INTRODUCTION
Mammalian population maintenance and perpetuation on a
certain environment relies on sexually mature individuals. Androgen production,
particularly testosterone (T) in adult males with fully functional reproductive
physiology, regulates such a complex and contrasting functions like spermatogenesis
and mating behavior patterns (Komori et al., 2007; Nelson, 2005). In
seasonal species, testes are actively involved in sperm and androgens production
only during the reproductive season and can be affected by environmental
conditions (Sadleir, 1969; van Tienhoven, 1983). Androgen
production and secretion is necessary for
other functions besides the reproductive processes,
such as territory delimitation and defense,
or group and kin recognition (Wyatt, 2009). Endocrine
function of testis can be determined in males of various species by evaluating T
contents in blood (plasma or serum), and
associating its circulating content to particular events in the animal reproductive
biology, such as the increased size of testicles
known as recrudescence (Aire, 2007; Bentley, 1998; Norris, 2000).
Testis recrudescence is a phenomenon
representing a flurry increase in testicular gamete production
(spermatogenesis) associated with a
general increase in both volume and cell numbers. In mammals, this phenomenon
is associated to the activity of gonadotrophin
hormones affecting both germinal and somatic testis cells, giving rise to a
volume increase, as well as the descending of the organ to the scrotum (Aire, 2007; Clay &
Clay, 1992; Pelletier & Almeida, 1987).
In wild mice of genus Peromyscus, population dynamics
of reproduction (reproductive pattern) have been customarily reported, based on
the abundance of males with scrotal and
large testes, together with pregnant/lactating females, along
time (Kirkland & Layne, 1989; Kunz et al.,
1996), especially on high latitudes. On the other hand, scrotal recrudescent
testes have been related to production and content of T, only in a few males of
Black-eared deermouse (Castro-Campillo et al., 2012; Salame-Méndez et al., 2008; Salame-Méndez et al., 2005; Salame-Méndez et al., 2004), even
though relevant information of such processes of gonadal
physiology must be documented to really link position, size of testes, and content
of T.
In order to learn more about
the reproductive biology of Peromyscus, inhabiting mid
latitude, temperate forests, we have used two species as study models,
in periurban forested zones of Mexico City at “Cumbres del Ajusco” and “Desierto de los
Leones” Nationals Parks (Castro-Campillo et
al., 2012; Castro-Campillo et al., 2008; Salame-Méndez et al.,
2008; Salame-Mendez et al., 2005; Salame-Mendez et al.,
2004). One of such species, the Black-eared deermouse, Peromyscus melanotis, is a quasi-endemic species of Mexico (Álvarez-Castañeda, 2005; Castro-Campillo et
al., 2014; Castro-Campillo et al., 2005), whose reproductive activity occurs all year round in both studied
areas, but with two distinctive peaks, occurring during the summer and trough
autumn-winter, respectively (Castro-Campillo et
al., 2014; Castro-Campillo et al., 2008; Salame-Méndez et al.,
2008; Salame-Méndez et al., 2004).
From our previous studies,
we know that both production and intra-testicular androgen contents are higher
during summer and decrease from autumn to winter in adult males of P. melanotis
in both forested areas (Castro-Campillo et
al., 2012; Castro-Campillo et al., 2005; Salame-Méndez et al.,
2004). Therefore, to relate physiological evidence to a particular
morphological response, we wondered how the profile of T was related to
recrudescence processes of testis along a year in this species. To address this
question, we documented monthly circulating contents of T, together with
changes in testicular volume, in adult
males of Peromyscus melanotis,
along a year.
