Elsevier

Theriogenology

Volume 131, June 2019, Pages 177-181
Theriogenology

Hyperthermia is more important than hypoxia as a cause of disrupted spermatogenesis and abnormal sperm

https://doi.org/10.1016/j.theriogenology.2019.03.040Get rights and content

Highlights

  • Exposing mice to 36 °C for two, 12-h intervals, had deleterious effects on spermatogenesis, sperm quality and testicular histology.

  • Effects of increased temperature were neither broadly replicated by hypoxia nor mitigated by hyperoxia.

  • Hyperthermia and not secondary hypoxia was the fundamental cause of heat-induced effects on spermatogenesis and sperm.

  • These findings inform evidence-based approaches to mitigate effects of testicular hyperthermia.

Abstract

We tested the hypothesis that hypoxia replicates effects of hyperthermia on reducing number and quality of sperm produced, whereas hyperoxia mitigates effects of hyperthermia. Forty-eight CD-1 mice (∼50 d old), inspired air with 13, 21, or 95% O2 and were exposed to ambient temperatures of 20 or 36 °C (3 × 2 factorial, six groups) twice for 12 h (separated by 12 h at 20 °C and 21% O2), with euthanasia 14 or 20 d after first exposure. Combined for both post-exposure intervals, there were primarily main effects of temperature; mice exposed to 20 vs 36 °C had differences in testis weight (110.2 vs 96.9 mg, respectively; P < 0.0001), daily sperm production (24.7 vs 21.1 × 106 sperm/g testes, P < 0.03), motile sperm (54.5 vs 41.5%, P < 0.002), morphologically normal sperm (59.9 vs 45.4%, P < 0.002), morphologically abnormal heads (7.3 vs 22.0%, P < 0.0001), seminiferous tubule diameter (183.4 vs 176.3 μm, P < 0.004) and altered elongated spermatids (2.2 vs 15.9, P < 0.001). Increasing O2 (from 13 to 95%) affected morphologically abnormal heads (15.4, 10.8 and 17.6%, respectively; P < 0.03), seminiferous tubule diameter (175.7, 185.6 and 178.4 μm, P < 0.003) and total altered spermatids (8.3, 3.3 and 15.2, P < 0.05). Our hypothesis was not supported; hypoxia did not replicate effects of hyperthermia with regards to reducing number and quality of sperm produced and hyperoxia did not mitigate effects of hyperthermia. We concluded that hyperthermia per se and not secondary hypoxia was the fundamental cause of heat-induced effects on spermatogenesis and sperm. These findings are of interest to develop evidence-based efforts to mitigate effects of testicular hyperthermia, as efforts should be focused on hyperthermia per se and not on hyperthermia-induced hypoxia.

Introduction

Testes must remain cooler than body temperature for production of normal, fertile sperm in mammals [1,2], with increased testicular temperature having deleterious effects on sperm production, sperm motility and proportion of morphologically normal sperm [3,4]. The long-standing explanation is the testis operates on the brink of hypoxia under physiological conditions (testicular temperature a few degrees cooler than core body temperature), elevated testicular temperature increases metabolism with a concurrent need for more O2, but blood flow does not significantly increase and consequently hypoxia, secondary to increased testicular temperature, is the major cause of heat-induced changes in spermatogenesis [[5], [6], [7]].

Effects of hyperthermia on testicular function and fertility have been studied in laboratory and farm animals, whereas effects of hypoxia have been studied within the contexts of disrupted blood flow and hypobaric hypoxia, as models of testicular torsion and living at high altitudes, respectively [8,9]. Despite an apparent paucity of studies concurrently examining both conditions, there is impetus for doing so. Male mice exposed to an ambient temperature of 36 °C had increases of ∼1 °C in body temperature and ∼5 °C in testicular temperature at 12 h after the onset of exposure [10]. In a subsequent study [11], male mice exposed to 36 °C for two 12-h intervals (on successive days) and mated to females 10 or 14 d after exposure had significantly lower pregnancy rates and litter sizes. Regarding O2 concentrations, atmospheric air is ∼21% O2, whereas 10.8 and 16.0% O2 in inspired air constituted hypoxia and mild hypoxia, respectively, in rats [12]. Mice breathing air with 12.5, 15.0, 21.0, and 100% O2 had testicular 02 concentrations of 16, 24, 36, and 102 μmol/L [13]. Similarly, breathing 100% O2 reportedly doubled O2 saturation in rat testes [14]. Based on those data, there is an apparent association between O2 content of inspired air and O2 content of the testes, enabling testicular O2 content to be varied from approximately 50 to ≥200% of physiologic concentrations.

