Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006.

The effects of heat on the testes of mammals1

B.P. Setchell2

Discipline of Anatomical Sciences, University of Adelaide, Adelaide 5005, Australia.

Abstract increased the time at which infertility began following

testicular heating. Treatment with vitamin E or PBN (N-
Advances since my last review in 1998 on our tert-butyl-α−phenylnitrone, a free radical spin trap but
understanding of the effects of heat on the testis are also a possible source of NO) decreased the effects of
summarized. Techniques used to study these effects local testicular heating on testis weight, while ascorbic
have included exposing the whole animal to a hot acid had no effect; in contrast treatment with PBN
environment, insulation of the whole scrotum or just its increased the effect of temporary induced
neck, immersing the testes in a warm water bath, cryptorchidism. The most surprising finding was that
surgically returning one or both testes to the abdominal induced cryptorchidism actually improved
cavity (induced cryptorchidism) or exposing them to spermatogenesis in juvenile spermatogonial depletion
microwave radiation. Most techniques have been shown (jsd) mice and to some extent in previously irradiated
to affect principally primary spermatocytes and early mice and rats treated with hexanedione.
spermatids, but there is some evidence for effects on
spermatogonia and Sertoli cells. The Leydig cells and Keywords: testis; heat effects.
androgen secretion do not appear to be directly affected.
There is considerable variation in susceptibility Introduction
between individual animals and between different
strains, which appears to be due to differences in the The literature on the effects of heat on the testis
ability of the animals to maintain testis temperature as has already been recently reviewed (Kandeel and
well as in inherent susceptibility of the different germ Swedloff, 1988, Setchell, 1998, Morgenthaler et al.,
cells. The mechanism of cell death appears to be 1999) but as a great deal of new information has been
apoptosis, not necrosis, and may involve reactive obtained since 2000, an update would seem to be
oxygen species (ROS), the tumour suppressor protein justified. The testes of many species of mammals
p53, nitric oxide synthase (NOS), the translocation of descend either during fetal life or shortly after birth into
the proapototic factor Bax from the cytoplasm to a a scrotum, where the temperature is appreciably lower
perinuclear position, the release of cytochrome c from than in the abdomen. However, in some mammals,
mitochondria and several caspases. Animals deficient in notably elephants, hyraxes, edentates, insectivores and
Fas and Fas ligand appear still to be susceptible, making cetaceans, the testes remain inside the abdomen (see
it unlikely that these factors are involved. Setchell and Breed, 2006), although in dolphins, the
Although many authors believe that the effects testes appear to be cooled by venous blood from the tail
of heat are fully reversible, there is now evidence of running into the abdominal cavity and next to the
long-term effects, following either locally applied heat arterial supply for the testes (Rommel et al., 1992;
or temporary induced cryptorchidism. These long-term 1994). In bulls, as in other animals with scrotal testes,
effects appear to be different from those seen following the testes are cooled by countercurrent exchange of heat
irradiation, and may indicate a failure of Sertoli cell between the venous and arterial blood in the spermatic
function. Sperm produced by mice which had been cord (Waites and Moule, 1961; Brito et al., 2004). Heat
exposed to a hot environment bind to ova normally but is lost from the testis and scrotum to the environment
are less able to fertilize in vivo and in vitro, even when through the scrotal skin, which is well endowed with
motile sperm are selected by a swim-up procedure, and sweat glands (see Setchell and Breed 2006). The
many of the resultant embryos do not develop normally. temperature on the surface of the scrotum is lower at its
Blocking caspase activity or release of cytochrome c base than near the neck, but the temperature inside the
reduced the effect of heat. Mice lacking iNOS were testis is almost uniform, even slightly warmer at the
more resistant to the effects of local testicular heating, base (Kastelic et al., 1996; 1997). In rams, the tunica
and inhibitors of xanthine oxidase (an enzyme important dartos muscle is involved in moving the testes closer to
in the production of ROS) reduced the effects of making the body by contracting under cold conditions and
the testis cryptorchid, while mice in which the gene for further by relaxing when hot. The body-scrotal
superoxide dismutase (SOD1, which is involved in the temperature difference is greater in an environment of
breakdown of ROS) had been knocked out were more 6oC and less at 40oC, although during fever induced by
sensitive. Treatment of rats with pregnant mare serum lipopolysaccharide injection or during exercise, scrotal
gonadotropin, thyroxin or low doses of testosterone temperature remains constant while body temperature
__________________________________________________________________
1
Conference paper. International Symposium on Animal Biology of Reproduction, Nov. 15-18, 2006, Belo Horizonte, Brazil.
2
Corresponding author: [email protected]
Phone: +61(8) 8303 5965; Fax: +61(8) 8303 4398
 Setchell. Heat and the testis.

rises (Maloney and Mitchell, 1996). Scrotal surface spermatids are the cells in the testis which are most
temperature in stallions is increased during exercise, susceptible to heat (see Setchell 1998 for earlier
particularly if the animal is fitted with a scrotal references). These findings have since been confirmed
suspensory (Staempfli et al., 2005). using flow cytometry and confocal microscopy of
isolated seminiferous tubules after making the testis
Techniques cryptorchid in hamsters (Vigodner et al., 2003).
However, even when these cells are not killed, there is
The techniques for studying the effects of heat now evidence that some of them may complete their
on scrotal testes are varied and include: (1) exposure of development, but appeared as spermatozoa with
the whole animal to a hot environment; (2) intermittent damaged DNA, as revealed by COMET and sperm
(Mieusset et al., 1992; Arman et al., 2006) or chromatin structure analyses (SCSA, Karabinus et al.,
continuous (Vogler et al., 1991; 1993; Fleming et al, 1997; Sailer et al., 1997; Banks et al., 2005). In bulls,
2004; Walters et al., 2005a; b; 2006) insulation of the these effects are most obvious during the period 12 to
scrotum, or just the neck of the scrotum (Kastelic et al., 21 days after a 48 hour scrotal insulation, so the affected
1996); (3) surgically returning one or both testes to the sperm would have been from cells still inside the testis
abdomen, referred to here as induced cryptorchidism at the time of heating; however, some changes are seen
(Yin et al., 1997; 1998; 2002; Mu et al., 2000; Setchell between 3 and 9 days post-heating, indicating some
and Wahab-Wahlgren, 2001; Zhang et al., 2002; 2004; sensitivity of sperm already in the epididymis
Vigodner et al., 2003; Setchell and de Rooij, 2006); (4) (Karabinus et al., 1997). Likewise in mice, sperm from
immersing the scrotum in a warm water bath (Lue et al, the cauda epididymidis show maximal changes between
1999; 2000; 2002; Lee et al. 1999; Rockett et al., 2001; 10 and 14 days after heating, again these cells would
Setchell et al., 2001; 2002; Zhang et al., 2005); (5) have been still in the testis at the time of heating. There
exposing the testes to microwave radiation (Imig et al., were however some changes at 3 days, and these cells
1948; Gunn et al., 1961; Saunders and Kowalczuk, again would have already been in the epididymis when
1981; Saunders et al., 1983; Kowalczuk et al, 1983). heated (Sailer et al., 1997). Even clearer indications of
The results using this last technique were not included an epididymal effect were seen by Banks et al. (2005),
in my previous review, as it was thought then that the who showed that while COMET changes were apparent
effects may not have be just due to the increase in in caudal mouse sperm at 21, 24, 28 and 32 days post-
testicular temperature, which rose to about 41oC during heat, even greater rises were found between 1 and 24
a 5 minute exposure and to 44oC during a 10 minute hours, with normal values again at 7 and 14 days post
exposure, then cooled to 36oC over the next 6 and 15 heat, showing that sperm in the epididymis were
minutes respectively. Damage to scrotal skin was also affected, as well as cells in the testis. A similar pattern
reported (Gunn et al., 1961) and this may have reduced was seen with SCSA assays, which were elevated
the ability of the animal subsequently to keep its testes between 1 and 6 hours and between 14 and 32 days
cool. Later work has shown that exposure to the post-heat, but normal at 24 hours and 7 days. There was
microwave emission from a mobile phone had about a 30% reduction in the percentage of ram sperm
negligible effects on the testis, although rectal immunoreactive for the post-meiotically expressed
temperature was increased slightly (Dasdag et al., 1999; sperm surface protein PH20 between 17 and 31 days
2003). after 24 h scrotal insulation, although there were
minimal effects on the distribution of the activity on the
Systemic effects sperm head (Fleming et al., 2004). In a follow-up to this
study, SCSA assays showed an increase in the
It must be remembered that heating the proportion of sperm with DNA damage after either 24
scrotum may have systemic effects as well as direct or 48 scrotal insulation, but there was also a spectral
effects on the testes. It has been known for some years shift in the total population of cells staining in the
that heating the scrotum in sheep increased respiration semen, possibly indicating an increase in immature cells
rate even to the extent of cooling the rest of the body (JF Smith and RM McDonald, personal
including the hypothalamus, so that the animal began to communication). A similar shift was also seen in human
shiver as soon as the scrotal heating ceased (Waites, sperm collected after a 24h fever (Evenson et al., 2000).
1962). More recent studies have shown that panting and Even ejaculated human sperm with poor capacitation
reduction in body temperature following scrotal heating characteristics contained fragile DNA after heat
occurred in rams with a fever as well as under normal treatment (40oC for 4 hours) in vitro (Mann et al.,
conditions (Maloney et al., 2003). 2002).
There is also some evidence that Sertoli cells
Cells in testis affected by heat may be affected, in terms of decreased secretion of
androgen binding protein (Hagenas and Ritzen, 1976;
From histological studies, it has been Karpe et al., 1981) and expression of intermediate
concluded that pachytene spermatocytes and early filaments in Sertoli cells is disrupted in cryptorchid