MATERIALS AND METHODS
Collecting rodents
Adult males of Peromyscus melanotis
were monthly collected at Cumbres del Ajusco National Park (0.85 Km N, 3.5 Km W Ecuanil, 3180 msnm, CDMEX, 19° 13´ 37” N, 99° 15´ 37” W),
using Sherman traps (8 x 9 x 23 cm, Tallahassee, FL, USA),
baited with oat flakes, along two years. Selection of adult individuals was
made using conspicuous somatic and diagnostic characters of the species, such
as size and pattern of pelage color (Ávarez-Castañeda, 2005; Castro-Campillo et
al., 2014; Castro-Campillo et al., 2005). Trapped mice were transferred to laboratory facilities at UAM-Iztapalapa, and killed by cervical dislocation the same
day. Each individual was conventionally sexed, measured, and weighted (Kunz et al.,
1996; Ramírez-Pulido et al., 1989). Capture of rodents was
made according to the Scientific Collector Permit from the National Ministry of
Natural Resources (SEMARNAT) and all animal manipulations
were made according to international standards (NIH,
2011) and approved by the UAM-I CBS Ethic Commission.
Samples
Blood samples were taken from each adult mouse from the heart by cardiac
puncture and poured into EDTA tubes. Plasma was obtained by centrifugation of each
tube to 3000 x g x 5 mn at room
temperature, and then transferred into Eppendorf
tubes to be stored at -20 ºC, until androgen quantification. Testes were removed
and were measured (width x length, mm) conventionally to the nearest 0.01 mm.
Their volume was calculated using the geometric formula for a prolate spheroid: V = 4/3 pa2b, where a
and b are the respective semiaxes, or half axes, of the minor (width) and the major (length)
axes, respectively (Castro-Campillo et
al., 2012). Corpses of mice were also used in another study, therefore, their remains were
prepared as skull and skeleton (Ramírez-Pulido et al., 1989) to be housed as voucher osteological
specimens in the mammal scientific collection at Universidad Autónoma Metropolitana-Iztapalapa
(UAMI). After skulls were biologically cleaned with dermestid beetles (Salame-Méndez et
al., 2008), each individual was assigned to an age category, using wear of the occlusal surface of cheek-teeth sensu (Hoffmeister, 1951).
Quantification of testosterone (T)
Methods for valuation of T in plasma by radioimmunoassay (RIA) have been made according
to Salame-Méndez et
al. (2005) with some modifications. Briefly, 50 µL aliquot was taken from each
plasma were transferred to an Eppendorf tube and
added a phosphate buffer (0.25 M, pH 7, with sodium azide
and gelatin to 1%), which contained a diluted solution of specific antiserum
and tritiated T as a tracer; the tubes were kept at 4
ºC for 18 hrs. After this time, each tube was added 100 µL of a diluted
solution of activated charcoal-dextran, separating
the steroid bound to the antibody by centrifugation. The supernatant was
decanted to vials and these were added Instagel (Packard). Amount of free radioactive steroid was
measured in a liquid scintillation spectrophotometer (Beckman, LS-7000), with a maximum efficiency for tritium of 53%. RIA method was validated by means of a standard curve;
being the coefficient of variation intra-assay < 4%. Quality control of each
RIA was made, according to international
specifications of accuracy, precision, and sensitivity (Cekan, 1976; Rodbard, 1974).
Statistical analysis
To
determine possible monthly differences in the annual profile of plasma T
contents, and testicular volume, we used analysis of variance (ANOVA), followed
by Tukey’s multiple comparison tests. Both monthly
data of T contents and testis size (volume) were plotted, and a polynomial
regression model of second grade (parabole) was
fitted to their pattern; then the R2 was
calculated assuming that a good empirical fit was achieved when R2 ≥ 0.7. All statistical analyses were
carried out at α £ 0.05, using the algorithms of the statistical package
GraphPrisma (Motulsky,
1999) and NCSS Data Analysis (version 11, http:// www.ncss.com/ software/ncss/demo). The
former and Excel were used to plot results, beginning with December data, for
the sake of simplicity.
RESULTS AND DISCUSSION
We analyze 83 adult males of P. melanotis (January n = 12; February n = 11; March n =
6; April n = 5; May n = 6; June n = 2; July n = 6; August n = 6; September n =
5; October n = 4; November n = 8; December n = 12). There were no significant
monthly differences (P < 0.05), between mice of both years when their plasma
T contents were compared. Thus, monthly results of both years were pooled as
adult male mice.
