Abstract
Pigs are the only livestock species encoding a functional protein for both the second isoform of gonadotrophin-releasing hormone (GnRH-II) and its cognate receptor (GnRHR-II). Unlike the classical GnRH system (GnRH-I and GnRHR-I), GnRH-II and GnRHR-II are abundantly produced in porcine testes. Moreover, GnRH-II binding its receptor on Leydig cells stimulates luteinizing hormone-independent testosterone secretion. Interestingly, GnRHR-II is also localised to the connecting piece of mature, ejaculated spermatozoa, whereas GnRH-II is detected in seminal plasma, an interaction possibly influencing the function of sperm. To examine the role of GnRH-II and its receptor in the testis, we produced a swine line with reduced endogenous GnRHR-II levels (GnRHR-II KD). The objectives of this study were to (1) compare sperm characteristics between mature GnRHR-II KD and littermate control boars on the day of collection and following semen extension and (2) determine whether a GnRHR-I and GnRHR-II antagonist alters sperm characteristics after storage of extended semen. In Experiment 1, GnRHR-II KD (n=3) and littermate control (n=3) ejaculates were collected (Day 1) and computer-assisted sperm analysis (CASA) was performed (IVOS II Animal; Hamilton Thorne) to determine measures of sperm motion (motility, progressive motility, slow, and static), morphology (normal morphology, bent tail, coiled tail, distal droplet, proximal droplet (PD), distal midpiece reflex, elongation, and area), and kinematics (length of average path (DAP), length of straight line path (DSL), length of curvilinear path (DCL), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), straightness (STR), linearity (LIN), amplitude of lateral head displacement (ALH), beat-cross frequency, and wobble (WOB)). Next, 3 billion sperm were extended with Androstar Plus (80-mL doses; Minitube) and stored at 17°C until Day 7 CASA. Data were analysed with the MIXED procedure of SAS (SAS Institute Inc.). On Day 1, semen doses from GnRHR-II KD boars had reduced DSL, VSL, STR, LIN, and WOB (P<0.05), whereas sperm from control boars possessed more PD (P<0.01). Day 7 CASA revealed that transgenic sperm had reduced DAP, DCL, VAP, and VCL, although sperm from control boars were slower (P<0.05). In Experiment 2, control ejaculates (n=3) were extended as above, treated with increasing concentrations (0, 0.0001, 0.001, 0.01, 0.1, 1, and 10μM) of a GnRH antagonist inhibiting both GnRHR-I and GnRHR-II (SB-75, cetrorelix), and stored at 17°C until Day 7 and 9 CASA. On Day 7, only sperm characteristics in doses treated with 10μM SB-75 were significantly lower (normal morphology, DAP, DCL, VAP, VCL, and ALH) or higher (PD, WOB, and area) than controls. Similar differences (except ALH; P<0.10) for the 10μM SB-75 treatment were detected on Day 9; however, motility, slow, static, STR, and LIN were also reduced (P<0.05). Thus, these data suggest that GnRH-II and its receptor are important to sperm function, representing a potential avenue to improve semen preservation. This research was funded by USDA/NIFA AFRI (2017-67015-26508; BRW).
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