A <Emphasis Type="Italic">vasa</Emphasis> gene from green mud crab <Emphasis Type="Italic">Scylla</Emphasis><Emphasis Type="Italic">paramamosain</Emphasis> and its expression during gonadal development and gametogenesis

VASA is one of the important regulatory factors that determine the development of the reproductive system. However, no information on vasa gene from Pleocyemata Brachyura is available. By using Race, we obtained a full-length cDNA of Sp-vasa of the green mud crab Scylla paramamosain. The full-length (2,851 bp) cDNA of vasa encodes a peptide of 631 amino acids. Real-time PCR results indicated that the expression level of Sp-vasa in the growth stage of ovary was higher than in the maturation stage, and in stage I and II of testis, the expression level of Sp-vasa were higher than in stage III. By using in situ hybridization, Sp-vasa RNAs were detected in the large part of oocyte plasm in stage I, nucleus zone in stage III and perinuclear zone in stage V. As the size of oocytes increases during oogenesis, the signals change from strong to weak. In addition, in stage I and II of testis, the expression levels of Sp-vasa were higher than in stage III, and the hybridization intensity of Sp-vasa gene gradually increased during spermatogenesis from spermatogonia to spermatids. However, no hybridization signal was detected in spermatozoon. Real-time PCR and in situ hybridization were consistent. These findings suggest that Sp-vasa is likely to serve as a useful and specific marker for germ cell development of S. paramamosain.     

Expression pattern of vasa in gonads of sea cucumber Apostichopus japonicus during gametogenesis and reproductive cycle

The vasa gene is a reliable germline marker to study the origin and development of germ cells and gonads, although the gene product (mRNA or protein) varies between different species. However, there has been little study on vasa genes in holothuroids to date. Here we determined the expression characteristics of the Apostichopus japonicus vasa gene (Aj-vasa) during gametogenesis in the ovary and testis using in situ hybridization and immunohistochemistry. During oogenesis, the expression pattern of Aj-vasa coincided at the mRNA and protein levels. Intensive signals in oogonia decreased gradually with the development of oocytes. Interestingly, the pattern was different during spermatogenesis. The Aj-vasa mRNA level was the highest in spermatogonia, reduced in spermatocytes, low in spermatids and absent in spermatozoa, but the Aj-VASA protein was restricted to spermatogonia and early spermatocytes. These expression characteristics of Aj-vasa persisted in both male and female gonads throughout the reproductive cycle. Our findings show that Aj-vasa mRNA is a good marker for studying the origin and migration of germline cells; moreover, Aj-VASA is a useful tool to identify spermatogonia in A. japonicus. Our findings indicate that Aj-vasa is vital in the development and differentiation of germ cells.     

Differential expression of ubiquitin-conjugating enzyme E2r in the developing ovary and testis of penaeid shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

In order to identify genes involved in oogenesis and spermatogenesis in penaeid shrimp Marsupenaeus japonicus, a modified annealing control primer (ACP) system was adapted to identify genes differentially expressed in ovary and testis at different developmental stages. By using 20 pairs of ACP primers, 8 differentially expressed genes were obtained. One of these genes is ubiquitin-conjugating enzyme E2r (UBE2r). Bioinformatics analyses show that this gene encodes a protein of 241 amino acids with a predicted molecular mass of 27.4 kDa. Real time PCR analyses demonstrated that the expression level changed significantly in the developing testis and ovary. In the stage 2 of testis, it reached its highest expression level, the lowest expression level present in the stage 1 of ovary. The significantly different expression levels in developing testis and ovary suggest that UBE2r has an important role in oogenesis and spermatogenesis. This article is the first report of UBE2r in crustaceans and also is the first report showing that UBE2r is differentially expressed at different stages of the developing ovary and testis in an animal.     