Androgen plasma profile (Figure 1A) of adult mice followed a pattern of increase from the
colder and drier months to the milder and wettest ones in the year; this
profile showed significant differences among some monthly means (F = 22.95, df = 11, 71, 82, P < 0.0001, Table 1). Low contents of
plasma T lasted from December to February, without significant differences
among these months. Up onto March, there was a noticeable difference, since the
rising of plasma T was drastic and
significant. From then until July, there was a steady increase with plenty of overlap of the standard deviations of
T contents, with only low significant differences between March and the other
months. Contents of plasma T reached its highest peak in July, from which it
started to decrease gently towards August. However, lowering of T was both
drastic and statistically significant from August to September and from October
to November. Indeed, contents of plasma T
reached its lowest amount in the latter month, which is also statistically
different from all winter months.
As contents of plasma T, testicular
recrudescence (volume) of adult mice (Figure 1B,
Table 1), also showed an overall monthly pattern subjected to climate changes,
but with some noticeable differences (F = 5.21, df = 11, 71, 82, P < 0.001, Table 1). During the
three colder and drier months (December-February), and March, testes volume
fluctuated up and down with no major significant differences, except between
January and December or February. From March until May, the recrudescence of testes increased sharply with
noticeable significant differences between March and April, and between the
latter and May, when testes reached its largest size. Then, testes steadily
lost volume until August, with no significant differences. From August to
September, there was a drastic and statistically significant reduction of
volume; but from then on, reduction of testes
became steady again and without significant differences until November.
Both curves of raw means and its standard
deviation (Figure 2) were better-fitted (R ≥ 0.7), using a polynomial
regression model of second degree (parable, y = ax2+bx+c,
Figure 2). Equation for the resulting parable in monthly contents of plasma T
was y = 2.72 x2 + 36.63 x - 26.59 (R =
0.73), while that of testicular recrudescence was y = 5.87 x2
+ 74.29 x - 41.84 (R = 0.72). Parables (Figure 2) verified that both the
androgen profile and testicular recrudescence are processes of raise and fall during the year. However, there
is a decoupling between both parables since raising of plasma T, precedes that
of testicular recrudescence and keeps on going within a month of difference; e.g., testicular
volume is triggered by plasma T to reach its maxima in May, while the latter
reaches its own maxima in June.
Finally, it should be noted that it was
noteworthy that even during the harsh cold season; all animals had a good
morphological profile with no evidences
of fasting or other evident alterations in body
or coat morphology or texture that could indicate an alteration of the global
health. Reproductive activity of free-living wild male mice, is usually assumed
from considering only external somatic characters, such as size and location of
testes, which in turn implies that
individuals with increased testicular size are the ones in which falls
reproductive activity (Bronson & Heideman, 1994; Hirschenhauser & Oliveira, 2006; Kunz et al., 1996; Layne, 1968; Lee, 2004; Ramírez-Pulido et al., 1989; Romero-Almaraz et al., 2007).
In the study area, considering testicular
recrudescence, together with number of pregnant/lactating females and number of
implants, fetus, and newborns, Peromyscus melanotis has two reproductive peaks: a
main one during the summer and another, but minor, during autumn-winter (Castro-Campillo et al., 2012; Salame-Méndez et al., 2004). Since both contents of circulating T and
testicular recrudescence, lowered as months became colder and drier from
September to February, while raised as months became milder and more humid from
March to August, this behavior suggests
that gonadal endocrine function depends on
environmental conditions (Figures 1, 2, Table 1). That is, both processes are
more efficient during the milder-rainy season and less efficient during the
general cold-dry one. This is reinforced by our previous studies in which
testicular androgen production is related to the season of the year, being
higher during spring-summer and lower during autumn-winter (Salame-Méndez et
al., 2004). Therefore, circulating content of T is a reflection of gonadal steroidogenic activity in
adult mice of Peromyscus melanotis,
which in turn, is associated, though decoupled (Figure 2), with gonadal recrudescence.
