Morphological development of testes in ostrich (Struthio camelus) embryo

ORIGINAL ARTICLE

Morphological development of testes in ostrich (Struthio camelus)embryo

Belal Hassanzadeh • Abolghasem Nabipour •

Morteza Behnam Rassouli • Hesam Dehghani

Received: 27 June 2013 / Accepted: 26 September 2013 / Published online: 15 October 2013

� Japanese Association of Anatomists 2013

Abstract Although the histological structure of ostrich

testis has been studied, very little information is currently

available on the embryonic development of this organ. The

aim of this study was to determine the sequence of the

histological changes in diverse components of the testis in

ostrich embryo from embryonic day (E) 20 to E42. The

main findings were categorized into four histological fea-

tures, i.e., development of sex cords, interstitial tissue and

rete ducts, and the appearance of defective septa. While the

lumen of sex cords, tunica albuginea, capsular rete ducts

and Leydig cell precursors appeared at E26, the filum-

shaped defective septa were visible at E36. The emersion of

the lumen in the primary sex cords and formation of cap-

sular rete ducts in the ostrich embryo is considerably dif-

ferent from that in other birds. However, tunica albuginea

and Leydig cell precursors appeared in a similar pattern to

those of other birds. An interesting observation was that the

primordial germ cell (PGC)-like cells were completely

distinct, while the capsular rete ducts were formed by

trapping of some Sertoli cell aggregations in the tunica

albuginea. This suggests that similar to the primary sex

cords, the capsular rete ducts may originate from the Sertoli

cell aggregations which had corralled some PGCs.

Stereological estimations in the ostrich embryo testis

showed the major proportion of testis is occupied by the

seminiferous tubules, which is unlike the fowl embryo

testis.

Keywords Ostrich embryo � Primordial germ cells �Rete ducts � Seminiferous tubules � Tunica albuginea

Introduction

General stages of the testicular development have already

been determined in many avian species, and testes in avian

embryos are known to arise from the genital ridge at both

sides of the dorsal mesentery (Fujimoto et al. 1976; Van

Krey 1990). The genital ridges then develop to undiffer-

entiated gonads and subsequently to morphologically

identical testes (Smith 2007). During this process, Sertoli

cell progenitors and primordial germ cells (PGCs) emerge

in the primary sex cords where they become wavy and

anastomose to each other, forming the reticular features

seen in histological sections (Romanoff 1960; Morrish and

Sinclair 2002; Gonzalez-Moran and Soria-Castro 2010a).

In the fowl embryo, the seminiferous cords lack a lumen by

embryonic day (E) 13, but the central axis produces a slit

and the lumen appears by E20 (approximate time of

hatching) (Gonzalez-Moran and Soria-Castro 2010a).

Studies on the morphology and development of testis are

mostly performed on mammals and some known birds. To

date, the morphological development of testis has been

well studied in some avian species, such as domestic fowl

and quail (Gonzalez-Moran 1997; Gonzalez-Moran and

Soria-Castro 2010a, b; Chang et al. 2012), but pre-hatching

studies have been done only on chick and quail testis, with

the primary focus on early embryological events in the

B. Hassanzadeh � A. Nabipour (&) � H. Dehghani

Department of Basic Science, Faculty of Veterinary Medicine,

Ferdowsi University of Mashhad, Mashhad, Iran

e-mail: [email protected]; [email protected]

M. Behnam Rassouli

Department of Biology, Faculty of Science, Ferdowsi University

of Mashhad, Mashhad, Iran

H. Dehghani

Embryonic and Stem Cell Biology and Biotechnology Research

Group, Research Institute of Biotechnology, Ferdowsi

University of Mashhad, Mashhad, Iran

123

Anat Sci Int (2014) 89:129–139

DOI 10.1007/s12565-013-0207-9

testis (Zaccanti et al. 1990; Sekido and Lovell-Badge 2007;

Tagami et al. 2007) and early or late testicular development

(Csaba et al. 1980; Shahin and Torok 1982; Gonzalez-

Moran and Soria-Castro 2010a, b). There have been few

pre-hatching developmental studies in ostrich and other

ratite birds, and these have focused mainly on embryonic

timing or hatchability of the eggs (Gefen and Ar 2001;

Malecki et al. 2005; Nagai et al. 2011). We have recently

published a morphological study on the juvenile ostrich

testis (Hassanzadeh et al. 2013), and in a few other studies

the authors have explained the morphological features of

adult ostrich testis (Soley 1992; Aire and Soley 2003;

Ozegbe et al. 2006, 2008, 2010; Lan et al. 2007; Zhang

et al. 2011); however, developmental studies of ostrich

testis are still scant and limited to the post-hatching period

(Madekurozwa et al. 2002; Wei et al. 2011). Budras and

Meier (1981) have performed the only developmental

study on the testicular excurrent ducts in ratite birds. This

study covers both the pre- and post-hatching periods, and

the testis in the juvenile and adult ostrich was compared

with that in rhea and emu.

In the study reported here, we studied in detail the mor-

phological development of ostrich testis, including the for-

mation of sex cords, testicular capsule and capsular rete ducts.

We also evaluated the morphometrical changes in the testis

and its tubular compartments quantitatively using stereolo-

gical techniques. Our results provide novel information on the

developmental characteristics of the ostrich testis.

