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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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Expression patterns of post-vitellogenic mRNA's isolated by SSH from ovaries of B. germanica. Expression pattern of seven post-vitellogenic genes in ovaries of adult Blattella germanica during the first gonadotrophic cycle. 7-day-old females in the period of chorion formation were divided into three stages: EC (early choriogenesis), MC (mid choriogenesis) and LC (late choriogenesis), according to criteria shown in figure 2. qRT-PCR was normalized against BgActin-5c. Data represent copies of mRNA per copy of BgActin-5c, and are expressed as the mean ± SD (n = 3).
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Figure 3: Expression patterns of post-vitellogenic mRNA's isolated by SSH from ovaries of B. germanica. Expression pattern of seven post-vitellogenic genes in ovaries of adult Blattella germanica during the first gonadotrophic cycle. 7-day-old females in the period of chorion formation were divided into three stages: EC (early choriogenesis), MC (mid choriogenesis) and LC (late choriogenesis), according to criteria shown in figure 2. qRT-PCR was normalized against BgActin-5c. Data represent copies of mRNA per copy of BgActin-5c, and are expressed as the mean ± SD (n = 3).

Mentions: Results (Figure 3) showed that the seven genes studied are expressed differentially during post-vitellogenesis, and all of them have maximal expression in some stage of choriogenesis. Yellow-g and cuticular protein-like, are expressed very transiently during choriogenesis, and their maximum expression is 6- and 12-fold higher than that of B. germanica actin-5c (BgActin-5c), respectively. The mRNA of follicle cell protein 3c appears in 3-day-old females, their levels increase slightly as basal oocyte matures, peak at MC, and suddenly decrease in LC. Cathepsin-L and CG10407-PA-like mRNAs are present during the entire gonadotrophic cycle, though with changing levels. Cathepsin-L show low mRNA levels in pre-vitellogenic and vitellogenic ovaries, they peak on day 7, just before the onset of choriogenesis, and then decrease progressively until LC. Those of CG10407-PA-like are relatively high at the beginning of the cycle, decrease during vitellogenesis (subtraction days) and increase during post-vitellogenesis, peaking on EC stage.


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

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

Expression patterns of post-vitellogenic mRNA's isolated by SSH from ovaries of B. germanica. Expression pattern of seven post-vitellogenic genes in ovaries of adult Blattella germanica during the first gonadotrophic cycle. 7-day-old females in the period of chorion formation were divided into three stages: EC (early choriogenesis), MC (mid choriogenesis) and LC (late choriogenesis), according to criteria shown in figure 2. qRT-PCR was normalized against BgActin-5c. Data represent copies of mRNA per copy of BgActin-5c, and are expressed as the mean ± SD (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Expression patterns of post-vitellogenic mRNA's isolated by SSH from ovaries of B. germanica. Expression pattern of seven post-vitellogenic genes in ovaries of adult Blattella germanica during the first gonadotrophic cycle. 7-day-old females in the period of chorion formation were divided into three stages: EC (early choriogenesis), MC (mid choriogenesis) and LC (late choriogenesis), according to criteria shown in figure 2. qRT-PCR was normalized against BgActin-5c. Data represent copies of mRNA per copy of BgActin-5c, and are expressed as the mean ± SD (n = 3).
Mentions: Results (Figure 3) showed that the seven genes studied are expressed differentially during post-vitellogenesis, and all of them have maximal expression in some stage of choriogenesis. Yellow-g and cuticular protein-like, are expressed very transiently during choriogenesis, and their maximum expression is 6- and 12-fold higher than that of B. germanica actin-5c (BgActin-5c), respectively. The mRNA of follicle cell protein 3c appears in 3-day-old females, their levels increase slightly as basal oocyte matures, peak at MC, and suddenly decrease in LC. Cathepsin-L and CG10407-PA-like mRNAs are present during the entire gonadotrophic cycle, though with changing levels. Cathepsin-L show low mRNA levels in pre-vitellogenic and vitellogenic ovaries, they peak on day 7, just before the onset of choriogenesis, and then decrease progressively until LC. Those of CG10407-PA-like are relatively high at the beginning of the cycle, decrease during vitellogenesis (subtraction days) and increase during post-vitellogenesis, peaking on EC stage.

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