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From the Laboratory of Veterinary Physiology, Nippon Veterinary Life Science University, Tokyo, Japan.
| Correspondence to: Hiroetsu Suzuki, Laboratory of Veterinary Physiology, Nippon Veterinary Life Science University, 1-7-1 Kyonancho, Musashino-shi, Tokyo 180-8602, Japan (e-mail: hiroetsu{at}nvlu.ac.jp). |
| Received for publication February 8, 2008; accepted for publication July 19, 2008. |
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Abstract |
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Key words: Testis, spermatogenesis, apoptosis, FSH, LH, testosterone
In a previous histological study, we found many extracellular vacuoles in the CA1 region of the hippocampus and the amygdaloid body of the lde/lde brain at 28 days of age. These pathological lesions may be associated with epileptogenesis in lde/lde rats. In addition to these defects, lde/lde rats have lower relative weights of the testes at 28 days of age as compared with controls. Histologically, seminiferous tubules were poorly matured and contained a reduced number of germ cells, and the maturation of adult-type Leydig cells was also delayed (Suzuki et al, 2007).
Spermatogenesis is established through a complicated process including paracrine and endocrine regulation during prepubertal and pubertal periods (Sharpe, 1994). It is possible that the testicular phenotype of lde/lde rats may be caused by pleiotropic expression of the lde gene in the testis. Alternatively, as inputs to the hypothalamus from the hippocampus and amygdala influence the activity of the hypothalamus-pituitary axis (Morrel and Montouris, 2004; Fawley et al, 2006), the testicular defects in lde/lde males may be mediated by the altered secretion of gonadotropins. Because the lde/lde rat is potentially useful as an animal model of pediatric epilepsy, it is important to characterize the testicular phenotype. In the present study, we examined postnatal alterations in reproductive organ weights, testicular pathogenesis, and plasma hormone levels in lde/lde rats after weaning.
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Materials and Methods |
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Collection of Blood Samples and Determination of Organ Weights
Blood samples were collected from the vena cava with a heparinized plastic
syringe around 1700 hours under light ether anesthesia. Plasma samples were
obtained and stored at –80°C until assayed. After collection of
blood samples, rats were sacrificed by an overdose of ether, and autopsied to
determine the weights of the male reproductive organs using an electric
balance. Because of the high lethality of immature lde/lde
rats (Suzuki et al, 2007), 3
to 9 mutant rats at each day were used. Organ weights are presented as means
and standard errors (SE), and differences between normal and mutant rats were
determined by the unpaired Student's t test.
Preparation of Testicular Sections
After weighing, all testes were fixed in Bouin solution overnight, embedded
in paraffin (paraffin pellets; Wako, Osaka, Japan), and cut into serial
sections at a thickness of 3 µm as described previously
(Suzuki et al, 2004). The
sections were deparaffinized in xylene, hydrated in a graded alcohol series,
and immersed in water or 0.01 M phosphate-buffered saline (PBS; pH 7.4). They
were then stained with hematoxylin-eosin, the TUNEL method was used to
identify apoptotic cells, and immunostaining was performed for
3β-hydroxysteroid dehydrogenase (3β-HSD), 11β-hydroxysteroid
dehydrogenase (11β-HSD), and vimentin.
Immunohistochemistry and TUNEL Staining for Testicular Tissue
Tissue sections were processed in a microwave (5 minutes x 1 for
3β-HSD; 3 minutes x 5 for 11β-HSD) or boiled (15 minutes for
vimentin) in 0.01 M citric acid buffer (pH 6.0) to reactivate their
antigenicity. Then, the sections were immersed in methanol containing 3%
periodic acid to inactivate internal peroxidases and soaked in PBS. After
soaking in PBS containing 2% bovine serum albumin or 10% skim milk to block
nonspecific antigen-antibody reactions, they were incubated with antibodies
against 3β-HSD (rabbit polyclonal, 1/4000 dilution, 4°C overnight;
generous gift from Dr Mason, University of Edinburgh School of Clinical
Sciences and Community Health; Doody et al,
1990), 11β-HSD (rabbit polyclonal, 20 µg/mL, room
temperature, overnight; Alpha Diagnostic International, San Antonio, Texas),
and Vimentin (monoclonal, 1/100, 1 hour, room temperature, Vimentin Ab-2,
clone V9; NeoMarkers, Fremont, California). Following immersion in PBS,
primary antibodies were detected using Histofine Simple Stain Rat MAX-PO
(MULTI; Nichirei Corporation, Tokyo, Japan) as reported previously (Suzuki et
al, 2004,
2006). Apoptotic cells were
detected with an In Situ Apoptosis Detection Kit (TaKaRa Bio Inc, Shiga,
Japan; Suzuki et al, 2004;
Yagi et al, 2006). The slides
were incubated with 3,3'-diaminobenzidine tetrahydrochloride,
counterstained with hematoxylin, and mounted with Mountquick (Daido Sangyo Co
Ltd, Tokyo, Japan). Microscopic images were obtained using a Penguin 600CL
digital camera system (Pixera Corporation, Osaka, Japan) attached to a
microscope (BX50; Olympus Corporation, Tokyo, Japan;
Suzuki et al, 2004).
