The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

Abstract

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.

Keywords: MSMB; PSP94; boar; capacitation; spermatozoa; ubiquitin-proteasome system; β-microseminoprotein.

Figure 1

Localization of porcine MSMB in ejaculated (A,A’) and in vitro capacitated (B,B´) spermatozoa with a specific polyclonal anti-MSMB antibody (green) by indirect immunofluorescent microscopy. Nucleus was counterstained with DAPI (4′,6-diamidino-2-phenylindole) (blue) and acrosome with PNA (Peanut agglutinin) lectin (red).

Figure 2

A representative flow cytometric histogram of MSMB changes during sperm in vitro capacitation without or under proteasomal (100 µM MG132)/E1 (50 µM PYR41) inhibiting conditions including vehicle control. The mean value of all flow cytometric measurements showed a higher fluorescence intensity in ejaculated spermatozoa (A). Representative image galleries of ejaculated spermatozoa (B), capacitated spermatozoa (B’), and negative control spermatozoa incubated with non-immune serum in place of anti-MSMB antibody (B”). Nuclei were counterstained with DAPI (blue); acrosomal integrity was monitored with lectin PNA (green) and binding of MSMB-Cy5 antibody (red). Every flow cytometric run represents 10,000 events. The experiment was replicated four times.

Figure 3

Quantification of the MSMB removal during in vitro capacitation (IVC). The baseline fluorescent intensity mean of ejaculated spermatozoa was defined as 100%, to which the other IVC sperm groups were compared. (A) The decrease in fluorescent intensity mean in IVC spermatozoa treatment groups, i.e., non-inhibited, proteasomally-inhibited, E1-inhibited, and vehicle control. (B) Graphic representation of fluorescent intensity means in all treatment groups. Results are presented as the mean ± SD of four independent biological replicates. Statistical significance (p < 0.05) is indicated by superscripts.

Figure 4

Western blot detection of porcine MSMB with specific polyclonal anti-MSMB antibody in the protein extracts from ejaculated and IVC spermatozoa under non-inhibiting, proteasomally-inhibited (100 µM MG132), and E1-inhibited conditions (50 µM PYR41), also including vehicle control (DMSO). The black arrow indicates the expected immunoreactive band of MSMB of approximately 12 kDa. Equal protein loads were confirmed by monoclonal antibody anti-α-tubulin DM1A. SDS-PAGE was run under reducing conditions and the experiment was replicated four times, see Figure 5 for densitometric quantification.

Figure 5

Densitometric quantification of 12 kDa immunoreactive MSMB bands from Figure 4 in protein extracts of ejaculated spermatozoa and all capacitated sperm treatment groups. The relative density of MSMB in the blot was calculated as the ratio of the optical density of anti- MSMB and anti-α-tubulin antibodies; the MSMB amount in the ejaculated sperm sample was defined as 100%, and all IVC sperm treatment groups were compared to ejaculated spermatozoa. Results are presented as the mean ± SD of four independent biological replicates. Statistical significance (p < 0.05) is indicated by superscripts.

Figure 6

Immunodetection of porcine MSMB in (poly)ubiquitinated protein sample isolated from the extract of whole ejaculated and IVC spermatozoa using the Signal-Seeker^TM Ubiquitination Detection kit (Ubiq. proteins—Binding fraction) with control detection of polyubiquitinated proteins and reciprocal detection of ubiquitinated proteins in MSMB immunoprecipitate (IP MSMB) from the extract of ejaculated spermatozoa with control detection of MSMB. Black arrows show MSMB (12, 17 and 22 kDa); asterisk indicates antibody heavy chains.