Materials and methods

Animals and tissue samples

A total of 50 fertile ostrich eggs obtained from commercial

farms were incubated at 36–37 �C and 25 ± 2 % relative

humidity with tilting to 90� at 4-h intervals for 20, 26, 36 and

42 days. Due to the coincidence of E42 with hatching day

and in order to minimize the number of animals sacrificed,

hatched chicks were sexed using the method of Malago et al.

(2002). After decapitation of male chicks and embryos and

the drainage of blood, the testicles were anatomically ana-

lyzed, photographed and separated from the body and placed

in Bouin’s solution. Three samples of testes at each of E26,

36 and 42 (size approx. 1 mm3) were taken and placed in 2 %

glutaraldehyde in 1 M cacodylate buffer for embedding in

resin. All procedures were approved by the Ethics Scientific

Committee of the Ferdowsi University of Mashhad.

Morphometry

Stereological techniques were employed to determine the

amount of testicular volume occupied by seminiferous

tubules (volume fraction or volume density of seminiferous

tubules in the testis). After fixation, samples (whole testes)

were dehydrated in increasing degrees of ethanol, cleared in

xylene and embedded in paraffin. Due to the existence of a

directional asymmetry (Moller 1994), the stereological

experiments were done only on the left testicles. Tissue

shrinkage was calculated by measuring testicle diameter just

before fixation and embedding in paraffin. Paraffin-embed-

ded samples were cut into 5-lm-thick serial sections and

sampled (at least 11 sections per sample) according to the

unbiased systematic random sampling (SRS) method. After

staining of the sections with Periodic acid–Schiff (PAS)

stain, several fields from large sections were selected and

imaged according to SRS method. The images were placed

under a stereology grid (with 300 intersection points without

any direction), and when the points were positioned on the

whole section, the reference area and seminiferous tubules

were analyzed and counted. The total volume of the testis

(Tes.V) was estimated according to the Cavalieri’s principle.

The volume fraction of seminiferous tubules in each testis

(Vv) was calculated using Eqs. 1, 2 and 3 based on the De-

lesse principle (Delesse 1848; Howard and Reed 2005):

Vv ¼ �a�A

ð1Þ

�a ¼ a1 þ a2 þ � � � þ ak

kð2Þ

�A ¼ A1 þ A2 þ � � � þ Ak

kð3Þ

Where a, A and k are the area occupied by seminiferous

tubules, the reference area and the number of images,

respectively. The volume of testis was multiplied by Vv to

estimate the total volume of seminiferous tubules (S.T.V).

The results were analyzed by one-way analysis of variance,

and the different groups were determined by Tukey’s

multiple comparison test. Differences among mean Vv of

the seminiferous tubules were considered to be statistically

significant at P \ 0.05.

Histology

The glutaraldehyde-fixed samples were re-fixed in 1 %

osmium tetroxide in 1 M cacodylate buffer. These samples

were dehydrated by the progressive lowering temperature

(PLT) method, embedded in epoxy resin (TAAB Labora-

tories Equipment Ltd, UK) and cut in 1-lm-thick sections.

The sections were stained with toluidine blue in order to

identify the lumen of the sex tubules and the lipid granules

of the Leydig cells (Kuo 2007). Some of the 5-lm-thick

sections from different parts of the right and left testes,

near two poles and the equatorial border, were selected for

staining with PAS, hematoxylin and eosin (H&E), Mas-

son’s trichrome (MT) and Alcian blue (AB) to clarify the

130 B. Hassanzadeh et al.

123

details of seminiferous tubules, interstitial tissue and the

testicular capsule. PGCs were recognized based on their

morphological characteristics, which have been described

previously (Ginsburg and Eyal-Giladi 1986; Ishiguru et al.

2009; Gonzalez-Moran and Soria-Castro 2010a; Wei et al.

2011). Images were collected on a digital camera (model

SX210 IS; Canon Inc., Tokyo, Japan) and using a light

microscope (models BX51 and 60; Olympus, Tokyo,

Japan) equipped with a digital camera (model DP12;

Olympus). The acquired images were prepared by Image J

(National Institutes of Health, Bethesda, MD), Paint

(Microsoft, Redwood, WA), and Snagit Editor (TechSmith

Corp., Okemos, MI) software.

Results

Gross anatomical changes

In the 20-day-old embryo, testes having a dirty-white color

were located on the ventral surface of mesonephros kid-

neys. The testes extended from the cranial edge to the mid-

portion of kidneys, where they inclined toward the median

plane of body and were located close to each other. At this

stage, the testes had a curved tortuous or elongated ovoid

shape and were directly attached to the kidneys (Fig. 1a).

In the 26-day-old embryo, the testes had become brighter

and were clearly distinguishable from the kidneys in tex-

ture and color. Their dimensions had increased and the

connection to the kidney had become narrower and distinct.

Their shape changed to elongated ovoid and became reg-

ular relative to the previous stage (Fig. 1b). In the 36-day-

old embryo, the testes had become larger and due to the

regression of mesonephros and growth of metanephros

their location changed caudally toward the metanephros

middle lobe (Fig. 1c). In the 42-day-old embryo or newly

hatched ostrich, the testes extended from the mid-portion of

the cranial lobe of the metanephros to its caudal edge

(Fig. 1d). In all cases the longitudinal axis of testes made

an acute angle with the sagittal axis of the body which was

greater on the right side. The shapes of the testicular

transverse sections changed from elongated in the 20-day-

old embryo to bean-shaped in the 26-day-old embryo, and

then to ovoid in the 36-day old embryo and subsequently to

a circular shape in the 42-day-old embryo.