Cell Counts and Morphometric Analysis
At least 10 round sections of seminiferous tubules of each testis were
randomly selected from testicular sections immunostained with antibody to
vimentin, and the numbers of vimentin-positive Sertoli cells and
vimentin-negative germ cells in the tubules were counted under a light
microscope. The diameters of the tubules used for cell counts were also
measured using NIH Image (NIH Image version 1.62;
http://rsb.info.nih.gov/nih-image/).
The area for the counts of TUNEL-positive cells was defined by a square field
(0.3 mm2) in the finder of the charge-coupled device (CCD) camera.
At least 10 areas were selected at random from histological sections of each
testis, and TUNEL-positive cells in the area were counted. After the values
were averaged as representative values of individual rats, representative
values were averaged for each group and were compared between the
phenotypically normal and lde/lde rats using the unpaired
Student's t test.
Plasma Follicle-Stimulating Hormone, Luteinizing Hormone, and Testosterone Assay
Plasma concentrations of luteinizing hormone (LH), follicle-stimulating
hormone (FSH), and testosterone were measured using the Rat Follicle
Stimulating Hormone Biotrak Enzyme Immunoassay (EIA) System (Amersham
Biosciences, Buckinghamshire, UK), Rat Luteinising Hormone (rLH) Enzyme
Immunoassay Biotrac (EIA) System (Amersham Biosciences), and Rodent
Testosterone ELISA Test Kit (Endocrine Technologies, Inc, Newark, California),
respectively. Each assay was performed in duplicate in accordance with the
manufacturer's recommendations. Data are presented as mean and SE, and
differences between normal and mutant rats were determined by Mann-Whitney
U test.
Immunohistochemical Detection of FSH and LH in Pituitary Tissue
Three normal and 3 mutant pituitary glands at 28 days of age were fixed in
Bouin solution for 1 hour, and paraffin sections were cut as described for the
preparation of testicular sections. After the sections were immersed in
methanol containing 3% periodic acid to inactivate internal peroxidases and
soaked in PBS, the polyclonal antibodies to rat FSH and LH (1/12 800, room
temperature, overnight; Biogenesis Ltd, Poole, United Kingdom) were applied to
the sections as the first antibodies. Detection of first antibodies and
imaging of the immunohistological pictures were performed as described for
testicular sections.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Postnatal Alteration of Testicular Histology
The areas of seminiferous tubular cross-sections and the number of cells in
the tubules were apparently smaller in the lde/lde
(Figure 2B, D, F, and H) than
in the normal testis (Figure 2A, C, E, and
G) on all days examined. In the normal testis, from 21
(Figure 2A) to 28
(Figure 2C) days of age, the
number of spermatogonia underlying the circumference of the tubular section
increased, and early spermatocytes differentiated into late spermatocytes with
large cytoplasm. Round spermatids appeared at 35 days of age
(Figure 2E), and differentiated
into elongated spermatids and sperm by 56 days of age
(Figure 2G). Therefore, the
initial round of spermatogenesis was completed before 56 days of age. In the
lde/lde testis, however, the increases in tubular diameter
and cellular number within the tubules were severely affected. Although
considerable numbers of spermatogonia and early spermatocytes were found in
the seminiferous tubules at 21 days of age
(Figure 2B), spermatocyte
numbers had decreased markedly at 28 days of age
(Figure 2D). Instead of normal
spermatocytes, many apoptotic cells with condensed nuclei were detected in
lde/lde seminiferous tubules at 21 and 28 days of age
(Figure 2B and D).