The ability to independently alter testicular temperature and testicular O2 content provided a novel opportunity to critically test effects of hyperthermia and hypoxia on spermatogenesis. In a recent ram study [15], insulating the scrotum (testicular hyperthermia) caused expected decreases in motile and morphologically normal sperm. However, it was noteworthy that these effects were neither replicated by hypoxia nor prevented by hyperoxia. To our knowledge, a similar study has apparently never been conducted in rodents. The objective was to determine relative effects of hypoxia versus hyperthermia on sperm quality and production. We tested the hypothesis that hypoxia replicates effects of hyperthermia on reducing number and quality of sperm produced, whereas hyperoxia mitigates effects of hyperthermia.

Section snippets

Experimental design

There were six treatment groups in a 3 × 2 complete factorial design; the factors were O2 concentration (13, 21 and 95% O2) and temperature (20 versus 36 °C). Note that 21% O2 and 20 °C represent atmospheric air and room temperature, respectively, whereas 13% O2 and 36 °C represent hypoxia and hyperthermia. Since inspired air containing 100% O2 is somewhat toxic for mammals [16], CO2 was added to reduce O2 concentration to ∼95%.

Mice and exposure conditions

Male mice (CD-1, ∼50 d of age; n=48) were used. To minimize

Results

There were effects of temperature on most end points involving testes and sperm, including reductions in weights of testis (P < 0.0001) and cauda epididymis (tendency) and in total and progressive motility, percentages of morphologically normal sperm and those with head or tail defects, epididymal sperm reserves (total and per gram), and daily sperm production (Table 1). Only a few histological end points were significantly affected (Fig. 1, Fig. 2). Seminiferous tubule diameter was lower at 13

Discussion

Our hypothesis was not supported; sperm quality and production were not consistently disrupted by hypoxia, nor were hyperthermia-induced disruptions prevented by hyperoxia. Similarly, in a recent ram study [15], insulating the scrotum (testicular hyperthermia) caused expected decreases in motile and morphologically normal sperm. However, as in the present study, in general, these effects were neither prevented by hyperoxia nor were they broadly replicated by hypoxia.

In the present study, mice

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

We thank the animal caretakers at the Lethbridge Research Centre vivarium for their care of the mice and other assistance.

References (36)

  • B.P. Setchell et al.

    Effect of testicular temperature on vasomotion and blood flow

    Int J Androl

    (1995)
  • G.M. Waites et al.

    Effect of local heating on blood flow and metabolism in the testis of the conscious ram

    J Reprod Fertil

    (1964)
  • E. Bustos-Obregón et al.

    Effects of chronic simulated hypobaric hypoxia on mouse spermatogenesis

    Int J Morphol

    (2006)
  • J.G. Reyes et al.

    The hypoxic testicle: physiology and pathophysiology

    Oxidative Med Cell Longevity

    (2012)
  • B. Zhu et al.

    Effect of paternal heat stress on the development in vitro of preimplantation embryos in the mouse

    Andrologia

    (2004)
  • J. Yaeram et al.

    Effect of heat stress on the fertility of male mice in vivo and in vitro

    Reprod Fertil Dev

    (2006)
  • X.Q. Chen et al.

    Effects of hypoxia on glucose, insulin, glucagon, and modulation by corticotropin-releasing factor receptor type 1 in the rat

    Endocrinology

    (2007)
  • D.J. Baker et al.

