In vitro germ cell differentiation during sex differentiation in a teleost fish

Abstract

To clarify the sexually dimorphic mechanisms of gonadal sex differentiation, we established an in vitro culture system for gonadal sex differentiation using the teleost fish Oreochromis niloticus. In vivo, the entry of germ cells into meiosis occurs around 35 days after hatching (dah) in XX gonads, whereas in XY gonads, meiotic cells became differentiated around 85 dah. In our in vitro culture system using gonads from young fry at 23 dah, the meiotic cells in the XX gonads appeared after 21 days of culture. In contrast, in the XY gonads, no meiotic cells were detected after 21 days. These results indicate that germ cell differentiation in this culture system progresses in a manner similar to that in vivo. To identify the gene products that are involved in the entry of germ cells into meiosis or in the arrest of germ cells at the gonial stage of gonadal sex differentiation, we performed subtractive hybridization screening with this in vitro culture system. From the screening process, we identified the female-related gene, FR-3, which is a homolog of zebrafish nanos-related gene (nos). The nos gene was expressed after gonadal formation around 35 dah in XX gonads, but not in XY gonads. In situ hybridization indicated that nos is expressed in oogenic meiotic cells, but not in spermatogenic meiotic cells. Further examination revealed that nos was expressed in oogenic meiotic cells after gonadal formation, specifically in teleost fish. Together, nos may be also involved in oogenic meiosis, with the exception of primordial germ cell migration.

Molecular Aspects of Gonadal Differentiation in a Teleost Fish, the Nile Tilapia

“…In contrast, no meiotic cells were detected after 21 days in the XY gonads. These results indicate that germ cell differentiation in this culture system progresses in a manner similar to that in vivo [Kobayashi, 2008]. This in vitro culture system for gonadal sex differentiation is a powerful tool for clarifying the mechanisms of gonadal sex differentiation in tilapia.…”

“…By means of RT-PCR, in situ hybridization, and immunohistochemical analyses, we demonstrated that the expression of P450arom (cyp19a1) in XX fry precedes any morphological gonadal sex differentiation (5 dah) and that transcripts are localized in the stromal cells near the blood vessels located in the interstitium on the side facing the mesentery, but not in the parenchyma/medullary cells including germ cell-surrounding cells [Kobayashi et al, 2003[Kobayashi et al, , 2008Wang et al, 2007]. Other steroidogenic enzymes required for estrogen synthesis, such as P450scc (cyp11a1) , P450c17 (cyp17a1) , 3 ␤ -HSD , and 17beta-HSD , were also expressed in XX gonads [Kobayashi et al, 2003;Ijiri et al, 2008].…”

“…To date, dmrt1 has been cloned and its expression pattern has been reported in several vertebrates [mouse: Raymond et al, 1999;chicken: Smith et al, 1999chicken: Smith et al, , 2003tilapia: Guan et al, 2000;turtle: Kettlewell et al, 2000;trout: Marchand et al, 2000]. For the first time in fish, the pattern in tilapia clearly indicates that dmrt1 is expressed in the germ cellsurrounding cells and parenchyma/medullary cell mass cells which differentiate into Sertoli cells and the efferent duct during testicular differentiation, respectively, if the phenotypic sex is male [Guan et al, 2000;Kobayashi et al, 2008]. Studies in chickens have also reported that ZW sex reversal from female to male was accompanied by DMRT1 expression in medullary cells that developed into Sertoli cells [Smith et al, 2003].…”

“…Recently, we have established an in vitro culture system for gonadal sex differentiation using Nile tilapia [Kobayashi, 2008]. In vivo, the entry of germ cells into meiosis occurs around 35 days after hatching (dah) in XX gonads, whereas in XY gonads, meiotic cells become differentiated at 85 dah .…”

Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation

“…Using the same transgenic zebrafish line as we used in the present study, Wang and his colleagues demonstrated that although the so-called juvenile ovaries might exist in the zebrafish at early stage, there was a significant variation in the degree of such female commitment with some individuals developing straight to males from indifferent gonads without going through an obvious stage of juvenile ovary26. However, if we consider the early entry into meiosis as a female-specific marker6667, the widespread existence of meiotic cells in juvenile gonads would support the view that all individuals develop femaleness first (20–25 dpf) at cell and molecular levels with some individuals, but not all, showing significant morphological features of ovary such as perinucleolar oocyte (PO).…”