See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/247155295

Two periods of total testicular regression are peculiar events of the annual
reproductive cycle of the black Myotis bat, Myotis nigricans (Chiroptera:
Vespertilionidae)

Article  in  Reproduction Fertility and Development · July 2013

DOI: 10.1071/RD13109 · Source: PubMed

CITATIONS READS

36 307

4 authors:

Mateus R Beguelini Rejane Góes

Universidade Federal do Oeste da Bahia São Paulo State University
43 PUBLICATIONS   504 CITATIONS    115 PUBLICATIONS   1,908 CITATIONS   

SEE PROFILE SEE PROFILE

Sebastião R Taboga Eliana Morielle-Versute

Univ. Estadual Paulista - IBILCE/UNESP São Paulo State University
339 PUBLICATIONS   4,822 CITATIONS    71 PUBLICATIONS   934 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

The effects of perinatal bisphenol A in the mammary gland of Mongolian gerbils View project

Melatonin action on prostate of diabetic rats View project

All content following this page was uploaded by Mateus R Beguelini on 09 December 2014.

The user has requested enhancement of the downloaded file.

CSIRO PUBLISHING

Reproduction, Fertility and Development

http://dx.doi.org/10.1071/RD13109

Two periods of total testicular regression are peculiar

events of the annual reproductive cycle of the black Myotis
bat, Myotis nigricans (Chiroptera: Vespertilionidae)

Mateus R. Beguelini A, Rejane M. Góes A, Sebastião

R. Taboga A,C and Eliana Morielle-Versute B
A
Department of Biology, UNESP – Univ Estadual Paulista, São José do Rio Preto,
São Paulo 15054-000, Brazil.
B
Department of Zoology and Botany, UNESP – Univ Estadual Paulista,
São José do Rio Preto, São Paulo 15054-000, Brazil.
C
Corresponding author. Email: taboga@ibilce.unesp.br

Abstract. Myotis nigricans presents few and controversial reproductive data, which indicate geographical variation in
reproduction. Thus, this study aimed to evaluate the seasonal modifications in testicular and epididymal morphologies in a
tropical environment, submitting these organs to morphometric and immunohistochemical analysis. The observations
revealed that this species presents two peaks of spermatogenic activity followed by two periods of total testicular
regression (a quiescent pre-pubertal-like morphology, where only Sertoli cells and spermatogonia could be observed), in
the same annual reproductive cycle, which seem to be only indirectly influenced by abiotic factors. This testicular
behaviour seems to be synchronised with the caput and corpus epididymidis, but not with the cauda epididymidis, which
presents aspects of sperm storage in May–June. The control of this variation seems to be directly linked to the expression of
the androgen receptor, since, throughout the year, it is high in periods of testicular recrudescence and low in periods
of deactivation. It is not thought to be directly linked to apoptosis, which is more pronounced in periods of recrudescence
than in periods of regression.

Additional keywords: recrudescence, reproduction, seasonality, testes.

Received 4 April 2013, accepted 14 May 2013, published online 8 July 2013

Introduction
Studies have shown that most bat species reproduce seasonally
(Gustafson 1979; Krutzsch 1979; Crichton and Krutzsch 2000).
The reproductive patterns in bats are intimately dependent on
both physiological and environmental factors (Racey and
Entwistle 2000). Temperate bats are seasonal breeders and this
characteristic is directly associated with hibernation and low
temperatures (Krutzsch 1979); however, summer food avail-
ability and precipitation intensity also influence this pattern
(Grindal et al. 1992; Lewis 1993).
Bats from the tropics do not hibernate and their reproduction
is strongly linked to the habitat in which they are found; thus,
tropical bats are directly influenced by seasonal temperature
changes, latitude, rainfall and feeding resources (Fleming et al.
1972; Taddei 1976). Thus, tropical bats may exhibit great varia-
tion in reproductive patterns, with either monoestrus or polyestrus
patterns or continuous reproduction throughout the year in some
species (Taddei 1980; Zortéa 2003; Beguelini et al. 2009).
Myotis nigricans is an endemic species of insectivorous
vespertilionid bat from the Neotropical region, with records
from the southern edge of the Mexican plateau to just below the
Tropic of Capricorn. Its known elevation range is from sea level
to 3150 m, with specimens occurring in virtually every tropical
and subtropical forest, as well as in areas of savanna and scrub
(Wilson and Laval 1974).
Data from females indicate that it has a unique reproductive
cycle, presenting three parturition peaks in Panama (Wilson and
Findley 1970). The gestation period is ,60 days and the first
parturition peak occurs in February. This is followed by post-
partum oestrus and repetitions of the cycle resulting in birth
peaks in April–May and August. The third peak is followed by
a period of declining reproductive activity until late December
when a new annual cycle begins (Wilson and Laval 1974).
The young remain attached to their mother only for the first
two or three days, then are left behind in large groups when the
mothers leave to feed at night. Juveniles acquire adult propor-
tions, weights and measurements, with fusion of the epiphyses
of the long bones to the diaphyses, at the thirteenth week of life
(Wilson 1971); males become reproductively active at Weeks
15 to 17 (Wilson and Findley 1971).
Wilson and Findley (1971) found that the males undergo
a spermatogenic cycle that is similar to the female cycle, with

