Dataset Information

Proteomic Landscape of Human Spermatozoa: Optimized Extraction Method and Application

ABSTRACT: Human spermatozoa proteomics exposed to some physical, biological or chemical stressors is being explored. However, there is a lack of systematic comparative study on protein extraction methods to achieve in-depth coverage. Meanwhile, it is not clear if antibiotics can regulate proteins to affect sperm quality. Here we systematically compared and optimized the sperm protein extraction methods. It showed that the UA_Ultrasonication extraction method can make more kinds of protein to obtain higher protein extraction rate. Applying the optimized method, a largest human sperm proteome (5685 protein groups) from healthy human sperm samples was described. Besides, we continually investigated for the first time the differential sperm characteristics between medication (amoxicillin and clarithromycin) period and withdrawal period’s sperm samples obtained from a patient with Helicobacter pylori infection using semen examination, morphological analysis, bioinformatic analysis and data-independent acquisition tandem mass spectrometry (DIA-MS/MS). The longitudinal study result indicated that antibiotics can disrupt proteome expression in sperm, leading quantitative coverage of 159 differentially expressed proteins. The reliability of the results was verified by four proteins (ACTB, SCRN2, FDFT1, PLPP1) quantified by western blot. These proteins were mainly located at cytosol, involved in phosphorylation process, adenosine triphosphate (ATP) binding and metabolic pathways using bioinformatic analysis. This work provides an optimized extraction method to characterize the in-depth human sperm proteome and to extend its clinical applications. In addition, antibiotics should be avoided when it is necessary to preserve sperm quality for reproductive purpose.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Testis

SUBMITTER: Yong Zhang

LAB HEAD: Yong Zhang

PROVIDER: PXD036023 | Pride | 2022-12-18

REPOSITORIES: Pride

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			Action	DRS
	20220521_Sperm_M-A-R1.raw	Raw
	20220521_Sperm_M-A-R2.raw	Raw
	20220521_Sperm_M-A-R3.raw	Raw
	20220521_Sperm_M-B-R1.raw	Raw
	20220521_Sperm_M-B-R2.raw	Raw

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Project description:Background: The cryopreservation of semen is crucial for conserving genetic diversity and reproductive success in endangered species like the Siberian sturgeon. However, this process can cause cryodamage, affecting quality and protein profile of spermatozoa. While cryoprotectants like dimethyl sulfoxide (DMSO) and methanol (MeOH) facilitate post-thaw motility recovery, DMSO-preserved spermatozoa exhibit reduced fertilizing ability. This study investigates how DMSO and MeOH impact the proteome of Siberian sturgeon spermatozoa and examines semen quality parameters. Two complementary approaches of quantitative proteomics, liquid chromatography-mass spectrometry (LC-MS) and two-dimensional difference in gel electrophoresis (2D-DIGE), were used to analyze the proteomic profiles of fresh and cryopreserved spermatozoa, as well as the extracellular medium (EM; n=7 for each group). Results: Cryopreservation led to a decline in motility parameters (MOT, VCL, PROG) and viability, along with an increase in ROS levels, membrane fluidity, and acrosome damage. Despite similar quality parameters between DMSO and MeOH-preserved sperm, DMSO-preserved sperm showed dramatically lower fertilization success (6.2% vs 51.2%). A total of 224 and 118 differentially abundant proteins in spermatozoa cryopreserved with MeOH and DMSO, respectively, were identified compared to fresh samples, with 342 and 363 proteins released into the EM. The most affected proteins by cryopreservation included H2A, CABYR, PYGM, ENO3, DBI and LTA4H. Additionally, 36 and 39 uniquely altered sperm-leakage proteins were identified for MeOH and DMSO cryopreserved samples, respectively. Bioinformatic analysis showed that MeOH-specific proteins were related to chromosomal structure and mitochondrial functionality, while DMSO-specific proteins were mainly involved in acrosome reaction, zona pellucida binding, flagella structure, and nuclear pore organization. These proteins are potentially involved in sturgeon sperm fertilizing ability. The expression of six proteins was verified by western blot analysis. Conclusions: This study provides the first comprehensive proteomic characterization of Siberian sturgeon spermatozoa after cryopreservation with DMSO and MeOH, revealing insights into proteomic changes that affect fertilizing ability and aiding conservation efforts for this endangered species.

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Proteomic Landscape of Human Spermatozoa: Optimized Extraction Method and Application

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