Glutathione Peroxidase 5 Is Expressed by the Entire Pig Male Genital Tract and Once in the Seminal Plasma Contributes to Sperm Survival and In Vivo Fertility

Abstract

Glutathione peroxidase-5 (GPX5) is an H2O2-scavenging enzyme identified in boar seminal plasma (SP). This study attempted to clarify its origin and role on sperm survival and fertility after artificial insemination (AI). GPX5 was expressed (Western blot and immunocytochemistry using a rabbit primary polyclonal antibody) in testes, epididymis and accessory sex glands (6 boars). SP-GPX5 concentration differed among boars (11 boars, P < 0.001), among ejaculates within boar (44 ejaculates, P < 0.001) and among portions within ejaculate (15 ejaculates). The first 10 mL of the sperm rich fraction (SRF, sperm-peak portion) had a significantly lower concentration (8.87 ± 0.78 ng/mL) than the rest of the SRF and the post-SRF (11.66 ± 0.79 and 12.37 ± 0.79 ng/mL, respectively, P < 0.005). Sperm motility of liquid-stored semen AI-doses (n = 44, at 15-17°C during 72h) declined faster in AI-doses with low concentrations of SP-GPX5 compared to those with high-levels. Boars (n = 11) with high SP-GPX5 showed higher farrowing rates and litter sizes than those with low SP-GPX5 (a total of 5,275 inseminated sows). In sum, GPX5 is widely expressed in the boar genital tract and its variable presence in SP shows a positive relationship with sperm quality and fertility outcomes of liquid-stored semen AI-doses.

Fig 1. Variation among boars and among ejaculates within boar in the concentration of Glutathione Peroxidase 5 in seminal plasma (SP-GPX5).

Scatterplot showing the concentration of SP-GPX5 of entire ejaculates collected from 11 boars (four ejaculates per boar). Circles show the SP-GPX5 concentration measured in each ejaculate and the lines show the mean for each boar.

Fig 2. Variation in the concentration of Glutathione Peroxidase 5 (SP-GPX5) in seminal plasma among the three different portions of the boar ejaculate.

Box-whisker plot showing variation in the concentration of SP-GPX5 in the three-ejaculate portions: the first 10 mL of spermatozoa-rich ejaculate fraction (SRF, sperm-peak portion), the rest of SRF and the post-SRF of 15 ejaculates (one per boar). Boxes enclose the 25th and 75th percentiles; the line is the median; and the whiskers extend to the 5th and 95th percentiles. (a,b) indicate significant differences (P < 0.005) among ejaculate portions.

Fig 3. Western blot detection of GPX-5 in boar genital tract.

L1: protein ladder; L2: blood vessel (positive control); L3: testis. L4: epididymis. L5: cauda epididymis. L6: seminal vesicle. L7: prostate. L8: bulbourethral gland.

Fig 4. Immunolocalization of glutathione peroxidase-5 (GPX5) in boar genital organs.

Fig 4a depicts a representative positive control section depicting immunostained blood vessels with a major restricted endothelial GPX-5 localization (arrows). A representative negative control prostate section (primary Ab omitted) is depicted in Fig 4b. Fig 4c shows a representative section of boar testis, showing immunostaining in the interstitium (blood vessels and Leydig cells, asterisk) and the seminiferous tubules, with a marked immunostaining in the elongated spermatids (arrows). The immunostaining was more marked in the lining epithelium of the ductus epididymides (Fig 4d, cauda segment) where both the principal epithelial cells (arrows point to the apical cell region, base of the stereocilia) and the luminal spermatozoa (spz) appeared stained, while the surrounding smooth muscle appears only faibly stained (m), lu: lumen. Fig 4e-g depict accessory sexual glands secretory epithelia of the prostate (4e), the seminal vesicles (4f) and the bulbourethral gland (4g). While prostate depicted both nuclear and cytoplasmic staining (4e), the seminal vesicles depicted mainly cytoplasmic staining (arrows) and a clear staining in the luminal secretion (lu), lp: lamina propria. The bulbourethral gland (4g) was immunostained in the blood vessel dominated interstitial with some staining in the latero-basal epithelial membrane (arrows). Not-counterstained sections, viewed with phase-contrast optics. Bars: 50μm.

Fig 5. Relationship between the boar seminal plasma concentration of Glutathione Peroxidase 5 (SP-GPX5) and total sperm motility.

Box-whisker plot showing total sperm motility of artificial insemination semen doses stored for 24 and 72 h at 15–17°C from boar ejaculates hierarchically grouped according to the concentration of SP-GPX5 as high (from 20.74 to 30.14 ng/mL, 15 ejaculates) and low (from 9.64 to 20.04 ng/mL, 29 ejaculates). (*) and (**) indicate significant differences (P < 0.01) and (P < 0.001), respectively, among different SP-GPX5 groups in each storage time.

Fig 6. Potential boar fertility biomarker of boar seminal plasma Glutathione Peroxidase 5 (SP-GPX5).

Nonparametric Receivers Operating Characteristic (ROC) curves showing the ability of boar SP-GPX5 concentration to predict farrowing rates and litter size of artificial insemination semen doses stored 24–72 h at 15–17°C. AUC: area under the ROC curve.