Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset,a model primate

Small RNAs mediate gene silencing by binding Argonaute/Piwi proteins to regulate target RNAs. Here, we describe small RNA profiling of the adult testes of Callithrix jacchus, the common marmoset. The most abundant class of small RNAs in the adult testis was piRNAs, although 353 novel miRNAs but few endo-siRNAs were also identified. MARWI, a marmoset homolog of mouse MIWI and a very abundant PIWI in adult testes, associates with piRNAs that show characteristics of mouse pachytene piRNAs. As in other mammals, most marmoset piRNAs are derived from conserved clustered regions in the genome, which are annotated as intergenic regions. However, unlike in mice, marmoset piRNA clusters are also found on the X chromosome, suggesting escape from meiotic sex chromosome inactivation by the X-linked clusters. Some of the piRNA clusters identified contain antisense-orientated pseudogenes, suggesting the possibility that pseudogene-derived piRNAs may regulate parental functional protein-coding genes. More piRNAs map to transposable element (TE) subfamilies when they have copies in piRNA clusters. In addition, the strand bias observed for piRNAs mapped to each TE subfamily correlates with the polarity of copies inserted in clusters. These findings suggest that pachytene piRNA clusters determine the abundance and strand-bias of TE-derived piRNAs, may regulate protein-coding genes via pseudogene-derived piRNAs, and may even play roles in meiosis in the adult marmoset testis.     

Expression of anti-Müllerian hormone (AMH) in the equine testis

Anti-Müllerian hormone (AMH) induces regression of Müllerian ducts during male fetal development; in the human male, it is expressed in Sertoli cells during fetal development (and through puberty). The objective was to characterize expression of AMH in the fetal, neonatal, prepubertal, and adult equine testis, as well as in equine cryptorchid testes, in select testicular neoplasms, and in intersex gonads, based upon immunohistochemistry (IHC). Testes were removed from equine fetuses at 5.5, 10, and 11 months of gestation, at 12 months of age, and from adult stallions. In addition, cryptorchid testes, testis tumors (teratomas, seminomas, Sertoli cell tumors), and male intersex gonads were examined by IHC for expression of AMH using a goat polyclonal primary antibody (alpha-AMH) directed against a C-terminal peptide antigen from human AMH. Immunolabeling with alpha-AMH was localized to Sertoli cells within the developing seminiferous tubules of fetal, neonatal and prepubertal equine testes, with no expression detected in Sertoli cells from normal adult equine testes. Furthermore, expression was detected in cryptorchid testes (in animals up to 3-4 years of age) and in Sertoli cell tumors and male intersex gonads. In conclusion, AMH was strongly expressed by Sertoli cells in fetal, neonatal and prepubertal equine testes, but not in normal adult testes. That AMH was expressed in cryptorchid testes may provide a useful biomarker for detection of cryptorchid testes, as well as for immunohistochemical characterization of testicular tumors and intersex gonads in the horse.     

Gonadal development and diandric protogyny in two populations of Dascyllus reticulatus from Madang, Papua New Guinea

Two Dascyllus reticulatus populations from Madang, Papua New Guinea exhibited diandric protogyny. In both populations, gonads began as undifferentiated, and then developed oocytes in the primary growth stage and an ovarian lumen. From this ovarian state or from more developed ovaries containing oocytes beyond the primary‐growth stage, some gonads developed into testes. The first sign of testicular development was degeneration of oocytes, degeneration of oocytes in the primary growth stage in ovarian gonads and degeneration of oocytes of all growth stages present including the primary growth stage in ovaries, which was then followed by development of spermatogenic tissue. In both populations, most of the fish that had gonads with degenerating oocytes were smaller than the smallest mature females, indicating that development towards testes was mostly initiated in immature gonads containing only pre‐vitellogenic oocytes. On some occasions, however, females as large as other mature females also had gonads with degenerating oocytes, suggesting that development towards testes may have occurred in mature ovaries as well. This latter notion is further strengthened by the discovery of a fish having a gonad that contained both degenerating vitellogenic oocytes and developing spermatogenic tissue. Taken together, these results suggest that D. reticulatus can exhibit diandric protogyny, because testes in D. reticulatus developed from juvenile gonads as well as from mature ovaries.     

