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Histology and structure of the testicles in three species of Atelopus

frogs (Anura: Bufonidae) endemic to the Sierra Nevada

de Santa Marta, Colombia

Arantxa Sánchez-Ferreira

; https://orcid.org/0000-0001-7571-7952

Edgar Javier Rincón-Barón

; https://orcid.org/0000-0003-1347-171X

Luis Alberto Rueda-Solano

1,3

; https://orcid.org/0000-0001-6968-0719

1. Universidad del Magdalena, Facultad de Ciencias Básicas, Grupo de Investigación en Biodiversidad y Ecología

Aplicada, calle 32 No 22-08, Santa Marta, Colombia; biologaarantxasanchez@gmail.com (Correspondence*)

2. Universidad de Santander, Facultad de Ciencias de la Salud, Grupo de Investigación Agroambiente y Salud-

MICROBIOTA, calle 70 No 55-210, Campus Universitario Lagos del Cacique, Bucaramanga, Colombia;

ed.rincon@mail.udes.edu.co

3. Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia; biologoluisrueda@gmail.com

Received 19-XI-2020. Corrected 11-V-2021. Accepted 20-VII-2021.

ABSTRACT

Introduction: Testicular histology constitutes one of the least explored aspects in frogs of the genus Atelopus.

This taxonomic group shows an alarming population decline; therefore, its reproductive biology is one of the

greatest topics of interest for its conservation.

Objective: To describe the testicular morphology and the spermatogenetic lineage cells in adult males of

Atelopus laetissimus, Atelopus nahumae, and Atelopus carrikeri in the Sierra Nevada de Santa Marta, Colombia.

Methods: During June – July 2017 and 2018, sampling was conducted in the localities of San Lorenzo and

Páramo Cebolletas, Sierra Nevada de Santa Marta (SNSM), to collect 15 adult males, 5 per species. Testes

samples were fixed in Bouin to be processed by the standard paraffin-embedding technique. Histological sec-

tions (3 μm) were stained with Hematoxylin-eosin and Mallory-Heidenhain-Azan-Gomori’s. For the description

and photographic register of the germ cells, the photonic microscopy technique was used with the differential

interference contrast system.

Results: The testes are oval organs, compact, light yellow color, and with little vascularization. Externally, they

are surrounded by a thin albuginea tunic constituted by regular dense connective tissue. Inside this layer, they are

composed of numerous seminiferous tubules of hexagonal contour, in which germ cell cysts are distinguished at

different stages of spermatogenesis (spermatogonia I and II, spermatocyte I and II, and early and late spermatids)

and spermiogenesis (spermatozoa in fascicles and free spermatozoa). Separating the seminiferous structures is

the interstitial tissue in which Leydig cells and blood vessels stand out. Additionally, in the cranial part of the

testis, the Bidder’s organ was found, formed by two distinguishable regions, the cortex and the medulla. In the

cortex, there are previtellogénic oocytes of different sizes surrounded by a monolayer of flat follicular cells.

For its part, the medullary region is the connective tissue that nourishes the oocytes and is constituted by blood

capillaries.

Conclusions: The gonads of the three species analyzed present a cystic cellular organization similar to other

anurans, where all stages of spermatogenesis and spermiogenesis were identified, possibly indicating a con-

tinuous reproductive activity. Likewise, the Bidder’s organ is reported for the first time in the three Atelopus

species, which allows suggesting a possible sexual reversion in case of a population decrease of females as a

reproductive strategy.

Key words: spermatogenic cells; histology; bidder’s organ; sexual reversal; testes.

Sánchez-Ferreira, A., Rincón-Barón, E. J., & Rueda-Solano,

L. A. (2021). Histology and structure of the testicles

in three species of Atelopus frogs (Anura: Bufonidae)

endemic to the Sierra Nevada de Santa Marta, Colombia.

Revista de Biología Tropical, 69(3), 811-828. https://doi.

org/10.15517/rbt.v69i3.44727

https://doi.org/10.15517/rbt.v69i3.44727

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In anurans, the male reproductive system is

made up of the testes and a group of ducts that

conduct spermatozoa to the outside (Oliveira

et al., 2002). The testes are paired organs

with an ovoid shape that are located in the

abdominal cavity (Oliveira & Vicentini, 1998;

Pierantoni et al., 2002). Internally they present

two compartments, the germinal and the inter-

stitial. In the first, they are organized by semi-

niferous tubules (Duellman & Trueb, 1994)

where in turn there are cysts of spermatogonia,

spermatocytes, spermatids, and spermatozoa

morphofunctionally associated with Sertoli

cells (Chavadej et al., 2000; Oliveira et al.,

2002). For its part, the interstitium is the tissue

between the seminiferous structures formed by

collagen fibers, blood vessels, Leydig cells,

and other connective tissue elements (Pudney,

1995; Yoshida, 2016). There are several find-

ings related to the morphophysiology of the

reproductive system in anurans. Among these,

some have focused on aspects such as sperm

cell morphology (Carezzano & Cabrera, 2010;

Carezzano et al., 2013), the reproductive cycle

(Ortiz & Díaz-Páez, 2001), plasma androgen

level (Delgado et al., 1989), hormonal stimu-

lants (Aguilar-Miguel et al., 2009), and their

relationship with the development of second-

ary sexual characteristics (Rastogi et al., 1986;

Kaptan & Murathanoğlu, 2008).