Journal compilation Ó CSIRO 2013 www.publish.csiro.au/journals/rfd

B Reproduction, Fertility and Development
slow or stopped spermatogenesis in September, October and
November, and no sperm storage. However, they observed that
specimens from Mexico more closely resemble temperate zone
bats in their reproductive condition at certain times of the year.
Thus, these authors proposed that M. nigricans is geographically
variable with regard to its reproductive cycle and that the
controlling factor in the seasonality in this species lies with
the males.
Due to these interesting characteristics and the lack of
information on the reproduction of this species in South America,
the aim of this study was to evaluate the annual variations on the
testicular and epididymal parameters of this species in south-east
Brazil and, based on these data, to try to understand how the
reproduction of this species is regulated in this environment.
Materials and methods
Study area, capture, climatic conditions and licenses
The animals were collected in the city of São José do Rio Preto,
in north-west São Paulo State, Brazil, (49W220 450 20S490 110 ),
which is located at 500 m above sea level. This is a semi-flat
region, in a degraded Cerrado biome, with a mesothermal
climate (with rainy summers and dry winters).
The capture was performed at night, using five mist-nets
(3 m  6 m) set to intercept bats flying 1–3 m above the ground.
The nets were precisely set on possible flight paths or exits from
shelters. At least five collections were performed each month,
except for nights with a full moon.
The climatic conditions (temperature, rainfall and photo-
period) during the period of collection were measured by
the Integrated Center for Agrometeorological Information
(CIIAGRO-Brazil: http://www.ciiagro.sp.gov.br).
The capture and captivity of bats were authorised by
the Brazilian institution responsible for wild animal care
(Instituto Brasileiro do Meio Ambiente, IBAMA – Processes:
02027.001957/2006–02 and 21707–1), whereas the ethics com-
mittee at São Paulo State University (UNESP) authorised all
experimental procedures (Process: 013/09 – CEEA). The
animals were treated according to the recommendations of
the Committee on Care and Use of Laboratory Animals from
the Institute of Laboratory Animal Resources, National Research
Council, ‘Guide for the Care and Use of Laboratory Animals’.
Species, aging and experiment
The species analysed was the exclusively Neotropical Vesper-
tilionidae bat, Myotis nigricans. This species is not listed as
endangered on the International Union for Conservation of
Nature (IUCN) Red List of Threatened Species.
The bats were aged as adults based on bodyweight, complete
ossification of the metacarpal–phalangeal epiphyses, wear of
the teeth (Dinerstein 1986; De Knegt et al. 2005), positioning
of the testes and the presence of spermatozoa inside the testes or
cauda epididymidis.
Despite the wide geographical distribution, M. nigricans is
a species whose occurrence is restricted to certain specific
habitats, which are infrequent in our region. Adding to these,
the small size of their colonies associated with their harem
structure (with a single male mated to multiple females) makes
M. R. Beguelini et al.
Table 1. Number of animals analysed in each month
Month Number of animals analysed
September 2008 3
October 2008 2
November 2008 3
December 2008 2
January 2009 2
February 2009 2
March 2009 2
April 2009 3
May 2009 2
June 2009 3
July 2009 2
August 2009 2
November 2009 2
June 2010 2
the collection of males specimens even more difficult. Thus, the
number of specimens used was the maximum obtained from
a balance between a huge collection effort (up to five weekly
collections, sometimes with two collections on the same day
in different locations) and the care to not break up the few
colonies present in the region. Similarly, we took care not to
disturb the females.
Thus, twenty-eight sexually mature male specimens were
used in this study (Table 1), due to the difficulty in the capture
of the animals, with at least two specimens collected each
month between September 2008 and August 2009. One testis
and one epididymis of each animal were subject to morpho-
metric analysis and the others were analysed by immunohisto-
chemistry. Four specimens were collected later, two in
November 2009 and two in June 2010, to confirm the periods
of testicular regression.
Processing of animals and histological analyses
The animals were sacrificed by cervical dislocation and, after
the bodyweight was measured, the testes and epididymides were
removed for analysis. The testes were also weighed.
The testes and epididymides were immersed in Bouin fixa-
tive solution for at least 24 h, dehydrated in a graded series of
ethanol, embedded in glycol methacrylate (Historesin; Leica
Instruments, Nussloch, Germany), sectioned (1-mm thickness),
stained with haematoxylin–eosin (Ribeiro and Lima 2000) and
morphometrically analysed. Alternatively, the organs were
immersed in a methanol : chloroform : acetic acid (6 : 3 : 1) fixa-
tive solution for three hours at 48C, dehydrated in a graded series
of ethanol, clarified in xylene, embedded in paraffin, sectioned
(4-mm thickness) and submitted to immunohistochemical proce-
dures for localisation of the androgen receptor (AR), of prolifer-
ating cell nuclear antigen and the detection of apoptotic cells.
All the specimens are housed in the Chiroptera collection at
the São Paulo State University (DZSJRP- UNESP).
Morphometric–stereological analyses
The following measurements were performed in testicular and
epididymidal (caput, corpus and cauda) cross-sections using
Total testicular regression in Myotis nigricans
Image Pro-Plus-Media Cybernetics, version 6.1 for Windows
(Media Cybernetics, Rockville, MD, USA) computer software
for image analysis: the relative percentage of tissues (epithelial,
luminal and interstitial tissues), the tubular and luminal dia-
meters and the epithelium height.
The relative percentages of the epithelium, lumen and
interstitial tissue were estimated according to the procedure of
Weibel et al. (1966) using a 168-point grid test system. The data
were obtained from 30 random microscopic fields per animal
at 200 magnification. The relative percentage (%) was calcu-
lated after counting the number of points that coincided with
each of the tissue compartments (epithelium, lumen of the ducts
and interstitial tissue).
The analysis of the tubular and luminal diameters and the
epithelium height were performed in 200 tubule cross-sections
per animal at 400 magnification. Only transverse sections
were included in this study. The epithelium height was taken
as the linear length from the base of the epithelium (basal
lamina) to the apical edge (excluding the stereocilia); the
luminal diameter was taken as the more elongated measurement
from one apical edge to the other and the tubular diameter was
taken as the more elongated distance between basal laminae.
Immunohistochemistry
After microwaving for antigen retrieval, non-specific antibody
binding was blocked using 3% bovine serum albumin (BSA)
before incubation with primary antibodies against the androgen
receptor (rabbit polyclonal IgG, N-20; Santa Cruz Biotechnol-
ogy, Dallas, TX, USA; 1 : 75) and a cell proliferation marker
(PCNA – monoclonal anti-mouse, PC10 sc-56; Santa Cruz
Biotechnology; 1 : 100) and for the detection of apoptotic
cells by TUNEL (TdT-Fragel-Calbiochem; Merck KGaA,
Darmstadt, Germany, according to the manufacturer’s instruc-
tions). The immunoreaction was visualised by the reaction with
diaminobenzidine (DAB) and sections were counterstained with
Harry’s haematoxylin. For negative controls, the sections
received phosphate-buffered saline (PBS) in place of the
primary antibody or the enzyme (TUNEL). Immunofluores-
cence was assessed using a fluorescein isothiocyanate (FITC)-
conjugated secondary antibody followed by counterstaining
with 4-6-diamidino-2-phenylindole (DAPI).
The incidence of AR-positive cells, apoptosis and cell
proliferation in the testes was estimated according to Dadhich
et al. (2010), with modifications, by calculating the mean
number of AR-positive, apoptotic or proliferating cells per
mm2 of seminiferous tubule, according to the formula: x ¼
CST/AST, where CST is the number of cells counted per semini-
ferous tubule section and AST is the area of the seminiferous
tubule section (expressed in mm2). It is important to consider that
the Sertoli cell and the germ cell in division (spermatogonia and
early spermatocytes – pre-leptotene and leptotene) are the only
cell types within the seminiferous tubule stained by antibodies to
the AR and PCNA, respectively, so only those cell types were
counted. On the other hand, all apoptotic cells were counted.
The incidence of AR-positive cells and cell proliferation in
the epididymis was estimated by calculating the proportion of
AR or proliferating positive cells per tubule section. Only the
secretory cells were evaluated.
Reproduction, Fertility and Development C
Statistical analysis
Means and standard deviations were calculated for all datasets.
Differences between groups were evaluated using one-way
analysis of variance (ANOVA), followed by pairwise compar-
isons with the Tukey test, using the program Statistica 7.0
(Statsoft Inc., Tulsa, OK, USA). A P value of #0.05 was
accepted as statistically significant.
Results
Climatic conditions
The mean temperature varied little throughout the year, staying
close to 258C (Fig. 1a). The period from December–March had the
highest average temperature; however, a reduction in temperature
was observed in June–July (minimum temperature ¼ 38C).
The mean monthly rainfall recorded in the analysed area is
shown in Fig. 1b. The data indicate an increase in rainfall
from December to March (more than 150 mm) and low
rainfall from May–July.
The mean monthly daylength recorded to the analysed area is
shown in Fig. 1c. We observed a maximum daylength in the
period from November–January and a minimum in June–July.
With these data, we confirmed the abiotic pattern of the
studied region, observing the presence of two marked and
different seasons: a rainy summer, with high temperatures and
long daylengths; and a dry winter, with low temperatures
and short daylengths.
Body and gonad weights and the gonad–somatic index
The bodyweights of the bats studied showed a marked variation
(Fig. 1d ), presenting a significant high bodyweight in May
(4.6  0.25 g), preceded (March, 3.63  0.08 g) and also followed
(June, 3.74  0.09 g and July, 3.69  0.02 g) by a lower value.
The mean bodyweight in the other months was close to 4 g.
In contrast to the bodyweight, the gonad weights presented
two significant peaks (Fig. 1e), one in September (46  24.8 mg)
and another in April (56  39.7 mg). We observed that both
peaks were followed by a reduction in gonad weight, with some
months presenting very low weights (November, 7.2  1.6 mg
and June, 15.6  3.5 mg).
The gonad–somatic index presented a similar pattern to that
observed for the gonad weight, with two peaks followed by very
low values (Fig. 1f ).
Testicular reproductive cycle
The testes of M. nigricans undergo considerable changes in
morphology (Fig. 2) and morphometric patterns during the
year. They underwent a profound involution in October (Fig. 2b)
and were totally regressed in November (Fig. 2c), exhibiting
a quiescent pre-pubertal-like morphology, where only Sertoli
cells and spermatogonia could be observed. Afterwards, in
December, they began a recrudescence process (Fig. 2d–f ) that
extended to the production of the first spermatozoa in March
(Fig. 2g). In April, the testes reached their highest level of
development and activity during the year (Fig. 2h). In May,
another process of total testicular regression occurred (Fig. 2i),
which culminated in the regressed testes of June (Fig. 2j). In
July, the testes were again active (Fig. 2k), showing that the
D Reproduction, Fertility and Development M. R. Beguelini et al.

