**5.1. FTIR microspectroscopy supported by PCA-LDA for the characterization of SN and NSN murine oocytes**

Recently, we applied FTIR microspectroscopy supported by PCA-LDA to the study of murine oocytes characterized by two different types of chromatin organization, namely surrounded nucleolus (SN) oocytes in which the chromatin is highly condensed and forms a ring around the nucleolus, and the not surrounded nucleolus (NSN) type where chromatin is dispersed and less condensed around the nucleolus [7, 75]. Interestingly, only SN oocytes are capable to complete the embryonic development after fertilization, while the NSN type, if fertilized, arrests at the two cell stage. To try to get new insights on the mechanisms that drive the different chromatin organization in the two kinds of oocytes, crucial for their embryonic development after fertilization, we studied the infrared absorption of single intact cells at different maturation stages, namely antral germinal vesicle (GV), metaphase I (MI, matured for 10 hours in vitro), and metaphase II (MII, matured for 20 hours in vitro).

Indeed, as we will show in the following, the FTIR spectra of the oocytes taken at the different maturation stages are very complex, since they provide information on different processes that were taking place simultaneously within the cells. For this reason, beside a fundamental visual inspection of the data, enabling the identification and assignment of the different spectral bands, it was crucial the application of PCA-LDA that made it possible to draw out the most significant spectral information responsible for the different cell behavior. Moreover, PCA-LDA allowed to identify the stage at which the separation between the SN and NSN oocytes took place, leading to their well distinct cell destinies.

As we discussed in paragraph 2, since the FTIR spectrum of cells is due to the overlapping contributes of the main biomolecules (see Figure 2), we analysed the second derivative spectra to identify the band peak positions and to assign them to the different biomolecule vibrational modes. The spectral analysis, strongly supported by PCA-LDA, allowed us to disclose the most important spectral differences between the two types of oocytes, at each maturation stage, that were found to occur mainly in the lipid and nucleic acid absorption regions, as we will discuss below. For a full discussion of the results see [7].

#### *5.1.1. Lipid analysis*

#### *5.1.1.1. NSN oocytes*

The analysis between 3050 and 2800 cm-1, mainly due to the lipid carbon-hydrogen stretching vibrations [29], disclosed significant variations in the lipid content of NSN oocytes during their maturation up to MII. Indeed, besides an increase of the CH2 band intensity up to MII, respectively at 2922 cm-1 and 2852 cm-1, important changes concerned mainly the unsaturated fatty acid composition, as indicated by variations of the band between 3020 and 3000 cm-1 due to the olefinic group absorption. Indeed, as shown in Figure 3A, a single peak around 3013 cm-1 was present at GV and MI stages, while a splitting in two components at ~ 3016 cm-1 and at ~ 3010 cm-1 characterized the MII stage (see the inset of Figure 3A). These results could reflect important changes in membrane fluidity, which in turn could confer to the oocyte a different division ability after fertilization [8].

Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes 209

The results obtained by the direct inspection of second derivative spectra were confirmed by PCA-LDA analysis performed on raw spectra. Firstly, the analysis was made on each type of oocyte taken at the different maturation stages. For the SN oocytes, the component carrying the highest discrimination weight resulted that at 2938 cm-1, likely due to cholesterol and / or phospholipids [76, 77], in agreement with what found by the direct

Concerning the NSN oocytes, on the other hand, the wavenumbers with the highest discrimination weight were the 2922 cm-1, due to the CH2 stretching vibration, which increases up to MII, and the 3018 cm-1, assigned to the olefinic group =CH of polyunsaturated fatty acids, whose absorption was observed to vary during the oocyte

We, then, compared the two types of oocyte at each maturation stage - as illustrated in Figure 4 - and we found that at the antral and MII stages the spectral components with the highest discrimination weight were those due to cholesterol and /or phospholipids, while at MI was that due to the olefinic group. Furthermore, to support the crucial role played by lipids in determining at some extent the oocyte developmental capacity, we should add that when we compared by PCA-LDA the spectra of the two oocyte types at the same maturation stage in the 1800-1500 cm-1 spectral range, dominated by the amide I and amide II absorption, the wavenumber with the highest discrimination weight was the 1739 cm-1,

**Figure 4.** PCA-LDA analysis of SN and NSN oocytes in the lipid acyl chain absorption region (3050 – 2800 cm-1). The separation between the two types of oocytes at each maturation stage is reported as average of PCA-LDA scores. The height of the boxes and the whiskers corresponds to 1 and 1.5 standard deviations from the mean values, respectively. The analysis has been performed on the

due to the carbonyl stretching vibration of esters [7, 29].

