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Identifying genes related to choriogenesis in insect panoistic ovaries by Suppression Subtractive Hybridization.

Irles P, Bellés X, Piulachs MD - BMC Genomics (2009)

Bottom Line: The sequences were compared against non-redundant NCBI databases using BLAST.We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries.The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Biologia Evolutiva (UPF-CSIC), Passeig Marítim de la Barceloneta, Barcelona, Spain. paula.irles@ibe.upf-csic.es

ABSTRACT

Background: Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH) library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model.

Results: We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis.

Conclusion: The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For most of the genes, functions related to choriogenesis are postulated. The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.

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Localization of Bg30001 and Bg30017 expression. In situ hybridization using antisense Bg30001 and Bg30017 RNA probes. mRNA of Bg30001 (A) and Bg30017 (E) is detected in the follicular epithelium of basal oocytes from 7-day-old females in the period of chorion formation, but not in sub-basal oocytes or in the germarium. Detail of the follicular cells showing that Bg30001 (C) and Bg30017 (G) transcripts localize in the cytoplasm of the follicular cells. Control ovarioles tested with sense probes of Bg30001 (B, D) and Bg30017 (F, H) were not labelled. Scale bars: 200 μm in panels A, B, E, F; and 50 μm in panels C, D, G, H.
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Figure 5: Localization of Bg30001 and Bg30017 expression. In situ hybridization using antisense Bg30001 and Bg30017 RNA probes. mRNA of Bg30001 (A) and Bg30017 (E) is detected in the follicular epithelium of basal oocytes from 7-day-old females in the period of chorion formation, but not in sub-basal oocytes or in the germarium. Detail of the follicular cells showing that Bg30001 (C) and Bg30017 (G) transcripts localize in the cytoplasm of the follicular cells. Control ovarioles tested with sense probes of Bg30001 (B, D) and Bg30017 (F, H) were not labelled. Scale bars: 200 μm in panels A, B, E, F; and 50 μm in panels C, D, G, H.

Mentions: In order to assess whether Bg30001 and Bg30017 are specific from the ovary, we studied its expression by semiquantitative RT-PCR in different tissues of adult females. RNA from muscle, midgut, epidermis, fat body, colleterial glands and ovaries from newly emerged females and 7-day-old females were included in the study. As shown in figure 4, the expression of Bg30001 and Bg30017 is restricted to mature ovaries. Furthermore, in situ hybridization studies showed that both genes are specifically expressed in the follicular cells cytoplasm of basal oocytes during choriogenesis (Figure 5). Interestingly, neither the sub-basal oocytes nor the germarium were labelled, and optical sections of the basal oocyte showed that the ooplasm and the oocyte nucleus were also free of label. These spatial data and the temporal data afforded by expression studies strongly suggest that Bg30001 and Bg30017 are involved in the process of chorion formation.


Identifying genes related to choriogenesis in insect panoistic ovaries by Suppression Subtractive Hybridization.

Irles P, Bellés X, Piulachs MD - BMC Genomics (2009)

Localization of Bg30001 and Bg30017 expression. In situ hybridization using antisense Bg30001 and Bg30017 RNA probes. mRNA of Bg30001 (A) and Bg30017 (E) is detected in the follicular epithelium of basal oocytes from 7-day-old females in the period of chorion formation, but not in sub-basal oocytes or in the germarium. Detail of the follicular cells showing that Bg30001 (C) and Bg30017 (G) transcripts localize in the cytoplasm of the follicular cells. Control ovarioles tested with sense probes of Bg30001 (B, D) and Bg30017 (F, H) were not labelled. Scale bars: 200 μm in panels A, B, E, F; and 50 μm in panels C, D, G, H.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683872&req=5

Figure 5: Localization of Bg30001 and Bg30017 expression. In situ hybridization using antisense Bg30001 and Bg30017 RNA probes. mRNA of Bg30001 (A) and Bg30017 (E) is detected in the follicular epithelium of basal oocytes from 7-day-old females in the period of chorion formation, but not in sub-basal oocytes or in the germarium. Detail of the follicular cells showing that Bg30001 (C) and Bg30017 (G) transcripts localize in the cytoplasm of the follicular cells. Control ovarioles tested with sense probes of Bg30001 (B, D) and Bg30017 (F, H) were not labelled. Scale bars: 200 μm in panels A, B, E, F; and 50 μm in panels C, D, G, H.
Mentions: In order to assess whether Bg30001 and Bg30017 are specific from the ovary, we studied its expression by semiquantitative RT-PCR in different tissues of adult females. RNA from muscle, midgut, epidermis, fat body, colleterial glands and ovaries from newly emerged females and 7-day-old females were included in the study. As shown in figure 4, the expression of Bg30001 and Bg30017 is restricted to mature ovaries. Furthermore, in situ hybridization studies showed that both genes are specifically expressed in the follicular cells cytoplasm of basal oocytes during choriogenesis (Figure 5). Interestingly, neither the sub-basal oocytes nor the germarium were labelled, and optical sections of the basal oocyte showed that the ooplasm and the oocyte nucleus were also free of label. These spatial data and the temporal data afforded by expression studies strongly suggest that Bg30001 and Bg30017 are involved in the process of chorion formation.

Bottom Line: The sequences were compared against non-redundant NCBI databases using BLAST.We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries.The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Biologia Evolutiva (UPF-CSIC), Passeig Marítim de la Barceloneta, Barcelona, Spain. paula.irles@ibe.upf-csic.es

ABSTRACT

Background: Insect ovarioles are classified into two categories: panoistic and meroistic, the later having apparently evolved from an ancestral panoistic type. Molecular data on oogenesis is practically restricted to meroistic ovaries. If we aim at studying the evolutionary transition from panoistic to meroistic, data on panoistic ovaries should be gathered. To this end, we planned the construction of a Suppression Subtractive Hybridization (SSH) library to identify genes involved in panoistic choriogenesis, using the cockroach Blattella germanica as model.

Results: We constructed a post-vitellogenic ovary library by SSH to isolate genes involved in choriogenesis in B. germanica. The tester library was prepared with an ovary pool from 6- to 7-day-old females, whereas the driver library was prepared with an ovary pool from 3- to 4-day-old females. From the SSH library, we obtained 258 high quality sequences which clustered into 34 unique sequences grouped in 19 contigs and 15 singlets. The sequences were compared against non-redundant NCBI databases using BLAST. We found that 44% of the unique sequences had homologous sequences in known genes of other organisms, whereas 56% had no significant similarity to any of the databases entries. A Gene Ontology analysis was carried out, classifying the 34 sequences into different functional categories. Seven of these gene sequences, representative of different categories and processes, were chosen to perform expression studies during the first gonadotrophic cycle by real-time PCR. Results showed that they were mainly expressed during post-vitellogenesis, which validates the SSH technique. In two of them corresponding to novel genes, we demonstrated that they are specifically expressed in the cytoplasm of follicular cells in basal oocytes at the time of choriogenesis.

Conclusion: The SSH approach has proven to be useful in identifying ovarian genes expressed after vitellogenesis in B. germanica. For most of the genes, functions related to choriogenesis are postulated. The relatively high percentage of novel genes obtained and the practical absence of chorion genes typical of meroistic ovaries suggest that mechanisms regulating chorion formation in panoistic ovaries are significantly different from those of meroistic ones.

Show MeSH
Related in: MedlinePlus