(a) Temperature (b) 250 Rainfall

45
40 200
Temperature (°C)

35

Rainfall (mm)
30 150
25
20 100
15
10 50
5
0 0
p /08 ct/08 ov/08 c/08 n/09 b/09 ar/09 pr/09 ay/09 n/09 ul/09 g/09 8 8 8 8 9 9 9 9 9 9 9
p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0
9
Se O N De Ja Fe M A M Ju J Au Se N D F M M A
Maximum Average Minimum

Photoperiod Body weight

(c) 14 (d ) 5.0 e
4.8
4.6
13
Body weight (g)
Photoperiod (h)

4.4
a a
4.2 a a
a,b a,d
12 4.0 b,c
b,c,d b,c b,c
3.8 c
3.6
11
3.4
3.2
10 3.0
8 8 8 8 9 9 9 9 9 9 9 9 8 8 8 8 9 9 9 9 9 9 9 9
p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0 p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0
Se N D F M M A Se N D F M M A

Gonad weight Gonad-somatic index

(f )
a 25 a
(e)
Gonad-somatic index (⫻10⫺4)

100
20
Gonad weight (mg)

80 a,b
a,b
15
60

10
40 a,b,c a,b,c a,b,c
a,b,c b,c b,c b,c
b,c c
b,c c b,c c c
20 c c 5 c
c c c

0 0
8 8 8 8 9 9 9 9 9 9 9 9 8 8 8 8 9 9 9 9 9 9 9 9
p/0 t/0 v/0 c/0 n/0 b/0 r/0 r/0 y/0 n/0 Jul/0 ug/0 p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0
Se Oc No De Ja Fe Ma Ap Ma Ju A Se N D F M M A

Fig. 1. Seasonal changes in climatic conditions of the studied area and in the body and gonad weights and the gonad–somatic index of Myotis nigricans
from south-east Brazil from September 2008 to August 2009. (a) Interannual monthly variations in temperature. (b) Interannual monthly variations in
rainfall. (c) Interannual monthly variations in daylength. (d ) Seasonal variations in the mean bodyweight. (e) Seasonal variations in the mean gonad
weight (both testes). ( f ) Seasonal variations in the gonad–somatic index. Different letters indicate statistically significant differences (ANOVA at
P , 0.05). Circular dots in (d–f ) indicate the absolute values of each sample.

process of recrudescence in July occurred rapidly. In August and
September, the testes showed an active pattern (Figs 2l and 2a,
respectively). The occurrence of both processes of total
regression (a quiescent pre-pubertal-like morphology, where
only Sertoli cells and spermatogonia could be observed) was
confirmed in the subsequent year by the observation of totally
regressed testes in November 2009 and June 2010.
The process of testicular regression in June seemed to be
less accentuated than in November. Despite this, both months
presented only Sertoli cells and spermatogonia in their semini-
ferous epithelium, with no other cell type being seen; these cells
had similar morphologies in both months. In contrast, they
presented discrepant differences in stereology, with a large
increase in the amount of interstitial tissue and a consequent
Total testicular regression in Myotis nigricans Reproduction, Fertility and Development E

September – 2008 October – 2008 November – 2008 December – 2008

(a) (b) (c) (d )

January – 2009 February – 2009 March – 2009 April – 2009

(e) (f ) (g) (h)

May – 2009 June – 2009 July – 2009 August – 2009

(i) (j ) (k) (l )

Fig. 2. Seasonal changes in testicular morphology of Myotis nigricans from south-east Brazil from September
2008 to August 2009. Haematoxylin–eosin stain. (a) September 2008. (b) October 2008. (c) November 2008.
(d ) December 2008. (e) January 2009. ( f ) February 2009. (g) March 2009. (h) April 2009. (i) May 2009. ( j) June
2009. (k) July 2009. (l ) August 2009. Note that the testes underwent a profound involution in October (b) and were
totally regressed in November (c). Afterwards, in December, they began a recrudescence process (d–f ) that
extended to the production of the first spermatozoa in March (g). In April, the testes reached their highest level of
development and activity during the year (h). In May, a rapid process of testicular regression occurred (i), which
culminated in the regressed testes of June ( j). In July, the testes were reactivated (k) and in August and September
the testes showed an active pattern (l and a, respectively). Scale bars ¼ 20 mm.

decrease in epithelium in November (Fig. 3a), which was not
observed in June, where the tubular size did not seem to decrease
sharply and the amount of interstitial tissue did not appear to
increase (Fig. 3a).
The stereological and morphometric analyses of the testes
confirmed the presence of two periods of testicular regression,
one in October–November and another in May–June, as well as
peaks of spermatogenic activity in September–October and
April (Fig. 3a–b). The morphometric data clearly indicate two
periods of total testicular regression (Fig. 3b); however, the
stereology clearly indicates only the October–November period,
where there was an inversion in the proportion of tissues, and
a sharply decreased percentage of epithelium (30.1  5%)
and greatly increased interstitial tissue (65.8  5.6%), which
was not observed in May–June (Fig. 3a). These data show that
the testicular regression observed in May–June differed from
that in October–November and was less accentuated.
The immunohistochemistry for proliferating cells (PCNA)
showed two peaks of spermatogonial proliferation (Fig. 4a), one
in November–December (Fig. 4b) and other in June (Fig. 4c).
These periods corresponded to the quiescent periods, when the
testes were at the lowest regressive point and began to reactivate.
The data also indicate two periods of sparse proliferation,
showing that October (Fig. 4d ) and May were periods
of testicular deactivation, which preceded the two periods of
quiescence. Fig. 4e is the negative control of the reaction.
PCNA also showed considerable seasonal changes with three
peaks in Leydig cell proliferation (Fig. 4f ) – November
(Fig. 4g), January and June (Fig. 4h) – and two periods of sparse
proliferation – in August–October (Fig. 4i) and May (Fig. 4j).
The TUNEL method showed two significant peaks of apop-
tosis in the seminiferous tubules (Fig. 4k), one in December
(Fig. 4l–m) and other in June (Fig. 4n). The proportion of
apoptotic cells in other months remained at basal levels
(Fig. 4o). Fig. 4p is the positive control of the reaction.
The expression of the androgen receptor (AR) in Sertoli
cells showed considerable seasonal variation (Fig. 4q), not
only quantitatively but also qualitatively. There were two
peaks of strong expression – one in December (Fig. 4r) and
other in June (Fig. 4s) – and two periods of low expression – in
September (Fig. 4t) and March–May. Fig. 4u is the negative
control of the reaction. On the other hand, expression of the
AR in Leydig cells was more qualitative, with consistent
expression or lack of expression in each month. AR expression
in Leydig cells was completely opposite to that seen in Sertoli
cells, i.e. when AR expression in Sertoli cells was high, the
Leydig cells did not express the receptor and when AR
expression in Sertoli cells was low, the Leydig cells expressed
the receptor. Thus, expression was absent in December–
January (Fig. 4v), high from February to May (Fig. 4x), absent
in June and high from July to November (Fig. 4y). Another
interesting feature was that the expression of the AR in Leydig
F Reproduction, Fertility and Development M. R. Beguelini et al.