82 Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006

 Setchell. Heat and the testis.

monkeys (Zhang et al., 2004). Dedifferentiation of adult of normal tubules also remained low, at about 20 % for
Sertoli cells has been induced in monkeys by heat the heated and 70% for the previously induced
treatment (Zhang et al., 2006b) cryptorchid testes (Setchell and de Rooij, 2006).
The boundary tissue surrounding the However, there is an important difference between the
seminiferous tubules may also be affected (Kanwar et damage caused by heat and by irradiation; the latter is
al., 1974), but it is not clear if this is a direct effect of thought to result from an arrest of spermatogonial
the heat or a consequence of the disrupted recruitment from Aal to A1 (Porter et al., 2006). Of the
spermatogenesis as the changes were apparent only after affected tubules in the previously induced cryptorchid
7 days. Insulation of the whole scrotum or just the testes, about 35% contained no germ cells, and in the
scrotal neck of Bos indicus bulls for 4 days was others, the most advanced cell types were A
sufficient to produce falls in sperm production and spermatogonia (35%), B gonia or preleptotene
motility without affecting the echotexture of the testis spermatocytes (19%), leptotene or zygotene
(Brito et al., 2003). The sensitivity of cells in the testis spermatocytes (4%), pachytene spermatocytes (9%) or
other than pachytene spermatocytes and early round spermatids (3%); the corresponding values for the
spermatids is apparent from the results of experiments heated testes were 22, 26, 33, 7, 12 and 1% respectively
in which testis weight was reduced after heating even (Setchell and de Rooij, 2006). Similarly, in adult rabbits
when spermatogenesis had been arrested by treatment of made cryptorchid for 13 weeks, the numbers of B
rats with a GnRH agonist and an antiandrogen (Setchell spermatogonia, the various classes of spermatocytes and
et al., 2002). Spermatogonia are generally believed to round spermatids were still below normal 7 weeks after
be unaffected, but there is some evidence that their orchidopexy, with still no elongated spermatids,
susceptibility to radiation is increased by heat treatment although there had been a substantial rise in the
30 minutes previously (Reid et al., 1981). Furthermore, numbers of A spermatogonia at this time (Zhang et al.,
the numbers of A spermatogonia are drastically reduced 2002). These findings suggest that there are local
in rabbits made cryptorchid for 13 weeks but recovered factors, perhaps originating from the Sertoli cells, which
partially 7 weeks after orchidopexy (Zhang et al., 2002) allow spermatogenesis to proceed in some areas, but
In induced cryptorchid mouse testes, which are deficient in other sites where proliferation of
differentiation of A-spermatogonia was blocked, but the spermatogonia is inhibited, but not recruitment from
could be restored if the testis was returned to the Aal gonia.
scrotum (Nishimune et al., 1978; Nishimune and
Haneji, 1981) or if tubule fragments from cryptorchid Individual and strain variation
testes were cultured at scrotal temperature (32.5oC,
Nishimune and Komatsu, 1972; Nishumune and There is considerable variation between
Aizawa, 1978, Aizawa and Nishimune, 1979; Haneji individual animals in their response to heat exposure. Of
and Nishimune, 1982). the six bulls subjected to scrotal insulation by Vogler et
It has been generally accepted that the effects al., (1991; 1993), two showed a large increase in
of heat on the testis are fully reversible, but some recent abnormal spermatozoa (to more than 60%) whereas
studies suggest that this is not so. In a number of studies others had as few as 23% abnormal cells. Likewise, 4
reviewed previously, testis weight had not returned to bulls used for semen collection for in vitro fertilization
control values even 60 days after a single heat exposure showed widely variable effects of 48h scrotal insulation
(Setchell, 1998). Rats, about 50 days old at the start of on pronuclear formation, embryo development and
the experiment, were followed for about 6 months after apoptosis, with two bulls classed as severe responders,
a single exposure of their testes for 30 minutes to 43oC. one a moderate responder and one showing no response
There was an initial decline in testis weight and sperm to scrotal insulation (Walters et al., 2005a;b; 2006). There
numbers but then they showed a return of testis weight was also considerable individual variation in the
and sperm numbers to about 70 and 50% respectively of percentage of PH-20 positive sperm between 4 rams
control after 97 days, followed by a second fall to about following scrotal insulation for 24h (Fleming et al., 2004).
50 and 5% at 182 days. This was accompanied by a In a study on the effects of intermittent scrotal insulation in
decrease in the percentage of histologically normal rams, percent motile sperm at 21 days after the start of
tubules cross-sections from 51% to 6% (Setchell et al., insulation varied from 9 to 35% in 4 animals. In a
2001). In a second experiment, testis weight was still second study, two rams were subjected to intermittent
only 70% of control at 105 days after a single exposure scrotal insulation in two successive years; one showed a
of the testes to heat (Setchell et al, 2002). Making one severe reduction in percent motile sperm on both
rat testis cryptorchid for 48 hours reduces testis weight occasions, while the other was much less severely
to about 60% of control after 21 days (Setchell and affected, even though the temperatures produced in their
Wahab-Wahlgren, 2001), compared with 35% for a testes were similar (Arman et al., 2006).
single 30 minute 43 oC exposure (Galil and Setchell, Bos indicus bulls are less sensitive to the
1988), but the testes after both treatments remained at effects of high temperatures than Bos taurus or
about the same size for up to 185 days. The percentage crossbred bulls, but as they are actually more sensitive

Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006 83

 Setchell. Heat and the testis.

to the effects of scrotal insulation (Brito et al, 2003); eliminated (Yin et al, 1998). In the testis, p53 is
this would appear to be due to the greater ability of confined to primary spermatocytes (Almon et al, 1993;
indicus animals to keep their testes cool (Brito et al, Schwartz et al, 1993) and in cryptorchid monkey testes,
2002). Bos indicus bulls have greater testicular artery p53 is thought to repress the expression of the orphan
length to testicular volume ratios, and smaller testicular receptor TR2, which is localized in germ cells (Mu et
artery wall thickness and arterial to venous distances, al., 2000). However, the earliest observations in this
which may be responsible for greater cooling of the study were made only after one month, when germ cell
arterial blood in the spermatic cord (Brito et al., 2004). loss would have probably been quite extensive.
Differing capacity to keep their testes cool was Furthermore, following heating of monkey testes, p53
also found in mice of two strains, one of which had been RNA is elevated 3 days later, but there is no change in
kept for many generations at 33 oC and bred TR2, while TR3 and TR4 are depressed (Zhang et al.,
successfully under these conditions, but their testes 2006a). A germ cell-specific heat shock protein hsp 105
remained just as sensitive to the effects of direct heating (Itoh and Tashima 1990; 1991) translocates from
(van Zelst et al., 1995). Mice of the MRL/MpJ and cytoplasm to nucleus within 2 days of the testis being
AKR strains are much more sensitive to the effects of moved to the abdominal cavity in rats and hsp105 binds
cryptorchidism than those of A/J, BALB/c, C3H/He or to p53 in cultured testis cells at scrotal temperature
C57BL/6 strain (Kon and Endoh, 2001; Kazusa et al, (32.5oC) but not at 37 or 42oC (Kumagai et al., 2000).
2004; Kon, 2005; Namiki et al, 2005), but these authors Hsp105 mRNA and protein in spermatids fell within 3
did not present any evidence on the testicular days and rose again by day 30 after two exposures of
temperatures achieved. monkey testes to 43 oC for 30 minutes, while hsp60 in
There is also some evidence for the Sertoli cells and spermatogonia rose about 5-fold over
development of thermotolerance in the testes of mice the same period. It was concluded that hsp105 and its
(Marigold et al, 1985) and rats (Shilkina, 1976). complex with p53 might be involved in cell cycle arrest
However, repeated exposure of rat testes to heat or the induction of apoptosis (Zhang et al., 2005). Other
produced a progressive decrease in testis size and an heat shock proteins (hsp 27 and 90, which are found in
increase in the percentage of severely damaged tubules Sertoli cells, spermatogonia, spermatocytes and
(Bowler, 1972), which was attributed to an effect on spermatids), also increase and move from cytoplasm to
spermatogonia. nucleus in isolated testis cells from mice when the
temperature is increased from 37 to 42 oC (Biggiogera et
Mechanism of cellular damage caused by heat al., 1996) although these authors seem to believe that
the mouse testes are normally abdominal! Hsp 70-2,
It was originally claimed that cell death which is uniquely expressed in meiotic phase
following heating was not apoptosis but necrosis, spermatocytes (Dix 1997; Dix et al., 1996; 1997) is not
although this was not accompanied by inflammation induced by increases in temperature, but an increase in
(Allan et al., 1987). However, subsequent studies have the mRNAs for the related proteins hsp 70-1 and 70-3
shown that apoptosis was involved (Shikone et al., could be detected within 4 hours and the proteins
1994; Ohta et al, 1996;Yin et al., 1997; Lue et al., 1999; themselves were increased within 16 hours after
2000; 2002; Rockett et al., 2001; Sinha Hikim et al., exposure of mouse testes for 20 min at 43 oC,
2003a; b). Isolated cells from 40 day-old rats also predominantly in spermatocytes (Rockett et al., 2001).
showed apoptosis when the culture temperature was These authors also employed DNA microarrays to
raised from 32.5oC to 43oC for one hour, followed by a measure expression of genes in the testes from heat-
return to 32.5oC for another 24h. Similar apoptosis shocked mice. Of the 2208 genes studied, 27 were
could be induced in these cells by generating reactive upregulated and 151 downregulated. Among those
oxygen species with xanthine and xanthine oxidase, and upregulated were genes involved in stress responses,
the effects of heat were reduced in the presence of DNA repair and cell adhesion, while those
catalase. A temperature-dependent increase in downregulated included genes involved in cell cycle
intracellular peroxide production by testicular cells was regulation, both in mitosis and meiosis, protein folding,
detected using the fluorescent probe DCFH/DA (Ikeda DNA repair, stress responses and apoptosis, giving an
et al., 1999). Xanthine oxidase inhibitors suppress overall pattern of cellular shutdown. In particular, the
apoptosis induced by experimental cryptorchidism meiotic cell cycle of the spermatocytes appears to be
(Kumagai et al., 2002). It is also relevant that mice in arrested at the G2/M transition, as indicated by changes
which superoxide dismutase had been knocked out were in the G2/M-specific cyclins, A1, B1 and B2 and the M-
more sensitive to the effects of induced cryptorchidism phase inducer phosphatase CDC251 genes (Rockett et
(Ishii et al., 2005). al., 2001). The activity of DNA polymerases
The tumour suppressor protein p53 also α and β was decreased in induced cryptorchid rat testes,
appears to be involved. Mice deficient in this protein while DNA polymerase γ and topoisomerase did not
show a decreased response to induced cryptorchidism, change (Fujisawa et al., 1988). There is also cleavage of
although the fall in testis weight is only delayed, not poly(ADP)ribose polymerase (Sinha Hikim et al.,

84 Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006

 Setchell. Heat and the testis.