The morphology of spermatogenic cells is

widely described within the order Anura and

has been found to be similar (Delgado et al.,

1989; Ko et al., 1998; Oliveira & Zieri, 2005).

For the Neotropics, little has been studied about

the processes of spermatogenesis and spermio-

genesis in this group of organisms (Carezzano

et al., 2013). It has been evidenced that the

majority of anurans present a testis with cystic

cellular organization, although with differences

in aspects such as pigmentation and thickness

of the albuginea tunic (Carlos & Matta, 2009;

Guillama et al., 2014). Dark pigmentation in

the testes has been described in some species

and is caused by cells called melanocytes that

are randomly distributed in the albuginea tunic

and the interstitium (Aoki et al., 1969; Oliveira

et al., 2002; Guillama et al., 2014). These cells

present association with some organs and struc-

tures mainly of the vascular system and the

reproductive system, which generates intra-and

inter-specific differences, for this reason, little

is known to date about the function and role of

these pigments at the gonadal level (Oliveira &

Zieri, 2005; Franco-Belussi et al., 2009).

A unique aspect of the family Bufonidae

is the presence of Bidder’s organ (BO) in the

cranial part of the testis (Vitale-Calpe, 1969;

Petrini & Zaccanti, 1998), whose existence

has led to think of males as possible hermaph-

rodites (Ponse, 1927). This structure has been

described in several species of neotropical

distribution (King, 1907; Ponse, 1927; Alex-

ander, 1932; Brown et al., 2002; Farias et al.,

2002; Calisi, 2005). Historically this organ has

been considered a rudimentary ovary (Petrini

& Zaccanti, 1998) in which only previtello-

génic oocytes are formed under natural condi-

tions; on the contrary, laboratory experiments

have shown that oocytes can reach mature

vitellogénic stages (Pancak-Roessler & Norris,

1991; Tanimura & Iwasawa, 1992; Scaia et

al., 2011). The BO has been reported in males

and females of the same species with a similar

histological morphology (Pancak-Roessler &

Norris, 1991; Tanimura & Iwasawa, 1992;

Duellman & Trueb, 1994; Farias et al., 2002);

in the case of females, it is lost during the

early phase of development while in males

it remains throughout their life (King, 1908;

Ponse, 1927; McDiarmid, 1971). Likewise, it is

indicated that it is better developed in juveniles

than in adults, where in some species it can

degenerate during sexual maturity (McDiar-

mid, 1971). Although Bidder’s organ has been

extensively studied, little is known about its

functionality (Caroli, 1925; Brown et al., 2002;

Silberschmidt-Freitas et al., 2015).

The genus Atelopus (Duméril & Bibron,

1841), toads, or harlequin frogs, include about

100 species distributed in moist lowland and

highland forests, from Costa Rica to Bolivia

(Frost, 2021). In Colombia there are about

45 spp, which are mostly endemic, being the

country with the greatest number and diversity

of this taxon (Frost, 2021). In Latin America,

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previous studies within the genus focus on

distribution patterns (La Marca et al., 2005;

Cusi et al., 2017), population dynamics (Tarvin

et al., 2014), and aspects of its natural history

such as ecology (Lötters, 1996), reproductive

biology (Lynch, 1986), territoriality (Crump,

1988), and emerging diseases (Lampo et al.,

2017). In addition, some works on phylogenetic

relationships and biogeography have included

this genus (Lötters, 1996; Pramuk et al., 2008;

Lötters et al., 2011). In Colombia, the issues

addressed in the genus Atelopus have focused

on ecological aspects such as foraging behavior

(Rueda-Solano & Warkentin, 2016), substrate

use (Granda-Rodríguez & Del Portillo-Mozo,

2007), thermal ecology (Rueda-Solano et al.,

2016a), coloration type (Rueda-Solano, 2008;

Rueda-Solano, 2012), tadpole morphology

(Rueda-Solano et al., 2015; Pérez-González

et al., 2020), monitoring (Gómez-Hoyos et

al., 2014), and diseases caused by the chytrid

fungus (Rueda-Solano et al., 2016b; Flechas et

al., 2017). Several authors have studied impor-

tant aspects of reproductive biology associated

mainly with the breeding season, reproductive

effort, and the interspecific amplexus (Pérez-

González et al., 2017; Rocha-Usuga et al.,

2017). However, other issues such as gonadal

morphology (McDiarmid, 1971; Siqueira et al.,

2013), sexual maturity (La Marca et al., 1989),

and mating patterns (Barrantes-Cartín, 1986;

Crump, 1988; Lötters, 1996; Karraker et al.,

2006) are still unknown to most representatives

of the genus. This group of harlequin frogs is

one of the most endangered in the world (La

Marca et al., 2005), whose populations have

decreased in high mountain habitats, including

areas with little intervention (Pounds & Crump,

1994; Pounds et al., 2006). Turning the five

endemic frog species that inhabit the Sierra

Nevada de Santa Marta (SNMS) into objec-

tives of great interest for the conservation of

the genus (IUCN, 2021), among which are the

three species under study.