Morphometrical changes

Stereological techniques were used for evaluating mor-

phological changes in the testis, including the Tes.V, Vv of

the seminiferous tubules in the testis, and S.T.V. in each

testis. Evaluations showed that body weight (B.W.)

increased from E20 to E36 and thereafter slightly

decreased up to the hatching day (Fig. 2a). The Tes.V. and

S.T.V. increased linearly up to the hatching day (Fig. 2a).

In general, the ratio of Tes.V. and S.T.V. to the BW

decreased from E20 to E42 (Fig. 2b). The estimated Vv

decreased from E20 to E26 but increased slowly up to E42

(Fig. 2c).

Histological changes

The 20-day-old embryo

Sertoli cell precursors surrounding the PGCs formed Ser-

toli cell aggregations which extended toward each other

and in some cases came into contact and anastomosed

(Fig. 3). PGCs were distinguishable by their relatively

large, pale, spherically shaped nucleus (Fig. 3a, c). In MT-

and AB-stained sections, the nucleus of mitotic PGCs

appeared as a dark floccus of chromatin (Fig. 3b, d). Sertoli

cell precursors had elongated ovoid or triangular nuclei

which were smaller and more heterochromatic than the

PGC nuclei, whereas when treated with AB stain the nuclei

of the former stained pale relative to those of the PGCs

(Fig. 3). The Sertoli cell precursors had a polar epithelial

Fig. 1 Ventral view of ostrich testes in different embryonic days (E).

a Testes (dashed lines) at E20 are located in the ventral surface of

mesonephros kidneys (Ms). Note that they are inclined toward the

median plane of the body and are located close to each other. b Testes

at E26 are attached to the mesonephros. c Morphology and

topography of the testes at E36 relative to the mesonephros and

metanephros kidneys. d Testes at E42 in the vicinity of metanephros

cranial lobe (Mtc). Note the change in color, shape and location of

testes during in ovo development. Mt Metanephros kidney, R right,

L left. Scale bar 3 mm

Embryonic development of ostrich testis 131

123

cell-like morphology with numerous secretary granules at

the apical pole and the nucleus at the basal pole. The apical

pole was more ramshackle than the basal pole which was

supported by a delicate basement membrane (Fig. 3c).

Interstitial tissue contained only PAS-positive fibers that

appeared pink or purple in color, while Leydig cell pre-

cursors were undistinguishable from mesenchymal cells

(Fig. 3). Testicular capsules consisted of a single layer of

cuboidal cells as a covering epithelium and its underlying

blood vessels (Fig. 3b). Attachment to the kidney was

devoid of covering epithelium, where a few rete ducts

paved by flattened cells were present (Fig. 3a).


Elongating and anastomosing cords superseded Sertoli cell

aggregations causing a net-like structure (Fig. 4b, c) to

develop. The number of PGCs was clearly increased rela-

tive to E20, but their morphology was the same except for

their completely eccentric location in the cords (Fig. 4a, b).

Fig. 2 Morphometrical analysis. a Logarithmic plot to show changes

in body weight (B.W.), left testis volume (Tes.V) and total volume of

seminiferous tubules (S.T.V) from E20 to E42. Note that B.W.

increased with age until E36, following which it slightly decreased

due to weight loss during the hatch, while Tes.V. and S.T.V.

constantly increased. b Ratio of S.T.V. and Tes.V. to B.W. from E20

to E42. This logarithmic plot shows that these ratios follow a pattern

opposite to that for B.W. c Estimated volume fraction of seminiferous

tubules in testis (Vv) decreased from E20 to E26, following which it

increased. Data are shown as the mean ± standard deviation (SD)

Fig. 3 Sertoli cell aggregations (some of them defined by broken

lines) in the 20-day-old ostrich embryo. a Illustration of the rete ducts

(R) paved by flattened cells at the attachment site of testis and kidney.

Note the differences between the rete ducts and blood vessels (V).

Hematoxylin and eosin (H&E) staining. b Sertoli cell aggregations

came into contact and anastomosed to each other. Note the

anastomosing site (asterisks), testicular covering epithelium (Ep)

and its underlying blood vessels (V). Masson’s trichrome (MT)