Medaka dead end encodes a cytoplasmic protein and identifies embryonic and adult germ cells

dead end (dnd) was identified in zebrafish as a gene encoding an RNA-binding protein essential for primordial germ cell (PGC) development and gametogenesis in vertebrates. The adult dnd RNA expression has been restricted to the ovary in Xenopus or to the testis in mouse. Its protein product is nuclear in chicken germ cells but both cytosolic and nuclear in mouse cell cultures. Here we report the cloning and expression pattern of Odnd, the medakafish (Oryzias latipes) dnd gene. Sequence comparison, gene structure, linkage analysis and expression demonstrate that Odnd encodes the medaka Dnd orthologue. A systematic comparison of Dnd proteins from five fishes and tetrapod representatives led to the identification of five previously unidentified conserved regions besides the RNA recognition motif. The Odnd RNA is maternally supplied and preferentially segregated with PGCs. Its adult expression occurs in both sexes and is restricted to germ cells. In the testis, Odnd is abundant in spermatogonia and meiotic cells but absent in sperm. In the ovary, Odnd RNA persists throughout oogenesis. Furthermore, we developed a dual color fluorescent in situ hybridization procedure allowing for precise comparisons of expression and distribution patterns between two genes in medaka embryos and adult tissues. Importantly, this procedure co-localized Odnd and Ovasa in testicular germ cells and PGCs. Surprisingly, by cell transfection and embryo RNA injection we show that ODnd is cytoplasmic in cell cultures, cleavage embryos and PGCs. Therefore, medaka dnd encodes a cytoplasmic protein and identifies embryonic and adult germ cells of both sexes.     

Vasa Identifies Germ Cells and Critical Stages of Oogenesis in the Asian Seabass

Germ cells produce sperm and eggs for reproduction and fertility. The Asian seabass (Lates calcarifer), a protandrous marine fish, undergoes male-female sex reversal and thus offers an excellent model to study the role of germ cells in sex differentiation and sex reversal. Here we report the cloning and expression of vasa as a first germ cell marker in this organism. A 2241-bp cDNA was cloned by PCR using degenerate primers of conserved sequences and gene-specific primers. This cDNA contains a polyadenylation signal and a full open reading frame for 645 amino acid residues, which was designated as Lcvasa for the seabass vasa, as its predicted protein is homologous to Vasa proteins. The Lcvasa RNA is maternally supplied and specific to gonads in adulthood. By chromogenic and fluorescent in situ hybridization we revealed germ cell-specific Lcvasa expression in both the testis and ovary. Importantly, Lcvasa shows dynamic patterns of temporospatial expression and subcellular distribution during gametogenesis. At different stages of oogenesis, for example, Lcvasa undergoes nuclear-cytoplasmic redistribution and becomes concentrated preferentially in the Balbiani body of stage-II~III oocytes. Thus, the vasa RNA identifies both female and male germ cells in the Asian seabass, and its expression and distribution delineate critical stages of gametogenesis.     

Gonads in Histiostoma mites (Acariformes: Astigmata): Structure and development

The development of male and female gonads in arrhenotokous and thelytokous species of Histiostoma was studied using transmission electron microscopy (TEM). All instars were examined: larvae, protonymphs, facultative heteromorphic deutonymphs (=hypopi), tritonymphs, and adults. In testis primordium, spermatogonia surrounding a testicular central cell (TCC) with a gradually enlarging, branched nucleus are present already at the larval stage. Spermatogonia and the TCC are connected via narrow, tubular intercellular bridges revealing that the TCC is a germline cell. Spermatocytes appear at the protonymphal stage. At the heteromorphic deutonymph stage, the testis primordium is similar to that of the protonymph, but in the tritonymph it is much larger and composed as in the adult: spermatids as well as sperm cells are present. The latter are congregated ventrally in the testis at the entrance of the deferent duct.In the larval ovary, an eccentrically located ovarian nutritive cell (ONC) is surrounded by oogonia which are connected with the ONC via tubular intercellular bridges. In later stages, the ovary grows and oocytes appear in the protonymph. Meiotic synaptonemal complexes in oocytes occur from the tritonymph stage. At about the time of the final molting, tubular intercellular bridges transform into peculiar diaphragm-crossed bridges known only in Histiostoma mites. In the adult female, growing oocytes at the end of previtellogenesis lose intercellular bridges and move ventro-laterally to the ovarian periphery towards the oviduct entrance. Vitellogenesis occurs in oviducts.Germinal cells in both the testis and ovary are embedded in a few somatic stroma cells which may be well discernible already in the larval ovary; in the testis, somatic stroma cells are evident not earlier than the end of the tritonymphal stage. The ovary has a thin wall of flat somatic cells, whereas the testis is covered by a basal lamina only.The obtained results suggest that gonads in Histiostoma and other Astigmata originate from two primordial cells only.     