Stereology Morphometry
(a) 90 ∗
(b) 200 ∗ ∗
80 ∗ ∗ 180
∗ ∗ ∗ ∗ ∗ 160 ∗ ∗
70
140
Stereology (%)

60 ∗
120
50
Testes

μm
100 ∗ ∗
40 ∗ ∗ ∗ ∗ ∗
80 ∗ ∗ ∗ ∗ ∗
30 ∗ ∗ ∗ ∗ ∗
∗ ∗ ∗
60
∗ ∗ ∗ ∗
20
∗
40
∗ ∗
10 ∗ ∗ 20
0 0
8 8 8 8 9 9 9 9 9 9 9 8 8 8 8 9 9 9 9 9 9 9 9
p/0 ct/0 ov/0 ec/0 an/0 eb/0 ar/0 pr/0 ay/0 un/0 ul/09 ug/0 p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0
Se O N D J F M A M J J A Se N D F M M A
Epithelium Lumen Interstitial tissue Tubular diameter Epithelium height Luminal diameter
(c) 80 (d ) 80
∗ ∗
70 ∗ ∗ 70
∗ ∗ ∗ ∗
∗
Caput epididymis

60
∗ 60 ∗ ∗ ∗
Stereology (%)

∗ ∗
50 50
∗ ∗
∗
μm
40 ∗ ∗ 40
∗ ∗ ∗∗ ∗ ∗ ∗ ∗
30 ∗ ∗ 30 ∗ ∗ ∗ ∗ ∗ ∗
∗ ∗
20 20 ∗
∗ ∗ ∗
10 10 ∗
∗
0 0
8 8 8 8 9 9 9 9 9 9 9 9 8 8 8 8 9 9 9 9 9 9 9 9
p/0 t/0 v/0 c/0 n/0 b/0 r/0 r/0 y/0 n/0 Jul/0 ug/0 p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0
Se Oc No De Ja Fe Ma Ap Ma Ju A Se N D F M M A
Epithelium Lumen Interstitial tissue Tubular diameter Epithelium height Luminal diameter
(e) 80 (f ) 100
∗ ∗ ∗ ∗ ∗
90 ∗
70
∗ ∗
Corpus epididymis

80
60 ∗ ∗ ∗
Stereology (%)

70 ∗
50 60 ∗
∗ ∗ ∗∗ ∗
μm

40
∗ ∗ 50
∗ ∗
30 ∗ ∗ 40 ∗
30 ∗ ∗
∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗
20 ∗ ∗
20 ∗
10 ∗ 10
0 0
8 8 8 8 9 9 9 9 9 9 9 9 8 8 8 8 9 9 9 9 9 9 9 9
p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0 p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0
Se N D F M M A Se N D F M M A
Epithelium Lumen Interstitial tissue Tubular diameter Epithelium height Luminal diameter
(g) 100 ∗
(h) 180 ∗ ∗
90 ∗ 160 ∗
80 ∗ ∗
∗ 140 ∗ ∗ ∗
Cauda epididymis

∗ ∗ ∗
Stereology (%)

70 120 ∗∗
60 ∗ ∗ ∗ ∗ ∗
∗ ∗ 100 ∗ ∗
∗ ∗ ∗
μm

50 ∗
80
40 ∗ ∗
∗ 60 ∗ ∗
30 ∗ ∗ ∗
20 ∗ ∗ 40
∗ ∗ ∗ ∗ ∗
10 20 ∗ ∗
0 0
8 8 8 8 9 9 9 9 9 9 9 9 8 8 8 8 9 9 9 9 9 9 9 9
p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0 p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/0 ug/0
Se N D F M M A Se N D F M M A
Epithelium Lumen Interstitial tissue Tubular diameter Epithelium height Luminal diameter

Fig. 3. Seasonal changes in stereology (amount of epithelial, luminal and interstitial tissue) and morphometry (tubular and luminal diameters and
epithelial height) of the testes and epididymis of Myotis nigricans in south-east Brazil from September 2008 to August 2009. (a–b) Testes. (c–d ) Caput
epididymidis. (e–f ) Corpus epididymidis. (g–h) Cauda epididymidis. (* indicates significant difference with respect to the value of the previous month;
ANOVA at P , 0.05).
Total testicular regression in Myotis nigricans Reproduction, Fertility and Development G

PCNA
(a) a b (b) (c)
5

(PCNA⫹ cells μm⫺2) . 10⫺3

b
c
4
d
d,e
3 e,f f
f,g f
g
2 (d ) (e)
1

0
8 8 8 8 9 9 9 9 9 9 9 9
p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 un/0 Jul/0 ug/0
Se N D F M M J A
(f ) 70 a (g) (h)
60
Leydig cells PCNA⫹ (%)

50
b b,c
40 c
d d,e
30
f,g e,f (i ) (j )
20 g,h
i,j h,i
10 j
0
⫺10 /08 /08 /08 /08 /09 /09 /09 /09 /09 /09 /09 /09
p Oct ov ec Jan eb ar Apr ay un Jul ug
Se N D F M M J A

(k) 0.25 a
TUNEL (l) (m)
(TUNEL⫹ cells μm⫺2) . 10⫺3

0.20
a
0.15

0.10
(n) (o) (p)
0.05 b b
b b b b b b b
b
0
/09
/08

/08
/08

/09
r/09
/09
/09

/09
/08

/09

09

−0.05
Jul/
May
Sep

Dec
Nov

Aug
Feb
Jan

Jun
Oct

Apr
Ma

(q) 5.0 a AR (r) (s)

4.5
(AR⫹ cells μm⫺2) . 10⫺3

4.0
3.5
3.0
2.5 b b
2.0
1.5 (t) (u)
c c
1.0 d,e d d d,e
e d,e
0.5 e
0
Ju 9
08

08
08

09
9
M 9
09

09
8

9
/0
/0
0
/0

r/0

l/0
p/

c/
v/

g/
b/
n/

n/
ay
ar
ct

Ju
Ap
No

De
Se

Au
Fe
Ja
O

(v) (x) (y) (z)

Fig. 4. Seasonal changes in amount of (a–j) proliferating cells (PCNA), (k–p) apoptosis (TUNEL) and in
(q–z) expression of androgen receptor (AR) in testes of Myotis nigricans from south-east Brazil from
September 2008 to August 2009. (a) Graph showing the number of PCNAþ cells by testicular area. (b–d )
Expression of PCNA in animals from (b) December, (c) June and (d ) October. (e) Negative control of the
PCNA immunoreaction. ( f ) Graph showing the number of PCNAþ Leydig cells. (g–j) Expression of PCNA
in the Leydig cells of animals in (g) November, (h) June, (i) October and ( j) May. (k) Graph showing the
number of TUNELþ cells by testicular area. (l–o) TUNEL reaction in the testes of animals in (l–m) December,
(n) June and (o) October. (p) Positive control of the TUNEL immunoreaction. (q) Graph showing the number
of ARþ cells by testicular area. (r–t) Expression of AR in Sertoli cells of animals in (r) December, (s) June and
(t) September. (u) Negative control of the AR immunoreaction. (v–y) Expression of AR in Leydig cells of
animals in (v) December, (x) May and (y) September. (z) Immunofluorescence showing cytoplasmic
expression of AR in Leydig cells. Different letters indicate statistically significant differences (ANOVA at
P , 0.05). Scale bars ¼ 10 mm.
H Reproduction, Fertility and Development M. R. Beguelini et al.