This investigation describes for the first

time the testicular morphology in adult males

of Atelopus laetissimus Ruiz-Carranza et al.,

1994, Atelopus nahumae Ruiz-Carranza et al.,

1994, and Atelopus carrikeri Ruthven, 1916

to know part of their reproductive cycle, pro-

viding information on the histological and

structural characteristics of the spermatogenic

cells. Additionally, possible intersexuality is

suggested by the presence of Bidder’s organ

in the cranial part of the testis. It is necessary

to highlight that currently, these species are at

threat of extinction that is why knowing part

of its reproductive biology will contribute

to its conservation.

MATERIALS AND METHODS

Study area: Three sites were sampled at

different altitudes in the SNSM in the depart-

ment of Magdalena in Northern Colombia

(Fig. 1A).

Initially the locality of A. carrikeri which

corresponds to the Páramo de Cebolletas (3

500 m.a.s.l) located on the Sevilla River to the

West of the SNSM (10°54’03” N & 73°55’05”

W) and to the East in the San Pedro de la Sierra

district in Ciénaga-Magdalena (Fig. 1A, Fig.

1B). The popuation of A. laetissimus, located

in the Pascual stream (2 100 - 2 200 m.a.s.l.),

San Pedro de la Sierra, SNSM (10°54’3.70” N

& 73°55’4.50” W) (Fig. 1A, Fig. 1C). Finally,

the A. nahumae specimens that occur at the

headwaters of the Gaira river (1 560 m.a.s.l.),

Serranía de San Lorenzo, SNSM (11°10’2.0” N

& 74°10’41.5” W) (Fig. 1A, Fig. 1D).

Field sampling and specimen data: Five

field studies were carried out during June-July

2017 and 2018 according to the reproductive

season of the species (Carvajalino-Fernández

et al., 2008; Granda-Rodríguez et al., 2008;

Rocha-Usuga et al., 2017), locating adult males

by body size, vocalizations, and the presence

of nuptial pad (Sinsch, 1988; Duellman &

Trueb, 1994; Carezzano et al., 2013). To find

the organisms, the active search method and

the recording by visual encounter were applied

(Crump & Scott, 1994), with an exploration

time that ranged from 6 pm to 11 pm for 3

days in the San Lorenzo sector and 4 days in

the Cebolletas sector. The capture was carried

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out manually with the permission of the Cor-

poración Autónoma Regional del Magdalena,

resolution No 0425 of 2015. The 15 adult males

collected (Atelopus laetissimus N = 5; Atelopus

nahumae N = 5, and Atelopus carrikeri N =

5) were transported to the laboratory in plastic

containers with little water to avoid desicca-

tion (Carezzano et al., 2013). The sacrifice of

the organisms occurred by anesthesia, through

abdominal rubbing with Garhocaine gel (based

on 20 % Benzocaine) (Phuge & Gramapurohit,

2013). Subsequently, an incision was made

in the abdominal wall to remove the gonads

for anatomical and morphological analysis. It

is worth mentioning that each specimen was

treated as established by the Ethics Committee

for the correct handling of organisms, approval

CICUAL No C.FUA. 17_006 of 2017, Uni-

versidad de los Andes, Bogotá-Colombia. The

supporting vouchers were deposited at the

Universidad del Magdalena in Santa Marta-

Colombia with catalogue number CBUMAG:

ANF:01019 - CBUMAG: ANF:01023, CBU-

MAG: ANF:01028, CBUMAG: ANF:01036.

Histological analysis: Testes were

removed and fixed in Bouin’s solution for 48 h

at room temperature (Phuge & Gramapurohit,

2013); subsequently preserving them in 70

% ethyl alcohol. After this period, they were

washed in distilled water to remove the excess

Boiun. They were then dehydrated in the grad-

ual series of ethanols (70 %, 80 %, 90 %, 96 %)

for 4 hours at each step and two final steps in

100 % ethanol, a 4-hour change, and a 12-hour

change (Hopwood, 1990; Pikle et al., 2017).

Rinsing was carried out in two steps by xylol

also at 4 h and 12 h (Guillama et al., 2014).

The infiltration-inclusion was performed in

Paraplast plus (Mc Cormick ®) for 12 h at

Fig. 1. Geographical location of the sampled localities in the Sierra Nevada de Santa Marta- Magdalena, Northern Colombia,

and images of the study species. A. Altitudinal distribution of the localities: San lorenzo-location of Atelopus nahumae,

San Pedro-location of Atelopus laetissimus and Páramo Cebolletas-location of Atelopus carrikeri. B. Atelopus carrikeri. C.

Atelopus laetissimus. D. Atelopus nahumae.