GnRH在性成熟高白鲑神经系统及性腺中的分布定位

摘要：应用免疫组织化学方法,系统观察性成熟期高白鲑(Coregonus peled)神经系统及性腺中的促性腺激素释放激素( GnRH)的分布情况。结果表明,GnRH在大脑、小脑、中脑、脊髓、延髓中免疫阳性反应明显,且主要分布在神经元内。GnRH免疫阳性细胞在卵巢和精巢中均有分布,而且其阳性部位在卵巢主要分布于小生长期卵...     

Immunohistochemical Study of Expression of Sohlh1 and Sohlh2 in Normal Adult Human Tissues

The expression pattern of Sohlh1 (spermatogenesis and oogenesis specific basic helix-loop-helix 1) and Sohlh2 in mice has been reported in previous studies. Sohlh1 and Sohlh2 are specifically expressed in spermatogonia, prespermatogonia in male mice and oocytes of primordial and primary follicles in female mice. In this report, we studied the expression pattern of Sohlh1 and Sohlh2 in human adult tissues. Immunohistochemical staining of Sohlh1 and Sohlh2 was performed in 5 samples of normal ovaries and testes, respectively. The results revealed that Sohlh genes are not only expressed in oocytes and spermatogonia, but also in granular cells, theca cells, Sertoli cells and Leydig cells, and in smooth muscles of blood vessel walls. To further investigate the expression of Sohlh genes in other adult human tissues, we collected representative normal adult tissues developed from three embryonic germ layers. Compared with the expression in mice, Sohlhs exhibited a much more extensive expression pattern in human tissues. Sohlhs were detected in testis, ovary and epithelia developed from embryonic endoderm, ectoderm and tissues developed from embryonic mesoderm. Sohlh signals were found in spermatogonia, Sertoli cells and also Leydig cells in testis, while in ovary, the expression was mainly in oocytes of primordial and primary follicles, granular cells and theca cells of secondary follicles. Compared with Sohlh2, the expression of Sohlh1 was stronger and more extensive. Our study explored the expression of Sohlh genes in human tissues and might provide insights for functional studies of Sohlh genes.     

Identification and migration of primordial germ cells in Atlantic salmon,Salmo salar: Characterization of Vasa,Dead End,and Lymphocyte antigen 75 genes

No information exists on the identification of primordial germ cells (PGCs) in the super‐order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full‐length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi‐quantitative RT‐PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole‐mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid‐blastula stage, vasa‐expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp‐rt‐vasa 3′‐UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

Rhipicephalus appendiculatus: characterization of a testis-associated protein

A novel gene coding for Rhipicephalus appendiculatus Male-specific Protein (RAMP) was identified in a cDNA library constructed from the testis/vas deferens of R. appendiculatus ticks. This gene encodes a secreted protein exclusively expressed in the testis/vas deferens. The putative RAMP amino acid sequence contains a signal peptide and has 29% amino acid identity with male-specific Is5 gene of Ixodes scapularis. Gene expression studies revealed that RAMP mRNA was up-regulated in male ticks during blood feeding. RAMP was detected not only in the testis/vas deferens of males but also in postcoitum female ticks based on Western blotting, indicating that this protein is transferred to the female tick during copulation. Virgin female ticks, microinjected with recombinant RAMP, had significantly prolonged attachment duration during feeding, but there was no effect on fed weight. These results suggest that RAMP is a male-specific molecule in the spermatophore, and is related to female attachment behavior in R. appendiculatus ticks.     

Development of the reproductive system of Echinostoma paraensei in Mesocricetus auratus analyzed by light and confocal scanning laser microscopy

This study was performed to gain insight into the maturation of the reproductive system of Echinostoma paraensei worms grown in an early infection of Mesocricetus auratus. Hamsters were infected with 100 metacercariae and necropsied on days 3, 5, 7, 10 and 14 post infection (dpi). Recovered flukes stained with hydrochloric carmine were preserved as whole mounts and analyzed by light and confocal scanning laser microscopy. The average worm recovery was 43.7 per host. Images of the male and female reproductive systems were taken. The ovary and anterior and posterior testis were evidenced on day 3, while the ootype and cirrus sac were present on day 5. Confocal imaging showed primordium testis and ovary as a cluster of primordial cells from day 3 onward. The testes, ovary, cirrus sac and uterus organs were already present during the first week of life. The two testes were seen as individual structures on 7 dpi while the cirrus sac and vitelline glands were in development. The ovary was connected to the uterus while the ootype was adjacent to it. Both testes were larger than the ovary, showing cells at different stages of development, but with few bundles of functional spermatozoa in 10 day-old worms. On day 14, eggs and spermatozoa were seen in the uterus and seminal vesicle, respectively, while oocytes appeared in the ootype as fertilized eggs. We conclude that the reproductive system of E. paraensei was functional on 14 dpi in the hamsters.     