2003a; b; Vera et al., 2005). apoptosis induced by experimental cryptorchidism

Apoptosis 4 or 6 h following heating of the (Ishikawa et al., 2005).
mouse testis is greater if the clusterin/ApoJ gene is
knocked out, but similar to wild type after 12 h and less Male-mediated effects induced by heat on fertility
at 24h, while testis weight falls earlier (48h vs 72h post- and embryonic development
heat) in wild type than knockout mice, suggesting that
clusterin delays the initial kinetics of the heat-stress There were a number of suggestions in the
pathway, but clears the damaged cells more quickly earlier literature that males exposed to heat can produce
(Bailey et al, 2002). sperm which do not produce normal offspring in
It has been suggested that Fas is responsible for unexposed females (see Setchell, 1998). These findings
the second phase of apoptosis beginning about 10 days have been extended in two further studies involving
after the testis is made cryptorchid (Yin et al., 2002). male mice exposed in a microclimate chamber to 36oC
Increased expression of Fas, but not Fas ligand RNA for 24 hours (Zhu and Setchell, 2004; Zhu et al., 2004)
can be detected following heat exposure of rat testes or 35 oC for two periods of 12 hours on consecutive
(Lee et al., 1999). Fas expression in germ cells was days (Yaeram et al., 2006). In the former, there were no
increased following induced cryptorchidism, but testis changes in the proportion of eggs showing two polar
weight was still decreased in Fas-deficient lpr/lpr mice, bodies as an indication of fertilization 14 to 16 hours
although not to the same extent as in BALB/c mice (Ogi after mating when this took place with oestrus-
et al., 1998). However, in Fas ligand-defective gld mice, synchronized females on days 7 or 35 days after
heat-induced apoptosis is not blocked, casting more heating, although there was a fall at 21 days. However,
doubt on the involvement of the Fas system in this the proportion of zygotes progressing to 2-cell stage by
process (Sinha Hikim et al., 2003a; b; Vera et al., 2004). 34 to 39 h after mating was reduced when mating
The pro-apoptotic factor Bax is translocated from a occurred 21 or 35 days after heating, and likewise, the
cytoplasmic to a paranuclear localization within half an proportion progressing to 4-cell or morulae by 61 to 65
hour of heating the testis, while total Bax levels in the testis hours was reduced when mating occurred 7 or 21 days
remained constant. The relocation of Bax is accompanied after heating. The proportion reaching blastocyst stage
by cytosolic translocation of cytochrome c and is by 85 to 90 hours after mating was reduced when
associated with activation of the initiator caspase 9 and the mating occurred 7 or 21 days after heating, and the
executor caspases 3, 6 and 7 (Yamamoto et al., 2000; Vera proportion of expanded blastocysts was reduced by
et al., 2004). However, Rockett et al. (2001) detected a mating on day 35. The proportion of abnormal embryos
reduction in Bax expression following heating. at 61 to 65 hours and 85 to 90 hours was increased with
Inhibitors of caspases reduced apoptosis, mating at 21 days post-heat (Zhu and Setchell, 2004).
independently of the cytosolic translocation of When embryos were collected 25 to 28 h after hCG
cytochrome c, and protection was also provided by from mated superovulated females and cultured in vitro,
minocycline, a second generation tetracycline that development to 2-cell stage after 24h of culture was
effectively inhibits cytochrome c release, and in turn normal with mating on day 3 or day 42, but reduced for
caspase activation (Matsuki et al., 2003;Vera et al., days 7, 14, 21, 28 and 35; the 4-cell or morulae after
2005). Ectopic expression of testis-specific calpastatin 48h culture, the 8-cell or blastocysts after 72h, and
(a naturally occurring inhibitor of calpain, a protease blastocysts after 96 or 120 h culture were reduced with
involved in apoptosis) in spermatocytes reduces apoptosis mating on the same days post-heat. The percentage of
in heated mouse testes (Somwaru et al., 2004). abnormal embryos was increased after 48, 72, or 96 h of
Nitric oxide synthase (NOS) appears to have a culture with mating on days 14 to 35, and the proportion
functional role in heat-induced apoptosis since in adult of degenerating embryos after 72, 96 or 120 h culture
inducible NOS (iNOS) deficient mice, the testes were was increased with mating on days 7 to 28, 7 to 35 and
about one-third larger than normal, and furthermore 3 to 35 post-heat respectively (Zhu et al., 2004).
showed much less apoptosis in early (I to IV) and late In the second study, the proportion of ova
(XI to XII) stages of spermatogenesis following reaching 2-cell stage by 24h after mating with
exposure of the testes to 43oC for 15 minutes (Lue et al., superovulated females had fallen slightly when mating
2003). Endothelial NOS (eNOS) is increased in rat occurred7 days post-heat and appreciably with mating
testes made cryptorchid between 20 and 34 days of age, after 10 or 14 days. Using in vitro fertilization, it was
and examined at 60 days old, and appears in the found that the same number of motile sperm from the
degenerating cells as well in its normal location in epididymides of males heated 7, 10 or 14 days earlier
endothelial, Sertoli and Leydig cells (Zini et al., 1999). were less effective in terms of the proportion of ova
In Hoxa11 knockout mice, which have congenitally fertilized, with no effect 3 days after heating. Even
cryptorchid testes, treatment with L-NAME (an when motile sperm were separated from immotile sperm
inhibitor of NOS) reduces the apoptosis and improves in the sample by a swim-up procedure, a similar result
spermatogenesis (DeFoor et al., 2004). Overexpression was obtained. This was not due to a reduction in the
of eNOS in transgenic mice accelerates germ cell number of sperm binding to the zona pellucida, but

Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006 85

 Setchell. Heat and the testis.

there were reductions in the proportion of ova with like gonadotropin or thyroxin, or with a low dose of
sperm in the perivitelline space and in the cytoplasm of testosterone; a higher dose of testosterone was without
the eggs (Yaeram et al., 2006). effect, while no treatments affected the time at which
In another study, bulls were subjected to scrotal fertility returned at between 60 and 85 days after
insulation for 48 hours and semen collected and heating. The beneficial effects of caspase inhibitors and
cryopreserved 2 or 3 weeks later. Following IVF with drugs which inhibit the release of cytochrome c have
swum-up sperm from these samples, there were been mentioned above.
decreased rates of sperm penetration, pronuclear The effects of antioxidants and the free radical
formation (Walters et al., 2006), embryo cleavage, spin trapping agent PBN (α-phenyl-N-t-butylnitrone)
development and blastocyst formation (Walters et al., on the testes were studied by treating rats before and
2005a) with semen collected from two of the bulls three during local heating of the testes or making one testis
weeks after the insulation, but not with semen from two cryptorchid for 48 hours (Setchell and Wahab-
other bulls, or with semen collected after two weeks. The Wahlgren, unpublished results). Antioxidants have been
same two bulls produced embryos with increased caspase shown to reduce the damage to DNA in infertile patients
activity after 8 days in culture, but there was no effect on (Kodama et al., 1997) and PBN reduces the free radical
apoptosis, as judged by percentage of cells positive in the oxidative damage to DNA in thalidomide toxicity
TUNEL procedure (Walters et al., 2005a). (Parman et al., 1999). Vitamin E and PBN significantly
Further work is needed to determine whether reduced the effect on the weight of the testes of heating
these effects are due to changes in the DNA in the 21 days earlier, while the effect if Vitamin C was not
sperm or to a change in other factors, such as the significant. In unilaterally induced cryptorchid rats,
cytosolic phospholipase C introduced into the egg at the
time of fertilization (see Saunders et al., 2002). Vitamin E was ineffective, whereas PBN caused a
greater fall in testis weight than in the controls (Table
Factors affecting sensitivity of testes to heat 1). This may have been because under hyperthermic
conditions, PBN releases nitric oxide (Cui et al., 2006),
The results of Elfving (1950) have already which may have contributed to the apoptosis caused by
been summarised in my earlier review, but no-one the abdominal temperature.
seems to have followed up his observations that the Treatment of rats with adrenaline enhances the
period of infertility following local heating of the testes effect of heating the testes (Murashev, 1984),
of rats can be delayed from about 10 days post-heat to presumably by reducing testicular blood flow (see
more than 20 days by treating the animals with FSH- Setchell and Breed, 2006).