Spermatocytes appeared again at 35 days of age
(Figure 2F) and differentiated
into spermatids at 56 days of age (Figure
2H). No sperm were found in the lde/lde testis
(Figure 2H).
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Diameter of Seminiferous Tubules and Cell Number in the Tubules
To distinguish Sertoli cells from germ cells in seminiferous tubules,
testicular sections were immunostained for vimentin
(Suzuki et al, 2004). Because
immunostaining of vimentin was located close to the nuclei of Sertoli cells in
both lde/lde and normal testes
(Figure 3, upper panel),
Sertoli cells were easily detected in their tubules, and vimentin-negative
cells in seminiferous tubules were regarded as germ cells. The diameters of
the tubules were significantly smaller in the testes of
lde/lde rats than in normal controls on all days examined
(P < .01; Figure 3,
lower panel, left). The numbers of vimentin-positive Sertoli cells in
seminiferous tubular sections were significantly smaller in the testes of
lde/lde rats than in normal controls at 21 and 28 days of
age (P < .01; Figure
3, lower panel, right). In both groups, the number of Sertoli
cells was almost constant through the pubertal period. In contrast, germ cell
numbers increased by about 4- and 3-fold from 21 to 56 days of age in normal
and lde/lde testes, respectively. The number of germ cells
was significantly smaller in the testes of lde/lde rats than
in normal controls at all days examined (P < .01;
Figure 3, lower panel,
right).
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Postnatal Differentiation of Leydig Cells
Instead of fetal-type Leydig cells, adult-type Leydig cells differentiate
from peritubular mesenchymal cells after around 10 days of age, and gradually
increase in number with age during prepubertal and pubertal periods
(Mendis-Handagama and Ariyaratne,
2001). 3β-HSD is a specific marker for both fetal- and
adult-type Leydig cells. 11β-HSD is a specific marker for immature or
mature adult-type Leydig cells
(Mendis-Handagama and Ariyaratne,
2001). In the normal testis, fetal-type Leydig cells were rarely
observed after 21 days of age, and 3β-HSD–positive adult-type
Leydig cells gradually increased in number with age
(Figure 6A, C, and E). In the
testis of lde/lde rats, although fetal-type Leydig cells
showed intense immunostaining with an antibody to 3β-HSD, the intensity
of 3β-HSD immunostaining was weak in developing adult-type Leydig cells
at 21 and 28 days of age, and the increase in number of
3β-HSD–positive adult-type Leydig cells was delayed from 21 to 35
days of age (Figure 6B, D, and
F). In the normal testis, most Leydig cells were positive for
11β-HSD at 56 days of age (Figure
6G). In the lde/lde testis, the number of
11β-HSD–positive cells was lower than that in the normal controls
(Figure 6H).
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Plasma Hormone Levels and Pituitary Localization of Gonadotropins
Plasma concentrations of both FSH and LH increased gradually from 21 to 35
days of age in normal males. In lde/lde rats, plasma
concentrations of both hormones remained low at 21 and 28 days of age, and the
concentrations were significantly lower in lde/lde rats than
in normal controls at 28 days of age (P < .05). However, the
concentrations of both hormones became equivalent to those of normal rats at
35 days of age, and there were no significant differences between the
concentrations in normal and lde/lde rats at 35 and 56 days
of age (Figure 7, upper panel).
To confirm the production of these hormones in the anterior lobe of the
pituitary gland at 28 days of age, immunohistochemical analyses were carried
out for in situ detection of FSH and LH. Both FSH-positive cells and
LH-positive cells were detected in the pituitary sections of both normal and
lde/lde males. FSH-positive cells showed weak
immunostaining, and LH-positive cells had less cytoplasm stained with the
antibody to LH in lde/lde males
(Figure 7, lower panel). In
normal males, plasma testosterone level was almost constant until 35 days of
age but increased rapidly at 56 days of age. In lde/lde
males, plasma testosterone level was significantly lower than that in normal
controls at 28 days of age (P < .01). Although the level increased
gradually from 28 to 56 days of age, it was significantly lower than the
normal level (P < .05; Figure
8).