September – 2008 October – 2008 November – 2008 December – 2008

(a) (b) (c) (d )

January – 2009 February – 2009 March – 2009 April – 2009

(e) (f ) (g) (h)

May – 2009 June – 2009 July – 2009 August – 2009

(i ) (j ) (k) (l )

Fig. 5. Seasonal changes in the morphology of the cauda epididymidis of Myotis nigricans from south-east Brazil from September 2008 to August 2009.
Haematoxylin–eosin stain. (a) September 2008. (b) October 2008. (c) November 2008. (d ) December 2008. (e) January 2009. ( f ) February 2009.
(g) March 2009. (h) April 2009. (i) May 2009. (j) June 2009. (k) July 2009. (l ) August 2009. Note that the cycle of the cauda epididymidis started in
November, where it presented its minimum size (c); from December to February, the cauda epididymidis tubules developed gradually (d–f ); however, the
first spermatozoa were observed within it only in March (g). From April (h), the cauda epididymidis started to store spermatozoa, reaching the maximum
size in June ( j). The storage of spermatozoa extended until October (b) and in November no spermatozoa were observed inside. Scale bars ¼ 20 mm.

cells was only cytoplasmic (Fig. 4x–y), a fact confirmed by
immunofluorescent staining (Fig. 4z).
Epididymal reproductive cycle
The caput and corpus epididymidis showed changes in mor-
phology and morphometric patterns throughout the year similar
to the testes, and presented two clear periods of regression by
morphometry, in October–November and May–June (Fig. 3d
and f, respectively), and only one by stereology, November
(Fig. 3c and e, respectively). The two peaks of development,
September–October and April, were also observed in both
regions (Fig. 3c–d and e–f, respectively). The data indicate
that the caput and corpus epididymidis vary in a similar way to
the testes; however, changes in the cauda epididymidis were
different.
The cycle of the cauda epididymis of M. nigricans started in
November, when it presented its minimum size (Fig. 5c). From
December to February, the cauda epididymidis tubules devel-
oped gradually (Fig. 5d–f ); however, the first spermatozoa were
observed within it only in March (Fig. 5g). From April, the
cauda epididymidis started to retain or store spermatozoa,
reaching its maximum size in June (Fig. 5j). The retention or
storage of spermatozoa extended until October (Fig. 5b), and in
November no spermatozoa were observed.
The stereological and morphometric data confirmed the
regression in the cauda epididymidis in October–November
and showed maximum development in June (Fig. 3g–h). During
the period of regression, the percentage of interstitial tissue
predominated over the other tissues (Fig. 3g), and the morpho-
metric measurements presented their lowest values for luminal
and tubular diameters, with the absence of spermatozoa inside
(Fig. 3h). On the other hand, with the storage of spermatozoa, the
lumen increased greatly, predominating over the other tissues
(Fig. 3g), and the morphometric measurements presented their
greatest values (Fig. 3h).
The immunohistochemistry for PCNA showed a similar
variation for the three epididymal regions throughout the year,
with two peaks of proliferation (Fig. 6a–c) – one in September
(Fig. 6d ) and other in January (Fig. 6e) – and two periods of
sparse proliferation – one in October and other in June–July
(Fig. 6f ). Fig. 6g is the negative control of the reaction.
Expression of the AR in the epididymis showed considerable
differences between the three regions. The caput presented
one period of low expression in September–October; however,
Total testicular regression in Myotis nigricans Reproduction, Fertility and Development I

PCNA
(a) 100 a Caput epididymis (b) 100 a Corpus epididymis
90 90
80 b 80
PCNA⫹ cells (%)

PCNA⫹ cells (%)

70 c 70
b
60 d,e,f d,e d,e,f,g c,d 60 c
f,g d,e c c
50 e,f,g g 50 d
c,d
40 40 e
30 h 30 f f
20 20 g
g
10 10
0 0

9
8

8
09

09

09

09
09

09
9

09

9
08

08

08

08

08

09
08
9

9
l/0

l/0
/0

r/0

/0

r/0
/0

/0
/0

/0
n/

n/

n/

n/
b/

b/
g/
p/

v/

c/

v/

c/

g/
p/
ct

ct
ar

ar
ay

ay
Ju

Ju
Ap

Ap
No

No
De

De
Ja

Ju

Ja

Ju
Fe

Fe
Au

Au
Se

Se
O

O
M

M
M

M
(c) 100 a Cauda epididymis (d ) (e)
90 b
80
PCNA⫹ cells (%)

70 c c
c
60
50 d
d,e e,f
40 f,g
30 e,h (f ) (g)
20 h,i i
10
0
9
8

9
9

9
9

9
08

08

08

09

/0
/0

r/0
/0

/0
/0

/0

/0
p/

v/

c/

l
ct

n
b

ar

g
ay

Ju
Ap
No

De

Ja

Ju
Fe

Au
Se

AR
(h) 100 Caput epididymis (i ) 80 Corpus epididymis
90 a,b a a,b b,c a,b a,b a a,b a a a
c 70 b,c a,b
c c c c,d c,d
80 d c,d d
60
AR⫹ cells (%)

AR⫹ cells (%)

70
60 e 50
f
50 40
40 30
30
20
20
10 10
0 0
8 8 8 8 9 9 9 9 9 9 09 g/09 8 8 8 8 9 9 9 9 9 9 09 g/09
p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/ p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/
Se N D F M M Au Se N D F M M Au

(j) 70 Cauda epididymis (k) (l )

a a,b a,b,c
a,b
60 b,c c,d
d,e d,e
50 e
AR⫹ cells (%)

40 f f
f
30
(m) (n)
20

10

0
8 8 8 8 9 9 9 9 9 9 09 g/09
p/0 Oct/0 ov/0 ec/0 Jan/0 eb/0 ar/0 Apr/0 ay/0 Jun/0 Jul/
Se N D F M M Au