Table 1. The effects of antioxidants on the loss of weight of rat testes 21 days after local heating to 42oC for 20
minutes or of making one testis cryptorchid for 48 hours. Values are means + SEM with numbers of rats in
parentheses.
Treatment Testis weight (mg)
Control unheated 1557 + 38 (5)
Control heated 1143 + 23 (8)###
Vitamin E heated 1239 + 30 (8)**
PBN heated 1211 + 28 (8)*
Vitamin C heated 1198 + 35 (14)
Testis weight as fraction of scrotal testis of same animal
Cryptorchid control 0.606 + 0.016 (8)###
Cryptorchid + Vitamin E 0.599 + 0.043 (8)
Cryptorchid + PBN 0.519 + 0.015 (8)***
Doses used were 200 mg Vitamin E S/C before heating or at time of cryptorchidectomy (0900h), then every 12
hours, 16 mg PBN I/V before heating or I/P at the same times at Vitamin E in the cryptorchid rats and 200 mg
Vitamin C intravenously at start and 10mg/min during testis heating.
*, **, ***: P< 0.05, 0.01 or 0.001 different from heated or cryptorchid controls;
###: P< 0.001 different from control unheated or contralateral scrotal testis.

Interaction between heat and other effects on the testosterone in the testis. The detailed effects on
testis spermatogenesis are different from those after heat, and
it is interesting that a combination of the two treatments
Low doses of testosterone suppress has a greater effects that either alone, leading to the
spermatogenesis in rat, (Lue et al., 1999) and monkeys concept of a “Two-hit” approach to male contraception.
(Lue et al., 2006), paradoxically by suppressing LH Induced cryptorchid testes in rats are less
secretion and thus reducing the concentration of affected than scrotal testes by treatment of the animals

86 Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006

 Setchell. Heat and the testis.

with 2,5-hexanedione, but this appears to be due to a Bailey RW, Aronow B, Harmony JAK, Griswold
reduction in the formation in the testes of pyrroles, a MD. 2002. Heat shock-initiated apoptosis is accelerated
required intermediate step in the hexanedione-induced and removal of damaged cells is delayed in the testis of
injury (Boekelheide et al., 2000). However, the most clusterin/ApoJ knock-out mice. Biol Reprod, 66:1042-
surprising finding is that spermatogonial differentiation 1053.
arrest in juvenile spermatogonial depletion (jsd) mice is Banks S, King SA, Irvine DS, Saunders PTK. 2005.
virtually eliminated by making the testes cryptorchid, Impact of a mild scrotal heat stress on DNA integrity in
whether this was done in animals 4, 12 or 62 weeks old. murine spermatozoa. Reproduction, 129:505-514.
A similar but smaller effect was seen in improving the Biggiogera M, Tanguay RM, Marin R, Wu Y,
tubule differentiation index in irradiated rats from 0.1% Martin TE, Fakan S. 1996. Localization of heat shock
in scrotal testes to 2.1% in those made cryptorchid for 8 proteins in mouse male germ cells: an immunoelectron
weeks, beginning 4 weeks after irradiation. It appears microscopical study. Exp Cell Res, 229:77-85.
that spermatogonial differentiation may be sensitive to Boekelheide K, Eveleth J, Hall SJ. 1990.
reduced temperatures or is inhibited by testosterone Experimental cryptorchidism protects against long-term
only at scrotal but not abdominal temperatures (Shetty 2,5-hexanedione testicular germ cell loss in the rat. J
and Weng, 2004). Androl, 11:105-112.
Bowler K. 1972. The effect of repeated applications of
Conclusions heat on spermatogenesis in the rat: a histological study.
J Reprod Fértil, 28:325-333.
Recent studies on the effects of heat on the Brito LFC, Silva AEDF, Barbosa RT, Kastelic JP.
testis, either applied directly, by insulating the scrotum 2004. Testicular thermoregulation in Bos indicus,
or by making the testis cryptorchid have provided much crossbred and Bos taurus bulls: relationship with
new information on the mechanism of the damage scrotal, testicular vascular cone and testicular
caused to spermatogenesis. They also provide hope that morphology, and effects on semen quality and sperm
a treatment may soon be devised which may protect the production. Theriogenology. 61:511-528.
testes by blocking the apoptosis. Brito LFC, Silva AEDF, Marbosa RT, Unanian MM,
However, other recent evidence has been Kastelic JP. 2003. Effects of scrotal insulation on
obtained that there may be much more long-term effects sperm production, semen quality and testicular
of heat than previously thought. Furthermore, the effects echotexture in Bos indicus and Bos indicus x Bos
of heat may not be confined to cell death in the testis Taurus bulls. Anim Reprod Sci, 79:1-15.
and the consequent fall in sperm numbers in semen, but Brito LFC, Silva AEDF, Rodrigues LH, Vieira FV,
the sperm produced may be less capable of fertilization Deragon LAG, Kastelic JP. 2002. Effect of age and
and the production of normal embryos. In these times of genetic group on characteristics of the scrotum, testes
global warming, these effects may have more serious and testicular vascular cones, and on sperm production
consequences for both the human population as well as and semin quality in AI bulls in Brazil.
for domestic and wild animals. Theriogeneology, 58:1175-1186.
Nevertheless, the main question still remains Cui ZG, Kondo T, Matsumoto H. 2006. Enhancement
unanswered. Why do the testes of some, but not all of apoptosis by nitric oxide release from -phenyl-tert-
mammals require a temperature lower than body butyl nitrone under hyperthermic conditions. J Cell
temperature for normal spermatogenesis to proceed. Physiol, 206:468-476.
Dasdag S, Ketani MA, Akdag Z, Ersay AR, Sari I,
References Demirtas OC, Celik MS. 1999. Whole body
microwave exposure emitted by cellular phones and
Aizawa S, Nishimune Y. 1979. In-vitro differentiation testicular function of rats. Urol Res, 27:219-223.
of type A spermatogonia in mouse cryptorchid testis. J Dasdag S, Akdag MZ, Aksen F, Yilmaz F, Bashan
Reprod Fertil, 56:99-104. M, Dasdag MM, Cellik MS. 2003. Whole body
Allan DJ, Harmon BV, Kerr JFR. 1987.Cell death in exposure of rats to microwaves emitted from a cell
spermatogenesis. In: Potten CS. (Ed.). Perspectives on phone does not affect the testes. Bioelectomagnetics,
mammalian cell death. Oxford: Oxford University 24:182-188.
Press. pp.229-258. DeFoor WR, Kuan CY, Pinkerton M, Sheldon CA,
Almon E, Goldfinger N, Kapon A, Schwartz D, Lewis AG. 2004. Modulation of germ cell apoptosis
Levine AJ, Rotter V. 1993. Testicular tissue-specific with a nitric oxide synthase inhibitor in a murine model
expression of the p53 suppressor gene. Dev Biol, of congenital cryptorchidism. J Urol, 172:1731-1735.
156:107-116. Dix DJ. 1997. Hsp70 expression and function during
Arman C, Quintana Casares PI, Sanchez Partida gametogenesis. Cell stress Chaperones, 2:73-77.
LG, Setchell BP. 2006. Ram sperm motility after Dix DJ, Allen JW, Collins BW, Mori C, Nakamura
intermittent scrotal insulation evaluated by manual and N, Poorman-Alen P, Goulding EF, Eddy EM. 1996.
computer-assisted methods. Asian J Androl, 8:411-418. Targeted gene disruption of Hsp70-2 results in failed

Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006 87

 Setchell. Heat and the testis.