*5.1.1.3. PCA-LDA analysis* 

inspection of the spectra.

maturation.

measured spectra.

#### *5.1.1.2. SN oocytes*

SN oocytes were found to be characterized - during maturation up to MII - by a significant increase of the 2937 cm-1 component that could be likely due to cholesterol and/or phospholipids (Figure 3B) [76, 77]. As discussed for NSN oocytes, the observed changes could reflect variations in the membrane properties, again highlighting the crucial role of lipids as markers of oocyte developmental competence [8, 78].

**Figure 3.** Second derivative absorption spectra of NSN (A) and SN (B) oocytes in the lipid absorption region. The second derivatives of the FTIR absorption spectra of single oocytes, measured at the antral (continuous line), MI 10 H (dotted line), and MII 20 H (dashed line) stages, are reported in the acyl chain absorption region, after normalization at the tyrosine peak (~1516 cm-1). In the inset a magnification of the olefinic group band is shown.

#### *5.1.1.3. PCA-LDA analysis*

208 Multivariate Analysis in Management, Engineering and the Sciences

The analysis between 3050 and 2800 cm-1, mainly due to the lipid carbon-hydrogen stretching vibrations [29], disclosed significant variations in the lipid content of NSN oocytes during their maturation up to MII. Indeed, besides an increase of the CH2 band intensity up to MII, respectively at 2922 cm-1 and 2852 cm-1, important changes concerned mainly the unsaturated fatty acid composition, as indicated by variations of the band between 3020 and 3000 cm-1 due to the olefinic group absorption. Indeed, as shown in Figure 3A, a single peak around 3013 cm-1 was present at GV and MI stages, while a splitting in two components at ~ 3016 cm-1 and at ~ 3010 cm-1 characterized the MII stage (see the inset of Figure 3A). These results could reflect important changes in membrane fluidity, which in

SN oocytes were found to be characterized - during maturation up to MII - by a significant increase of the 2937 cm-1 component that could be likely due to cholesterol and/or phospholipids (Figure 3B) [76, 77]. As discussed for NSN oocytes, the observed changes could reflect variations in the membrane properties, again highlighting the crucial role of

**Figure 3.** Second derivative absorption spectra of NSN (A) and SN (B) oocytes in the lipid absorption region. The second derivatives of the FTIR absorption spectra of single oocytes, measured at the antral (continuous line), MI 10 H (dotted line), and MII 20 H (dashed line) stages, are reported in the acyl chain absorption region, after normalization at the tyrosine peak (~1516 cm-1). In the inset a

turn could confer to the oocyte a different division ability after fertilization [8].

lipids as markers of oocyte developmental competence [8, 78].

magnification of the olefinic group band is shown.

*5.1.1. Lipid analysis* 

*5.1.1.1. NSN oocytes* 

*5.1.1.2. SN oocytes* 

The results obtained by the direct inspection of second derivative spectra were confirmed by PCA-LDA analysis performed on raw spectra. Firstly, the analysis was made on each type of oocyte taken at the different maturation stages. For the SN oocytes, the component carrying the highest discrimination weight resulted that at 2938 cm-1, likely due to cholesterol and / or phospholipids [76, 77], in agreement with what found by the direct inspection of the spectra.

Concerning the NSN oocytes, on the other hand, the wavenumbers with the highest discrimination weight were the 2922 cm-1, due to the CH2 stretching vibration, which increases up to MII, and the 3018 cm-1, assigned to the olefinic group =CH of polyunsaturated fatty acids, whose absorption was observed to vary during the oocyte maturation.

