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The E?ects of Increased Testicular Temperature on Testis-Speci?c Isoform of Na+ ?K+ -ATPase in Sperm and its Role in Spermatogenesis and Sperm Function JC Thundathil1 , GD Rajamanickam1 , JP Kastelic1,2 and LD Newton1

1 Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, Canada;

2 Agriculture and Agri-Food Canada, Lethbridge Research Centre, Lethbridge, AB, Canada Contents Impaired testicular thermoregulation is commonly implicated in abnormal spermatogenesis and impaired sperm function in animals and humans, with outcomes ranging from subclinical infertility to sterility.

Bovine testes must be maintained 4C5°C below body-core temperature for normal spermatogenesis. The e?ects of elevated testicular temperature have been extensively studied in cattle using a scrotal insulation model, which results in abnormal spermatogenesis and impaired sperm morphology and function. Using this model and proteomic approaches, we compared normal and abnormal sperm (from the same bulls) to elucidate the molecular basis of impaired function. We identi?ed a cohort of sperm functional proteins di?erentially expressed between normal vs abnormal sperm, including a testis-speci?c isoform of Na+ ? K+ -ATPase. In addition to its role as a sodium pump regulating sperm motility, Na+ ? K+ - ATPase is also involved as a signalling molecule during sperm capacitation. In conclusion, because of its involvement in regulation of sperm function, this protein has potential as a fertility marker. Furthermore, comparing normal vs abnormal sperm (induced by scrotal insulation) is a useful model for identifying proteins regulating sperm function. Regulation of Testicular Temperature In mammals with a scrotum, testes are maintained 2C6°C (Waites 1970;

Setchell 1978) below core body tempera- ture to ensure normal spermatogenesis;

this is achieved by the combined action of the scrotum, testes and testicular vasculature (Cook et al. 1994). The highly coiled testicular artery and the associated venous network (pampiniform plexus), which are collectively termed the testicular vascular cone, enable counter- current heat exchange between arterial and venous blood vessels, thereby cooling arterial blood before it enters the testis (Cook et al. 1994). In addition, the scrotal muscu- lature (Setchell and Carrick 1973) and tunica dartos keep the testis away from the body, scrotal sweat glands provide evaporative cooling (Blazquez et al. 1988), and radiation of heat from the scrotal surface (Coulter et al. 1988) provides additional cooling. Increased ambient temperature and seasonal climatic changes (Chacon et al. 2002) decrease semen quality and sperm production in cattle (Meyerhoe?er et al. 1985;

Coulter et al. 1997) and swine (Wettemann et al. 1976;

Suriyasomboon et al. 2005). In humans, several envi- ronmental, lifestyle-related risk factors and pathological conditions of the testis (e.g. varicocele) have been implicated in the elevation of scrotal?testicular temper- ature and subsequent deterioration of semen quality (Chen and Chen 2011, Sheykin et al. 2005, Hjollund et al. 2000). The current dogma is that the testis operates on the brink of hypoxia under physiological conditions (Waites and Setchell 1964) and in situations of increased scrotal?testicular temperatures, metabolism and oxygen utilization increase, but blood ?ow to the testis remains constant, resulting in frank hypoxia (Setchell 1998). We tested this hypothesis by exposing mice to environmen- tal temperatures of

20 and 36°C and concurrently exposing them to hyperoxic conditions to prevent hypoxia induced by elevated testicular temperature. Hyperoxia (known to increase testicular oxygen satura- tion) did not protect against hyperthermia-induced deterioration in sperm quality. Furthermore, because sperm characteristics were not signi?cantly di?erent between mice exposed to hypoxic vs normoxic or hyperoxic conditions at 20°C, we concluded that the e?ects of increased testicular temperature on semen quality were because of hyperthermia per se and not hypoxia (Kastelic et al. 2008). It was noteworthy that similar results were obtained with a scrotal insulation model in rams (Kastelic J, Wilde R, Bielli A, Genovese P, Bilodeau-Goeseels S, Thundathil J, unpublished). Abnormal Spermatogenesis Induced by Scrotal Insulation as an Experimental Model for Investigation of Spermatogenesis and Sperm Function in Bulls Effect of scrotal temperature on spermatogenesis and sperm function Increase in testicular temperature, because of elevated ambient temperature or scrotal insulation, impairs spermatogenesis and reduces semen quality and sperm production. Pachytene spermatocytes, spermatids and epididymal sperm are the germ cells most susceptible to heat (Rockett et al. 2001;