Figure
1. Monthly profile of plasma testosterone contents (A), and testicular
recrudescence (B) in adult males of Peromyscus melanotis from a mid-latitude temperate forest, along a
year. Vertical lines depict a standard deviation at both sides of the mean
(points on profile line). Number (n) of adult males for month: January n = 12;
February n = 11; March n = 6; April n = 5; May n = 6; June n = 2; July n = 6;
August n = 6; September n = 5; October n = 4; November n = 8; December n = 12.
Table 1. Significant differences among monthly mean
values of plasma testosterone (above diagonal) and testicular volume (below
diagonal) in adult males of Peromyscus melanotis from a middle
latitude, temperate forest.
|
Dic |
Jan |
Feb |
Mar |
Apr |
May |
Jun |
Jul |
Aug |
Sep |
Oct |
Nov |
|
|
Dic |
ns |
ns |
*** |
*** |
*** |
*** |
*** |
*** |
ns |
ns |
ns |
|
|
Jan |
ns |
ns |
*** |
*** |
*** |
*** |
*** |
*** |
ns |
ns |
ns |
|
|
Feb |
ns |
ns |
** |
*** |
*** |
*** |
*** |
*** |
ns |
ns |
ns |
|
|
Mar |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
*** |
|
|
Apr |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
*** |
|
|
May |
* |
*** |
ns |
* |
ns |
ns |
ns |
ns |
ns |
ns |
*** |
|
|
Jun |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
ns |
*** |
|
|
Jul |
ns |
* |
ns |
ns |
ns |
ns |
ns |
ns |
** |
** |
*** |
|
|
Aug |
ns |
* |
ns |
ns |
ns |
ns |
ns |
ns |
* |
* |
*** |
|
|
Sep |
ns |
ns |
ns |
ns |
ns |
* |
ns |
ns |
ns |
ns |
** |
|
|
Oct |
ns |
ns |
ns |
ns |
ns |
* |
ns |
ns |
ns |
ns |
* |
|
|
Nov |
ns |
ns |
ns |
ns |
ns |
*** |
ns |
* |
* |
* |
* |
Abbreviations:
ns, no significant; > number of asterisks > significant P level.
As a rule, it is claimed that environmental
conditions influence reproductive biology during the cold-dry seasons, being
lack of food a limitation (Merritt et al.,
2001; Sadleir, 1969; Wolff & Sherman, 2008). However, in our study area, circulating
levels of T had detectable values in P. melanotis during October to February
(colder and dryer conditions). Moreover,
even when the testicular size decreased significantly in these mice, as
compared to testicular size reached during the milder and more humid conditions
of May to August (t = P < 0.0033; 48.92 ± 12.98 vs. 185.9 ± 64.11, respectively), these mice also showed
spermatogenesis and gametes in the epididymis (Salame-Méndez et al.,
2008). Taken together, these facts allow us to confirm that under such
conditions in the study area, adult males of P. melanotis may be able to
reproduce during this unfavorable period (second reproductive peak, (Castro-Campillo et al., 2012; Salame-Méndez et al., 2004) due to its adaptive plasticity, as has been
reported for other Peromyscus species (Bronson & Heideman 1993; Kaufman & Kaufman, 1989;
Munshi-South & Richardson, 2017).
Figure 2. Raw data above and regression curves (Parables)
below for plasma testosterone contents (continuous line R = 0.73), and
testicular recrudescence (discontinuous line, R = 0.72) in adult males of Peromyscus melanotis
from a middle latitude, temperate forest, showing the relationship of these two
physiological processes. Triggering of testicular recrudescence (testis volume)
by plasma T, occurs in the coldest months (December-February), together with a
steady rising until May to a steady fall from then on. Notice decoupling of
plasma T that precedes testicular recrudescence and how it remains higher by a
month of difference.