We, then, compared the two types of oocyte at each maturation stage - as illustrated in Figure 4 - and we found that at the antral and MII stages the spectral components with the highest discrimination weight were those due to cholesterol and /or phospholipids, while at MI was that due to the olefinic group. Furthermore, to support the crucial role played by lipids in determining at some extent the oocyte developmental capacity, we should add that when we compared by PCA-LDA the spectra of the two oocyte types at the same maturation stage in the 1800-1500 cm-1 spectral range, dominated by the amide I and amide II absorption, the wavenumber with the highest discrimination weight was the 1739 cm-1, due to the carbonyl stretching vibration of esters [7, 29].

**Figure 4.** PCA-LDA analysis of SN and NSN oocytes in the lipid acyl chain absorption region (3050 – 2800 cm-1). The separation between the two types of oocytes at each maturation stage is reported as average of PCA-LDA scores. The height of the boxes and the whiskers corresponds to 1 and 1.5 standard deviations from the mean values, respectively. The analysis has been performed on the measured spectra.

#### *5.1.2. Nucleic acid analysis*

#### *5.1.2.1. NSN oocytes*

We then analyzed the nucleic acid IR response of NSN and SN oocytes during their maturation, exploring the spectral region between 1000 and 800 cm-1, where RNA and DNA vibrational modes mainly occur [31, 32].

Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes 211

Furthermore, the analysis of the low frequency range, between 840-820 cm-1, allowed us to obtain information on DNA methylation. In particular, in this spectral range, bands due to DNA S-type sugar puckering modes occur, which are sensitive to changes in the DNA sugar conformation induced by cytosine methylation [32]. The possibility to monitor changes in the profile of this spectral region in whole intact cells makes it possible, therefore, to obtain information on the variation of global DNA methylation in the CpG islands. In this way, we found that in the NSN oocytes DNA methylation was high at the antral stage, while it became very low, almost negligible at MII, in agreement with what found for the

Finally, significant spectral differences were found between 890 and 850 cm-1, where four different bands due to adenine and uracil vibrational modes occur (see Figure 5) [79]. Interestingly, the relative variation of these bands enables to monitor the mRNA polyadenylation extent, a crucial mechanism that regulates transcription. We found, in particular, that NSN oocytes were characterized during maturation by a low level of mRNA polyadenylation, being the polyadenylic acid band at 884 cm-1 absent at MII, while a new band at 854 cm-1 - likely due to adenine possibly not involved in polyA tail [80] – appeared. These results seem to suggest that an inadequate level of mRNA polyadenylation could preclude the possibility to resume meiosis, leaving the NSN oocytes in an unsuccessful

The analysis of SN oocytes (Figure 5B) in the spectral range between 1000 and 800 cm-1 led to very different results compared to NSN oocytes (see Figure 5A). Briefly, during all the studied maturation stages, the SN oocyte transcriptional activity was found to be maintained at lower levels than NSN oocytes, as revealed by the analysis of the CC stretching of the DNA backbone (980-950 cm-1) and the monitoring of the ribose (~ 922 cm-1) and deoxyribose (895-898 cm-1) vibrations. These results were supported by the temporal evolution of the DNA methylation bands that suggested a partial CpG methylation at the antral and MI stages, which dramatically increased at MII, contrary to what observed for

Noteworthy, while no evidence of mRNA polyadenylation was observed for SN oocytes at the antral stage - as indicated by the absence of the two polyadenylic acid bands around 884 cm-1 and 860 cm-1 - starting from MI the adenine and uracile bands at 870 cm-1 and 850 cm-1 appeared, to then dramatically increase up to MII. These findings likely indicate that SN MII oocytes are characterized by an adequate level of maternal polyadenylated mRNAs, making

The above results overall indicate that the IR spectra of oocytes at different maturation stages are very informative in the nucleic acid absorption region, allowing to obtain information on several cell processes simultaneously, including transcriptional activity, DNA methylation, and RNA polyadenylation. For this reason, PCA-LDA analysis was

them ready to sustain a proper embryo development, contrary to NSN oocytes.

transcriptional activity pattern at the different maturation stages.

transcriptional state.

*5.1.2.2. SN oocytes* 

NSN oocytes.

