Histomorphologic features of neonatal and juvenile uro-genital development in sprague-dawley rats
Ancuta Apreutese 1,2, Cedric Gordon 1, Roy Forster 3, Andrew Graham 1, Bernard Palate 3, Julius Haruna 1 and Marie-Odile Benoit-Biancamano 2
1 CiToxLAB in North America, Laval, Qc, Canada - 2 Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Qc, Canada - 3 CiToxLAB in France, Evreux CEDEX, France
Abstract
This study describes the key histomorphologic postnatal developmental events occurring
in the uro-genital system of rat pups from birth to postnatal day (PND) 30. Tissues were
collected from 51 rats, using equal numbers from each sex whenever possible, at PND1, 2,
4, 6, 8, 10, 14, 17, 21, 24, 26, 28, and 30. The testes and kidney weight and the organ weight
relative to body weight ratios were also calculated. Our study revealed that nephrogenesis
in rats ceased by PND14, while few mitoses were still observed at the cortico-medullary
junction at PND21. Individual cell death and mitoses were abundant at birth and followed a
centripetal pattern (from outer cortex to medulla), accompanying developing renal structures.
At PND26, the ovary exhibited a sharply demarcated cortico-medullary junction. Between
PND21-PND26 numerous apoptotic ova were present. The first uterine glands became visible
around PND14, whereas scattered apoptotic epithelial cells were still present up to PND26.
By PND24, the superficial layer of the vaginal epithelium was multifocally composed of large
cells containing a mucinous material mixed with few apoptotic cells. At birth, the seminiferous
tubular epithelium was 1 to 2 layers thick, composed of spermatogonia and Sertoli cells. A
seminiferous lumen was rarely seen and the interstitium was abundant and hypocellular.
The pachytene spermatocytes were observed around the second week of life, while the first
round spermatids became visible on PND26. The description of these major histological
features of the male and female uro-genital systems from birth to PND30 will serve as a
valuable histological historical database that will be useful in pediatric drug development.
Introduction
Preclinical juvenile toxicity studies are required when the pediatric population is the intended
target of a new drug. The rat is one of the most commonly used small animal species in
developmental and juvenile toxicity studies. When compared to its human counterpart,
the glomerular filtration or onset of puberty underline some of the scientific challenges of
underdeveloped organs in newborn and juvenile animals. Time-course histomorphological
background changes (programmed cell death, increase mitoses) are part of the normal
anatomic development processes during the postnatal period and should be differentiated
from treatment-related lesions. The focus of this study was to provide a concise outline
of histomorphological features of postnatal uro-genital development in Sprague-Dawley
rats during the first month of life.
nephrons ended in small and hypercellular corpuscles which were located beneath the
nephrogenic zone (figure 3). Perinatally, the cuboidal epithelium lining the luminal surface
of the urinary bladder consisted of two distinct cell layers and became 3 to 4 layers thick
by PND21. On the first postnatal day, the ovarian parenchyma was diffusely composed by
pockets of several oocytes enveloped in stromal cells (figure 4). By PND 26, necrotic ova
were primarily observed in the medulla and the ovary exhibited a sharp cortico-medullary
separation (figure 5). The uterine glands became visible two weeks after birth and leukocyte
infiltrates were entirely absent over the investigated period. The vaginal epithelium showed
multifocal mucification starting on PND24 (figure 6). At birth, the seminiferous tubular
epithelium was 1 to 2 layers thick, and composed of spermatogonia and Sertoli cells
(figure 7). A seminiferous lumen was rarely seen and the interstitium was abundant and
hypocellular. The pachytene spermatocytes were observed around the second week of
life, while the first round spermatids became visible on PND26 (figure 8). No elongated
spermatids were observed within the testes and spermatozoa were systematically absent
from the epididymis and seminiferous tubules. Tissue remodelling during the rat postnatal
development occurred by concurrent processes of programmed cell death and increased
mitoses. Both histological features accompanied the developing structures showed a diffuse
pattern and peaked on the first four days of life. Within the ovary, apoptotic granulosa cells
and necrotic ova were consistent with follicle atresia and were most prominent between
PND24-PND26. In kidneys, the subcapsular nephrogenic zone and the developing papilla
were the main target regions of programmed cell death and increased mitoses, which were
mostly abundant in the first week of life (figure 9). The urothelium multifocally sloughed
off due to epithelial cell desquamation, individually or in groups of superficial cells (figure
10), occasionally accompanied by apoptosis. These background changes achieved a peak
at the end of the first week of life and between PND21 and PND26.
Figure 3 : Histological aspect of immature kidney (Sprague-Dawley rat PND1).
Within the subcapsular nephrogenic zone (in parenthesis), note the presence
of the renal vesicle (black star), comma-shaped body (red star) and S-shaped
body (red arrow). The corpuscles are hypercellular (black arrow) and arise
from lower indentation of S-shaped (red arrow). H&E 200x.
Figure 4 : Histological aspect of immature ovary (Sprague-Dawley PND1). The
ovarian parenchyma is hypercellular and diffusely composed of pockets of
oocytes enveloped in stromal cells (arrow). Col. H&E 100x.
Figure 5 : Light micrograph of ovary (Sprague-Dawley PND26). Note the
sharply demarcated cortico-medullary separation and presence of tertiary
follicles. H&E 40x.
Figure 6 : Light micrograph of vagina (Sprague-Dawley rat PND24).
Multifocally, the superficial layer of the vagina contains pale cytoplasmic
material corresponding to mucous (arrows). H&E 200x.