staining. c The primordial germ cell (PGCs, P) with a large, pale,

spherical nucleus are located between the Sertoli cells (Sr). The color

of interstitial tissue (I) is because of its Periodic acid–Schiff (PAS)-

positive fibers. Arrows Basal pole of Sertoli cells closed to the

basement membrane, arrowheads apical pole. PAS staining. d Inter-

stitial tissue fibers were distinctly stained by Alcian blue (AB) while

the nuclei of the PGCs stained as dark flocci. Ms mesonephric tubule,

Bg blood globule. Scale bar 30 lm


123

The central portion of the cords had a different texture from

the other parts, and a PAS-positive line had circled this

portion which made it look like a lumen (Fig. 4c). Exis-

tence of this lumen was completely distinct in resin sec-

tions (Fig. 5a). A few Leydig cell precursors were

distinguishable based on their lipid granules (Fig. 6a). Two

types of connective tissue fibers, namely, red and green

fibers, were distinct only in the MT-stained sections

(Fig. 4b). The tunica albuginea appeared between the

covering epithelium and its underlying blood vessels,

causing increasing thickness of the capsule (Fig. 4b). In

MT-stained sections, similar to the interstitial tissue, fibers

were mostly red and only rarely green, (Fig. 4b). The

thickness of this layer at the cranial and caudal pole of

testis was more than that at the equatorial area. Some cell

aggregations similar to Sertoli cells were trapped in the

tunica albuginea, but a number of larger cells with the

morphology of PGCs were also present (Fig. 4d). These

aggregations were transforming into a tubule or duct, and

in some areas they were located inside the larger cisterns

(Fig. 4a, d). The marginal blood vessels were following a

distinct model which was clear only in the sections of the

equatorial part of the testis (Fig. 4c). In the hilar portion,

the primary sex tubules were directed toward the hilus and

terminated in the lacunar rete ducts (Fig. 4d).


The primary sex cords became wavy and formed a net-like

structure. The PGCs had the same morphology, but they had

Fig. 4 Network of anastomosing primary sex cords, tunica albuginea

and capsular rete appear in 26-day-old ostrich embryo. Transverse

sections from the cranial pole (a), caudal pole (d) and equatorial area

(b, c) of the testis. a Cell aggregations (black arrowheads) trapped in

the capsule containing mostly Sertoli-like (Sl) cells and only rarely

PGC-like (Pl) cells. H&E staining. b Anastomosing primary sex

tubules (broken lines) formed a net-like appearance. Remnants

(H) remained after loss of the tubules lumen (asterisks) contents.

MT staining. c Capsular blood vessel canalization; longitudinal

vessels (Vl) connected by transverse vessels (Vt). PAS-positive lines

(Ps) define the boundary of lumens. PAS staining. d In some areas

capsular cell aggregations (black arrowheads) were located in the

larger cisterns (arrows). The magnified images show their transfor-

mation to rete ducts (d0) and morphological details of PGCs (d0 0). AB

staining. Ep Covering epithelium, P PGC, Pm mitotic PGC, Sr Seroli

cell, Ca capsule, I interstitial tissue, R lacunar rete ducts, V blood

vessel, Ms mesonephric tubule, Mg mesonephric glomerulus. White

arrowhead Basement membrane. Scale bar 50 lm


123

increased in number, and most of them were mitotic. Leydig

cell precursors were widespread throughout the interstitial

tissue. The polymorphic nucleus of these cells, as well as

the presence of a few lipid granules, distinguished them

from the fibroblasts and blood cells (Fig. 6b). The testicular

capsule, which completely coated the testis, had acquired a

Fig. 5 Appearance of lumen in the primary sex cords. Transverse

semithin sections of the ostrich testis stained by toluidine blue

indicate the presence of a lumen (arrows) in the primary sex cords on

E26 (a), E36 (b) and E42 (c). Note that the apical pole of Sertoli cells

contains numerous secretory granules and that the basal pole usually

contains the nucleus. d Circling fibroblasts (F), around the seminif-

erous cords, which are presumably the progenitors of the myoid cells.

Ca Testicular capsule, I interstitial tissue. Asterisks PGCs, broken

lines boundary of sex cords. Scale bar 10 lm

Fig. 6 Appearance of Leydig cell lipid granules. Transverse semithin

sections of ostrich testis stained by toluidine blue shows the presence

of lipid granules (arrowheads) on E26 (a), E36 (b) and E42 (c).

Periphery of sex cords (S) are marked by broken lines. Bl Blood cells,

I interstitial tissue. Asterisks PGCs. Scale bar 10 lm


123

tree-partite structure that included the tunica serosa, tunica

albuginea and tunica vasculosa (Fig. 7a, b). Extending from

the tunica albuginea, some meandering rays penetrated the

interstitial tissue, while embedded testicular internal arter-

ies had the typical structure of a muscular artery (Fig. 7b0,d00). Lacunar rete ducts in the hilar portion were connected

to the capsular rete system which extended up to the free

surface of the testis (Fig. 7a–d). The capsular rete system

consisted of large cisterns, duct-like structures and trapped

cell aggregations which were obviously transforming into

the duct-like structures (Fig. 7a, c).


In the hatching chick, the primary sex cords became wavier

and formed a reticular structure while the confines of their

lumen became more distinct (Fig. 5c). It appeared that the

number of PGCs had not increased relative to E36. The

tree-partite structure of the capsule had become more dis-

tinct and completely coated the testis (Fig. 8a). The cap-

sular rete ducts had expanded toward the free surface of the

testis (Fig. 8a, b) and the thickness of the capsule was

increased due to intensification of fibers in the tunica

Fig. 7 Histology of testis in the 36-day-old ostrich embryo. Trans-

verse sections from the cranial pole (a, c) and equatorial area (b, d) of

ostrich testis on E42 stained by H&E (a), MT (b), PAS (c) and AB

(d). a Lacunar rete ducts (R) were present in the testicular hilus, while

capsular rete ducts (arrowheads) inside the large cisterns (arrows)

extended up to the testicular free surface. Magnified parts (a0 and a0 0)illustrate details of the capsular rete cuboidal epithelium which is

different from a blood vessel endothelium. b Meandering rays from

the tunica albuginea (Ta), defective septa (Ds), penetrate the

interstitial tissue (I). In the central part of the testis some arteries

can be seen to be embedded inside the defective septa (b0).c Distribution of capsular rete (arrowheads) and large cisterns