*5.1.2.3. PCA-LDA analysis* 

We found that NSN oocytes maintain, in all the studied stages, an appreciable transcriptional activity as indicated mainly by the simultaneous presence of the RNA ribose component around 921 cm-1 and of the DNA deoxyribose between 895-898 cm-1 - indicative of a DNA/RNA hybrid - whose relative intensities were seen to vary during maturation (see Figure 5A). In particular, the intensity of these two components is higher at the antral stage, while it decreases at MI, to increase again up to MII. These results were also supported by the response of the complex band between 980-950 cm-1, mainly due to the CC stretching vibration of DNA backbone. Indeed, the profile of this band varies depending on the DNA structure that, in turn, could reflect a different nucleic acid activity. In particular, for the NSN oocytes we found that at the antral stage DNA is mainly in A-form - with a triplet at 975 cm-1, 966 cm-1 and 951 cm-1 - typical of the DNA/RNA hybrid during transcription. At MI, the reduction of the 975 cm-1 and 966 cm-1 bands and the appearance of that at 969 cm-1 indicate that DNA is mainly in the B-form, suggesting a sort of transcriptional "stand by state", further supported by the reduction extent of the DNA/RNA hybrid, as discussed above. From this "stand by state" NSN oocytes seem to resume their transcriptional activity at MII, where a coexistence of DNA A and B forms was observed, as indicated by the increase of the ~ 975 cm-1 band and again in agreement with the simultaneous increase of the ribose (921 cm-1) and deoxyribose (898 cm-1) components.

**Figure 5.** Second derivative absorption spectra of NSN (A) and SN (B) oocytes in the nucleic acid absorption region. The second derivatives of the FTIR absorption spectra of single oocytes, measured at the antral (continuous line), MI 10 H (dotted line), and MII 20 H (dashed line) stages, are reported in the 1000-800 cm-1 absorption region, after normalization at the tyrosine peak (~1516 cm-1).

Furthermore, the analysis of the low frequency range, between 840-820 cm-1, allowed us to obtain information on DNA methylation. In particular, in this spectral range, bands due to DNA S-type sugar puckering modes occur, which are sensitive to changes in the DNA sugar conformation induced by cytosine methylation [32]. The possibility to monitor changes in the profile of this spectral region in whole intact cells makes it possible, therefore, to obtain information on the variation of global DNA methylation in the CpG islands. In this way, we found that in the NSN oocytes DNA methylation was high at the antral stage, while it became very low, almost negligible at MII, in agreement with what found for the transcriptional activity pattern at the different maturation stages.

Finally, significant spectral differences were found between 890 and 850 cm-1, where four different bands due to adenine and uracil vibrational modes occur (see Figure 5) [79]. Interestingly, the relative variation of these bands enables to monitor the mRNA polyadenylation extent, a crucial mechanism that regulates transcription. We found, in particular, that NSN oocytes were characterized during maturation by a low level of mRNA polyadenylation, being the polyadenylic acid band at 884 cm-1 absent at MII, while a new band at 854 cm-1 - likely due to adenine possibly not involved in polyA tail [80] – appeared. These results seem to suggest that an inadequate level of mRNA polyadenylation could preclude the possibility to resume meiosis, leaving the NSN oocytes in an unsuccessful transcriptional state.

#### *5.1.2.2. SN oocytes*

210 Multivariate Analysis in Management, Engineering and the Sciences

ribose (921 cm-1) and deoxyribose (898 cm-1) components.

We then analyzed the nucleic acid IR response of NSN and SN oocytes during their maturation, exploring the spectral region between 1000 and 800 cm-1, where RNA and DNA

We found that NSN oocytes maintain, in all the studied stages, an appreciable transcriptional activity as indicated mainly by the simultaneous presence of the RNA ribose component around 921 cm-1 and of the DNA deoxyribose between 895-898 cm-1 - indicative of a DNA/RNA hybrid - whose relative intensities were seen to vary during maturation (see Figure 5A). In particular, the intensity of these two components is higher at the antral stage, while it decreases at MI, to increase again up to MII. These results were also supported by the response of the complex band between 980-950 cm-1, mainly due to the CC stretching vibration of DNA backbone. Indeed, the profile of this band varies depending on the DNA structure that, in turn, could reflect a different nucleic acid activity. In particular, for the NSN oocytes we found that at the antral stage DNA is mainly in A-form - with a triplet at 975 cm-1, 966 cm-1 and 951 cm-1 - typical of the DNA/RNA hybrid during transcription. At MI, the reduction of the 975 cm-1 and 966 cm-1 bands and the appearance of that at 969 cm-1 indicate that DNA is mainly in the B-form, suggesting a sort of transcriptional "stand by state", further supported by the reduction extent of the DNA/RNA hybrid, as discussed above. From this "stand by state" NSN oocytes seem to resume their transcriptional activity at MII, where a coexistence of DNA A and B forms was observed, as indicated by the increase of the ~ 975 cm-1 band and again in agreement with the simultaneous increase of the

