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. 2022 Apr 3;11(7):1208.
doi: 10.3390/cells11071208.

Analysis of Secreted Proteins from Prepubertal Ovarian Tissues Exposed In Vitro to Cisplatin and LH

Serena Marcozzi  1 , Fabiola Ciccosanti  2 , Gian Maria Fimia  2   3 , Mauro Piacentini  2   4 , Cinzia Caggiano  5   6 , Claudio Sette  5   6 , Massimo De Felici  1 , Francesca Gioia Klinger  7
Affiliations

Analysis of Secreted Proteins from Prepubertal Ovarian Tissues Exposed In Vitro to Cisplatin and LH

Serena Marcozzi et al. Cells. .

Abstract

It is well known that secreted and exosomal proteins are associated with a broad range of physiological processes involving tissue homeostasis and differentiation. In the present paper, our purpose was to characterize the proteome of the culture medium in which the oocytes within the primordial/primary follicles underwent apoptosis induced by cisplatin (CIS) or were, for the most part, protected by LH against the drug. To this aim, prepubertal ovarian tissues were cultured under control and in the presence of CIS, LH, and CIS + LH. The culture media were harvested after 2, 12, and 24 h from chemotherapeutic drug treatment and analyzed by liquid chromatography-mass spectrometry (LC-MS). We found that apoptotic conditions generated by CIS in the cultured ovarian tissues and/or oocytes are reflected in distinct changes in the extracellular microenvironment in which they were cultured. These changes became evident mainly from 12 h onwards and were characterized by the inhibition or decreased release of a variety of compounds, such as the proteases Htra1 and Prss23, the antioxidants Prdx2 and Hbat1, the metabolic regulators Ldha and Pkm, and regulators of apoptotic pathways such as Tmsb4x. Altogether, these results confirm the biological relevance of the LH action on prepuberal ovaries and provide novel information about the proteins released by the ovarian tissues exposed to CIS and LH in the surrounding microenvironment. These data might represent a valuable resource for future studies aimed to clarify the effects and identify biomarkers of these compounds' action on the developing ovary.

Keywords: LH; chemotherapy; cisplatin; microenvironment; ovarian follicles; ovary; secretome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Heatmap combined with hierarchical clustering and principal component analysis (PCA) of FCM after 2, 12, and 24 h of treatment. In (A) a heatmap comparison of the 148 secreted metabolites, with two or more unique peptides and a 100% confidence, is depicted. Protein iBAQ values were log2-normalized, and cluster analysis was performed using Z-score protein intensities. Red indicates a high expression level; green indicates a low expression level. In (B) PCA analysis plot of FCM at 2, 12, and 24 h after treatment.
Figure 2
Figure 2
Violin plot representation of the metabolites expressed in each FCM after 2, 12, and 24 h of treatment. Mean log2 iBAQ values of the 148 secreted metabolites were considered at each time point in the analysis. Statistically significant differences between indicated groups * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.
Figure 3
Figure 3
Metabolites’ percentage trend during culture time in CTRL, LH, CIS, and CIS + LH. Appeared: proteins that were not expressed at 2 h but appeared during culture time (red). Disappeared: proteins whose expression disappeared during the culture period (green). Accumulated: proteins already expressed at 2 h time point and whose expression increased during the culture time (yellow). Decreased: proteins expressed at 2 h of culture and whose expression decreased during the 24 h of analysis (black). Variable: proteins with a variable expression mode over time (blue). Stable: proteins whose expression did not change during the period of culture analyzed (purple).
Figure 4
Figure 4
Venn diagrams showing unique and shared proteins between CTRL and the other experimental conditions. The numbers indicate the metabolites counts in the indicated area. (A) Overlaps between CTRL and CIS at 2, 12, and 24 h of treatment. (B) Overlaps between CTRL and LH at 2, 12, and 24 h of treatment. (C) Overlaps between CTRL and CIS + LH at 2, 12, and 24 h of treatment.
Figure 5
Figure 5
Venn diagram showing unique and shared proteins between all experimental conditions. The numbers indicate the metabolites counts in the indicated area. The metabolites are indicated below the diagram for each category, except for those shared among all groups (reported in Table S3). (A) Overlaps between CTRL, LH, CIS, and CIS + LH at 2 h of treatment. (B) Overlaps between CTRL, LH, CIS, and CIS + LH at 12 h of treatment. (C) Overlaps between CTRL, LH, CIS, and CIS + LH at 24 h of treatment.
Figure 6
Figure 6
Venn diagram showing unique and shared proteins between indicated groups. The numbers indicate the metabolites counts in the indicated area. The metabolites are indicated below the diagram for each category, except for those shared among groups considered (reported in Table S3). (A) Overlaps between CTRL, LH, and CIS at 2, 12, and 24 h of treatment. (B) Overlaps between CTRL, CIS, and CIS + LH at 2, 12, and 24 h of treatment. (C) Overlaps between CTRL, LH, and CIS + LH at 2, 12, and 24 h of treatment.
Figure 7
Figure 7
Heatmap comparison of the Mean log2 iBAQ values of metabolites hypothetically implicated in the protective effect of LH against CIS-induced apoptosis. LH was able to (A) inhibit the expression and/or the release in the microenvironment of probable proapoptotic factors induced by CIS in the ovarian tissues (Inhibited); (B) reactivate those of several antiapoptotic factors inhibited by CIS (Reactivated); (C) anticipate the induction of other (Induced early).
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