Histological and Histomorphometric Changes of the Rabbit Testis During Postnatal Development
Yasser A. Ahmed,
Mahmoud M. Abd-Elhamied
Gamal K.M. Ali
The aim of the current study was to follow up the histological and histomorphometrical changes of descending rabbit testis during different postnatal-developmental stages (1-day and 1-4-, 8-, 12-, 16-, 20- and 24-weeks). The testes were found at the sublumbar region just after birth, moved into the inguinal canal after 4 weeks, located near the scrotum after 8 weeks and were completely within the scrotum after 12-16 weeks. Paraffin and semi-thin sections were taken from the testis and examined with light microscopy. The testis was surrounded by a fibrous capsule with no smooth muscle fibers until 12 weeks, but smooth muscle fibers were seen after 16 weeks. Testicular parenchyma was made up of solid seminiferous cords with no lumina and the cords had only 2 types of cells; gonocytes and Sertoli cells until 8 weeks. Lumination of the testicular cords started after 12 weeks forming seminiferous tubules. Seminiferous tubules were lined by spermatogonia, spermatocytes and round spermatids, in addition to Sertoli cells. After 16-24 weeks, spermatogenesis was very active and sperms were seen. Leydig cells were small and less active at 1-day postnatal but became large and more active after 16-24 weeks. Many growth parameters of seminiferous cords or tubules including their diameter and the numbers of germ cells and Sertoli cells were recorded. The current study widely covered different points of rabbits testicular maturity including the anatomical location and the histological and histomorphometrical changes of descending rabbit testis.
to cite this article:
Yasser A. Ahmed, Mahmoud M. Abd-Elhamied and Gamal K.M. Ali, 2012. Histological and Histomorphometric Changes of the Rabbit Testis During Postnatal Development. Research Journal of Veterinary Sciences, 5: 42-50.
Received: March 31, 2012;
Accepted: April 28, 2012;
Published: June 21, 2012
The testis, the male gonad, consists mainly of seminiferous tubules and interstitial
tissue containing Leydig cells, demarcated from outside by a thick vascular
connective tissue tunica albuginea, sending septa that subdivide the cavity
of the testis into smaller incomplete compartments. Seminiferous tubules are
the site of spermatogenesis, while Leydig cells are responsible for secreting
male sex hormones, testosterone to maintain spermatogenesis (Eurell
and Frappier, 2006).
Mammalian spermatogenesis is a developmental complex process that occurs in
the seminiferous tubules producing spermatozoa or sperms able to penetrate and
fertilize a healthy oocyte produced by the ovary of corresponding female species
(Miles and Western, 2012). The seminiferous epithelium
contains numerous stages in the developmental germ cells; spermatogonia, spermatocytes
and spermatids that lead to mature spermatozoa (Curtis and
Amann, 1981). Spermatogonia, the diploid stem cell from which the rest of
the cell types are derived, are found peripherally and can be identified by
their densely stained round centrally located nuclei. Some spermatogonia remain
as undifferentiated stem cells and continually supply new spermatogonia, while
the others divide by meiosis to give the haploid primary spermatocytes. Primary
spermatocytes, the initial product of spermatogonial maturation, are large cells
with a round nucleus filled with clumped chromatin material and usually seen
in various stages of the meiotic division. Secondary spermatocytes are haploid
and very short-lived rarely seen cells that quickly undergo meiosis to give
spermatids. Spermatids are haploid cells observed in different stages of maturation.
Some are square cells with nuclei beginning to get quite dense. Others more
advanced have tails beginning to develop. In some stages, the excess cytoplasm
is being cast off. The spermatids complete differentiation process, spermiogenesis,
to become mature sperms with its characteristic structure; head and tail releasing
their tails into the lumen (Amann, 2008).
