Exploring OR2H1-Mediated Sperm Chemotaxis: Development and Application of a Novel Microfluidic Device

Abstract

Microfluidic platforms have emerged as critical technologies for exploring sperm chemotaxis, providing precise gradient control, and facilitating in-depth behavioral assessment. We designed a novel, user-friendly microfluidic device that is optimized for human sperm morphology and motility. The device was validated using two well-established sperm chemoattractants, progesterone and bourgeonal, demonstrating its reliability and reproducibility. Given the key role of olfactory receptors (ORs) in mediating sperm chemotaxis, the newly developed device was employed to identify additional receptors that may contribute to sperm behavior. Using the Atlas database, we identified OR2H1 as a candidate receptor. It is enriched in testis-derived cells, particularly in early and late spermatids, and it is broadly expressed across human spermatozoa. We demonstrated that OR2H1's ligand, methional, a sulfur-containing aldehyde naturally found in vaginal fluid and biosynthesized by Lactococcus lactis, significantly enhances sperm migration and progressive motility. Methional stimulation also triggered increased intracellular calcium levels, indicating receptor activation. Computer-assisted sperm analysis revealed that methional treatment improved sperm linearity, straightness, and wobble without affecting the average velocity, suggesting enhanced directional movement. These findings provide evidence that methional promotes sperm chemotaxis via OR2H1 and highlight the potential role of the vaginal microbiome in influencing human fertility.

Keywords: Lactococcus lactis; OR2H1; chemotaxis; methional; microfluidic device; olfactory receptor.

Figure 1

Schematic representation of the sperm device. The central well serves as the main site for sperm activation and swimming. Well S is used for sperm loading. Wells A and B lead to the respective channels and are designated to introduce the testing solutions (the control and the chemoattractant solution, respectively, in our experiment). The chamber wells are labeled (A, B, S) to facilitate tracking the loading and collection procedures. Further details and a step-by-step protocol are provided in the Materials and Methods section. The ready-to-print 3D file is available in the Supplementary Material, as well as a detailed project of the sperm device.

Figure 2

(A) The histogram shows the migrated spermatozoa recovered in well A (CTR) and well B (P, progesterone). Data are presented as the fold of recovered sperm upon progesterone treatment relative to the CTR untreated well. The number of sperm cells (CTR) recovered in the untreated well was arbitrarily set to 1. The mean ± SDs of five independent experiments are shown (N = 5, * p < 0.05, one-sample t-test). (B) The histogram shows the progressive motility of spermatozoa recovered in well A (CTR) and well B (P, progesterone). Data are presented as the fold of recovered sperm progressive motility upon progesterone treatment relative to the CTR untreated well. The motility of sperm cells (CTR) recovered in the untreated well is arbitrarily set to 1. The mean ± SDs of five independent experiments are shown (N = 5, ** p < 0.01, one-sample t-test). (C) The histogram shows the migrated spermatozoa recovered in well A (CTR) and well B (B, bourgeonal). Data are presented as the fold of recovered sperm upon bourgeonal treatment relative to the CTR untreated well. The number of sperm cells (CTR) recovered in the untreated well is arbitrarily set to 1. The mean ± SDs of five independent experiments are shown (N = 5, * p < 0.05, one-sample t-test). (D) The histogram shows the progressive motility of spermatozoa recovered in well A (CTR) and well B (B, bourgeonal). Data are presented as the fold of recovered sperm progressive motility upon bourgeonal treatment relative to the CTR untreated well. The motility of sperm cells (CTR) recovered in the untreated well is arbitrarily set to 1. The mean ± SDs of five independent experiments are shown (N = 5, ** p < 0.01, one-sample t-test).

Figure 3

(A) OR2H1 tissue-specific enriched RNA expression in the Atlas database (https://www.proteinatlas.org/ENSG00000204688-OR2H1/tissue#rna_expression; accessed on 10 March 2024). The consensus dataset presents normalized expression levels (nTPM, normalized transcripts per million) across 55 tissue types. (B) OR2H1 single-cell type specific RNA expression in the Atlas database (https://www.proteinatlas.org/ENSG00000204688-OR2H1/single+cell; accessed on 10 March 2024). The consensus dataset presents normalized expression levels (nTPM) across 81 cell types.

Figure 4

(A) The histogram shows the migrated spermatozoa recovered in well A (CTR) and well B (M, methional 10 µM). Data are presented as the fold of recovered sperm upon methional treatment relative to the CTR untreated well. The number of sperm cells (CTR) recovered in the untreated well is arbitrarily set to 1. The mean ± SDs of five independent experiments are shown (N = 5, ** p < 0.01, one-sample t-test). (B) The histogram shows the progressive motility of spermatozoa recovered in well A (CTR) and well B (M, methional). Data are presented as the fold of recovered sperm progressive motility upon methional treatment relative to the CTR untreated well. The motility of sperm cells (CTR) recovered in the untreated well is arbitrarily set to 1. The mean ± SDs of five independent experiments are shown (N = 5, * p < 0.05, one-sample t-test). (C,D) Time–course curve of Ca²⁺ influx in seminal fluids treated with PBS (CTR) or methional (M). The fluorescent signals recorded at 0 (starting time point) and 20 s (final time point) are obtained for statistical analysis in comparison to the untreated samples (CTR). The mean ± SDs of three independent experiments are shown (N = 3, p-value = 0.0096, one-sample t-test).

Figure 5

The histograms show the kinematic parameters obtained by the CASA system relative to the sperm cells incubated in the control medium (CTR) or a medium containing methional for 1 and 3 h at 37 °C. In (A), the average curvilinear velocity (VCL, μm/s) is shown; in (B), the average straight-line velocity (VSL, μm/s) is shown; in (C), the average path velocity (VAP, μm/s) is shown; in (D), the average beat cross frequency (BCF, Hz) is shown; in (E), the average amplitude lateral head displacement (ALH, μm) is shown; in (F), the linearity parameter (LIN, %) is shown; in (G), the straightness parameter (STR, %) is shown; in (H), the wobble index parameter (WOB, %) is shown. In (F–H), the kinematic values are represented as the percentage of methional (M)-treated cells with respect to untreated sperm (CTR, arbitrarily set to 100). The mean ± SDs of 5 independent experiments are shown (N = 5. * p < 0.05, ** p < 0.01, *** p < 0.001, one-sample t-test).

Figure 6

Proposed schematic pathway of ectopic OR2H1 in sperm: the binding of methional to the OR2H1 receptor leads to the interaction of the receptor with a GTP-binding protein, Golf. This interaction, in turn, leads to the release of a GTP-coupled Gαolf subunit, which then stimulates adenylyl cyclase III (ACIII) to produce elevated levels of cyclic AMP (cAMP). The increase in cyclic AMP opens the Ca²⁺ channel (CatSper?), leading to an increase in intracellular Ca²⁺ concentration and depolarization of the cell membrane. This intracellular cascade modulates sperm chemotaxis. LIN: the linearity parameter; STR: the straightness parameter; WOB: the wobble index parameter.