(arrows) in the testicular free surface and their histological details (c0

and c0 0). Note the presence of PGC-like (Pl) and Sertoli-like (Sl) cells

in the structure of capsular rete ducts, and the cuboidal epithelium

(Co) of the large cisterns. d Histological details of capsular cell

aggregation (arrowheads) which still had not acquired a duct-like

shape (d0). Note the typical structure of a muscular artery (d00)including the tunica intima (In), tunica media (Md) and a fibrous

tunica adventitia (Ad) which merged with the fibrous texture of their

container defective septum. Bg Blood globule, Ca testicular capsule,

P PGC, Pm mitotic PGC, S seminiferous tubule, Sr Sertoli cell, Ts

tunica serosa, Tv tunica vasculosa, V blood vessel in tunica vasculosa.

Asterisk Lumen of seminiferous tubules, arrows large cisterns,

arrowheads capsular rete ducts. Scale bar 50 lm


123

albuginea, while the inner vasculosa and outer serosa layers

were approximately constant (Fig. 8a). More and larger

lipid granules were found in the cytoplasm of Leydig cell

precursors (Fig. 6c). There were no morphologically dis-

tinct peritubular myoid cells, but some fibroblasts were

clearly circling around the seminiferous tubules (Fig. 5d).

Discussion

The main findings of this study were categorized into the

development of four histological features: (1) the devel-

opment of sex cords; (2) the development of interstitial

tissue; (3) the development of rete ducts; 4) the appearance

of defective septa. A general observation was that the

development of sex cords between E20 and E42 in the

ostrich was similar to that reported in other birds. Our

results indicate that the primary sex cords in ostrich orig-

inate from Sertoli cell aggregations which elongate, contact

and anastomose with each other during embryonic devel-

opment, similar to what occurs in other birds. Whereas the

formation of primary sex cords in fowl was completed by

E7.5 of the first half of the embryonic period (Morrish and

Sinclair 2002), in the ostrich embryo primary sex cord

formation was completed at E26 of the second half of the

embryonic period. According to the previous reports, the

appearance of a lumen in the primary sex cords is post-

poned until hatch in birds and until puberty in mammals

(Romanoff 1960; Skinner and Griswold 2005; McGeady

et al. 2006; Gonzalez-Moran and Soria-Castro 2010a; Sa-

dler 2010), but in the ostrich embryo the lumen began to

form on E26, and on E36 a distinct lumen was present;

consequently, these cords can be called ‘‘primary sex

tubules’’. One important feature of these cords is that they

contain filled lumens. Resin sections, but not paraffin

sections, demonstrated that the lumens were filled with a

semisolid fluid. Furthermore, the unoccupied spaces usu-

ally seen in the lumen of post-embryonic seminiferous

tubules were also due to the absence of its fluid contents

(Hassanzadeh et al. 2013).

The most important finding regarding the development

of the interstitial tissue was that fibroblast-like Leydig cells

appeared on E26. In ostrich testis, the appearance of

fibroblast-like Leydig cells began from E26 onwards, while

in fowl this event occurs at E8 (Gonzalez-Moran and Soria-

Castro 2010b) in the first half of the embryonic period. The

presence of these cells has been reported in the testis of

Japanese quail (Nicholls and Graham 1972) and 2-day-old

chicken (Connell 1972), as well as in mammals (Chris-

tensen and Fawcett 1961; Black and Christensen 1969),

and they transform from a fibroblast-like form, through a

transitional stage, to a mature androgen-producing Leydig

cell form (Narbaitz and Adler 1966; Connell 1972; Jorda-

nov et al. 1978; DeFalco et al. 2011). The presence of lipid

granules, which were dark in coloration in most of our

images, is an important characteristic by which to identify

these cells. This is in contrast to the white coloration of

these granules that has been reported by other authors

(Nicholls and Graham 1972; Gonzalez-Moran and Soria-

Castro 2010b). This difference might be related to the

specific methodology used in the study. The PLT method

used in our study preserves the lipid contents of Leydig cell

granules, which are then stained by osmium tetroxide. It

was not possible to define the exact boundaries of Leydig

Fig. 8 Detailed histological characteristics of testis on E42. Trans-

verse sections stained by MT (a) and H&E (d). a Presence of

developing capsular rete ducts (arrowheads) at the free surface of the

testis beside a defective septum (Df) are shown (a0 and a00). Note that

the central portion of seminiferous tubules and rete ducts have been

lost during the staining procedure. b Developing capsular rete ducts

(arrowheads), containing PGC-like (Pl) and Sertoli-like (Sl) cells in

the lateral surface of testis are still present. The magnified parts (b0

and b00) show their morphology in details. H Hilus of testis,

I interstitial tissue, P PGC, S seminiferous tubule, Ta tunica

albuginea, Ts tunica serosa, Tv tunica vasculosa. Arrows Large

cisterns, arrowheads capsular rete ducts. Scale bar 50 lm


123

cell precursors due to the compression of cells and the

randomness of the fibers in the interstitial tissue. At first,

the fibers of the interstitial tissue were not distinct collagen

fibers (Eurell and Van Sickle 2006), but during develop-

ment collagen fibers appeared, increased in number, and

finally caused the change in interstitial tissue color from

red to a mixed color of green and red. At the territorial

portions of the interstitial tissue, a group of interstitial

tissue fibroblasts similar to peritubular myoid cells were

observed encircling the seminiferous tubules. These cells,

however, did not have the distinct morphological charac-

teristics of the myoid cells which are seen in the juvenile

ostrich testis (Hassanzadeh et al. 2013). It might be possi-

ble that these encircling fibroblasts acquire the morphol-

ogy of myoid cells during post-hatching developmental

processes.