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55 °C (Asenjo at al., 2011) and from this,

cross-sections were obtained with LEICA RM

2125 ® rotary microtome, of 3 μm-thick. The

sections were stained with Hematoxylin-Eosin

(Luna, 1968) and Mallory-Heidenhain-Azan-

Gomori’s (Gomori, 1939; Clark, 1973). The

sections obtained were examined with a Nikon

80i eclipse® and Nikon eclipse Ni photonic

microscope equipped with a differential inter-

ference contrast (DIC) system. Also, some sec-

tions were analyzed with fluorescence, using

the DAPI-FITC-Texas triple band excitation

filter which incorporates an excitation filter

with narrow band pass windows in the violet

(395 to 410 nm), blue (490 to 505 nm), and

green (560 to 580 nm) spectral regions. The

images were digitized with a Nikon DS-Fi1®

camera using the NIS-Elements software ver-

sion 4.30.02. In the course of the study, it was

found that the processes of spermatogenesis

and spermiogenesis occurred similarly in the

three Atelopus species analyzed, therefore,

for the detailed description of the germ cells

the species that provided the best illustrations

were chosen.

RESULTS

The testes are located in the abdominal

cavity, ventral to the kidneys joined to them

by a membrane, the gonadal mesenterium or

mesorchium. In general, they are oval-shaped,

compact, light yellow organs, and with little

vascularization (Fig. 2A). Fatty bodies are

found in their cranial extremity, with several

thin extensions of color yellowish and slightly

translucent (Fig. 2B).

Histologically, the testes are lined by the

albuginea tunic, a thin layer of regular dense

connective tissue in which collagen fibers and

blood vessels predominate. The tunic is thin

and has no pigment cells. Inside this layer,

numerous seminiferous tubules of hexagonal

contour are distinguished in which germ cell

cysts can be observed at different times of

spermatogenesis and spermiogenesis (Fig. 3,

Fig. 4). In cysts, cells unite in the same state

of differentiation, indicating that they have

synchrony of development (Fig. 3A, Fig 4A).

Surrounding the seminiferous tubules is the

interstitial tissue consisting of loose connective

tissue formed by Leydig cells (testosterone-

producing) and loose blood capillaries (Fig.

3B). Leydig cells have a rounded or oval-

contour nucleus and the chromatin is slightly

condensed; at the same time, they can be seen

separately or in groups near the blood vessels

(Fig. 3B).

Germ cells were identified according

to morphology, degrees of nuclear material

Fig. 2. Anatomical aspect of the testis and Bidder’s organ in Atelopus laetissimus. A. Location of the testicle and Bidder’s

organ. B. Association with other organs present in the abdominal cavity. BO: bidder’s organ; FB: fatty bodies; L: lung; LI:

liver; S: stomach; T: Testicle.

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Fig. 3. Aspect of the testicular anatomy in Atelopus carrikeri during the breeding season A. General view of the male gonad

with abundant seminiferous tubules B. Detail of the interstitial tissue between two seminiferous tubules showing Leydig cells

and blood capillaries. Figures A and B stained with Hemtaoxilin-eosin (photon microscopy with the differential interference

contrast-DIC system). C. Primary spermatogonia, in the field with a DAPI-FITC-Texas triple band excitation filter. One per

cyst is seen near the wall of the seminiferous tubule. D-H. Seminiferous tubules with spermatogenic cell cysts at different

stages of development. Mallory-Heidenhain-Azan-Gomori’s stain (photon microscopy with the differential interference

contrast-DIC system). AT: albuginea tunic; BV: blood vessels; BZ: bundle of spermatozoas; C1: primary spermatocytes;

C2: secondary spermatocytes; G1: primary spermatogonia; G2: secondary spermatogonia; IT: interstitial tissue; L: leydig

cells; ST: seminiferous tubule; T1: early spermatids; T2: late spermatids; TW: tubular wall; Z: spermatozoa in the lumen.

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condensation, and cystic arrangement (Fig. 3C,

Fig. 3D, Fig. 3E, Fig. 3F, Fig. 3G, Fig. 3H, Fig.

4A, Fig. 4B, Fig. 4C, Fig. 4D, Fig. 4E, Fig. 4F,

Fig. 4G, Fig. 4H). Spermatogonia are the cells

that initiate the spermatogenic process and

undergo multiple mitotic divisions that give

rise to a large clonal population of germ cells.

Of these, two types were identified: primary

spermatogonia (I) and secondary spermatogo-

nia (II). The former are the largest cells with

a rounded contour, large polymorphic nucleus,

and mostly euchromatic (Fig. 3C). They are

usually very close to the seminiferous tubule

wall and are arranged one per cyst (Fig. 3C).

From their mitotic division, secondary sper-

matogonia originate, cells that do form cysts

and are smaller than the primary. Regarding

their morphocytological characteristics, they

present the basophilic cytoplasm, small spheri-

cal nuclei, and scarcely heterochromatic (Fig.

3D, Fig. 3E, Fig. 4G, Fig. 4H).

