex-specific DNA polymorphisms exist between the sexes.

Several experimental approaches derived from the fields of transmission, molecular, and cytogenetics were used to examine the nature of the sex determination process.

In the first series of experiments (Chapter 2) ploidy manipulation techniques were used  to produce gynogenetic and polyploid white sturgeon. A novel random amplified polymorphic DNA (RAPD)-based technique was developed to rapidly assess the overall success of treatments designed to induce gynogenesis, and measuring erythrocyte nuclei size with a Coulter Counter was found to be a rapid and accurate technique for ploidy analysis in sturgeon. Both sexes were observed in 23-24 month old gynogenetic progeny groups derived from four different females supporting the hypothesis that white sturgeon has a female heterogametic (ZW:ZZ) genetic sex determination system. The proportion of males in diploid (48%), gynogenetic (18%), and triploid (14%) progeny groups derived from the same female suggested that the sex-determining element on the W chromosome segregates independently of the centromere.

In the second series of experiments (Chapter 3) various molecular techniques were used in an attempt to isolate genetic markers for the identification of sex in white sturgeon. Subtractive hybridization, representational difference analysis and bulked segregant analysis were used, in conjunction with genetically unique groups of sturgeon, which were specifically developed  to increase the probability of isolating sex-specific DNA sequences. DNA sequences associated with sex in white sturgeon were not identified. These results suggest that either 1) there are no sex-specific sequences in sturgeon, or 2) the sex-specific DNA is comprised of unusual sequences which were not complementary to the 1200 random decamer primers or the recognition sequence of the restriction endonucleases used in this set of experiments, or 3) the sex-specific sequences comprise a very small portion of the genome.

Chapter 4 details a synaptonemal complex (SC) analysis of white sturgeon spermatocytes. Synaptonemal complex analyses have not been previously reported for any sturgeon species and this study was initiated to determine if it was possible to directly identify heteromorphic sex chromosomes in meiotic prophase SC spreads. No bivalent consistently exhibited asynaptic behavior or had axes of unequal length suggesting that heteromorphic sex chromosomes are not present in white sturgeon spermatocytes which concurs with the findings of Chapter 2. Pachytene nuclei had varying numbers of univalents, self-paired foldback elements with no obvious centromeric region (1-7), and other SC peculiarities. No consistent evidence of alignment or pairing involving more than two lateral elements was found indicating that the process of diploidization is now complete in this ancient polyploid. The SC number was 139 (± 3.4) with both between and within animal variation.

The final series of experiments (Chapter 5) involved mitotic analyses of metaphase chromosome spreads from four white sturgeon individuals of each sex. Conventional mitotic analyses provided no evidence for a heteromorphic sex chromosome pair or any sex-related chromosomal polymorphism. Likewise fluorescence in situ hybridization of labeled male and female sturgeon genomic DNA to metaphase chromosome spreads of each sex did not reveal a chromosome that was seen to be specifically hybridizing only to the genomic DNA of one sex. The average chromosome number was 271 (range 265-276) which concurred with the meiotic count found in Chapter 4. An  improved C-banding technique using propidium iodide and epifluorescence microscopy revealed between animal variation (2-7) in the number of entirely heterochromatic metacentric chromosomes. These heterochromatic chromosomes and the self-pairing elements observed in  Chapter 4 were approximately equal in number and relative size and they may represent accessory B chromosomes. In combination these results suggest that white sturgeon has a female heterogametic ZW:ZZ genetic sex determination system, and that the sex-determining element segregates independently ofthe centromere. No evidence of a heteromorphic sex chromosome pair was found in meiotic analyses of white sturgeon spermatocytes supporting the hypothesis of male homogamety in this species.  The inability to identify sex-specific DNA sequences despite evidence of a genetic sex determination system suggests that sex-specific DNA is rare, making up a very small portion of the genome. Mitotic analyses provided no evidence for a heteromorphic sex chromosome pair suggesting that the accumulation of sex chromosomal rearrangements is insufficient to be visible at the cytogenetic level. All of these results support the contention that white sturgeon sex chromosomes are at an early stage of differentiation.