Rete ducts are other noticeable structures of ostrich

testis that consist of three parts of intratesticular, ex-

tratesticular or lacunar, and intracapsular rete ducts or true

rete with large cisterns (Budras and Meier 1981; Aire and

Soley 2003). We recently investigated the capsular rete, the

important part of this system, and reported its extension up

to the free surface of the testis in the juvenile ostrich

(Hassanzadeh et al. 2013). In accordance with that report,

our results also show that the true rete is present in the

medial, lateral and free surfaces of the testis from the pre-

hatching period. Since rete ducts were frequently seen in

the capsule of the polar parts, we can conclude that these

ducts might first appear in the polar parts of the testis and

subsequently extend toward the equatorial parts. Budras

and Meier (1981) proposed that the buds of glomerular

capsules of mesonephros are the origin of rete testis, but

our results indicate that the capsular rete develops from the

cell aggregations trapped inside the tunica albuginea which

are comprised of two cell types: Sertoli-like and PGC-like

cells. Thus, it is a plausible speculation that the capsular

rete ducts may originate from the primary sex cords.

Despite the presence of primary sex cords inside the testis,

the thickness of the epithelium in these capsular aggrega-

tions decreases during embryo development and ultimately

forms a squamous or flattened cuboidal epithelium (Has-

sanzadeh et al. 2013). However, regarding the structural

differences between different parts of the rete system

(Budras and Meier 1981; Aire 1982; Barker and Kendall

1984; Aire and Soley 2003), it is possible that its different

parts develop from diverse origins.

As in other birds, in the ostrich the covering epithelium

and its underlying blood vessels are present from the early

stages of testis formation, while its third layer, the tunica

albuginea, appears later (Gonzalez-Moran and Soria-Castro

2010a). It has been shown that tunica albuginea, the main

part of the testicular capsule, is considerably different in

the ostrich (Ozegbe et al. 2008; Hassanzadeh et al. 2013).

In the ostrich embryo, this layer first consists of a loose

connective tissue, but due to intensification of collagen

fibers (which stain green by MT), changes to a dense

connective tissue in the newly hatched ostrich (Nicholls

and Graham 1972; Eurell and Van Sickle 2006). Initially

this layer had a loose structure, and its internal compo-

nents, such as the capsular rete ducts and blood vessels,

appeared in their real shapes in the sections. After densi-

fication, which forces these structures to collapse, it was

difficult to find these internal components in the sections.

Hardening and thickening of the tunica albuginea has

another outcome, i.e., the appearance of defective septa. In

mammals, testicular septa branch from the inner part of the

capsule and conduct blood vessels and nerves into and out

of the testicular substance (Davis et al. 1970). In earlier

studies, it was believed that these septa were absent in birds

(Lake 1971; Hodges 1974), but their presence were sub-

sequently reported in ostrich (Soley and Groenewald 1999;

Hassanzadeh et al. 2013). Our observations confirm the

presence of defective septa and suggest that the mamma-

lian lamellar septa are the evolutionary remnants of the

defective septa in ostrich which have a filum-like shape.

The defective septa penetrated the interstitial tissue in a

meandering way while conducting blood vessels and

nerves into and out of the testis.

While classical measurements are routinely used in

anatomical and embryological studies, new methods such

as stereology have been applied recently in studies on avian

developmental biology (Gonzalez-Moran 1997, 2011;

Gonzalez-Moran and Soria-Castro 2010a, b). In addition to

measurements of B.W. and testis diameter, we used ster-

eological techniques to estimate the Tes.V and Vv of

seminiferous tubules. The weight of embryos increased in a

linear manner during the period leading up to the hatching

day, but it decreased during the day of hatching (Fig. 2a).

This is a common finding in newborns in both mammals

and birds (Mortola 2009; Eidelman et al. 2012). Our results

show that testis and seminiferous tubule volumes increased

during the embryonic period and that this pattern of

increase was similar to that of the respectivegrowth curves.

Thus, the ratios of Tes.V/B.W. and S.T.V/B.W. fell on E26

(Fig. 2b). Considering that the Vv of seminiferous tubules

generally decreases from E20 to E42 and reaches about

0.56 in the juvenile ostrich (Hassanzadeh et al. 2013), it is

possible to conclude that there is no direct relationship

between the B.W. increase and changes in the Vv of

tubular compartments. However, a decrease in Vv between

E20 and E26 (Fig. 2c) could be related to histological

changes in the components of the testis other than semi-

niferous tubules, including changes in the testis capsule

(appearance of tunica albuginea) and the development of

new blood vessels. In comparison with other animals,

increased Vv of seminiferous tubules from prenatal to


123

pre-pubertal period is very different from the increasing

pattern in domestic fowls and bonnet monkeys (Macaca

radiata) reported previously (Gonzalez-Moran and Soria-

Castro 2010a; Prakash et al. 2008).