Fig. 6. Seasonal changes in (a–g) the number of proliferating cells (PCNA) and in (h–n) expression of the androgen receptor (AR) in epididymis
of Myotis nigricans from south-east Brazil from September 2008 to August 2009. (a) Graph showing the number of PCNAþ cells in the caput
epididymidis. (b) Graph showing the number of PCNAþ cells in the corpus epididymidis. (c) Graph showing the number of PCNAþ cells in the
cauda epididymidis. (d–f ) Expression of PCNA in the cauda epididymidis of animals in (d ) September, (e) January and ( f ) July. (g) Negative
control of the PCNA immunoreaction. (h) Graph showing the number of ARþ cells in the caput epididymidis. (i) Graph showing the number of
ARþ cells in the corpus epididymidis. ( j) Graph showing the number of ARþ cells in the cauda epididymidis. (k–m) Expression of AR in the cauda
epididymidis of animals in (k) May, (l ) January and (m) July. (n) Negative control of the AR immunoreaction. Different letters indicate statistically
significant differences (ANOVA at P , 0.05). Scale bars ¼ 10 mm.
J Reproduction, Fertility and Development
the other months showed high expression, around 80% (Fig. 6h).
Interestingly, the expression of the AR in this region was higher
than in others. The expression in the corpus varied little
throughout the year, with three periods of a slight reduction in
expression, in September, December and May. Its expression
was intermediate between the caput and cauda, remaining at
,60% in most months (Fig. 6i). The cauda had the lowest
expression, around 50% (Fig. 6j), presenting one period of low
expression in March–May (Fig. 6k), which was preceded and
also followed by peaks of high expression, in January (Fig. 6l )
and July (Fig. 6m). Fig. 6n is the negative control of the reaction.
Discussion
The Vespertilionidae family presents the widest distribution of
any living chiropteran family: north to the Arctic Circle in the
Palearctic and Nearctic regions, south to the southern parts
of Africa, Australia and South America and including many
oceanic islands, such as Hawaii, Indonesia, Madagascar and
New Zealand (Krutzsch 1979; Simmons 2005). Within this wide
distribution, these bats inhabit a great variety of habitats, colo-
nising natural and urban environments, and thus are exposed to
a large variety of abiotic factors, including latitude, temperature,
rainfall, photoperiod etc. (Fleming et al. 1972; Beguelini et al.
2012).
In response to all these factors, vespertilionid bats have
evolved some unique reproductive cycles. The majority of
vespertilionid bats are seasonally monoestrous, mainly in the
temperate zones (Gustafson 1979; Krutzsch 1979; Crichton and
Krutzsch 2000), as the prolonged hibernation period during
the winter restricts the reproductive process to the summer,
with spermatogenesis and copulation occurring during this
period (Krishna and Singh 1997). However, there are several
non-hibernating species that also exhibit this pattern: Scoto-
philus heathi (Krishna and Singh 1997) and S. wroughtoni
(Gopalakrishna 1948) in India, Nycticeius schlieffenii (van der
Merwe and Rautenbach 1987) and Pipistrellus rusticus (van der
Merwe and Rautenbach 1990) in South Africa, Lasiurus ega in
Paraguay (Myers 1977) and Corynorhinus mexicanus in Mexico
(León-Galván et al. 2005), among others. These species are
usually found in regions where winter temperatures are mild
or in regions that present reduced seasonal variation in abiotic
patterns, and frequently present an annual reproductive cycle
synchronised with female reproductive events (Krutzsch 1979).
On the other hand, vespertilionid species that inhabit low
latitudes, in tropical or subtropical regions, tend to be polyes-
trous, presenting two or three parturition peaks annually
(Krutzsch 1979).
Species of the genus Myotis also present great variation in
their reproductive cycles; from a seasonally monoestrous cycle
in hibernating species of temperate regions, as in M. daubentonii
(Encarnação et al. 2004), M. lucifugus (Gustafson and Shemesh
1976; Gustafson and Damassa 1985), M. macrodactylus
(Lee and Mori 2004), M. thysanodes (O’Farrell and Studier
1973) and M. velifer (Krutzsch 2009) to polyestrous cycles in
species that inhabit low latitudes, in tropical or subtropical
regions. Myotis adversus demonstrates two male reproductive
peaks with a reduced or regressed period between them in
Australia (Dwyer 1970), while M. albescens breeds two or three
M. R. Beguelini et al.
times annually, with spermatogenesis occurring only from
autumn to spring in Paraguay (Myers 1977).
Data concerning the reproductive cycle of M. nigricans are
scarce, with only a few earlier studies available (Wilson and
Findley 1970; Wilson 1971; Wilson and Laval 1974). Wilson
and Findley (1971) and subsequently Krutzsch (1979) proposed
that the reproductive cycle of M. nigricans is geographically
variable, with animals from Paraguay presenting an active
pattern throughout the year and those from Panama presenting
an active pattern for most of the year, but with reproductive
quiescence for approximately three months (September–
November); animals from Mexico more closely resemble
temperate zone bats in their reproductive patterns.
Despite the geographic proximity to Paraguay, our study
demonstrated that specimens from São Paulo State, Brazil, are
not reproductively active throughout the year, since during the
period from November–February there are no spermatozoa in
their reproductive tract (testes and epididymis) to inseminate
females. In comparison with specimens from Panama, we also
observed a period of total testicular regression in October–
November; however, conversely, our specimens presented not
one but two periods of total testicular regression in the same
annual reproductive cycle, a pattern never before observed in
any other species or study.
The observation of periods of total testicular regression
(a quiescent pre-pubertal-like morphology, where only Sertoli
cells and spermatogonia could be observed) from 1 to 6 months
in hibernating species is common, but the observation of two
periods in the same annual reproductive cycle, one of which
presents considerable sperm storage in the cauda epididymidis,
in a tropical species, seems to be specific to M. nigricans.
Thus, similar to Wilson and Findley (1971), we propose that
this species has a unique pattern of reproduction that is geo-
graphically variable, as it presents a hibernating pattern in some
regions, an active pattern throughout the year in others and two
peaks of spermatogenic activity followed by two periods of total
testicular regression in south-east Brazil.
The gonad weight and morphometric data show that
M. nigricans presents two peaks of spermatogenic activity,
one in September and the other in April, which seem to be
synchronised in the testes, caput and corpus epididymidis,
indicating that, possibly, they are regulated in a similar fashion.
However, the cauda epididymidis presents a different pattern,
presenting a late peak (June). This difference may be explained
by sperm storage from March to October. The presence of
spermatozoa in the cauda epididymidis of M. nigricans from
March to October shows that this species is capable of reprodu-
cing seven to eight months per year, with the number of breeding
episodes and fertilisations depending on the reproductive cycle
of the females.
Our data show that the annual reproductive cycle of
M. nigricans can be subdivided into four different phases
(active, regressing, regressed and recrudescence) that repeat in
the same order twice during the year, all presenting distinct
characteristics (August–September, active; October, regressing;
November, regressed; December–February, recrudescence;
March–April, active; May, regressing; June, regressed; late
June–early July, recrudescence; late July, active).
Total testicular regression in Myotis nigricans
The two peaks of spermatogenic activity were followed by
two periods of total testicular regression (October–November
and May–June) that also seem to be synchronised with a
decrease in the caput and corpus epididymidis, but not in
the cauda epididymidis in May–June (sperm storage). The two
periods of regression present different patterns: (1) in October–
November, regression occurs with a decrease in the epithelium
and an increase in interstitial tissue proportions; however, in
May–June, the interstitial tissue does not show an increase;
(2) in October–November, regression seems to occur by deacti-
vation of spermatogenesis that gradually regresses the epitheli-
um; however, in May–June, regression seems to be accelerated