Figure 7 : Histological aspect of immature testes (Sprague-Dawley rat PND1).
The testicular parenchyma is hypocellular and the seminiferous tubular
epithelium is composed of spermatogonia and Sertoli cells (black arrow). Note
the abundance of mitoses (red arrow). H&E 400x.
Figure 1: Diagram of the polynomial regression of the ratio kidney weight relative to body weight
in Sprague-Dawley rats. Note that, at PND10, the ratio of kidney weight relative to body weight
attained the maximum value.
Figure 8 : Light micrograph of testes (Sprague-Dawley rat PND26). At PND26,
the first round spermatids became visible (arrows). H&E 400x.
Figure 9 : Photomicrograph of immature papilla of kidney (Sprague-Dawley
rat PND4). Note the increased single cell necrosis within the papilla (arrows).
H&E 200x.
Materials and methods
The 51 pups in this study were the progeny of 6 time-mated Sprague-Dawley rats females
Crl:CD (SD) purchased from Charles River Laboratories Canada Inc. (St-Constant, QC) at
Day 0 of pregnancy. The tissues were obtained from pups at different time points from
post-natal day (PND) 1 to PND30. When possible, equal number of females and males
were used for each occasion. The body weight and the absolute testes and kidneys weight
were recorded for each time point. The organ weight to body weight ratio was calculated
and the results were statistically evaluated using a dispersion diagram and a polynomial
regression. The kidney, urinary bladder, ovaries, testes, epididymides, cervix, uterus and
vagina were collected and placed in 10% buffered formalin. After 24hrs, the tissues were
rinsed and stored in 70% ethanol until histological processing. The fixed samples were
trimmed, processed and paraffin-embedded. Sections were cut at a 4µm thickness and
stained with hematoxylin and eosin (H&E).
Figure 10 : Histological aspect of immature urinary bladder (Sprague-Dawley
rat PND24). Note the desquamated urothelial cells (black arrows) within the
bladder lumen. H&E 200x.
Conclusion
Figure 2: Diagram of the polynomial regression of the ratio of testes weight relative to body weight
in Sprague-Dawley rats. Note that, at the end of the first month, the testes are still developing and
gaining weight proportionally with body weight.
Results and Discussion
Our study revealed morphological dissimilarities between developing organs when
compared with adult ones. Throughout the first month of life, the kidney and testes weights
proportionally increased with age and body weight, especially after PND21. The organ
weight/body weight ratio reached a maximum value between PND10-PND15 for kidney and
on PND30 for testes. Those data suggest that, at the end of the first month, the testes are
still developing and gaining weight proportionally with the increase of body weight (figure
1) while the kidney increased at a less steep rate than body weight on PND30 (figure 2).
Generally, at birth, the kidneys showed an increased cellularity due to a mixture of blastemal
epithelial cells and undifferentiated, loosely arranged mesenchymal cells, within an edematous
interstitium. At birth, the histological features of kidney immaturity were consistent with
the presence of a distinct nephrogenic zone in outer one-fourth of the cortex. There,
the actively growing portion of the ureteral buds (ampullae) induced the metanephritic
mesenchyme differentiation and lead to different stages of forming nephrons into either
oval masses, renal vesicles or S-shaped structures. The lower indentation of the S-shaped
The urogenital system of Sprague-Dawley rats is immature at birth. After the first month
of life, the male rats had not reached puberty, while female rats showed evidence of estrus
around PND28. Nephrogenesis in rats ceased by PND14 and the urothelium multifocally
sloughed off around the end of the first postnatal week as well as between PND21-PND26.
Programmed cell death and increased mitoses coexisted, accompanied the developing
structures and should be differentiated from treatment-related changes. These results
will serve as database of background age-related changes of neonatal and juvenile rats
in preclinical toxicologic studies.
references
1. David A. Beckman, and Maureen Feuston (2003) – Landmarks in the development of the female reproductive system, Birth defects research
(Part B) 68:137-143 – 2. Harriet S. R. Coles, Julia F. Burne and Martin C. Raff (1993) - Large-scale normal cell death in the developing rat kidney and
its reduction by epidermal growth factor, Development, 118: 777-784 – 3. Melissa H. Little, Jane Brennan, Kylie Georgas, Jamie A. Davies, Duncan
R. Davidson, Richard A. Baldock, Annemiek Beverdam, John F. Bertram, Blanche Capel, Han Sheng Chiu, Dave Clements, Luise Cullen-McEwen,
Jean Fleming, Thierry Gilbert, Doris Herzlinger, Derek Houghton,Matt H. Kaufman, Elena Kleymenova, Peter A. Koopman, Alfor G. Lewis, Andrew
P. McMahon, Cathy L. Mendelsohn, Eleanor K. Mitchell, Bree A. Rumballe, Derina E. Sweeney, M. Todd Valerius, Gen Yamada, Yiya Yang, Jing Yu
(2007) - A high-resolution anatomical ontology of the developing murine genitourinary tract, Gene expression patterns, 7: 680-699 – 4. M. Sue
Marty, Robert E. Chapin, Louise G. Parks, and Bjorn A. Thorsrud (2003) – Development and maturation of the male reproductive system, Birth
Defects Research (Part B) 68: 125-136 – 5. Catherine A. Picut, Amera K. Remick, Midori G. Asakawa, Michelle L. Simon, and George A. Parker (2013)
– Histologic features of prepubertal and pubertal reproductive development in female Sprague-Dawley rats, Toxicologic pathology 42 (2), 403-413.
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