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components. Given the unique myriad of biochemical changes associated with oocyte growth,
maturation, embryonic development and differentiation, the application of FT-IR can provide
a deeper understanding at the single cell level, merging morphological and molecular assays
(Singh and Sinclair 2007; Wood
et al.
2008). Recently, FT-IR analysis of average spectra
of zebrafish oocytes at different developmental stages (I-II, IIIa, IIIb and IV) has revealed
specific vibrational patterns, correlated with the selective uptake of vitellogenin and with the
maturation process (Figure 12.4; Carnevali
et al.
2009). In particular, ongoing from I-II to IV
class oocytes, an increase in the lipidic and glucidic components, an increase in hydration
and phosphorylation processes and a modification in protein secondary structures were
evidenced by analysing specific vibrational modes (Figure 12.4). In addition, by comparing
representative spectra of vitellogenin and lipovitellin with the chemical map acquired on
a sample of zebrafish ovary, it was possible to visualize the distribution of these two yolk
components (Figure 12.5; Carnevali
et al.
2009).
This technique was recently utilized with a bidimensional focal plane array (FPA) detec-
tor to study the effects of probiotic
Lb. rhamnosus
on zebrafish oogenesis. For all oocyte
classes isolated from both probiotic treated and control zebrafish females, total absorbance
cartograms and the relative chemical maps were obtained (Figure 12.6; Giorgini
et al.
2010).
The results indicated that the probiotic administration mainly influenced oocytes at the later
stages of development, whose representative spectra showed changes in the glucidic and phos-
phate content as well as changes in protein secondary structures. In addition, earlier initiation
of hydration and phosphorylation processes, concomitantly with modifications in protein sec-
ondary structures, were registered at the IIIa follicle stage, indicating that
Lb. rhamnosus
acts
at this follicle stage affecting the maturation phase (Gioacchini
et al.
2012).
In a recent study, the effects of
Lb. rhamnosus
on maturational competence acquiring were
evidenced, with probiotic administration inducing the responsiveness of incompetent follicles
(IIIa) to MIH and their
in vitro
maturation (Gioacchini
et al.
2012). Very few IIIa follicles iso-
lated from control or probiotic treated females underwent spontaneous maturation after 18 h
in the absence of MIH (Table 12.1). However, when exposed to MIH for 18 h a significantly
higher GVBD rate was achieved by IIIa follicles isolated from probiotic fed females compared
to IIIa follicles isolated from control fed females (Table 12.1). The acquiring of competence of
the IIIa follicles was further validated by changes of lhr, mprβ, activinβA1, tgfβ1, gdf9 and oct4
gene expression. From DNA array experiments and from the relative gene ontology (GO) data
analysis, numerous prominent and putatively regulated functions and processes were revealed
to be regulated by
Lb. rhamnosus
during the follicle development. In class IIIa follicles (mat-
urationally incompetent) the probiotic administration modulated several genes belonging to
biological processes (BP) involved in signal transduction and response to chemical stimuli.
Interesting also was its effect in regulating transcripts associated with development (regulation
of anatomical structure size) and regulation of biological quality. The GO analysis of molec-
ular function (MF) in class IIIa follicles revealed that
Lb. rhamnosus
regulated the expression
of two main groups of genes: (1) those involved in the selective interaction of a molecule with
one or more specific sites on another molecule, such as heme binding, tetrapyrrole binding,
iron ion binding, GTP binding and guanyl ribonucleotide binding; and (2) those involved in
oxidoreductase activity and monooxigenase activity. In class IIIb (maturationally competent)
follicles both BP and MF analysis highlighted the probiotic regulation of many genes involved
in transcription including regulation of transcription DNA-dependent (BP), transcription fac-
tor activity (MF), transcription regulator activity (MF) and sequence-specific DNA binding
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