The Role Of RA In Teleosts Meiosis Onset

Meiosis is a special type of cell division necessary for sexual reproduction in eukaryotes. In recent years, great progress has been achieved in the initiation of meiosis in vertebrate and implicates RA as a requirement for the initiation of meiosis in mammals, birds and amphibians, which involving the interplay of multiple substances, but which plays a leading role is controversial. In mouse, germ cells in the fetal ovary undergo sex-specific entry into meiosis, the initiation of which is thought to be mediated by mesonephros-derived retinoic acid (RA), whilst those in the fetal testis enter mitotic arrest, for the action of the RA-metabolising enzyme CYP26B1. The role of RA in initiation of germ cell meiosis in teleosts is remains to be illustrated. Therefore, in the present study, the expression patterns of components of the RA metabolism and signaling pathways were examined, and RA synthesis inhibitor citral and RA applied to5dah (dah, day after hatching) XX/XY in Nile tilapia (Oreochromis nilolicus).Based on Nile tilapia genome sequences and the8transcriptome sequences of male and female Nile tilapia gonads at different developmental stages obtained by our lab, a number of important factors in the RA signaling pathway, including RA synthase aldhla family(aldh1a1/1a2/1a3), RA degrading enzyme cyp26(cyp26a/b1/cl), RA receptors rar(two rara, one rarb, two rarg, one rxra), rxr(one rxra, two rxrb, one rxrg), cellular retinoid-binding proteins crabp(crabpla/lb, crabp2a/2b) were isolated. It implied that a complete RA signaling pathway existed in the tilapia gonad.cyp26b1and aldhla2were cloned from Nile tilapia by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). The full length cDNA of cyp26bl, aldh1a2were1926bp and1832bp, respectively. Analysis of the tissue distribution pattern of cyp26b1and aldhla2in tilapia by RT-PCR revealed that the genes were widely expressed in multiple tissues with aldhla2higher in ovary, cyp26b1showed no significant sex differences in gonads. Ontogenic analysis of cyp26blaldh1a2expression patterns in different developmental stages of tilapia gonads by real-time PCR showed:before the female and male meiosis onset(female10to20 dah, male60to70dah), aldh1a2was upregulated, while cyp26b1was down regulated. So we speculated increased RA was used for male and female meiosis initiation. We speculate the reason for meiosis was initiated in female at25dah but been postponed in male was that aldhla2was higher expressed in female while cyp26b1higher in male, which allowed higher level of RA in female than in male gonads at20dah5dah XX female tilapia was treated by citral (citral,1mM/kg) for70days, then normal fed. The treated tilapia has a characteristic of an undifferentiated gonad, while the control group with ovarian structures at1.5mah (mah, month after hatching). In addition, ovotestis with a vast majority of testis structure was observed at4mah. Our results implied meiosis initiation in the Nile tilapia ovary requires RA, RA-dependent meiosis entry could be a conserved mechanism in vertebrates.By immunohistochemistry, aromatase (Cyp19a1a) in citral treated-4-mat gonad showed lower expression compared with normal ovary, and the positive signals were only detected in the preserved ovarian structure of ovotestis. Therefore, it indicated that RA may affect estrogen level in gonad. cyp26b1was higher expressed in the treated group, while aldh1a2was down regulated from the beginning to45dat in the3-month-old tilapia ovary during fadrozole treatment by real-time PCR. It is speculated that RA levels may be related to resume meiosis and gonadal redifferentiation. The results indicated that estrogen may have a moderating effect of RA levels in the gonads.5dah XY male tilapia treated by RA (RA,60μM/kg) for70days, then normal fed. At2mah and3mah, we observed an undifferentiated gonad structure with few somatic cells in the treatment group, while the control group has developed into a testis structure by H.E staining. A testis with a large cavity appeared in the treatment group at5mah. Therefore, we can see that exogenous RA postponed the process of the male (XY) tilapia gonadal development, but did not make the time of meiosis advance, we continued to treat part of5mah-RA-treated fish by estradiol for another40days, and observed the gonad with a larger cavity, the process of spermatogenesis disordered. This indicates male tilapia sex reversal process may involved more complex mechanisms and it is not easy to make ahead of male meiosis.In summary, our data confirmed RA signaling system exists in tilapia gonad. cyp26b1and aldh1a2may work together to control the level of RA in teleosts gonad instead of single synthetic enzyme or decomposing enzyme. By controlling RA level in gonad to affect meiosis initiation, and then influence the direction of sex differentiation, indicated that RA is indispensable for male and female Nile tilapia meiosis initiation. Our results indicated the possibility for crosstalk between estrogen and RA signaling in the course of sex differentiation.