Adjacent to the seminiferous epithelium the elongated Sertoli cells located
perpendicular to the basal lamina of the seminiferous tubules. Sertoli cells
support the germline cells during different stages of spermatogenesis. They
are tall columnar cells with large pale oval nucleus containing distinct nucleolus
and attached to the basal lamina of the seminiferous tubules (Griswold,
1998). Sertoli cells have many cytoplasmic processes that wrap around the
germ cells forming blood-testis barrier through network of junction complex
between their cytoplasmic processes and the germ cells; this barrier divide
the seminiferous tubules into basal and adluminal compartments to separate the
haploid cells from the blood immune system (Merchant-Larios
and Taketo, 1991). Furthermore, Sertoli cells phagocytose excess cellular
materials and apoptotic bodies (Barone et al., 2004).
Unlike the germ cells, Sertoli cells are long lived cells and normally not dividing
(Johnson et al., 2008). Cytoplasm of Sertoli
cells has numerous mitochondria with tubular cristae, abundant smooth endoplasmic
reticulum, free ribosomes, prominent Golgi apparatus and many phagocytosed materials
(Lebelo and van der Horst, 2010).
Leydig cells, the androgen producing cells are located in the interstitial
vascular connective tissue between the seminiferous tubules (Diagone
et al., 2012). They have different shapes; they are cuboidal, polygonal
or elongated groups of cells with ovoid nucleus (Gondos
et al., 1977). Their cytoplasm is acidophilic and has many lipid
droplets (Diagone et al., 2012), abundance of
smooth endoplasmic reticulum, a few short cisternae of rough endoplasmic reticulum,
rod-like mitochondria with tubular cristae, vacuoles resulting from lipid extraction,
filaments, microtubules and glycogen (Kerr, 1991).
The aim of the current study was to investigate the histological and histomorphometrical
changes of the descending rabbit testis during the postnatal development.
MATERIALS AND METHODS
Animals: Testicular specimens were obtained from 27 healthy New Zealand
white rabbits during different stages of postnatal development; 1-day, 1-, 4-,
8-, 12-, 16-, 20- and 24-weeks. The animals were raised in the animal house
of the Faculty of Medicine, Assiut University, Assiut, Egypt. The study conducted
at the period between May 2010 to October 2011.
Anatomical localization of the descending rabbit testis: The position of the rabbit testis during different postnatal developmental stages (from 1-day until 24 weeks of age) was noticed and recorded.
Paraffin embedding: Dissected testes were rapidly fixed in either 4%
phosphate-buffered paraformaldehyde (pH; 7.4) or Bouin's solution. Testes were
sectioned crossly or longitudinally, dehydrated in ascending grades of ethyl
alcohol, cleared in methyl benzoate and embedded in paraffin. Sections (5 μm-thicknesses)
were cut, stained with hematoxylin and eosin (H and E) as a general stain and
Periodic Acid Schiffs (PAS) for mucopolysaccharides and examined with
a light microscope.
Spurrs resin embedding: Some specimens were fixed in 2.5% gluteraldehyde/4% buffered formaldehyde, post-fixed in 1% osmium tetroxide, dehydrated through ascending grades of acetone and embedded in Spurrs resin. Semi-thin sections (0.5 μm-thicknesses) were cut using an ultra-microtome (Leica Microsystems, Wetzlar, Germany), stained with toluidine blue and examined with a light microscope.
Histomorphometrical analysis: Histomorphometrical parameters related to testicular growth such as the diameter of seminiferous tubules and numbers of germ cells and Sertoli cells per seminiferous tubule during different developmental stages were evaluated using an image analyzer system.
Statistical analysis: Statistical differences between groups were evaluated using one way ANOVA. Measurements were taken from 3 different animals. For each comparison, a p-value of less than 0.05 was considered to be significant.
Descending of the rabbit testis: The testes of the neonatal rabbit were found at the sublumbar region on both sides of the descending colon. One week postnatal, the testes migrated slightly caudally to lie in both sides of the rectum just caudal to the kidneys. Four weeks postnatal, testes began to descend within the inguinal canal and were mostly extra-abdominal near to the scrotum after 8 weeks, but completely locating within the scrotum after 12-16 weeks (Fig. 1).
