ABSTRACTIn this study, all genetic female (XX) broods of Japanese flounder were produced artificially by mating the females with sex‐reversed males. The proliferation and migration of primordial germ cells (PGCs), formation of ovary and oogenesis were described in detail. After hatching, around 20 individual PGCs migrated from the lateral to the dorsal of trunk region. At 15 days posthatching (dph), a part of PGCs were covered by a single layer somatic cells and formed the genital ridge. By 22 dph, the elongated gonadal primordia appeared under the ventral kidney, where the PGCs were totally enclosed by somatic cells. During the process of migration, PGCs were presumed to be mitotically inactive. From 63 to 73 dph, somatic cells rearrangement resulted in the formation of a narrow crevice, which became deeper and formed ovarian lumen. However, at 52 dph, dramatic mitotic proliferation of germ cell occurred and germline nest formed before the appearance of ovarian lumen. The onset of intensive germ cell proliferation and appearance of cell nests could be accepted as a criterion of initial ovarian differentiation. Then germ cells and somatic epithelial cells were gradually delimited by basement membrane and formed the germinal epithelium. In this period, results from in situ hybridization revealed that the early forkhead box L2 (pofoxl2) was expressed in somatic cells and oocytes in primary growth, which indicated the prefollicle cells formed. Then oogonia or oocytes, follicle cells, basement membrane, and theca cells composed a follicle complex. Finally, oocytes underwent meiosis and developed into to mature eggs. Anat Rec, 301:727–741, 2018. © 2017 Wiley Periodicals, Inc.

This study investigated the relationship between semen quality and pH of turbot(Scophthalmus maximus) and the impact of glycerin on turbot sperm activation using turbot sperm from different hatcheries (A, B, C, and D) through the computer-assisted sperm analysis (CASA) system. The results showed that sperm motility rates were different in the four hatcheries, A2 > A1 > D > C > B. The predicted sperm longevity times were 11.7, 7.1, 6.8, 4.6, and 3.3 min, respectively. The motility rates of turbot sperm significantly positively correlated with VCL, VSL, VAP, ALH, and BCF (P <0.05), but no significant correlation was observed with LIN, WOB, and STR. We found that the pH of turbot sperm significantly positively correlated with motility, VCL, VSL, VAP, ALH, and BCF(P < 0.05), whereas no significant correlation was found with LIN, WOB, and STR. In addition, high-quality sperm exhibited a weak alkalinity with a pH of 8.0. We also found that an appropriate concentration of glycerol (100 mmol/L) in sea water was useful for main-taining the longevity.

To explore the function of progestin during spermatogonial proliferation and meiosis, we used histological methods to investigate the gonadal development process of the male turbot (Scophthalmus maximus) 6 to 22 months of age. We detected the expression of amh and sycp3 via the real-time quantitative polymerase chain reaction (qRT-PCR). Using enzyme-linked immunosorbent (ELISA) technology, qRT-PCR, and in-situ hybridization, we detected the serum P4 and DHP levels and the expression patterns of the nuclear progesterone receptor (pgr) and membrane progestin receptor alpha (mPRa) during different development periods. The results reveal that the P4 and DHP levels were high during spermatogonial proliferation and meiosis Ⅱ. The pgr expression was high from spermatogonial proliferation to meiosis I, whereas the mPRα expression was high during meiosis Ⅱ and spermiogenesis. Our in-situ hybridization results indicate pgr mRNA to be predominantly located in the Sertoli cells that were in contact with the proliferating spermatogonia and primary spermatocyte and that the mPRα mRNA was undetectable. Taken together, our data indi-cate the pgr expression to be involved in mediating the progestin stimulation of spermatogonia proliferation and meio-sis, whereas the mPRa plays an important role in meiosis Ⅱ and spermiogenesis.

Sex determination is not only resort to a single genetic cascade but also regulated along a continuum of environmental and heritable factors in many fish species. Temperature is one of the most important non-genetic sex differentiation elements. Previous studies regarding high temperature masculinization of flatfish typically focused on sex ratios and reproductive endocrinology, but little was known about germ cells during the process. In this study, we investigated the dynamic changes of germ cells in terms of morphology and number employing serial-sectioning and the molecular investigation of expression of dnd and amh genes by qRT-PCR for qualitative and quantitative analysis of germ cell proliferation during sexual differentiation. Moreover, the proliferation activity of primordial germ cells was investigated using PCNA (proliferating cell nuclear antigen) immunohistochemistry.Experimental results revealed that high temperatures caused juvenile olive flounder masculinization (male ratio, 95.24%, 27.5 degrees C +/- 0.5 degrees C) in comparison with controls (male ratio 5.56%, 18 degrees C +/- 0.5 degrees C). The proliferation pattern of germ cells presented sexual dimorphism, from 42 to 66 days post-hatching (dph). The experimental group presented specifically slow proliferation and greatly reduced cell numbers. No cyst clusters were apparent in comparison to the control group. Consistently, the proliferation activity of germ cells in the experimental group was significantly reduced in comparison to that observed in the control group at 50 dph by PCNA immunohistochemistry. Furthermore, the expression levels of poamh during the process of temperature-induced masculinization gradually increased and were significantly higher than that in the control group at the stage gonads have been considered still undifferentiated in particular from a histological point of view. Thus, elevated temperatures result in masculinization, poamh up-regulation, accompanied by germ cell detention proliferation.

为研究斑石鲷雌、雄鱼染色体的形态与结构,本文采用植物血球凝集素(PHA)及秋水仙碱体内注射法,取头肾细胞采用冷滴片结合空气干燥的方法获取了斑石鲷雌、雄鱼染色体的中期分裂相,分别对其进行了常规的吉姆萨和硝酸银染色并进行核型和带型分析.结果显示:(1)雄性斑石鲷核型为2n=47、1m+2sm+44t、NF=50,雌性斑石鲷核型为2n=48、2sm+46t、NF=50.(2)斑石鲷雌、雄鱼染色体相对长度分别为(2.30±0.37)—(6.87±0.42)及(2.28±0.30)—(10.33±0.86),雄性斑石鲷具有一条体型巨大的异形中部着丝粒染色体,其相对长度达到其它染色体的2-5倍.(3)染色体经银染后,发现含有2个Ag-NORs位点的分裂相最多,位点位于一对亚中部着丝粒染色体短臂末端,呈短棒状,且具有活性异形现象.(4)本研究中雄性斑石鲷具有一条异形性染色体,可能是性染色体Y,我们推测其染色体的性别决定类型可能为X1X1X2X2/X1X2Y型.本研究为鲈形目鱼类的细胞遗传学研究提供了基础资料,并将为进一步开展斑石鲷遗传育种等研究奠定基础.

The present study obtained a germcell-specific marker dead end (dnd) in olive flounder (Paralichthys olivaceus) named Podnd. The tissue-specific expressions of Podnd transcripts were present in testis and ovary but were not detectable in other somatic tissues detected. SISH showed that Podnd expressed only in germ cells at different developmental stages but not in surrounding somatic cells. The expression of Podnd during embryonic development at 16 different stages revealed that the relative expression of Podnd transcript fluctuated at a high level in the cleavage stages, gradually decreased through subsequent development, and reached the lowest at late gastrula stage till it was nearly undetectable. The Podnd transcripts localization and migration were similar to zebrafish. Further research on the specification migration mechanism of PGCs and the role of germ cell during gonadal development in olive flounder would improve our understanding of germline development.