In relation to the next stage of devel-

opment, two classes of spermatocytes were

identified: primary spermatocytes (I) and sec-

ondary spermatocytes (II). Primary spermato-

cytes originate from the mitotic differentiation

of secondary spermatogonia and constitute the

most abundant phase during the spermatogen-

esis process. These are cells with oval contour,

large euchromatic nucleus, without nucleolus,

and abundant cytoplasm (Fig. 3D, Fig. 3E, Fig.

3F, Fig. 3G, Fig. 3H, Fig. 4B, Fig. 4C, Fig. 4D,

Fig. 4E, Fig. 4F, Fig. 4H). Generally, they are

observed in different phases of the first meiotic

division, presenting various degrees of conden-

sation in the chromatin. These cells differ from

spermatogonia II because they are larger and

the cysts are larger. For their part, secondary

spermatocytes are haploid cells that are distin-

guished by being smaller than their predeces-

sors (spermatocytes I). They have basophilic

nuclei, with a rounded contour, condensed

chromatin, and the cytoplasm is translucent

without any granulations (Fig. 3D, Fig. 3E, Fig.

3F, Fig. 3G, Fig. 3H, Fig. 4B, Fig. 4C, Fig. 4D,

Fig. 4E, Fig. 4F, Fig. 4G, Fig. 4H).

Continuing with the development phases,

spermatids are the result of the second meiotic

division and their cell population has a wide

variety of shapes ranging from spherical to

elongated (Fig. 3E, Fig. 4D). In the analyzed

species were found in two stages: early sper-

matids (I) and late spermatids (II). Early sper-

matids have a round shape, spherical nucleus,

slightly compressed, and condensed chromatin

(Fig. 3E, Fig. 3H, Fig. 4D). These differ from

spermatocytes II because they are smaller and

with denser chromatin. Late spermatids are

elongated cells with an elongated nucleus and

condensed chromatin (Fig. 3D, Fig. 3E, Fig.

3F, Fig. 3G, Fig. 4D, Fig. 4E, Fig. 4G). In some

preparations, they are seen to have weakly

stained flagella (Fig. 3E, Fig. 4D). Spermatids

undergo the spermiogenesis process to give rise

to spermatozoa, which are the most elongated

and basophilic cells of the germ lineage; their

characteristics include a compact nucleus and

reduced cytoplasm. These were observed at

two moments of spermiogenesis, in the first

phase, they form compact groups like fascicles

due to their association with Sertoli cells, in

such that the proximal part that contains the

nucleus is associated with these nourishing

cells and the distal part that corresponds mainly

to the flagellum is located towards the lumen

of the cyst (Fig. 3F, Fig. 3G, Fig. 3H, Fig. 4B,

Fig. 4C, Fig. 4D, Fig. 4E, Fig. 4F). In the sec-

ond stage, they completely lose the fascicular

arrangement and are free towards the lumen of

the seminiferous tubules (Fig. 3F, Fig. 4B, Fig.

4F, Fig. 4G).

Additionally, the specimens of the three

analyzed species presented a Bidder’s organ in

the cranial part of the testis (Fig. 5A, Fig. 5B).

This organ in vivo appears yellow and

slightly translucent (data not illustrated). His-

tologically, it is made up of two regions: the

cortex and the medulla (Fig. 5C, Fig. 5D). The

medulla is the intermediate tissue that nourish-

es the oocytes, which constitutes a loose irregu-

lar connective tissue with numerous blood

capillaries (Fig. 5D). In the cortex, there are

abundant previthellogenic oocytes of different

sizes, surrounded by a layer of follicular cells

(Fig. 5C). Follicular cells form a simple flat

epithelium (Fig. 5E). For their part, oocytes

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Fig. 4. Aspect of the testicular anatomy in Atelopus nahumae during the breeding season. A. General view of the male

gonad with abundant seminiferous tubules. B-H. Seminiferous tubules with spermatogenic cell cysts at different stages of

development. Photomicrographs stained with Mallory-Heidenhain-Azan-Gomori’s (photon microscopy with the differential

interference contrast-DIC system). AT: albuginea tunic; BZ: bundle of spermatozoas; C1: primary spermatocytes; C2:

secondary spermatocytes; G2: secondary spermatogonia; ST: seminiferous tubule; T1: early spermatids; T2: late spermatids;

TW: tubular wall; Z: spermatozoa in the lumen.

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Fig. 5. Morphophysiology of Bidder’s organ in Atelopus carrikeri and Atelopus nahumae. A-B. Location of the Bidder’s

organ in the cranial portion of the testis. C. Cortex region with previtellogénic oocytes. D. Region of the medulla formed by

connective tissue and blood capillaries. E-F. Different-sized previtellogénic oocytes surrounded by a simple flat epithelium.

G-H. Atretic oocytes with nuclear and cytoplasmic disintegration (*). Sections stained with Mallory-Heidenhain-Azan-

Gomori’s and Hematoxylin-eosin staining (photon microscopy with the differential interference contrast-DIC system).