meiosis, germ cell apoptosis and male infertility. Proc Kandeel FR, Swerdloff RS. 1988. Role of temperature
Natl Acad Sci USA, 93:3264-3268. in regulation of spermatogenesis and the use of heating
Dix DJ, Allen JW, Collins BW, Poorman-Allen P, as a method for contraception. Fertil Steril, 49:1-23.
Mori C, Blizard DR, Brown PR, Goulding EH, Kanwar KC, Bawa SR, Singal PK. 1974. Testicular
Srong BD, Eddy EM. 1997. HSP70-2 is required for hyperthermic shocks and the boundary tissue of the
desynapsis of synaptonemal complexs during meiotic semiferous tubules in rats. J Reprod Fertil, 41:201-204.
prophase in juvenile and adult mouse spermatocytes. Karabinus DS, Vogler CJ, Saacke RG, Evenson DP.
Development, 124:4595-4603. 1997. Chromatin structural changes in sperm after scrotal
Elfving G. 1950. Effects of the local application of heat insulation of Holstein bulls. J Androl, 18:549-555.
on the physiology of the testis an experimental study in Karpe B, Ploen L, Hagenas L, Ritzen EM. 1981.
rats. Helsinki, Finland: Veterinary College. Thesis. Recovery of testicular functions after surgical treatment
Evenson DP, Jost LK, Corzett M, Balhorn R. 2000. of experimental cryptorchidism in the rat. Int J Androl,
Characteristics of human sperm chromatin structure 4:145-160.
following an episode of influenza and high fever: a case Kastelic JP, Coulter GH, Cook RB. 1996a. Scrotal
study. J Androl, 21:739-746. surface, subcutaneous, intratesticular, and
Fleming JS, Yu F, McDonald RM, Meters SA, intraepididymal temperatures in bulls. Theriogeneology,
Montgomery GW, Smith JF, Nicholson HD. 2004. 44:147-152.
Effects of scrotal heating on sperm surface protein PH- Kastelic JP, Cook RB, Coulter GH. 1996b.
20 expression in sheep. Mol Reprod Dev, 68:103-114. Contribution of the scrotum and testes to scrotal and
Fujisawa M, Matsumoto O, Kamidono S, Hirose F, testicular thermoregulation in bulls and rams. J Reprod
Kojima K, Yoshida S. 1988. Changes of enzymes Fertil, 108:81-85.
involved in DNA synthesis in the testes of cryptorchid Kastelic JP, Cook RB, Coulter GH. 1997a
rats. J Reprod Fertil, 84:123-130. Contribution of the scrotum, testes, and testicular artery
Galil KAA, Setchell BP. 1988. Effects of local heating to scrotal/testicular thermoregulation in bulls at two
of the testis on testicular blood flow and testosterone ambient temperatures. Anim Reprod Sci, 45:255-261.
secretion in the rat. Int J Androl, 11:73-85. Kastelic JP, Cook RB, Coulter GH, Saacke RG.
Gunn SA, Gould TC, Anderson WAD. 1961. The 1997b. Insulating the scrotal neck affects semen quality
effect of microwave radiation on morphology and and scrotal/testicular temperatures in the bull.
function of rat testis. Lab Invest, 10:301-314. Theriogeneology, 45:935-942.
Hagenas L, Ritzen EM. 1976. Impaired Sertoli cell Kazusa K, Namik Y, Asano A, Kon Y, Endoh D,
function in experimental cryptorchidism in the rat. Mol Agui T. 2004. Difference in spermatogenesis in
Cell Endocrinol, 4:25-34. cryptorchid testes among various strains of mice. Comp
Haneji S, Nishimune Y. 1982. Hormones and the Méd, 54:179-184.
differentiation of type A spermatogonia in mouse Kodama H, Yamaguchi R, Fujuda J, Kasai H,
cryptorchid testes incubated in vitro. J Endocrinol, Tanaka T. 1997. Increase oxidative deoxyribonucleic
94:43-50. acid damage in the spermatozoa of infertile male
Ikeda M, Kodama H, Fukuda J, Shimizu Y, Murata patients. Fertil Steril, 68:519-524.
M, Kumagai J, Tanaka T. 1999. Role of radical Kon Y. 2005. Morphogenetic invstigation of
oxygen species in rat testicular germ cell apoptosis metaphase-specific cell death in meiotic spermatocytes
induced by heat stress. Biol Reprod, 61:393-399. in mice. Anat Sci Int, 80:141-152.
Imig CJ, Thomson JD, Hines HM. 1948. Testicular Kon Y, Endoh D. 2001. Heat-shock resistance in
degeneration as a result of microwave irradiation. Proc experimental cryptorchid testis of mice. Mol Reprod
Soc Exp Biol Med, 69:382. Dev, 58:216-222.
Ishii T, Matsuki S, Iuchi Y, Okada F, Toyosaki S, Kowalczuk CI, Saunders RD, Stapleton HR. 1983.
Tomita Y, Ikeda Y, Fujii J. 2005. Accelerated Sperm count and sperm abnormality in male mice after
impairment of spermatogenic cells in SOD1-knockout exposure to 2.45 GHz microwave radiation. Mutation
mice under heat stress. Free Radic Res, 39:697-705. Res, 122:155-161.
Ishikawa T, Kondo Y, Goda K, Fujisawa M. 2005. Kumagai J, Fukuda J, Kodama H, Murata M,
Overexpression fo endothelil nitric oxide synthase in Kawamura K, Itoh H, Tanaka T. 2000.Germ cell-
transgenic mice accelerate testicular germ cell apoptosis specific heat shock protein 105 binds to p53 in a
induced by experimental cryptorchidism. J Androl, temperature-sensitive manner in rat testis. Eur J
26:281-288. Biochem, 267:3073-3078.
Itoh H, Tashima Y. 1990. A novel testis-specific 105- Kumagai A, Kodama H, Kumagai J, Fukuda J,
kDa protein related to the 90-kDa heat-shock protein. Kawamura K, Tanikawa H, Sato N, Tanaka T. 2002.
Eur J Biochem, 193:429-435. Xanthine oxidase inhibitors suppress testicular germ cell
Itoh H, Tashima Y. 1991.Different expression time of apoptosis induced by experimental cryptorchidism. Mol
the 105-kDa protein and 90-kDa heat-shock protein in Hum Reprod. 8:118-123.
rat testis. FEBS Lett, 289:110-112 Lee J, Richburg JH, Shipp EB, Meistrich ML,

88 Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006

 Setchell. Heat and the testis.

Boekelheide K. 1999. The Fas system, a regulator of perspective. J Androl, 20:189-195.