In summary, we report here developmental changes in

the testis of the ostrich embryo based on histological and

morphometrical results and highlight differences in the

development of capsular rete ducts, defective septa and

changes in the Vv of tubular compartments in testis of

ostrich embryo in comparison with other birds. Our results

provide new information on the appearance of the lumen in

the primary sex cords, which is unique to the ostrich among

birds and mammals.

Acknowledgments This research has been supported by a grant

(No. 881) from the Research Council of the Ferdowsi University of

Mashhad. We especially thank Mrs. Naseri (Central lab of FUM), Dr.

J. Sadeghinezhad and Dr. J. Ashrafi Helan for their invaluable help.

Conflict of interest None.

References

Aire TA (1982) The rete testis of birds. J Anat 135(1):97–110

Aire TA, Soley JT (2003) The morphological features of the rete testis

of the ostrich (Struthio camelus). Anat Embryol 207:355–361

Barker SG, Kendall MD (1984) A study of the rete testis epithelium in

several wild birds. J Anat 138(1):139–152

Black VH, Christensen AK (1969) Differentiation of interstitial cells and

Sertoli cells in fetal guinea pig testes. Am J Anat 124(2):211–237

Budras KD, Meier U (1981) The epididymis and its development in

ratite birds (ostrich, emu, rhea). Anat Embryol (Berl)

162(3):281–299

Chang G, Chen R, Qin Y, Zhang Y, Dai A, Chen G (2012) The

development of primordial germ cells (PGCs) and Testis in the

Quail Embryo. Pak Vet J 32(1):88–92

Christensen AK, Fawcett DW (1961) The normal fine structure of

opossum testicular interstitial cells. J Biophys Biochem Cytol

9:653–670

Connell CJ (1972) The effect of luteinizing hormone on the

ultrastructure of the Leydig cell of the chick. Z Zellforsch

128:139–151

Csaba G, Shahin MA, Dobozy O (1980) The overlapping effect of

gonadotropins and TSH on embryonic chicken gonads. Arch

Anat Histol Embryol 63:31–38

Davis JR, Langford GA, Kirby PJ (1970) The testicular capsule. In:

Johnson AD, Gomes WR, Vandemark NL (eds) The testis

development, anatomy and physiology. Academic Press, Lon-

don, pp 281–337

DeFalco T, Takahashi S, Capel B (2011) Two distinct origins for

Leydig cell progenitors in the fetal testis. Dev Biol 352:14–26

Delesse A (1848) Procede mecanique pour determiner la composition

des roches. Ann Min 13:379–388

Eidelman AI, Schanler RJ, Johnston M, Landers S, Noble L, Szucs K,

Viehmann L (2012) Breastfeeding and the use of human milk.

Pediatrics 129:e827–e841

Eurell JA, Van Sickle DC (2006) Connective and supportive tissues.

In: Eurell JA, Frappier BL (eds) Dellmann’s textbook of

veterinary histology. Wiley-Blackwell, London, p 37

Fujimoto T, Ukeshima A, Kiyofuji R (1976) The origin, migration

and morphology of the primordial germ cells in the chick

embryo. Anat Rec 185(2):139–145

Gefen E, Ar A (2001) Morphological description of the developing

ostrich embryo: a tool for embryonic age estimation. Isr J Zool

47:87–97

Ginsburg M, Eyal-Giladi H (1986) Temporal and spatial aspects of

the gradual migration of primordial germ cells from the epiblast

into the germinal crescent in the avian embryo. J Embryol Exp

Morphol 95:53–71

Gonzalez-moran G (1997) A stereological study of the different cell

populations in chicken testes treated with follicle-stimulating

hormone during embryonic development. Anat Histol Embryol

26:311–317

Gonzalez-Moran MG (2011) Histological and stereological changes

in growing and regressing chicken ovaries during development.

Anat Rec 294:893–904

Gonzalez-Moran MG, Soria-Castro E (2010a) Changes in the tubular

compartment of the testis of Gallus domesticus during develop-

ment. Br Poult Sci 51:296–307

Gonzalez-Moran MG, Soria-Castro E (2010b) Histological and

stereological studies on Leydig cells in the testes of Gallus

domesticus from pre-hatching to sexual maturity. Anim Reprod

Sci 120(1–4):129–135

Hassanzadeh B, Nabipour A, Rassouli M, Dehghani H (2013)

Microanatomical study of testis in juvenile ostrich (Struthio

camelus). Anat Sci Int 88(3):134–140

Hodges RD (1974) The histology of the fowl. Academic Press,

London

Howard V, Reed MG (2005) Unbiased stereology: three-dimensional

measurement in microscopy. Garland Science/BIOS Scientific

Publishers, New York

Ishiguru S, Minematsu T, Naito M, Kanai Y, Tajima A (2009)

Migratory ability of chick primordial germ cells transferred into

quail embryos. J Reprod Dev 55(2):183–186

Jordanov J, Angelova P, Boyadjieva-Michailova A, Bakalska M

(1978) Ultrastructure of developing interstitial cells in chick

embryonic gonad in relation to their genesis and steroidogenic

function. Z Mikrosk Anat Forsch 92(3):449–464

Kuo J (2007) Electron microscopy methods and protocols. Humana

Press, Totowa

Lake PE (1971) The male in reproduction. In: Bell DJ, Freeman BM

(eds) Physiology and biochemistry of the domestic fowl.