Photomicrographs A, F-H of A. carrikeri individuals and images B-E of A. nahumae specimens. BO: bidder’s organ; BV:

blood vessels; C: cytoplasm; CO: cortex; CT: connective tissue; FC: follicular cells; M: medulla; N: nucleus; n: nucleolus;

PO: previtellogénic oocytes; ST: seminiferous tubule. The scalloped nuclear envelope of atretic oocytes (↓).

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are large cells, with an acidophilic nucleus

and prominent nucleoli (Fig. 5C, Fig. 5D, Fig.

5G, Fig. 5H). There were also abundant atretic

oocytes with nuclear and cytoplasmic disinte-

gration (Fig. 5G, Fig. 5H). The nuclear mem-

brane of oocytes in the atresia process appears

undulating and the nucleus loses its oval shape

(Fig. 5G, Fig. 5H). In general, the absence of

some type of specialized tissue that separates

the Bidder’s organ from the testicular region is

appreciated, beyond the connective tissue that

nourishes them. Therefore, bidderian oocytes

are close to the seminiferous tubules with cysts

in different spermatogenic stages.

DISCUSSION

The testicular architecture of A. laetis-

simus, A. nahumae, and A. carrikeri presented

great similarity with the descriptions of other

species of the same family such as Rhinel-

la arenarum (Cavicchia & Moviglia, 1983),

Anaxyrus woodhousii (Atherton, 1974), and

Bufo bufo (Falconi et al., 2004). Germline cells

exhibited intense spermatogenic activity and

cyst arrangement, being a common appearance

in anurans and a predictive property of anamni-

otic testicular evolution (Atherton, 1974; Lofts,

1974). Cysts form a hematotesticular barrier

that provides an optimal microenvironment for

germ cell maturity and is a diagnostic feature of

the Anura order (Cavicchia & Moviglia, 1983).

In general, all stages of spermatogenesis were

identified, although there was a predominance

of spermatids (early and late) and spermatozoa

(in fascicles and free), which confirmed sexual

maturity and reproductive activity of the organ-

isms (Iturriaga et al., 2012; Pacheco-Florez

& Ramírez-Pinilla, 2014). This suggests that

the analyzed species may present continuous

spermatogenic activity (Burgos & Mancini,

1948; Oliveira et al., 2002), as occurs in most

species of neotropical distribution where all

cell stages are present throughout the year

(Rastogi, 1976; Lofts, 1987; Oliveira et al.,

2002). However, to corroborate this hypothesis,

it would be necessary to carry out samplings in

non-reproductive seasons.

The morphocytological characteristics of

the primary spermatogonia analyzed in this

work are similar to those reported in the lit-

erature for various groups of anurans (King,

1907; Cavicchia & Moviglia, 1983; Oliveira

& Vicentini, 1998). In contrast, for some spe-

cies like Peltophryne fustiger, Pelophylax ridi-

bunda, and Dendropsophus minutus primary

spermatocytes constitute the largest cells (Kap-

tan & Murathanoğlu, 2008; Sanz-Ochotorena

et al., 2008; Ferreira & Mehanna, 2012). In

relation, to the next stage of development that

includes secondary spermatogonia, a phase

of intense cell division that remains until the

formation of early spermatids (Lofts, 1974;

Rastogi et al., 1988) it was possible to verify

great similarity with the descriptions of other

species including Pseudis limellum (Ferreira et

al., 2008). These are generally more basophilic

cells than primary spermatogonia (Jamieson,

2003), which contrasts with what was observed

in R. arenarum (Burgos & Fawcett, 1956;

Cavicchia & Moviglia, 1983) and D. minutus

(De Souza-Santos & De Oliveira, 2008). At

the same time, secondary spermatogonia were

one of the scarcest cell types in the sections

analyzed and even in the testes of some species

it has not been found (Asenjo et al., 2011; Itur-

riaga et al., 2012).

In this research, it was observed that pri-

mary spermatocytes were commonly larger

than secondary spermatogonia, which is due

to the growth phase they experience dur-

ing prophase I (Salas & Martino, 2008). In

this period, chromatin presents a variation in

the degree of condensation, being a common

aspect with our observations and in the descrip-

tions of other anurans (Ortiz & Díaz-Páez,

2001; Villagra et al., 2014). Additionally, they

were distinguished in greater numbers than

secondary spermatocytes as reported in other

taxonomic groups (Ferreira et al., 2008). On the

other hand, the secondary spermatocytes were

observed smaller and with denser chromatin

than their congeners, the primary spermato-

cytes. These cytological characteristics are

similar to some representatives of the family

Bufonidae (Echeverría & Maggese, 1987) and

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other species like Calyptocephalella gayi (Her-

mosilla et al., 1983) and Ceratophrys cultripes

(Báo et al., 1991).