Histological structure of the descending rabbit testis: The testis of
the rabbit during early stages of development (1 day to 8 weeks) was surrounded
by a thick dense connective tissue tunica albuginea containing a deep vascular
layer and covered by flattened mesothelial cells. No smooth muscle cells could
be demonstrated during this developmental stage (Fig. 2a).
||A diagram of the anatomical location of the descending rabbit
testes, the testes were located in 1: Sublumbar region one-postnatal-day,
2: The inguinal canal 4-postnatal-weeks, 3: Near to the scrotum 8-postnatal-weeks,
4: Completely within the scrotum 12-16-postnatal weeks
||Photographs rabbit testis during early postnatal development
Paraffin (a, b, c, e) and semi-thin (d, f) sections stained with H&E
(a, b, c, e) and toluidine blue (d, f) were taken from the testis of 1-day-old
(a, b, c, d) and 12-week-old (e, f) rabbits, (a) Tunica albuginea (TA) containing
wide blood vessels (BV), (b) Seminiferous cords (SC) containing Gonocytes
(G) and Sertoli cells (S), separated by interstitial connective tissue (IT)
having Leydig cells (LC), (c) Gonocytes (G) in mitotic division (arrowhead)
and Sertoli cells (S), (d) Leydig cells (Lc) containing cytoplasmic vacuoles
(arrowheads) surrounding blood capillaries (bc), (e) Seminiferous tubules
having lumen (ST) surrounded by tunica albuginea (TA) with large blood vessels
(bv) sending testicular septae (TS), (f) Spermatogenic cells; spermatogonia
(Sg), primary spermatocytes (Ps), rounded spermatids (Rs) surrounded by
interstitial Leydig cells (Lc), Bars = 10 μm in a, c, d, f, 50 μm
in b and 100 μm in E
Testicular parenchyma consisted of solid straight testicular cords with no
lumina, widely separated from each others by an abundant interstitial tissue
and the cords were demarcated by a well developed basal lamina (Fig.
2b). The testicular cords contained two types of cells; pre-spermatogenic
cells or gonocytes and Sertoli cells (Fig. 2c). The pre-spermatogenic
cells were large rounded cells with rounded nucleus having two distinct nuclei
and pale stained cytoplasm and frequently seen in mitotic division (Fig.
2c). These cells were seen in the center of the testicular cords and had
no contact to the basal lamina. While, the Sertoli cells were tall columnar
with oval or spindle-shaped nucleus and pale cytoplasm and their boundaries
were less clear (Fig. 2c). The interstitial tissue contained
small singly scattered or grouped spindle, oval, triangular, polyhedral or elongated
Leydig cells, which contained small deeply stained nucleus and pale vacuolated
cytoplasm due to the presence of lipid droplets (Fig. 2d).
After 12 weeks, seminiferous cord lumination started and the cords termed as
seminiferous tubules (Fig. 2e). Seminiferous tubules had no
gonocytes, but were lined with stratified germinal epithelium including spermatogonia,
spermatocytes and spermatids, in addition to Sertoli cells (Fig.
2f). Spermatogonia were peripherally located small, rounded or oval shaped
cells with relatively large, vesicular nucleus containing one or two distinct
nucleoli. Primary spermatocytes were the largest cells among the seminiferous
epithelium and had rounded nucleus with distinct nucleolus. Secondary spermatocytes
were smaller than primary spermatocytes and rarely seen. Spermatids were the
smallest germ cells and they are rounded cells with rounded nuclei.
After 16-24 weeks, when the animals reached sexual maturity, the rabbit testis
was surrounded by tunica albuginea containing smooth muscle fibers (Fig.
