Relationship between HSP90a, NPC2 and L-PGDS proteins to boar semen freezability

Abstract

Background: The purpose of this study was to determine the association of three proteins involved in sperm function on the freezability of porcine semen: the heat shock protein 90 alpha (HSP90a), the Niemann-Pick disease type C2 protein (NPC2), and lipocalin-type prostaglandin D synthase (L-PGDS). Six adult boars (each boar was ejaculated three times, 18 in total) were classified by freezability based on the percentage of functionally competent sperm. The male semen with highest freezability (MHF) and the male semen with lowest freezability (MLF) were centrifuged immediately after collection to separate seminal plasma and spermatozoa to make four possible combinations of these two components and to incubate them for 3 h, adjusting the temperature to 17 °C, to freeze them afterwards. The quantification of proteins was performed in two stages: at zero and at 3 h after incubation of the four combinations.

Results: The spermatozoa × incubation time (IT) interaction only had effect (P < 0.01) on HSP90a levels; this protein increased in seminal plasma, after 3 h of incubation, in larger quantity (P < 0.05) in combinations with MLF spermatozoa. In relation with the NPC2 protein, two isoforms of 16 and 19 kDa were identified. The 19 kDa isoform was affected (P < 0.01) only by the seminal plasma × IT interaction, with superior values (P < 0.01) both at zero and three hours of incubation, in the combinations with MHF seminal plasma; and 16 kDa isoform was affected (P < 0.01) only by the IT with reduction after 3 h of incubation. The levels of L-PGDS was affected (P < 0.01) only by the spermatozoa × IT interaction, which reduced (P < 0.01) in combinations with MLF spermatozoa after 3 h of incubation.

Conclusions: It is possible to consider that the three proteins evaluated were associated with freezability of boar semen due, especially, to the fact that mixtures with MLF spermatozoa showed greater increase levels of the HSP90a protein and reduction of L-PGDS in plasma. In addition, the seminal plasma of MHF had higher concentration of the NPC2 of 19 kDa protein, which was reduced by incubating with MHF spermatozoa.

Keywords: Boar freezability; HSP90a; L-PGDS; NPC2; Semen preservation; Seminal plasma.

Fig. 1

Effect of interaction spermatozoa per incubation time on the concentration of HSP90a in seminal plasma. RCU: relative concentration units. Different letters indicate highly significant differences (P < 0.01)

Fig. 2

A, Polyacrylamide gel. B, PVDF membrane with anti-HST90a antibody. WM: weight marker, 1: positive control, 2: seminal plasma of MHF, 3: seminal plasma of MLF, 4: HFS + HFSP, 5: LFS + HFSP, 6: HFS + LFSP, 7: LFS + LFSP, 8: negative control with BSA

Fig. 3

A, Polyacrylamide gel. B, PVDF membrane with anti-NPC2 antibody. WM: weight marker, 1: Positive control, 2: seminal plasma of MHF, 3: seminal plasma of MLF, 4: HFS + HFSP, 5: LFS + HFSP, 6: HFS + LFSP, 7: LFS + LFSP, 8: negative control with BSA

Fig. 4

Effect of incubation time on the concentration of NPC2 of 16 kDa. RCU: relative concentration units. Different letters indicate highly significant differences (P < 0.01)

Fig. 5

Effect of seminal plasma interaction per incubation time on the concentration NPC2of 19 kDa. RCU: relative concentration units. Different letters indicate highly significant differences (P < 0.01)

Fig. 6

A, Polyacrylamide gel. B, PVDF membrane with anti-L-PGDS antibody. WM: weight marker, 1: positive control, 2: seminal plasma of MHF, 3: seminal plasma of MLF, 4: HFS + HFSP, 5: LFS + HFSP, 6: HFS + LFSP, 7: LFS + LFSP, 8: negative control with BSA

Fig. 7

Effect of spermatozoa interaction times incubation time on L-PGDS concentration. RCU: relative concentration units. Different letters indicate highly significant differences (P < 0.01)