testicular germ cell apoptosis, is differentially up- Mu XM, Liu YX, Collins LL, Kim E, Chang C. 2000.
regulated in Sertoli cell versus germ cell injury in the The p53/retinoblastoma-mediated repression of
testis. Endocrinology, 140:852-858. testicular orphan receptor-2 in the rhesus monkey with
Lue Y, Sinha Hikim AP, Wang C, Leung A, cryptorchidism. J Biol Chem, 275:23877-23883.
Swerdloff RS. 2003. Functional role of inducible nitric Murashev AN. 1984. Effect of adrenaline on the post-
oxide synthase in the induction of male germ cell heat damage to the spermatogenic function in rats (in
apoptosis, regulation of sperm number and Russian). Biull Eksp Biol Med, 97:471-472
determination of testes size: Evidence from null mutant Namiki Y, Kon Y, Kazusa K, Asano A, Sasaki N,
mice. Endocrinology, 144:3092-3100. Agui T. 2005. Quantitative trait loci analysis of heat
Lue Y, Sinha Hikim AP, Wang C, Im M, Leung A, stress resistance of spermatocytes in the MRL/MpJ
Swerdloff RS. 2000. Testicular heat exposure enhances mouse. Mammal Genome, 16:96-102.
the suppression of spermatogenesis by testosterone in Nishimune Y, Aizawa S. 1978. Temperature sensitivity
rats: the “two-hit” approach to male contraceptive of DNA synthesis in mouse testicular germ cells in
development. Endocrinology, 141:1414-1424. vitro. Exp Cell Res, 113:403-408.
Lue Y, Lasley BL, Laughlin LS, Swerdloff RS, Sinha Nishimune Y, Haneji T. 1981. Testicular DNA
Hikim AP, Leung A, Overstreet JW, Wang C. 2002. synthesis in vivo: Comparison between unilaterally
Mild testicular hyperthermia induces profound cryptorchid testis and contralateral intact testis in
transitional spermatogenic suppression through mouse. Arch Androl, 6:61-65.
increased germ cell apoptosis in adult Cynomolgus Nishimune Y, Komatsu T. 1972. Temperature-
monkeys. J Androl, 23:799-805. sensitivity of mouse testicular DNA synthesis in vitro.
Lue Y, Sinha Hikim AP, Swerdloff RS, Im P, Taing Exp Cell Res, 75:514-517.
KS, Bui T, Leung A, Wang C. 1999. Single exposure Nishimune Y, Aizawa S, Komatsu T. 1978.Testicular
to heat induced stage-specific germ cell apoptosis in germ cell differentiation in vivo. Fertil Steril, 29:95-
rats: Rrole of intratesticular testosterone on stage 102.
specificity. Endocrinology, 140:1709-1717. Ogi S, Tanji N, Yokoyama M, Takeuchi M, Terada
Lue Y, Wang C, Liu YX, Sinha Hikim AP, Zhang N. 1998. Involvement of Fas in the apoptosis of mouse
XS, Ng CM, Hu ZY, Li YC, Leung A, Swerdloff RS. germ cells induced by experimental cryptorchidism.
2006. Transient testicular warming enhances the Urol Res, 26:17-21.
suppressive effect of testosterone on spermatogenesis in Ohta Y, Nishikawa A, Fukazawa Y, Urushitani H,
adult Cynomolgus monkeys (Macaca fascicularis). J Matsuzawa A, Nishina Y, Iguchi T. 1996. Apoptosis
Clin Endocrinol Metab, 91:539-545. in adult mouse testis induced by experimental
Maloney SK, Mitchell D. 1996. Regualtion of ram cryptorchidism. Acta Anat, 157:195-204.
scrotal temperature during heat exposure, cold exposure, Parman T, Wiley MJ, Wells PG. 1999. Free radical-
fever and exercise. J Physiol, 496:421-430. mediated oxidative DNA damage in the mechanism of
Maloney SK, Bonomelli JM, DeSouza J. 2003. Scrotal thalidomide teratogenicity. Nature Med, 5:582-585.
heating stimulates panting and reduces body Porter KL, Shetty G, Meistrich M. 2006. Testicular
temperature similarly in febrile and non-febrile rams edema is associated with spermatogonial arrest in
(Ovis aries). Comp Biiochem Physiol A, 135:565-573. irradiated rats. Eneodcrinology, 147:1297-1305.
Mann SL, Patton WC, King A, Chan PJ. 2002. Reid BO, Mason KA, Withers HR, West J. 1981.
Comparative genomic hybridisation analysis of sperm Effects pf hyerthermia and radiation on mouse testis
DNA apoptosis after exposure to heat shock. J Assist stem cells. Cancer Res, 41:4454-4457.
Reprod Genet, 19:195-2000 Rockett JC, Mapp FL, Garges JB, Luft JC, Mori C,
Marigold JCL, Hume SP, Hand JW. 1985. Dix DJ. 2001.Effects of hyperthermia on
Investigation of thermotolerance in mouse testis. Int J spermatogenesis, apoptosis, gene expression and
Radiat Biol, 48:589-595. fertility in adult male mice. Biol Reprod, 65:229-239.
Matsuki S, Iuchi Y, Ikeda Y. Sasagawa I, Tomita Y, Rommel SA, Pabst DA, McLellan WA, Mead JG,
Fujii J. 2003. Suppression of cytochrome c release and Potter CW. 1992.Anatomical evidence for a counter-
apoptosis in testes with heat stress by minocycline. current hest exchanger associated with dolphin testes.
Biochem Biophys Res Commun, 312:843-849 Anat Rec, 232:150-156.
Mieusset R, Qunitana Casares P, Sanchez Partida Rommel SA, Pabst DA, McLellan WA, Willimas
LG, Sowerbutts SF, Zupp JL, Setchell BP. 1992. The TM, Freidl WA. 1994. Temperature regulation of the
effects of heating the testes and epididymides of rams testes of the Bottlenose Dolphin (Tursiops truncates):
by scrotal insulation on fertility and embryonic Evidence from colonic temperatures. J Comp Physiol B,
mortality in ewes inseminated with frozen semen. J 164:130-134.
Reprod Fertil, 94:337-343. Sailer BL, Sarkar LJ, Bjordahl JA, Jost LK,
Morgenthaler A, Stahl BC, Yin Y. 1999. Testis and Evenson DP. 1997. Effects of heat stress on mouse
temperature: an historical, clinical and research testicular germ cells and sperm chromatin structure. J

Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006 89

 Setchell. Heat and the testis.