    Oxygen cathode measurements in the mouse testis

    Phys Med Biol

    (1970)
  • Cited by (18)

    • Changes in miRNA levels of sperm and small extracellular vesicles of seminal plasma are associated with transient scrotal heat stress in bulls

      2021, Theriogenology
      Citation Excerpt :

      Indeed, testicular degeneration disturbance is highly prevalent in many species since several factors are involved in triggering testicular hyperthermia and, thus, testicular degenerative processes, such as high environmental temperature, scrotal traumas, and infectious/febrile processes [7,8]. Although the paradigm referring to hypoxia as the mediator of the testicular heat stress effects has been recently questioned [9], it is postulated that testicular heat stress triggers an increase in cell metabolism associated with higher activity of the epithelial cells from seminiferous tubule [10]. From this perspective, the oxygen level arriving in testis is not enough to supply the increased demand, which creates an environment of hypoxia that results in oxidative stress, DNA fragmentation, and cell apoptosis [11,12].

    • Acute mild heat stress alters gene expression in testes and reduces sperm quality in mice

      2020, Theriogenology
      Citation Excerpt :

      Similarly, in a mouse study [26] with more intense testicular heat exposure (42 °C for 30 min), at 14, 21 and 28 d after exposure, there were reductions in both total motility (from ∼67% to 28, 8 and 37% respectively) and progressive motility (from ∼39% to 5.5, 6.5 and 6.6%). In other studies, when mice were exposed to temperatures of 36–40 °C for longer intervals (several hours or days), impairments in motility occurred at similar time frames [11,12]. In rats exposed to 43 °C for 15 min, apoptotic markers (in situ terminal deoxynucleotidyl transferase-mediated deoxy-UTP end labelling (TUNEL) assay) were highest in spermatids and spermatocytes [34].

    • Amelioration of heat stress-induced damage to testes and sperm quality

      2020, Theriogenology
      Citation Excerpt :

      Caspase-9 and Caspase-3, two hallmarks of apoptosis, are activated after HS in rat germ cells, whereas their pharmacological inhibition prevents germ cell death, implicating caspases in HS-induced death of germ cells [30,31]. Similarly, in a murine model, at 14 d after heat exposure, there was ∼4 fold upregulation (P < 0.05) of Casp 8 (initiator), plus reductions (P < 0.05) in paired testes weight and seminiferous tubules diameter (Rizzoto et al., unpublished data), with the latter two endpoints attributed to apoptosis [32]. One of the main factors involved in this pathway is the Heat Shock Factor 1 (HSF1) gene and its product, HSF1 protein; the latter regulates development of heat shock proteins (HSPs) that confer protection to cells after HS [33].

    • A new paradigm regarding testicular thermoregulation in ruminants?

      2020, Theriogenology
      Citation Excerpt :

      Furthermore, declines in sperm morphology and motility as consequences of heat exposure were not replicated by hypoxia nor prevented by hyperoxia [4,59]. Two studies were performed to study effects of hypoxia and hyperthermia on testes of mice [155] and rams [4]. The first study used 48 mice in a 2 × 3 factorial design involving two temperatures (20 and 36 °C) and three concentrations of O2 in inspired air (13, 21 and 95%).

    • Short-term testicular warming under anesthesia causes similar increases in testicular blood flow in Bos taurus versus Bos indicus bulls, but no apparent hypoxia

      2020, Theriogenology
      Citation Excerpt :

      However, we have substantial evidence challenging this paradigm. In rams [8] and mice [9], whereas hyperthermia (testicular or systemic, respectively) decreased percentages of morphologically normal and motile sperm, breathing hyperoxic air did not prevent effects of hyperthermia, nor did breathing hypoxic air replicate these effects. In Angus bulls, as ambient temperatures increased from 5 to 35 °C, there were increases in testicular temperature (mean ± SEM, 31.8 vs 34.9 °C; P < 0.01) and blood flow (2.45 vs 4.23 mL/min/100 g testis, P < 0.05; [10]).

    View all citing articles on Scopus
    View full text