Limits...
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.

Show MeSH

Related in: MedlinePlus

Stages of chorion formation. Apical pole of the basal oocyte of Blattella germanica ovaries in different stages of choriogenesis. A) day 7 just before the first symptoms of chorion secretion, B) day 7, early chorion stage (7EC), C) day 7, mid chorion stage (7MC), and D) late chorion stage (7LC). The process from 7EC to 7LC lasts 15 h approximately. Scale: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2683872&req=5

Figure 2: Stages of chorion formation. Apical pole of the basal oocyte of Blattella germanica ovaries in different stages of choriogenesis. A) day 7 just before the first symptoms of chorion secretion, B) day 7, early chorion stage (7EC), C) day 7, mid chorion stage (7MC), and D) late chorion stage (7LC). The process from 7EC to 7LC lasts 15 h approximately. Scale: 50 μm.

Mentions: In order to assess the efficiency and quality of the SSH results, the expression pattern of a selection of genes was investigated by quantitative real-time PCR (qRT-PCR). Expression was studied in the ovary of adult females from emergence (day 0) to oviposition (day 7). Ovaries on day 7 were further subdivided into four stages, depending on the degree of chorion formation: 7, no signs of choriogenesis, with the apical pole of the basal oocyte showing the basement membrane very close to the follicular epithelium (Figure 2A); 7EC, early choriogenesis (EC), with the basement membrane of the apical pole slightly pulled away from the follicle cells (Figure 2B); 7MC, mid choriogenesis (MC), with the basement membrane of the apical pole showing a concavity (Figure 2C); 7LC, late choriogenesis (LC), with the basement membrane of the apical pole showing a claviform aspect (Figure 2D). The process from 7EC to LC lasts 15 h approximately[7].


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

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

Stages of chorion formation. Apical pole of the basal oocyte of Blattella germanica ovaries in different stages of choriogenesis. A) day 7 just before the first symptoms of chorion secretion, B) day 7, early chorion stage (7EC), C) day 7, mid chorion stage (7MC), and D) late chorion stage (7LC). The process from 7EC to 7LC lasts 15 h approximately. Scale: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Stages of chorion formation. Apical pole of the basal oocyte of Blattella germanica ovaries in different stages of choriogenesis. A) day 7 just before the first symptoms of chorion secretion, B) day 7, early chorion stage (7EC), C) day 7, mid chorion stage (7MC), and D) late chorion stage (7LC). The process from 7EC to 7LC lasts 15 h approximately. Scale: 50 μm.
Mentions: In order to assess the efficiency and quality of the SSH results, the expression pattern of a selection of genes was investigated by quantitative real-time PCR (qRT-PCR). Expression was studied in the ovary of adult females from emergence (day 0) to oviposition (day 7). Ovaries on day 7 were further subdivided into four stages, depending on the degree of chorion formation: 7, no signs of choriogenesis, with the apical pole of the basal oocyte showing the basement membrane very close to the follicular epithelium (Figure 2A); 7EC, early choriogenesis (EC), with the basement membrane of the apical pole slightly pulled away from the follicle cells (Figure 2B); 7MC, mid choriogenesis (MC), with the basement membrane of the apical pole showing a concavity (Figure 2C); 7LC, late choriogenesis (LC), with the basement membrane of the apical pole showing a claviform aspect (Figure 2D). The process from 7EC to LC lasts 15 h approximately[7].

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