The importance of maintaining moderate T
content could be reflected on the health of the individual’s during colder and dryer conditions. Besides T function on
reproduction, it has been shown its activity on different key regulatory cells,
such as lymphocytes and macrophages. Macrophages and mononuclear white cells
have a positive response to intratesticular and
circulating T levels through their respective androgen receptors (Ahmadi & McCruden, 2006; Bebo et al., 1999) and regulate
the immunological environment of the testis (Chen et al., 2016). Also, relatively low T levels could explain the
contradictory results found by Bronson & Heideman (1993) that even cryptorchidic
Peromyscus
males show normal spermatogenesis and are capable to father normal size litters
indicating a full reproductive activity on the natural population. Besides that,
low T levels maintain active spermatogenesis (Spaliviero et
al., 2004; Walker, 2011; Zhang et al., 2003).
Gonadal recrudescence, involves cell division during
the proliferative spermatogenesis phase, testicular angiogenesis and fluid
production in the seminiferous tubules, and thus
promoting maximum testicular size (Li et al.,
2015; Seco-Rovira et al., 2015). On the other side, testicular size regression
implies the reverse processes, stopping partial or full cell division, fluid
loss, decrease of lumen of seminiferous tubules,
drastic vascularization reduction and apoptotic
processes of several cells, including germ cells (Alexandre-Pires et al., 2012; Carvalho et al., 2009; Sharpe et al., 1994). Therefore, at the cellular level, T plays an
important role on the complex processes involved in the testicular
recrudescence-regression cycle (Beguelini et al.,
2015; Bueno et al.,
2014; Han et al.,
2017; Pelletier & Almeida, 1987; Sun et al., 2011).
Therefore, the decoupled pattern of plasma T
and testicular recrudescence found in Peromyscus melanotis, also warns
us about inferring reproductive activity based only in testicular size and/or its
location within the scrotum. That is, a
captured male on August might have a large scrotal testis but it might also be
non-reproductive, since it is undergoing reduction and deactivation of
spermatogenesis. In addition to the above, Olivera et al. (1986) found reproductively active males in a laboratory colony of Neotomodon alstoni, even though they
had no scrotal testicles; a fact also reported by Boiani et al.
(2008) in Oligoryzomys flavescens
from a temperate boreal habitat.
CONCLUSION
We can conclude that there patterns of circulating T and testis
recrudescence are similar, but with a slight delay in testis volume, that
actively maintains the reproductive physiology necessary for the two
reproductive seasons of Peromyscus melanotis
along a year. This close association may be used as a first evaluation
characteristic of reproductive activity and individual contribution to the
maintenance of the community. Therefore, it is important to note that an adult Black-eared
mouse, with no conspicuous scrotal
testicles, might not be reproductively inactive and no contributing to the mating
population, especially during the second breeding season at this middle latitude, temperate forest. Therefore,
studies of the population dynamics of wild rodents in temperate forests should
be reinforced with physiological information. The above is important to
consider, since during autumn-winter, adult males of Peromyscus melanotis can produce androgens and
spermatozoa, although their testicles are not in the scrotum and/or have a
smaller volume with respect to spring-summer adult males; therefore, such males
cannot be considered reproductively
inactive. If so, then the reproductive dynamics of this species would be
underestimated.
Acknowledgement
Our
appreciation to the late Juan Patiño Rodríguez, technical collector and taxidermist in the UAM Collection of Mammals (UAMI),
for both its invaluable field and cabinet works, as well as to M. C. Joaquín Herrera Muñoz for his
technical aid for evaluating testosterone contents. This work was partially
supported by the Division of Biological Sciences and Health of the UAMI (CBS-144.03.07 for A.S.M., y
CBS-143.02.46 for ACC), by the National Council of Science and Technology (CONACyT-1253-9203 y 400200-5R29117N
for JRP.) and by Secretary of Public Education (SEP
94-01-00-002-247 for JRP).
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