大口黑鲈amh基因全长cDNA克隆、表达及多克隆抗体制备

为研究大口黑鲈(Micropterus salmoides)抗缪勒氏管激素(amh)基因的表达及其在性腺发育中的潜在作用,研究利用RACE技术克隆得到了大口黑鲈amh基因,并制备Amh多克隆抗体,通过qRT-PCR、Western Blot分析Amh在大口黑鲈不同组织和不同发育阶段性腺中的表达模式,最后利用HE染色法和免疫组化观察不同发育阶段性腺的形态组织学变化及其与Amh表达的潜在关系。结果显示:大口黑鲈amh基因cDNA序列全长2050 bp,由24 bp5′非编码区、394 bp3′非编码区和1632 bp的开放阅读框组成,共编码543个氨基酸。amh基因mRNA在大口黑鲈11个组织中均有表达,其中雄鱼精巢中表达量最高,肌肉次之,雌鱼卵巢中表达量最高,肌肉次之。amh基因在雌雄鱼不同发育阶段的性腺中表达存在显著差异,精巢中表达量均显著高于卵巢(P<0.05)。同时, Western Blot结果显示Amh蛋白在精巢中表达丰度较高。amh基因在精巢中的表达量呈先上升后下降的趋势,且在孵化后65d鱼精巢中其表达量达到最高(P<0.05),免疫组化结果显示Amh表达于早期精...     

Ultrastructural Morphology of the Male and Female Genital Tracts of <Emphasis Type="Italic">Psoroptes</Emphasis> spp. (Acari: Astigmata: Psoroptidae)

The structure of the male and female genital systems of the astigmatid mite Psoroptes ovis (Hering) is described. The male genital system is composed of a paired testis, fused at its proximal part, two vasa deferentia, an ejaculatory duct, into which a single accessory gland opens, and a copulatory organ. The testis is characterized by a peripheric syncytial cell surrounding spermatogonia, spermatocytes, spermatids and spermatozoa which are distributed regularly in the gonad according to the sequence of spermatogenesis. The female genital system consists of a copulatory pore (the bursa copulatrix), a seminal receptacle, paired ovaries and oviducts, a glandular uterus and an ovipositor which leads to the oviporus. Ovaries are composed of somatic cells, germ cells and a central cell, with a multilobular nucleus, connected to oocytes by a stalk. Similarities with other astigmatic mites belonging to Psoroptidia and Acaridia are also discussed.     

Cloning and expression analysis on a homolog of spermatogonial stem-cell renewal factor in Fenneropenaeus chinensis

The spermatogonial stem-cell renewal factor (SSRF) was named since its function in spermatogonial mitosis was reported in Japanese eel. Our previous study showed that a homolog of SSRF was highly expressed in the ovary of triploid shrimp, but not expressed in the ovary of diploid shrimp. To understand the function of SSRF in shrimp, the full-length cDNA of ssrf gene was cloned from Chinese shrimp Fenneropenaeus chinensis (Fcssrf) and its expression was analyzed. The full length of Fcssrf cDNA was 2588?bp and it contained an open reading frame encoding 450 amino acids. The predicted tertiary structure of FcSSRF was very similar to that of SSRF/eSRS34 from Anguilla japonica and TP/PD-ECGF from Homo sapiens. RT-PCR analysis showed that the Fcssrf was highly expressed in nerve, testis, hepatopancreas, gill, and stomach rather than in ovary. Expression of Fcssrf mRNA was not detected during embryonic stages and larval stages, from the nauplii to the post-larvae stage, in diploid, and triploid shrimp. However, it began to be expressed in juvenile stages (June–September) in diploid and triploid shrimp. Immunohistochemical analyses showed that FcSSRF was identified in both the diploid testis and triploid ovary. We inferred that the Fcssrf might be related to testis development.