In this work, two stages are described for

spermatids in a manner consistent with what

some authors have found (Ortiz & Díaz-Páez,

2001; Oliveira et al., 2002). Initially, they were

rounded cells that lengthened as they matured,

once in the late spermatid phase, they no longer

form cysts but are organized into fascicles as

observed in this study (Lofts, 1974; Oliveira

et al., 2002). However, in other taxa such as

P. ridibunda three stages of development have

been described, which includes an intermediate

phase (Kaptan & Murathanoğlu, 2008). The

morphological and histological characteristics

of the spermatozoa and their arrangement in

the seminiferous tubules did not vary in rela-

tion to that described in other works (Taboga

& Dolder, 1991; Scheltinga et al., 2002). In

this investigation, the Sertoli cells could not be

observed, perhaps due to the lack of detail in

the sections or of more histological sections,

a fact that contrasts with most studies where

they have been described morphofunctionally

associated with spermatogenic cells (Carez-

zano et al., 2013). Nevertheless, late spermatid

bundles were observed in the analyzed prepa-

rations, which indicates the presence of these

nourishing cells.

The albuginea tunic did not present pig-

ment cells in a common way with most anurans

(Duellman & Trueb, 1986; Franco-Belussi et

al., 2009). However, the presence of pigments

has been recorded in several species including

some of the genus Atelopus like A. varius, A.

longirostris, and A. senex (McDiarmid, 1971;

Franco-Belussi et al., 2009; Guillama et al.,

2014). Sanz-Ochotorena et al. (2011) describe

pigments in the testes of four Cuban anurans

and attribute them a protective function against

anthropogenic contamination and solar radia-

tion. Additionally, McDiarmid (1971) suggests

that they could be associated with the reproduc-

tive condition or with protection from ultravio-

let light. On the other hand, it is considered that

the melanin present in testicular melanocytes

can protect male gametes from oxidative stress

(Galván et al., 2011). However, this type of

hypothesis would need more studies, given

the testes are located in the abdominal cavity

protected by other tissues, which would not

imply the need to develop pigments to pro-

tect themselves from some cytotoxic effect

caused by ultraviolet radiation. Other findings

in anurans relate these pigments to an extracu-

taneous pigmentation system that is present in

several organs in addition to the primary sexual

characteristics, where the mode of appearance

and the number of melanocytes is highly vari-

able among species (Oliveira & Zieri, 2005).

Accordingly, Franco-Belussi et al. (2009) stud-

ied the testes of seventeen species of the Lep-

todactylidae, finding that the distribution of

melanocytes was variable, and therefore there

were differences in the degree of pigmentation

of the gonads, proposing that it could be an

intrinsic characteristic of the species or physi-

ological response. Therefore, the role of these

testicular pigments is still unknown, and more

exhaustive investigations are required taking

into account ecological, histological, and phy-

logenetic aspects in the genus Atelopus.

The presence of the Bidder’s organ is reg-

istered for the first time in the analyzed species,

which has been reported in other species of

the genus Atelopus (Griffiths, 1954; Griffiths,

1959; McDiarmid, 1971). Likewise, it is pres-

ent in most lineages of the family Bufonidae

(Ponse & Guyénot, 1927; Alexander, 1932;

Brown et al., 2002; Falconi et al., 2007) exclud-

ing Melanophryniscus (Echeverría, 1997; Pelo-

so et al., 2012) and Truebella (Graybeal &

Cannatella, 1995). Although certain authors

consider the Bidder’s organ a synapomorphy of

the clades derived from bufonids, the findings

in the species A. laetissimus, A. nahumae, and

A. carrikeri are contradictory since the genus

Atelopus constitutes an early divergent lineage

of bufonids (Pramuk et al., 2008). So, it is a

character shared only by part of the family

(Frost et al., 2006). Atelopus is a monophyletic

group (Lötters et al., 2011), related to other

genera of the family in which BO has been

described, such as Osornophryne, Peltophryne,

Bufo, and Rhinella (Graybeal & Cannatella,

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1995; Pramuk, 2006; Pyron & Wiens, 2011;

Pereyra et al., 2021). Therefore, the anatomical,

morphological, and histological characteristics

seen in BO are similar to those recorded in

other investigations where the presence of this

structure has been detected (Griffiths, 1954;

Griffiths, 1959; Silberschmidt-Freitas et al.,

2015; Copa, 2019). Our findings indicate that

the BO does not reach the formation of viable

oocytes and, on the contrary, they enter atresia

and are reabsorbed, which is similar to that

registered in Bufo bufo, Rhinella icterica, Rhi-

nella marina, and Peltophryne empusa (Zac-

canti & Gardenghi, 1968; Farias et al., 2002;

Abramyan et al., 2010; Copa, 2019). However,

some authors described the presence of vitel-

logénic oocytes in the BO of some bufonids as

R. marina and R. arenarum during the breeding

season (McCoy et al., 2008; Scaia et al., 2011).

Additionally, Lambert et al. (2019) reported

that some adult males of the Rana clamitans

changed sex and another group of organisms

presented oocytes in their testes (inter-sex) in

pristine conditions, despite this, the factors that

can cause this sexual reversal is unknown.