Academic Press, London, pp 1411–1447

Lan W, Ke-mei P, Lai-qiang L, Hui S, Yan W, Sheng-he L, Li T, An-

na D, Er-hui J, Jia-xiang W (2007) Morphological study on the

reproductive organs of the male ostrich chicks. Acta Vet Zootech

Sinica 38(8):851–855

Madekurozwa M-C, Chabvepi TS, Matema S, Teerds KJ (2002)

Relationship between seasonal changes in spermatogenesis in the

juvenile ostrich (Stuthio camelus) and the presence of the LH

receptor and 3b-hydroxysteroid dehydrogenase. Reproduction

123(5):735–742

Malago W Jr, Franco HM, Matheucci E Jr, Medaglia A, Henrique-

Silva F (2002) Large scale sex typing of ostriches using DNA

extracted from feathers. BMC Biotechnol 2:19

Malecki IA, Horbanczuk JO, Reed CE, Martin GB (2005) The ostrich

(Struthio camelus) blastoderm and embryo development follow-

ing storage of eggs at various temperatures. Br Poult Sci

46(6):652–660

McGeady TA, Quinn PJ, FitzPatrick ES, Ryan MT, Cahalan S (2006)

Veterinary embryology. Blackwell, London

Moller AP (1994) Directional selection on directional asymmetry:

testes size and secondary sexual characters in birds. Proc R Soc

Lond B 258:147–151


123

Morrish BC, Sinclair AH (2002) Vertebrate sex determination: many

means to an end. Reproduction 124(4):447–457

Mortola JP (2009) Gas exchange in avian embryos and hatchlings.

Comp Biochem Phys A 153(4):359–377

Nagai H, Mak S–S, Weng W, Nakaya Y, Ladher R, Sheng G (2011)

Embryonic development of the emu, Dromaius novaehollandiae.

Dev Dyn 240(1):162–175

Narbaitz R, Adler R (1966) Submicroscopical observations on the

differentiation of chick gonads. J Embryol Exp Morph

16(1):41–47

Nicholls TJ, Graham GP (1972) Observations on the ultrastructure

and differentiation of Leydig cells in the testis of the Japanese

quail (Coturnix coturnix japonica). Biol Reprod 6(2):179–192

Ozegbe PC, Aire TA, Soley JT (2006) The morphology of the efferent

ducts of the testis of the ostrich, a primitive bird. Anat Embryol

211(5):559–565

Ozegbe PC, Aire TA, Madekurozwa MC, Soley JT (2008) Morpho-

logical and immunohistochemical study of testicular capsule and

peritubular tissue of emu (Dromaius novaehollandiae) and

ostrich (Struthio camelus). Cell Tissue Res 332(1):151–158

Ozegbe PC, Kimaro W, Madekurozwa MC, Soley JT, Aire TA (2010)

The excurrent ducts of the testis of the emu (Dromaius

novaehollandiae) and ostrich (Struthio camelus): microstereol-

ogy of the epididymis and immunohistochemistry of its

cytoskeletal systems. Anat Histol Embryol 39(1):7–16

Prakash S, Prithiviraj E, Suresh S (2008) Developmental changes of

seminiferous tubule in prenatal, postnatal and adult testis of

bonnet monkey (Macaca radiata). Anat Histol Embryol

37(1):19–23

Romanoff AL (1960) The avian embryo. Structural and functional

development. Macmillan, New York

Sadler TW (2010) Langman’s medical embryology. Lippincott

Williams & Wilkins, Philadelphia

Sekido R, Lovell-Badge R (2007) Mechanisms of gonadal morpho-

genesis are not conserved between chick and mouse. Dev Biol

302:132–142

Shahin MA, Torok O (1982) Differentiation of male chick embryo

gonads during the last week of incubation in organ and tissue

culture. Z Mikrosk Anat Forsch 96(1):157–170

Skinner MK, Griswold MD (2005) Sertoli cell biology. Academic

Press, London

Smith CA (2007) Avian, sex determination and gonadal sex

differentiation. In: Jamieson BGM (ed) Reproductive biology

and phylogeny of birds. Science Publishers, Enfield, pp 479–506

Soley JT (1992) A histological study of spermatogenesis in the ostrich

(Struthio camelus). PhD thesis. University of Pretoria, Pretoria

Soley JT, Groenewald HB (1999) Reproduction. In: Deeming DC (ed)

The ostrich: biology production and health. CABI Publishing,

New York, pp 129–159

Tagami T, Kagami H, Matsubara Y, Harumi T, Naito M, Takeda K,

Hanada H, Nirasawa K (2007) Differentiation of female

primordial germ cells in the male testes of chicken (Gallus

gallus domesticus). Mol Reprod Dev 74(1):68–75

Van Krey H (1990) Reproductive biology in relation to breeding and

genetics. In: Crawford RD (ed) Poultry breeding and genetics.

Elsevier, Amsterdam, pp 61–90

Wei L, Peng KM, Liu H, Song H, Wang Y, Tang L (2011)

Histological examination of testicular cell development and

apoptosis in the ostrich chick. Turk J Vet Anim Sci 35(1):7–14

Zaccanti F, Vallisneri M, Quaglia A (1990) Early aspects of sex

differentiation in the gonads of chick embryos. Differentiation

43(2):71–80

Zhang Y, Ren Z, Tang L (2011) Anatomic study on the main male

reproductive organs of ostrich. Global J Health Sci 3(1):181–184


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Morphological development of testes in ostrich (Struthio camelus) embryo

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