Androl, 18:294-301. 2004. Heat-induced apoptosis of mouse meiotic cells is

Saunders RD, Kowalczuk CI. 1981. Effects of 2.45 suppressed by ectopic expression of testis-spedific
GHz microwave radiation and heat on mouse calpastatin. J Androl, 25:506-513.
spermatogenic epithelium. Int J Radiat Biol, Staempfli S, Janett F, Burger D, Kuendig H, Haessig
40:6230632. M, Thun R. 2005. Effect of exercise on scrotal surface
Saunders RD, Darby SC, Kowalczuk CI. 1983. temperature in the stallion. Anim Reprod Sci, 89:237-
Dominant lethal studies in male mice after exposure to 239.
2.45 GHz microwave radiation. Muta Res, 117:345-356. Van Zelst S, Zupp JL, Hayman DL, Setchell BP.
Saunders CM, Larman MG, Parrington J, Cox LJ, 1995. X-Y chromosome dissociation in mice and rats
Royse J, Blayney LM, Swann K, Lai FA. 2002. PLCζ: exposed to increased testicular or environmental
a sperm-specific trigger of Ca2+ oscillations in eggs and temperatures. Reprod Fertil Dev, 7:1117-1121.
embryo development. Development, 129:3533-3544. Vera Y, Diaz-Romero M, Rodriguez S, Lue Y, Wang
Schwartz D, Goldfinger N, Rotter V. 1993. C, Swedloff RS, Sinha Hikim AP. 2004. Mitchondria-
Expression of p53 protein in spermatocytes is confined dependent pathway is involved in heat-induced male
to the tetraploid pachytene primary spermatocytes. germ cell death: Lessons from mutant mice. Biol
Oncogene, 8:1487-1494. Reprod, 70:1534-1540.
Setchell BP. 1998. The Parkes lecture Heat and the Vera Y, Rodriguez, S, Castanares M, Lue Y, Ateinza
testis. J Reprod Fertil, 114:179-194. V, Wang C, Swerdloff RS, Sinha Hikim AP.
Setchell BP, Breed WG. 2006. Anatomy, vasculature 2005.Functional role of caspases in heat-induced
and innervation of the male reproductive tract. In: Neill testicular germ cell apaoptosis. Biol Reprod, 72:516-
JD (Ed.). Knobil and Neill’s physiology of reproduction, 522.
San Diego, USA: Elsevier. pp.771-825. Vigodner M, Lewin LM, Schochat L, Oschry I,
Setchell BP, de Rooij DG. 2006. Long-term effects on Lotan G, Kleen B, Golan R. 2003. Evaluation of
the testis of a short period of unilateral cryptorchidism damage to the testicular cells of golden hamsters caused
in rats. Proc Soc Reprod Biol, 37:253. (abstracts). by experimental cryptorchidism using flow cytometry
Setchell BP, Wahab-Wahlgren A. 2001. Effects of and confocal microscopy. Int J Androl, 26:84-90.
temporary cryptochidism on the testes and the Vogler CJ, Bame JH, DeJarnette JM, McGilliard
subsequent fertility of adult male rats. Proc Soc Reprod ML, Saacke RG. 1993. Effects of elevated testicular
Biol, 32:15. (abstracts). temperature on morphology characteristics of ejaculated
Setchell BP, Ploen L, Ritzen EM. 2001. Reduction of spermatozoa in the bovine. Theriogeneology, 40:1207-
long-term effects of local heating of the testis by 1219.
treatment of rats with a GnRH agonist and an anti- Vogler CJ, Saacke RG, Bame JH, DeJarnette JM,
androgen. Reproduction, 122:255-263. McGilliard ML. 1991.Effects of scrotal insulation on
Setchell BP, Ploen L, Ritzen EM. 2002. Effects of viability characteristics of cryopreserved bovine semen.
local heating of rat testes after suppression of J Dairy Sci, 74:3827-3835.
spermatogenesis by pre-treatment with a GnRH agonist Waites GMH. 1962. The effect of heating the scrotum
and an anti-androgen. Reproduction, 124:133-140. of the ram on respiration and body temperature. Q J Exp
Shetty G, Weng CCY. 2004. Cryptorchidism rescues Physiol, 47:314-323.
spermatogonial differentiaion in juvenile Waites GMH, Moule GR. 1961. Relation of vascular
spermatogonial depletion (Jsd) mice. Endocrinology, heat exchange to temperature regulation in the testis of
145:126-133. the ram. J Reprod Fertil, 2:213-224.
Shikone T, Billig H, Hsueh AJW. 1994. Walters, AH, Saacke RG, Pearson RE, Gwazdauskas
Experimentally induced cryptorchidism increases FC. 2005a. The incidence of apoptosis after IVF with
apoptosis in rat testis. Biol Reprod, 51:865-872. morphologically abnormal bovine spermatozoa.
Shilkima LA. 1976. Effect of hyperthemia on Theriogeneology, 64:1404-1421.
spermatogenesis in mice and the role of heat training in Walters, AH, Saacke RG, Pearson RE, Gwazdauskas
adaptation of the sex cells to high temperatures (in FC. 2006. Assessment of pronuclear formation
Russian) Biull Eksp Biol Med, 82:1489-1491. following in vitro fertilization obtained after thermal
Sinha Hikim AP, Lue Y, Diaz-Romero M, Yen PH, insulation of the testis. Theriogeneology, 65:1016-1028.
Wang C, Swerdloff RS. 2003a. Deciphering the Walters AH, Eyestone WE, Saacke RG, Pearson RE,
pathways of germ cell apoptosis in the testis. J Steroid Gwazdauskas FC. 2005b. Bovine embryo development
Biochem Mol Biol, 85:175-182. after IVF with spermatozoa having abnormal
Sinha Hikim AP, Lue Y, Yamamoto C, Vera Y, morphology. Theriogeneology, 63:1925-1937.
Rodriguez S, Yen PH, Soeng K, Wang C, Swerdloff Yaeram J, Setcheell BP, Maddocks S. 2006. Effect of
RS. 2003b. Key apoptotic pathways for heat-induced heat stress on the fertility of male mice in vivo and in
programmed germ cell death in the testis. vitro. Reprod Fertil Dev, 18:647-653.
Endocrinology, 144:3167-3175. Yamamoto CM, Sinha Hikim Ap, Huynh, PN,
Somwaru L, Li S, Doglio L, Goldberg E, Zirkin BR. Shapiro B, Lue Y, Salmeh WA, Wang C, Swerdloff

90 Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006

 Setchell. Heat and the testis.

RS. 2000. Redistribution of Bax is an early step in an germ cell apoptosis in the heat-treated testes of adult
apoptotic pathway leading to germ cell death in rats, cynomolgus monkeys (Macaca fascicularis). Front
triggered by mild testicular hyperthermia. Biol Reprod, Biosci, 10:3110-3121.
63:1683-1690. Zhang XS, Yuan JX, Liu T, Lue YH, Jin X, Tao SX,
Yin Y, DeWolf WC, Morgentaler A. 1998. Hu ZY, Sinha Hikim AP, Swerdloff RS, Wang C, Lie
Experimental cryptorchidism induces testicular germ YX. 2006a. Expression of orphan receptrs TR2, TR3
cell apoptosis by p53-dependent and –independent TR4 and p53 in heat-treated testis of cynomolgus
pathways in mice. Biol Reprod, 58:492-496. mokeys (Macaca fascicularis). J Androl, 27:405-413.
Yin Y, Hawkins KL, DeWolf WC, Morgentaler A. Zhang XS, Zhang ZH, Jin X, Wei P, Hu XQ, Chan
1997. Heat stress causes testicular germ cell apoptosis M, Lu CL, Lue YH, Hu ZY, Sinha Hikim AP,
in adult mice. J Androl, 18:159-165. Swerdloff RS, Wang C, Liu YX. 2006b.
Yin Y, Stahl BC, DeWolf WC, Morgentaler A. 2002. Dedifferentiation of adult monkey Sertoli cells through
p53 and Fas are sequential mechanism of testicular activation of extracellularly regulated kinase 1/2
germ cell apoptosis. J Androl, 23:64-70. induced by heat treatment. Endocrinology, 147:1237-
Zhang ZH, Hu, ZY, Song, XX, Xiao LJ, Zou RJ, Han 1245.
CS, Liu YX. 2004. Disrupted expression of intermediate Zhu BK, Setchell BP. 2004. Effects of paternal heat
filaments in the testis of the rhesus monkey after stress on the in vivo development of preimplantation
experimental cryptorchidism. Int J Androl, 27:234-239. embryos in the mouse. Reprod Nutr Dev, 44:617-629.
Zhang RD, Wen XH, Jong LS, Deng XZ, Peng B, Zhu BK, Walker SK, Oakey H, Setchell BP,
Huang AP, Wan Y, Yang ZW. 2002. A quantitative Maddocks S. 2004. Effect of paternal heat stress on the
(stereological) study of the effects of experimental development in vitro of preimplantation embryos in the
unilateral cryptorchidism and subsequent orchidopexy on mouse. Andrologia, 36:384-394.
spermatogenesis in adult rabbit testis. Reproduction, Zini A, Abitol J, Schulsinger D, Goldstein M,
124:95-105. Schlegel PN. 1999. Restoration of spermatogenesis
Zhang XS, Lue YH, Guo SH, Yuan JX, Hu ZY, Han after scrotal replacement of experimentally cryptorchid
CS, Sinha Hikim AP, Swerdloff RS, Wang C, Liu rat testis: Assessment of germ cell apoptosis and eNOS
YX. 2005. Expression of HSP105 and HSP60 during expression. Urology, 53:223-227.

Anim. Reprod., v.3, n.2, p.81-91, April/June. 2006 91