by a process of vacuolisation of the epithelium; (3) the reactiva-
tion process that follows the regression in October–November
occurs more slowly (December–March) than that in June, which
occurs rapidly (July).
Despite the impossibility of evaluating circulating plasma
testosterone levels due to the small size of the species (very little
blood), the expression of the AR indicated a strong link between
testosterone and the reproductive cycle of M. nigricans. AR
expression showed a connection with the periods of testicular
increase, as it was high in periods of testicular recrudescence and
low in periods of deactivation.
The regression in October–November seems to be more
drastic, with an accentuated decrease in the epithelium, which
could be caused by the large decrease in the stimulus (low AR
expression). With a gradual increase in AR expression, the testes
begin the recrudescence process that occurs slowly and extends
until March, with the production of the first spermatozoa.
This slow recrudescence process possibly occurs in response
to a slow and gradual increase in AR expression; however, the
detailed mechanisms that regulate this response still seem
uncertain. Another interesting question that still remains unan-
swered is the difference in the time of reactivation and occur-
rence of spermatogenesis observed between December–March
and June–July, since, in most mammals, the time from the start
of the spermatogenic cycle to the production of spermatozoa is
fixed: 64 days in humans (Heller and Clermont 1963), 40 days
in mice (Hess and França 2008), etc.
Wang et al. (2009) found that AR expression in Sertoli cells
plays the most important role in spermatogenesis during the
meiosis I stage; this was done using knockout mice with absent
AR expression in Sertoli cells in which the spermatogenesis
process was arrested at the diplotene primary spermatocytes
stage. Furthermore, they found that AR expression in Leydig
cells influences steroidogenic functions, as the lack of a func-
tional AR leads to arrest at the round spermatid stage. Similarly,
Holdcraft and Braun (2004) proposed that normal AR function
in Sertoli cells is also required for the terminal differentiation of
spermatids. As seen in our data, the stages of spermatogenesis
proceed according to those proposed by these authors: an
increase in the expression of AR in Sertoli cells (November–
January) stimulates spermatogonia to differentiate into sperma-
tocytes (December) and later to round spermatids (January), and
differentiation to elongated spermatids is linked to a peak in AR
expression in Leydig cells (February). However, the meaning of
the inverse relationship of AR expression in Sertoli and Leydig
cells remains unclear.
Reproduction, Fertility and Development K
On the other hand, the recrudescence process in June seems
to occur rapidly, possibly due to the strong and quick peak of the
stimulus (AR expression) and the less-accentuated regression
preceding it.
Apoptosis, the active, physiologically and genetically
controlled, signal-induced process of selective cell death
(Schwartzman and Cidlowski 1993), is normally conspicuous
during normal spermatogenesis in most mammalian species
(Sinha-Hikim and Swerdloff 1999; Print and Lakoski-
Loveland 2000); however, it appears to present different roles
in seasonally breeding species. It seems to directly contribute to
testicular regression in the Chinese soft-shelled turtle, ham-
sters, white-footed mice, the European brown hare (Zhang et al.
2008) and the bat Miniopterus inflatus (Onyango et al. 1995).
However, similar to what was observed by Dadhich et al.
(2010) in the Iberian mole, in M. nigricans, apoptosis is not
linked to periods of testicular regression; on the contrary, it
is linked to periods of spermatogenic peak, indicating that it is
not linked to testicular regression but with the elimination of
errors caused by the process of regression or by the rapid
reactivation of spermatogenesis. The detailed mechanisms that
regulate testicular regression remain unclear but can possibly
be linked to deactivation or the absence of activation by stimuli
for cell division.
Fleming et al. (1972) proposed that the reproductive cycles
of tropical bat species are directly influenced by climatic factors
(temperature, rainfall, photoperiod etc.) and also by the viability
of the food supply. In insectivorous bats, the temperature may
have a direct physiological influence on reproduction or present
an indirect role, acting on prey availability, which influences
energy resources (Racey et al. 1987). Similarly, continuous
rainfall negatively affects reproduction by reducing prey abun-
dance and increasing the energetic costs of homeothermy (Tuttle
and Stevenson 1982); however, it can indirectly influence
reproduction as well, with greater prey availability in the post-
rain period. It is predicted that the photoperiod enforces season-
ality in temperate zone bats (Heideman et al. 1992), acting in at
least two ways: turning the breeding season on or off and
influencing the endogenous circannual rhythm (Heideman
and Bronson 1994); however, little is known about its effects
at low latitudes.
Despite the low sampling number, comparing the reproduc-
tive data to the abiotic parameters, we observed that these
factors seemed to have only a minimal direct effect on the
male reproduction of M. nigricans, showing that its reproduc-
tive cycle is regulated by other factors (physiology, behaviour
etc.) or that it has not been properly regulated to the environ-
ment where the species lives (recent dispersion and a short time
of adaptation of the species to the new environment means that
the species shows patterns of its ancestral region, a temperate
region, yielding new patterns). Another possibility would be
that the male reproductive cycle is influenced by abiotic factors
only in an indirect way by variation in food resources, acting
directly in the male food intake, or regulating the female
reproductive cycle (acting on the breastfeeding period), and
that this (female cycle) would regulate the male cycle acting
via female receptivity to copulation (pheromones, sexual
behaviour etc.).
L Reproduction, Fertility and Development
Conclusion
These results indicate that M. nigricans, an exclusively Neo-
tropical species of vespertilionid bat, presents two peaks of
spermatogenic activity followed by two periods of total testi-
cular regression (a quiescent pre-pubertal-like morphology,
where only Sertoli cells and spermatogonia could be observed),
in the same annual reproductive cycle, which seem to be only
indirectly influenced by abiotic factors. This testicular behav-
iour seems to be synchronised with the caput and corpus
epididymidis, but not with the cauda epididymidis, which
present aspects of sperm storage in May–June. The control of
this variation seems to be directly linked to the expression of the
AR, since, throughout the year, it is high in periods of testicular
recrudescence and low in periods of deactivation. It is thought
not to be directly linked to apoptosis, which is more pronounced
in periods of recrudescence than in periods of regression.
Acknowledgements
Technical help from Luiz Roberto Falleiros Junior was highly appreciated.
The scholarship awarded to Mateus Rodrigues Beguelini by the Brazilian
Research Foundation (CAPES) is also gratefully acknowledged. This
research was supported by Grants 2009/16181–9 and 2009/03470–2 from
the São Paulo State Research Foundation (FAPESP) and Brazilian National
Research and Development Council (CNPq) Processes: 300163/2008–8
and 301596/2011–5 research fellowships to SRT and Process: 302008/
2010–1 to EMV.
References
Beguelini, M. R., Moreira, P. R. L., Faria, K. C., Marchesin, S. R. C., and
Morielle-Versute, E. (2009). Morphological characterization of
the testicular cells and seminiferous epithelium cycle in six species
of Neotropical bats. J. Morphol. 270(8), 943–953. doi:10.1002/
JMOR.10731
Beguelini, M. R., Taboga, S. R., and Morielle-Versute, E. (2012). Ultra-
structural characteristics of spermatogenesis in Pallas’s Mastiff bat,
Molossus molossus (Chiroptera: Molossidae). Microsc. Res. Tech.
75(7), 856–868. doi:10.1002/JEMT.22005
Crichton, E. G., and Krutzsch, P. H. (2000). ‘Reproductive Biology of Bats’.
(Academic Press: London.)
Dadhich, R. K., Real, F. M., Zurita, F., Barrionuevo, F. J., Burgos, M., and
Jiménez, R. (2010). Role of apoptosis and cell proliferation in the
testicular dynamics of seasonal breeding mammals: a study in the Iberian
mole, Talpa occidentalis. Biol. Reprod. 83, 83–91. doi:10.1095/
BIOLREPROD.109.080135