3a). Lumination of the seminiferous tubules increased, the tubules were
surrounded by a clear PAS-positive basal lamina and some of them appeared fused
together (Fig. 3b).
||Photographs of the rabbit testis during late postnatal development,
paraffin (a, b, c, e) and semi-thin (d, f) sections stained with H and E
(a, e), PAS and H and E (b, c) and toluidine blue (d, f) were taken from
the testis of 16-week-old (c, e, f), 20-week-old (B) and 24-week-old (a,
d) rabbits. (a) Smooth muscle fibers (arrowheads) in the tunica albuginea.
(B) Seminiferous tubules with PAS-positive basal lamina (ST), some of seminiferous
tubules fused together (arrows); note interstitial tissue (IT), blood vessels
and intratubular septa (IS). (c) Seminiferous tubules rest on PAS-positive
basal lamina and having PAS-positive spermatozoa (arrow). (d) Active spermatogenesis;
spermatogonia (Sg), primary spermatocytes (Ps), secondary spermatocytes
(Ss), rounded spermatids (R) and spermatozoa (arrow). (e) Seminiferous tubules
with wide lumen lined by spermatogonia (Sg), primary spermatocytes (Ps),
and spermatozoa (arrow), Sertoli cells (Sr) and Leydig cells (LC). (f) Large
active Leydig cells (Lc) containing numerous various sizes and shapes vacuoles
(arrowheads). Bars = 10 μm in A, D, E, 100 μm in B, 50 μm
in C and 25 μm in E
||Histomorphometric changes in the rabbit testis during postnatal
development, the diameter of seminiferous tubules (a), the numbers of spermatogenic
cells (b, c, d) including gonocytes (a), spermatogonia (b), Primary spermatocytes
(c) spermatids (d) Sertoli cells (f) were estimated using image analyzer
system. Each column represents mean±SE. Results were considered significant
Spermatogenesis was very active and all cell types including sperms were seen
during this stage (Fig. 3c, d). The elongated
Sertoli cells markedly increased in size (Fig. 3f) and Leydig
cells were rounded, oval, polyhedral or elongated in shape with large oval nucleus
and cytoplasm having a few and numerous small lipid vacuoles (Fig.
Histomorphometrical studies of the rabbit testis: The histometric data
of the present study showed that the diameter of the seminiferous cords of testis
of newborn rabbits was 133.3±44.7 μm and nearly remained unchanged
until the age of 8 weeks but it increased significantly increased after 24 weeks
to reach 450.78±150.53 μm (Fig. 4a). Gonocyte
number was 7.7±3.3/seminiferous cords at 1 day-postnatal, gradually decreased
to reach 1.7±0.4 after 8 weeks and then completely disappeared from the
seminiferous tubules after 12 weeks (Fig. 4b). Spermatogonia
had never been seen before the 8 weeks of postnatal development, but 22.7±2.4
spermatogonia were found after 12 weeks and then gradually decreased to 14.2±0.5
after 24 weeks (Fig. 4c). Primary spermatocytes (37.2±4.9)
were firstly detected after 12 weeks and increased later on reaching 76.4±3.67
after 24 weeks (Fig. 4d). Similarly, spermatids (7.6±4.3)
were firstly observed after 12 weeks and then increased to 111.4±23.6
by 24 weeks of age (Fig. 4e). Total spermatogenic cells (obtained
from summation of spermatogonia, primary spermatocytes and spermatids) increased
from 67.6±6.9 after 12 weeks of age to reach 202.2±27.2 after
24 weeks of postnatal development indicating increased spermatogenesis during
this time (data not shown). Sertoli cell number was 32±1.6 after birth
and gradually decreased to reach 5.4±0.7 after 24 weeks of age (Fig.
The current study was conducted to follow up the most dramatic histological and histomorphometrical changes associated with the descending process of the rabbit testis during postnatal development.