**Figure 5.** Second derivative absorption spectra of NSN (A) and SN (B) oocytes in the nucleic acid absorption region. The second derivatives of the FTIR absorption spectra of single oocytes, measured at the antral (continuous line), MI 10 H (dotted line), and MII 20 H (dashed line) stages, are reported in the

1000-800 cm-1 absorption region, after normalization at the tyrosine peak (~1516 cm-1).

*5.1.2. Nucleic acid analysis* 

vibrational modes mainly occur [31, 32].

*5.1.2.1. NSN oocytes* 

The analysis of SN oocytes (Figure 5B) in the spectral range between 1000 and 800 cm-1 led to very different results compared to NSN oocytes (see Figure 5A). Briefly, during all the studied maturation stages, the SN oocyte transcriptional activity was found to be maintained at lower levels than NSN oocytes, as revealed by the analysis of the CC stretching of the DNA backbone (980-950 cm-1) and the monitoring of the ribose (~ 922 cm-1) and deoxyribose (895-898 cm-1) vibrations. These results were supported by the temporal evolution of the DNA methylation bands that suggested a partial CpG methylation at the antral and MI stages, which dramatically increased at MII, contrary to what observed for NSN oocytes.

Noteworthy, while no evidence of mRNA polyadenylation was observed for SN oocytes at the antral stage - as indicated by the absence of the two polyadenylic acid bands around 884 cm-1 and 860 cm-1 - starting from MI the adenine and uracile bands at 870 cm-1 and 850 cm-1 appeared, to then dramatically increase up to MII. These findings likely indicate that SN MII oocytes are characterized by an adequate level of maternal polyadenylated mRNAs, making them ready to sustain a proper embryo development, contrary to NSN oocytes.

#### *5.1.2.3. PCA-LDA analysis*

The above results overall indicate that the IR spectra of oocytes at different maturation stages are very informative in the nucleic acid absorption region, allowing to obtain information on several cell processes simultaneously, including transcriptional activity, DNA methylation, and RNA polyadenylation. For this reason, PCA-LDA analysis was

crucial to disclose the most significant spectral response, enabling to identify the marker bands able to discriminate between the two kinds of oocytes.

Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes 213

We then compared by PCA-LDA the two types of oocytes taken at the same maturation stage. As reported in Figure 7, we found the largest spectral distance at MI (92% classification accuracy), with the components carrying the highest discrimination weight due to A-DNA, likely reflecting differences in the transcriptional activity. In this view, MI stage could be considered a sort of crucial checkpoint, when some molecular

**Figure 7.** PCA-LDA analysis of SN and NSN oocytes in the nucleic acid absorption region (1000 - 800 cm-1). The separation between the two types of oocytes at each maturation stage is reported as average of PCA-LDA scores. The height of the boxes and the whiskers corresponds to 1 and 1.5 standard deviations from the mean values, respectively. The analysis has been performed on the measured

These findings have been strongly supported by the comparison of the SN and NSN oocytes at each maturation stage, altogether. A very good discrimination accuracy (89%) was again found analyzing the nucleic acid absorption region, between 1000 and 800 cm-1, that led to a clear cut separation into two groups (see Figure 8): one containing only the MII SN oocytes, and the other containing all the other SN and NSN stages. In particular, the wavenumbers carrying the highest discrimination weight were found at 926 cm-1 (1.00), due to ribose vibration, and at 855 cm-1 (0.97), assigned to adenine vibration, indicating again that differences in the temporal evolution and extent of transcription and polyadenylation play a crucial role in determining the different oocyte fate: the MII SN oocytes, with their proper content of maternal mRNAs polyadenylated, ready to support successfully the embryonic development; on the other hand, the MII NSN oocytes, with their mRNA lacking the

appropriate polyadenylation, are kept in an unsuccessful transcriptional state.

rearrangements occur, deciding the oocyte fate.

spectra.