De Knegt, L. V., Silva, J. A., Moreira, E. C., and Sales, G. L. (2005). Bats
found in the city of Belo Horizonte, MG, 1999–2003. Arq. Bras. Med.
Zoo. 57(5), 576–583. doi:10.1590/S0102-09352005000500002
Dinerstein, E. (1986). Reproductive ecology of fruit bats and the seasonality
of fruit production in a Costa Rican cloud forest. Biotropica 18(4),
307–318. doi:10.2307/2388574
Dwyer, P. D. (1970). Latitude and breeding season in a polyestrus species of
Myotis. J. Mammal. 51(2), 405–410. doi:10.2307/1378506
Encarnação, J. A., Dietz, M., and Kierdorf, U. (2004). Reproductive
condition and activity pattern of male Daubenton’s bats (Myotis dau-
bentonii) in the summer habitat. Mamm. Biol. 69(3), 163–172.
doi:10.1078/1616-5047-00131
Fleming, T. H., Hooper, E. T., and Wilson, D. E. (1972). Three Central
American bat communities: structure, reproductive cycles and move-
ment patterns. Ecology 53, 555–569. doi:10.2307/1934771
M. R. Beguelini et al.
Gopalakrishna, A. (1948). Studies on the embryology of microchiroptera.
Part II. Reproduction in the male vespertilionida bat Scotophilus
wroughtoni (Thomas). Proceed. Indian Acad. Sci. B 27, 137–151.
Grindal, S. D., Collard, T. S., Brigham, R. M., and Barclay, R. M. R. (1992).
The influence of precipitation on reproduction by Myotis bats in British
Columbia. Am. Midl. Nat. 128, 339–344. doi:10.2307/2426468
Gustafson, A. W. (1979). Male reproductive patterns in hibernating bats.
J. Reprod. Fertil. 56(1), 317–331. doi:10.1530/JRF.0.0560317
Gustafson, A. W., and Damassa, D. A. (1985). Annual variations in plasma
sex steroid-binding protein and testosterone concentrations in the adult
little brown bat: relation to the asynchronous recrudescence of the testis
and accessory reproductive organs. Biol. Reprod. 33, 1126–1137.
doi:10.1095/BIOLREPROD33.5.1126
Gustafson, A. W., and Shemesh, M. (1976). Changes in plasma testosterone
levels during the annual reproductive cycle of the hibernating bat, Myotis
lucifugus lucifugus with a survey of plasma testosterone levels in adult
male vertebrates. Biol. Reprod. 15, 9–24.
Heideman, P. D., and Bronson, F. H. (1994). An endogenous circannual
rhythm of reproduction in a tropical bat, Anoura geoffroyi, is not
entrained by photoperiod. Biol. Reprod. 50, 607–614. doi:10.1095/
BIOLREPROD50.3.607
Heideman, P. D., Deoraj, P., and Bronson, F. H. (1992). Seasonal reproduc-
tion of a tropical bat, Anoura geoffroyi, in relation to photoperiod.
J. Reprod. Fertil. 96, 765–773. doi:10.1530/JRF.0.0960765
Heller, C. G., and Clermont, Y. (1963). Spermatogenesis in man: an estimate
of its duration. Science 140(3563), 184–186. doi:10.1126/SCIENCE.
140.3563.184
Hess, R. A., and França, L. R. (2008). Spermatogenesis and cycle of the
seminiferous epithelium. In ‘Molecular Mechanisms in Spermatogene-
sis’. (Ed. C. Y. Cheng.) pp. 1–15. (Landes Bioscience and Springer
Science þ Business Media: New York, NY.)
Holdcraft, R. W., and Braun, R. E. (2004). Androgen receptor function is
required in Sertoli cells for the terminal differentiation of haploid
spermatids. Development 131, 459–467. doi:10.1242/DEV.00957
Krishna, A., and Singh, K. (1997). The relationship between testicular
activity, accessory sex glands and circulating steroid concentration
during the reproductive cycle in a male Indian vespertilionid bat,
Scotophilus heathi. Can. J. Zool. 75, 1042–1050. doi:10.1139/Z97-125
Krutzsch, P. H. (1979). Male reproductive patterns in non-hibernating bats.
J. Reprod. Fertil. 56(1), 333–344. doi:10.1530/JRF.0.0560333
Krutzsch, P. H. (2009). The reproductive biology of the cave Myotis
(Myotis velifer). Acta Chiropterol. 11(1), 89–104. doi:10.3161/
150811009X465712
Lee, J. H., and Mori, T. (2004). Annual cycle of the seminiferous epithelium
of Myotis macrodactylus. J. Facul. Agric. Kyushu Univ. 49(2), 355–365.
León-Galván, M. A., López-Wilchis, R., Hernández-Pérez, O., Arenas-
Rı́os, E., and Rosado, A. (2005). Male reproductive cycle of Mexican
big-eared bats, Corynorhinus mexicanus (Chiroptera: Vespertilionidae).
Southwest. Nat. 50(4), 453–460. doi:10.1894/0038-4909(2005)050
[0453:MRCOMB]2.0.CO;2
Lewis, S. E. (1993). Effect of climatic variation on reproduction by pallid
bats (Antrozous pallidus). Can. J. Zool. 71, 1429–1433. doi:10.1139/
Z93-197
Myers, P. (1977). Patterns of reproduction of four species of vespertilionid
bats in Paraguay. Univ. Calif. Publ. Zool. 107, 1–41.
O’Farrell, M. J., and Studier, E. H. (1973). Reproduction, growth
and development in Myotis thysanodes and M. lucifugus (Chiroptera:
Vespertilionidae). Ecology 54(1), 18–30. doi:10.2307/1934371
Onyango, D. W., Gachoka, G. E., Otianga’a-Owiti, G. E., and Hendrickx,
A. G. (1995). Seasonally dependent testicular apoptosis in the tropical
Long-fingered bat (Miniopterus inflatus). Z. für Säugetierkunde, Inter-
nat. J. Mammal. Biol. 60, 206–214.
 Total testicular regression in Myotis nigricans
Print, C. G., and Lakoski-Loveland, K. (2000). Germ-cell suicide:
new insights into apoptosis during spermatogenesis. Bioessays
22, 423–430. doi:10.1002/(SICI)1521-1878(200005)22:5,423::AID-
BIES4.3.0.CO;2-0
Racey, P. A., and Entwistle, A. C. (2000). Life-history and reproductive
strategies of bats. In ‘Reproductive Biology of Bats’. (Eds E. G. Crichton
and P. H. Krutzsch.) pp. 363–414. (Academic Press: London.)
Racey, P. A., Speakman, J. R., and Swift, S. M. (1987). Reproductive
adaptations of heterothermic bats at the northern borders of their
distribution. S. Afr. J. Sci. 83, 635–638.
Ribeiro, M. G., and Lima, S. R. (2000). ‘Iniciação às Técnicas de Preparação
de Material Para o Estudo e Pesquisa em Morfologia’. (SEGRAC –
Editora e Gráfica Limitada: Belo Horizonte.)
Schwartzman, R. A., and Cidlowski, J. A. (1993). Apoptosis: the biochem-
istry and molecular biology of programmed cell death. Endocr. Rev. 14,
133–151.
Simmons, N. B. (2005). Order Chiroptera. In ‘Mammal Species of the
World: a Taxonomic and Geographic Reference’. (Eds D. E. Wilson and
D. M. Reeder.) pp. 312–529. (Johns Hopkins University Press:
Baltimore.)
Sinha Hikim, A. P., and Swerdloff, R. S. (1999). Hormonal and genetic
control of germ-cell apoptosis in the testis. Rev. Reprod. 4, 38–47.
doi:10.1530/ROR.0.0040038
Taddei, V. A. (1976). The Reproduction of Some Phyllostomidae (Chir-
optera) from the Northwestern Region of the State of São Paulo. Vol. 1.
Bol. Zool. Univ. São Paulo 1, 313–330.
Taddei, V. A. (1980). Biologia reprodutiva de Chiroptera: perspectivas e
problemas. Inter-facies Escrit. Doc. 6, 1–18.
Tuttle, M. D., and Stevenson, D. (1982). Growth and survival of bats.
In ‘Ecology of Bats’. (Ed. T. H. Kunz.) pp. 105–150. (Plenum Press:
New York.)
van der Merwe, M., and Rautenbach, I. L. (1987). Reproduction in
Schlieffen’s bat, Nycticeius schlieffenii, in the eastern Transvaal
www.publish.csiro.au/journals/rfd
View publication stats
Reproduction, Fertility and Development M
lowveld, South Africa. J. Reprod. Fertil. 81, 41–50. doi:10.1530/
JRF.0.0810041
van der Merwe, M., and Rautenbach, I. L. (1990). Reproduction in rusty bat,
Pipistrellus rusticus, in the northern Transvaal bushveld, South Africa.
J. Reprod. Fertil. 89, 537–542. doi:10.1530/JRF.0.0890537
Wang, R. S., Yeh, S., Tzeng, C. R., and Chang, C. (2009). Androgen receptor
roles in spermatogenesis and fertility: lessons from testicular cell-
specific androgen receptor knockout mice. Endocr. Rev. 30(2),
119–132. doi:10.1210/ER.2008-0025
Weibel, E. R., Kistler, G. S., and Scherle, W. F. (1966). Practical stereologi-
cal methods for morphometric cytology. J. Cell Biol. 30(1), 23–38.
doi:10.1083/JCB.30.1.23
Wilson, D. E. (1971). Ecology of Myotis nigricans (Mammalia: Chiroptera)
on Barro Colorado Island, Panama Canal Zone. J. Zool. 163, 1–13.
doi:10.1111/J.1469-7998.1971.TB04521.X
Wilson, D. E., and Findley, J. S. (1970). Reproductive cycle of a Neotropical
insectivorous bat, Myotis nigricans. Nature 225(5238), 1155.
doi:10.1038/2251155A0
Wilson, D. E., and Findley, J. S. (1971). Spermatogenesis in some Neotropi-
cal species of Myotis. J. Mammal. 52(2), 420–426. doi:10.2307/1378684
Wilson, D. E., and Laval, R. K. (1974). Myotis nigricans. Mammal. Spec. 39,
1–3. doi:10.2307/3503847
Zhang, L., Han, X. K., Qi, Y. Y., Liu, Y., and Chen, Q. S. (2008). Seasonal
effects on apoptosis and proliferation of germ cells in the testes of the
Chinese soft-shelled turtle, Pelodiscus sinensis. Theriogenology 69,
1148–1158. doi:10.1016/J.THERIOGENOLOGY.2008.01.028
Zortéa, M. (2003). Reproductive patterns and feeding habits of three
nectarivorous bats (Phyllostomidae: Glossophaginae) from the Brazilian
Cerrado. Braz. J. Biol. 63(1), 159–168. doi:10.1590/S1519-
69842003000100020