Although the sex-determining gene Sry has been identified in mammals, no comparable genes have been found in non-mammalian vertebrates. Here, we used recombinant breakpoint analysis to restrict the sex-determining region in medaka fish (Oryzias latipes) to a 530-kilobase (kb) stretch of the Y chromosome. Deletion analysis of the Y chromosome of a congenic XY female further shortened the region to 250 kb. Shotgun sequencing of this region predicted 27 genes. Three of these genes were expressed during sexual differentiation. However, only the DM-related PG17 was Y specific; we thus named it DMY. Two naturally occurring mutations establish DMY's critical role in male development. The first heritable mutant--a single insertion in exon 3 and the subsequent truncation of DMY--resulted in all XY female offspring. Similarly, the second XY mutant female showed reduced DMY expression with a high proportion of XY female offspring. During normal development, DMY is expressed only in somatic cells of XY gonads. These findings strongly suggest that the sex-specific DMY is required for testicular development and is a prime candidate for the medaka sex-determining gene.
The recent discovery of the DMY gene (DM domain gene on Y chromosome and one of the DMRT1 family genes) as a key determinant of male development in the medaka (Oryzias latipes) has led to its designation as the prime candidate gene for sex-determination in this species. This study focused on the sites and pattern of expression of DMY and DMRT1 genes during gonadal differentiation of medaka to further determine their roles in testis development. DMY mRNA and protein are expressed specifically in the somatic cells surrounding primordial germ cells (PGCs) in the early gonadal primordium, before morphological sex differences are seen. However, somatic cells surrounding PGCs never express DMY during the early migratory period. Expression of DMY persists in Sertoli cell lineage cells, from PGC-supporting cells to Sertoli cells, indicating that only DMY-positive cells enclose PGCs during mitotic arrest after hatching. DMRT1 is expressed in spermatogonium-supporting cells after testicular differentiation (20 -30 days after hatching), and its expression is much higher than that of DMY in mature testes. In XX sex-reversed testes, DMRT1 is expressed in the Sertoli cell lineage, similar to the expression of DMY in XY testes. These results suggest strongly that DMY regulates PGC proliferation and differentiation sex-specifically during early gonadal differentiation of XY individuals and that DMRT1 regulates spermatogonial differentiation. Developmental Dynamics 231:518 -526, 2004.
The field of environmental DNA (eDNA) analysis has rapidly developed over the past decade and the technique has become widely used for detecting aquatic macroorganisms in a variety of habitats. However, a variety of measurement protocols have been individually developed for different eDNA studies and this may lead to confusion for others who wish to incorporate eDNA analysis in their research. It is important therefore to synthesize the current status of—and future challenges to—the methodology of eDNA analysis. We here synthesized the protocols from total 438 published eDNA studies detecting aquatic macroorganisms were used to calculate the frequency of using each method in eDNA analysis steps. We found that the frequency of methods used converged to one or two methods for any analysis step. Furthermore, although the procedure with highest frequency is not always the best, it was shown that the eDNA collection by filtration and subsequent extraction/purification using a DNeasy Blood and Tissue DNA extraction kit (Qiagen, Hilden, Germany) or PowerWater DNA Extraction Kit (Qiagen) is the most common procedure. An understanding of the characteristics of commonly used methods can help those newly engaged in eDNA studies to understand the basic outline of eDNA analysis. Our review will be useful for the future improvement and development of analytical eDNA techniques of eDNA by sharing the recognition of methodological characteristic including advantages and disadvantages in major analytical techniques.
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