Firstly, we analyzed the different maturation stages of each kind of oocyte. In particular, NSN oocytes displayed a segregation into three separated clusters, each corresponding to a maturation stage, with a classification accuracy of about 80%. Noteworthy, the wavenumber with the highest weight (1.0) was that around 880 cm-1, due to polyadenylic acid, that, as revealed by second derivative analysis, was present only at the antral stage and disappeared upon maturation up to MII.

On the other hand, PCA-LDA analysis of SN oocytes led to an excellent discrimination accuracy (97%), with the wavenumbers with the highest discrimination weight at 817 cm-1 (1.0) and 859 cm-1 (0.83). While this last component is due to polyadenylic acid, the assignment of the 817 cm-1 band is not unequivocal, being due to overlapping contributions of DNA and polyadenylic acid.

The above results were then confirmed by the PCA-LDA analysis performed between 1400- 1000 cm-1, where contributions due to nucleic acids, such as sugar-phosphate vibrations, also occur [31]. In particular, for the NSN oocytes the wavenumber with the highest discrimination weight (1.0) was the 1305 cm-1, which is due to free adenine, possibly not involved in polyadenylation [79]. In agreement with the temporal pattern of the adenine band at 870 cm-1, discussed previously, the 1305 cm-1 component displayed a higher intensity at MII, confirming that an inadequate mRNA polyadenylation could preclude NSN oocytes from a successful embryonic development (see Figure 6).

**Figure 6.** Second derivative absorption spectra of NSN oocytes in the absorption region of "free" adenine. The second derivatives of the FTIR absorption spectra of single NSN oocytes, measured at the antral (continuous line), MI 10 H (dotted line), and MII 20 H (dashed line) stages, are reported in the 1330-1270 cm-1 spectral range, where "free" adenine absorbs, after normalization at the tyrosine peak (~1516 cm-1).

We then compared by PCA-LDA the two types of oocytes taken at the same maturation stage. As reported in Figure 7, we found the largest spectral distance at MI (92% classification accuracy), with the components carrying the highest discrimination weight due to A-DNA, likely reflecting differences in the transcriptional activity. In this view, MI stage could be considered a sort of crucial checkpoint, when some molecular rearrangements occur, deciding the oocyte fate.

212 Multivariate Analysis in Management, Engineering and the Sciences

upon maturation up to MII.

of DNA and polyadenylic acid.

(~1516 cm-1).

bands able to discriminate between the two kinds of oocytes.

crucial to disclose the most significant spectral response, enabling to identify the marker

Firstly, we analyzed the different maturation stages of each kind of oocyte. In particular, NSN oocytes displayed a segregation into three separated clusters, each corresponding to a maturation stage, with a classification accuracy of about 80%. Noteworthy, the wavenumber with the highest weight (1.0) was that around 880 cm-1, due to polyadenylic acid, that, as revealed by second derivative analysis, was present only at the antral stage and disappeared

On the other hand, PCA-LDA analysis of SN oocytes led to an excellent discrimination accuracy (97%), with the wavenumbers with the highest discrimination weight at 817 cm-1 (1.0) and 859 cm-1 (0.83). While this last component is due to polyadenylic acid, the assignment of the 817 cm-1 band is not unequivocal, being due to overlapping contributions

The above results were then confirmed by the PCA-LDA analysis performed between 1400- 1000 cm-1, where contributions due to nucleic acids, such as sugar-phosphate vibrations, also occur [31]. In particular, for the NSN oocytes the wavenumber with the highest discrimination weight (1.0) was the 1305 cm-1, which is due to free adenine, possibly not involved in polyadenylation [79]. In agreement with the temporal pattern of the adenine band at 870 cm-1, discussed previously, the 1305 cm-1 component displayed a higher intensity at MII, confirming that an inadequate mRNA polyadenylation could preclude

**Figure 6.** Second derivative absorption spectra of NSN oocytes in the absorption region of "free" adenine. The second derivatives of the FTIR absorption spectra of single NSN oocytes, measured at the antral (continuous line), MI 10 H (dotted line), and MII 20 H (dashed line) stages, are reported in the 1330-1270 cm-1 spectral range, where "free" adenine absorbs, after normalization at the tyrosine peak

NSN oocytes from a successful embryonic development (see Figure 6).