In the Bidder’s organ, abundant degenerat-

ed and atretic oocytes were identified similarly

with other bufonids, such as R. marina and

R. arenarum (Brown et al., 2002; Scaia et al.,

2011). This could be due to the lack of stimuli

to continue its development or to the absence

of a specialized tissue that separates the BO

from the testicles (Silberschmidt-Freitas et al.,

2015). The latter would be explained by the

hormonal regulation of some androgens that

can cause oocyte degeneration and at the same

time inhibit the uptake of estradiol for vitel-

logenesis to occurs (Calisi, 2005). Despite this,

different studies consensus that oogenesis is not

completed in males under natural conditions

(Silberschmidt-Freitas et al., 2015). Our find-

ings on the presence of the Bidder’s organ in

the three Atelopus species studied, allow us to

suggest that males may possibly present sexual

reversion because this organ could be used in

the case of a population decrease in females as

a possible strategy reproductive. Considering

that this taxonomic group is highly threatened,

further research in this direction could contrib-

ute to understanding the stability of their popu-

lations and their current conservation status.

Additionally, research on the morphohistology

of the testis is important to have a broader

understanding of the reproductive biology of

the genus Atelopus and of neotropical anurans

in general, for which literature is scarce.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

We thank the Universidad de Santander

(UDES), the Biotechnology Laboratory of the

University Research Headquarters -SIU- of

the Universidad de Antioquia (UdeA), and the

Biodiversity and Applied Ecology Research

Group (GIBEA) of the Universidad del Mag-

dalena. Thanks to Universidad de los Andes

(Colombia) internal scholarship INV-2018-34-

1281 and Colciencias Grant/Convocatoria 757

of 2016 for providing financial support to the

project ‘‘Adaptive Implications of the Mating

System and Female-Guarding in Harlequin

Frogs (Bufonidae: Atelopus)’’. This study was

part of Arantxa Sánchez-Ferreira’s Bachelor›s

thesis in Biological Sciences, directed by Luis

Alberto Rueda-Solano (Beto Rueda).

RESUMEN

Histología y estructura de los testículos en tres especies

de ranas Atelopus (Anura: Bufonidae) endémicas de

la Sierra Nevada de Santa Marta, Colombia

Introducción: La histología testicular constituye uno de los

aspectos menos explorados en las ranas del género Atelo-

pus. Este grupo taxonómico ostenta un declive poblacional

alarmarte, es por ello, que su biología reproductiva resulta

uno de los temas de mayor interés para su conservación.

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Objetivo: Describir la morfología testicular y las células

del linaje espermatogénico en machos adultos de Atelopus

laetissimus, Atelopus nahumae y Atelopus carrikeri en la

Sierra Nevada de Santa Marta, Colombia.

Métodos: Durante Junio - Julio de 2017 y 2018 se realiza-

ron muestreos en las localidades de San Lorenzo y Páramo

Cebolletas, Sierra Nevada de Santa Marta (SNSM), para

recolectar 15 machos adultos, 5 por especie. Las mues-

tras de testículo se fijaron en Bouin para ser procesadas

mediante la técnica estándar de inclusión en parafina. Las

secciones histológicas (3 μm) se tiñeron con Hematoxilina-

eosina y Mallory-Heidenhain-Azan-Gomori’s. Para la des-

cripción y registro fotográfico de las células germinales, se

utilizó la técnica de microscopía fotónica con el sistema de

contraste diferencial de interferencia.

Resultados: Los testículos son órganos ovalados, com-

pactos, de color amarillo claro y con poca vascularización.

Externamente, están rodeados por una delgada túnica

albugínea constituida por tejido conectivo denso regular.

Al interior de esta capa se componen por numerosos

túbulos seminíferos de contorno hexagonal, en los que se

distinguen quistes de células germinativas en diferentes

etapas de la espermatogénesis (espermatogonia I y II,

espermatocito I y II y espermátidas tempranas y tardías) y

espermiogénesis (espermatozoides en fascículos y esper-

matozoides libres). Separando las estructuras seminíferas

se halla el tejido intersticial en el que se destacan las células

de Leydig y los vasos sanguíneos. Adicionalmente, en la

parte craneal del testículo se encontró el órgano de bidder

formado por dos regiones diferenciables, la corteza y la

medula. En la corteza se aprecian ovocitos previtelogénicos

en diferente tamaño rodeados por una monocapa de células

foliculares planas. Por su parte, la región medular es el

tejido conectivo que nutre los ovocitos y está constituido

por capilares sanguíneos.

Conclusiones: Las gónadas de las tres especies analizadas

presentan una organización celular quística de manera

similar con otros anuros, donde se identificó todos los esta-

dios de la espermatogénesis y espermiogénesis indicando

posiblemente una actividad reproductiva continua. Así

mismo, se reporta por primera vez el órgano de bidder en

las tres especies de Atelopus, lo cual permite sugerir una

posible reversión sexual en caso de una disminución pobla-

cional de las hembras como una estrategia reproductiva.

Palabras clave: células espermatogénicas; histología;

órgano de bidder; reversión sexual; testículos.

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