Identification,chromosomal mapping and conserved synteny of porcine Argonaute family of genes

The Argonaute proteins are recently identified and evolutionarily conserved family with two subfamilies Ago and Piwi, which play important roles in small RNA pathways. Most species have eight Argonaute members in their genomes, ranging from 1 to 27. Here we report identification of six Argonaute genes in pig, four members of the Ago subfamily (Ago1, Ago2, Ago3 and Ago4) and two members of the Piwi subfamily (Piwil1 and Piwil2), which were predicted to encode proteins of 857, 860, 860, 861, 861 and 985 amino acids, respectively. Phylogenetic analysis showed that the porcine Ago and Piwi genes were clustered into relevant branch of mammalian Argonaute members. The porcine Ago4- Ago1-Ago3 genes are linked together at the p12 of the chromosome 6, while Ago2 is located at the p15 of the chromosome 4. The porcine Piwil1 and Piwil2 are mapped together onto the chromosome 14, at the q14 and q11 respectively. Comparatively mapping of the Argonaute members on chromosomes showed that linkage group of the Ago4-Ago1-Ago3 and several neighborhood genes is evolutionarily conserved from chicken to mammals. The genes Piwil1 and Piwil2 are separated onto different chromosomes from fish to mammals, with exception to this tendency in both pig and stickleback, indicating an opposite tendency of recombination together or non-disjunction of these two genes during speciation. Further expression analysis showed an ubiquitous expression pattern of Ago members, oppositely a restricted expression pattern in gonads of the Piwi members, suggesting distinct potential roles of the porcine Argonaute genes.     

In vitro maturation and fertilization of prepubertal goat oocytes

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.     

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.     

Characterization of Sus scrofa Small Non-Coding RNAs Present in Both Female and Male Gonads

Small non-coding RNAs (sncRNAs) are indispensable for proper germ cell development, emphasizing the need for greater elucidation of the mechanisms of germline development and regulation of this process by sncRNAs. We used deep sequencing to characterize three families of small non-coding RNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the small RNA fraction present in both male and female gonads. Although similar numbers of reads were obtained from both types of gonads, the number of unique RNA sequences in the ovaries was several times lower. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cell proliferation, and carcinogenesis. Unexpectedly, we detected a novel sncRNA type, the tRFs, which are 30–36-nt RNA fragments derived from tRNA molecules, in gonads. Analysis of S. scrofa piRNAs show that testes specific piRNAs are biased for 5′ uracil but both testes and ovaries specific piRNAs are not biased for adenine at the 10^th nucleotide position. These observations indicate that adult porcine piRNAs are predominantly produced by a primary processing pathway or other mechanisms and secondary piRNAs generated by ping-pong mechanism are absent.     

Novel perspectives in sexual lability through gonadal biopsy in larval sea lampreys

Gonadal biopsies of sea lamprey Petromyzon marinus larvae (118·5–142·5 mm) were examined quantitatively and categorized as ovaries, presumptive testes, or atypical gonads. Subsets of larvae were then killed 1, 2, 4, 8, 16 and 52 weeks following surgery at which time each larva was matched with its biopsy, and its gonad re–examined for quantifiable changes in composition. During 52 weeks, oocyte diameters increased significantly while numbers of oocytes per unit area significantly declined. Germ cell numbers remained within the range of typical presumptive testes. Overall abundance of typical ovaries decreased over the course of the experiment, whereas the number of presumptive testes increased. This was a direct result of presumptive testis development from atypical gonads, oocyte atresia and sex reversal. The results suggest an argument for environmental sex determination in lampreys, while indicating that they remain sexually labile throughout most of the larval period, despite primary differentiation.     

大鼠肝癌模型中Piwil4、Mael和Ddx4基因mRNA和蛋白表达水平及其与肿瘤检测的关系

PIWI和piRNA的表达水平与肿瘤类型密切相关。Piwil2 mRNA在肝癌中的表达量比肿瘤周围的肝脏组织的表达量高。PIWI/piRNA通路基因Piwil4、Mael和Ddx4为候选癌基因,在多种癌组织中表达,而在肝癌组织中的研究尚无报道。为探讨Piwil4、Mael和Ddx4基因在肝癌组织中表达水平变化的影响,建立了大鼠肝癌模型,并提取血清用ELISA方法检测肿瘤标记物,采用实时荧光定量PCR、蛋白质免疫印迹方法和免疫组织化学方法检测正常肝组织与模型组肝组织中Piwil4、Mael和Ddx4基因mRNA转录和蛋白表达水平。结果表明,大鼠肝癌动物模型血清中肿瘤标记物含量明显升高。Piwil4、Mael和Ddx4基因mRNA及蛋白在肝癌模型组织中高表达。研究表明,肝癌模型组织中Piwil4、Mael和Ddx4基因表达水平有望作为肝癌检测的一种分子标志物。