Imaging mass spectrometry and shotgun proteomics reveal dysregulated pathways in hormone induced male infertility

Abstract

ABBREVIATIONS MALDI-IMS -Matrix assisted laser desorption/ionisation imaging mass spectrometry LC-MS -Liquid chromatography mass spectrometry WT -Wild type AROM+ -Mice overexpressing human P450 aromatase E2/T -Estrogen/Testosterone ratio ECM -Extracellular matrix ER -Endoplasmic reticulum GO -Gene ontology UPR -Unfolded protein response AbstractSpermatogenesis is a complex multi-step process involving intricate interactions between different cell types in the male testis. Disruption of these interactions results in infertility.Combination of shotgun tissue proteomics with MALDI imaging mass spectrometry is markedly potent in revealing topological maps of molecular processes within tissues. Here, we use a combinatorial approach on a characterized mouse model of hormone induced male infertility to uncover misregulated pathways. Comparative testicular proteome of wildtype and mice overexpressing human P450 aromatase (AROM+) with pathologically increased estrogen levels unravels gross dysregulation of spermatogenesis and emergence of proinflammatory pathways in AROM+ testis. In situ MS allowed us to localize misregulated proteins/peptides to defined regions within the testis. Results suggest that infertility is associated with substantial loss of proteomic heterogeneity, which define distinct stages of seminiferous tubuli in healthy animals. Importantly, considerable loss of mitochondrial factors, proteins associated with late stages of spermatogenesis and steroidogenic factors characterise AROM+ mice. Thus, the novel proteomic approach pinpoints in unprecedented ways the disruption of normal processes in testis and provides a signature for male infertility.

MALDI MS Imaging of Chickpea Seeds (Cicer arietinum) and Crab’s Eye Vine (Abrus precatorius) after Tryptic Digestion Allows Spatially Resolved Identification of Plant Proteins

“…For chickpea sections, two different washing protocols were tested: The first, according to Grassl et al, includes two washing steps for 30 s with 70% ice-cold 2-propanol and a 15 s step with 95% ice-cold 2-propanol. The second was modified from Enthaler et al, using two cycles of 70 and 100% ice-cold ethanol for 30 s each, with 5 dips in ammonium bicarbonate buffer at pH 4.5 in between . The buffer was introduced instead of pure water for better protein fixation due to the isoelectric point of most chickpea proteins.…”

“…The second was modified from Enthaler et al, 46 using two cycles of 70 and 100% ice-cold ethanol for 30 s each, with 5 dips in ammonium bicarbonate buffer at pH 4.5 in between. 47 The buffer was introduced instead of pure water for better protein fixation due to the isoelectric point of most chickpea proteins. The comparison showed that with the latter protocol, some proteins are washed off the section, and the corresponding tryptic peptides could not be detected, as demonstrated for tryptic peptides of 40S ribosomal protein S4 (Accession No.…”

Exploring the Ion Channel TRPV2 and Testicular Macrophages in Mouse Testis

“…In a recent study [ 3 ], proteomes from both WT and AROM + testes of 11 month-old animals ( n = 3, each) were studied by mass spectrometry and the datasets were screened for macrophage markers, inflammation markers, TRPV2 and ROS generating enzymes ( Figure 3 ). The macrophage markers CAPG, CD68, and CD206 were readily found in all three animals of both genotypes with the mean iBAQ intensities being significantly higher in testes from AROM animals (CAPG: p = 0.0097; CD68: p = 0.0484; CD206: p = 0.0007).…”

“…Transgenic mice overexpressing aromatase (AROM + ) have been studied for many years [ 1 , 2 , 3 ]. The hormonal imbalance in these animals causes immunological changes and, in some organs, inflammation.…”

Biogenic amines in the testis: sources, receptors and actions

“…the integration of molecular (DNA, RNA, protein) data and lineage data, possibly with three-dimensional spatial resolution (or even fourdimensional, temporal resolution). Indeed, more and more single cell sequencing studies are already being published (125,126) and together with other approaches (127,128), they eventually will provide a clear and precise picture of the sources of biogenic amines, and their testicular receptors, as well as their changes during development, aging and diseases.…”