**Figure 7.** PCA-LDA analysis of SN and NSN oocytes in the nucleic acid absorption region (1000 - 800 cm-1). The separation between the two types of oocytes at each maturation stage is reported as average of PCA-LDA scores. The height of the boxes and the whiskers corresponds to 1 and 1.5 standard deviations from the mean values, respectively. The analysis has been performed on the measured spectra.

These findings have been strongly supported by the comparison of the SN and NSN oocytes at each maturation stage, altogether. A very good discrimination accuracy (89%) was again found analyzing the nucleic acid absorption region, between 1000 and 800 cm-1, that led to a clear cut separation into two groups (see Figure 8): one containing only the MII SN oocytes, and the other containing all the other SN and NSN stages. In particular, the wavenumbers carrying the highest discrimination weight were found at 926 cm-1 (1.00), due to ribose vibration, and at 855 cm-1 (0.97), assigned to adenine vibration, indicating again that differences in the temporal evolution and extent of transcription and polyadenylation play a crucial role in determining the different oocyte fate: the MII SN oocytes, with their proper content of maternal mRNAs polyadenylated, ready to support successfully the embryonic development; on the other hand, the MII NSN oocytes, with their mRNA lacking the appropriate polyadenylation, are kept in an unsuccessful transcriptional state.

Multivariate Analysis for Fourier Transform Infrared Spectra of Complex Biological Systems and Processes 215

*Center for Nanotechnology Innovation @NEST, Italian Institute of Technology (IIT), Pisa, Italy* 

D. A. is indebted to the University of Milano-Bicocca (I) for the supporting postdoctoral fellowship. P. M. acknowledges a postdoctoral fellowship from Italian Institute of Technology. S.M. D. acknowledges the financial support of the FAR (Fondo di Ateneo per la

The authors wish to thank Carlo Alberto Redi and his collaborators at the University of Pavia (I) for the collaboration on murine oocyte maturation, and Antonino Natalello of the

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[2] Ami D, Mereghetti P, Natalello A, Doglia SM. Fourier transform infrared microspectroscopy as a tool for embryonic stem cell studies. In: Atwood CS. (ed.) Stem

[3] Heraud P, Nga ES, Caine S, Yu QC, Hirst C, Mayberry R, Bruce A, Wood BR, McNaughton D, Stanley EG, Elefanty AG. Fourier transform infrared microspectroscopy identifies early lineage commitment in differentiating human

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[6] Sandt C, Féraud O, Oudrhiri N, Bonnet ML, Meunier MC, Valogne Y, Bertrand A, Raphaël M, Griscelli F, Turhan AG, Dumas P, Bennaceur-Griscelli A. Identification of spectral modifications occurring during reprogramming of somatic cells. PLoS ONE

[7] Ami D, Mereghetti P, Natalello A, Doglia SM, Zanoni M, Redi CA, Monti M. FTIR spectral signatures of mouse antral oocytes: molecular markers of oocyte maturation and developmental competence. Biochimica et Biophysica Acta, 2011; 1813(6) 1220–

[8] Wood BR, Chernenko T, Matthäus C, Diem M, Chong C, Bernhard U, Jene C, Brandli AA, McNaughton D, Tobin MJ, Trounson A, Lacham-Kaplan O. Shedding New Light on the Molecular Architecture of Oocytes Using a Combination of Synchrotron Fourier

Paolo Mereghetti

**7. References** 

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vibrational spectroscopy. Journal of Biophotonics 2009; 2(11) 656-668.

**Figure 8.** PCA–LDA analysis of SN and NSN oocytes in the nucleic acid absorption region. The PCA– LDA analysis has been carried out on measured FTIR absorption spectra obtained from SN and NSN oocytes at each maturation stage, between 1000 and 800 cm−1. The semi-axes of ellipsoids in the 3D score plot correspond to two standard deviations of the data along each direction.