It was noticed that the descending of the rabbit testis into the scrotum showed
4 sequential phases; in neonatal rabbit, the testis were located at the sublumbar
region. Testes were deviated caudally into the inguinal canal after 4 weeks,
near to the scrotum after 8 weeks and completely located within the scrotum
after 12-24 weeks. This normal way of the testicular descending explained in
the other studies and any abnormality in this descending pathway is clinically
evident as cryptorchidism (Kaleva and Toppari, 2003;
Amann and Veeramachaneni, 2007). Movement of two ligaments
appears to play an important role in determining the position of the gonads;
the gubernaculum, which develops below the gonad in the inguinal area of the
abdominal cavity and the cranial suspensory ligament which develops between
the gonads and the dorsal abdominal wall near to the last rib (Hutson
et al., 1997).
Histological examination of the rabbit testis during postnatal development showed that the testis of the was surrounded by a thick dense connective tissue capsule, tunica albuginea containing large blood vessels and covered with flattened mesothelial cells. No smooth muscle cells could be demonstrated until 12 weeks of age. However, capsule showed differentiated smooth muscle fibers after 16 weeks, when the testes were completely descended in the scrotum.
Seminiferous tubules were observed as solid cords without a central lumen until
the 8th week of postnatal development. Pre-spermatogenic or gonocytes, the only
germ cells seen, were located in the centre of the seminiferous cords and surrounded
by the Sertoli cells. Similar observation was seen in the sheep testis (Herrera-Alarcon
et al., 2007). It is known that the pre-spermatogonia enter waves
of mitotic division during the early testicular development and simultaneously
they undergo morphological differentiation to form other spermatogenic cells
(Curtis and Amann, 1981; De Rooij
and Russell, 2000). Lumination of the rabbit testicular cords was demonstrated
after 12 postnatal-weeks when the diameter of the seminiferous tubules significantly
increased. Appearance of the seminiferous tubule lumen is likely due to accumulation
of tubular fluid secreted by the Sertoli cells as an earlier event of spermatogenesis
(Russell et al., 1989). The seminiferous tubules
of the rabbit testis at 12 weeks of age were lined by a stratified germinal
epithelium characterized by the first appearance of the round spermatids in
addition to spermatogonia and spermatocytes. During the period 16-24 weeks of
postnatal development, the rabbit became sexually mature (Hafez
and Kodituwakku, 1970), spermatogenesis was very active as indicated by
the increased total number of the spermatogenic cells and sperms were seen.
In addition, the presence of contractile smooth muscle fibers within the capsule
during this stage of development is important for squeezing the testis allowing
moving of the sperms into the clearly formed lumen.
Histomorphometry revealed that the number of the Sertoli cells was higher during
the first 8 weeks, decreased at 12 weeks but remained unchanged until 24 weeks
postnatal. Similar observation was recorded in previous study (Herrera-Alarcon
et al., 2007). It is widely accepted that Sertoli cells are essential
in the development and differentiation of germ cells (Shinohara
et al., 2003). Sertoli cell proliferation occurs during a limited
period comprised between sexual differentiation and puberty as Sertoli cells
differentiate to functional adult cells, they loss the capacity of proliferation
(Sharpe et al., 2003). Sertoli cells have nutritive,
protective and supportive functions for spermatogenic cells phagocytose regressive
sperms and produce an androgen-binding protein and secret constituents of intratubular
fluid (Cigankova, 1983), which facilitates the lumination
of the seminiferous cords. Furthermore, they form the blood-testis barrier (Tindall
et al., 1975) to keep the developing haploid spermatogenic cells
protected from the action of immune cells of the blood.
Leydig cells were found just after birth and were numerous small scattered
or grouped of cells that became larger, more active and had many lipid droplets
in the testis of the adult rabbit. These cells are known to secret testosterone
to activate the hypothalamo-hypophyseal testicular axis for the continued development
of the male reproductive system (Ariyaratne and Mendis-Handagama,
2000; Habert et al., 2001).
In conclusion, the current study localized the anatomical position of the rabbit testis during postnatal development. This study has described the histological and histomorphometrical changes of the rabbit testis from 1 day to 24 weeks of postnatal development.
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