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Discussion |
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Although immature Sertoli cells proliferate actively for 2 weeks after birth, they do not proliferate once they enter the arrested phase (Sharpe et al, 2003). Consistent with this previous report, the number of Sertoli cells in the seminiferous tubules was constant in normal rats after 21 days of age in the present study, even though the diameter of seminiferous tubules increased gradually. Therefore, the increases in the area of tubular sections after weaning were mostly caused by the increased volume of each Sertoli cell and the increased number of germ cells. These processes are apparently delayed in the lde/lde testes with smaller seminiferous tubule diameter.
Embryonic and early postnatal proliferation of immature Sertoli cells are promoted by intratesticular factors (Baker and O'Shaughnessy, 2001). Subsequently, FSH plays critical roles in the proliferation and differentiation of immature Sertoli cells to establish the normal capacity of sperm production at the adult stage (Krishnamurthy et al, 2001; Sharpe et al, 2003; Meachem et al, 2005). Therefore, the reduced numbers of Sertoli cells in the lde/lde testis at 21 and 28 days of age may be caused by the low levels of plasma FSH, which may start before 21 days of age. Because the number of Sertoli cells in the seminiferous tubules became comparable between lde/lde rats and normal controls at 35 and 56 days of age, Sertoli cells with abnormally immature features in the lde/lde testes at 28 days of age may still have some proliferation capacity, similar to hypogonadotropic hypogonadal (hpg) mice (Baker and O'Shaughnessy, 2001).
In contrast to the constant number of Sertoli cells after 21 days of age, the number of germ cells increased rapidly by 56 days of age in the normal testes. This increase involves the meiotic division of spermatocytes, because each spermatocyte may produce 4 spermatids (sperm) (Clermont and Perey, 1967; Sharpe, 1994). In the present study, the appearance of sperm at 56 days of age in the normal testes clearly demonstrated completion of the initial round of spermatogenesis in normal rats before 56 days of age. It has also been reported that apoptosis occurs in a considerable fraction of germ cells, especially spermatocytes in the first wave of spermatogenesis (Jahnukainen et al, 2004). Here, as well as in the previous study, the number of apoptotic cells was greatest at 21 days of age, and tended to decrease with age in the normal testes.
In the testes of lde/lde rats, the numbers of apoptotic cells was greater than in normal controls at all stages examined, and the greatest number was detected at 28 days of age. In addition, the cellularity of the immature stage of spermatogenesis in lde/lde seminiferous tubules at 56 days of age resembled that of normal tubules at 35 days of age. This apparent retardation of spermatogenesis could not be explained simply by delayed initiation of the first round of spermatogenesis, because a considerable number of spermatocytes were already present in the lde/lde testis at 21 days of age. In the lde/lde testes, however, most of the early spermatocytes present at 21 days of age failed to differentiate to late spermatocytes, and they were apparently lost by an apoptotic process around 28 days of age. Therefore, spermatocytes that appeared at 35 days of age were considered to be derived from the next generation of spermatogonia, and progression of the initial round of spermatogenesis may be delayed by about 2 weeks. This transient interruption of spermatogenesis probably causes the retarded appearance of spermatids at 56 days of age.
Many mouse and rat strains with mutations in stage-specific genes of spermatogenesis have been established, and they often show developmental arrest and apoptotic death of germ cells at a specific stage of spermatogenesis (de Rooij and de Boer, 2003). Although we could not confirm whether spermiogenesis progresses normally or not in lde/lde males because of their premature death, spermatogenesis was seen to proceed to at least the spermatid stage. As the cause of the transient degeneration of early spermatocytes in lde/lde testes, it is therefore reasonable to postulate incomplete support of germ cells by somatic cells resulting from extratesticular (hormonal) and/or somatic cellular defects rather than mutations in spermatogenic genes expressed in spermatocytes.
The depletion of testosterone production is known to prevent the progression of spermatogenesis at spermatocyte and spermatid stages (Lei et al, 2004; Spaliviero et al, 2004). Although the low level of testosterone in lde/lde males at 28 days may contribute in part to the increased apoptosis of spermatocytes, the appearance of spermatids under conditions of continuously low levels of plasma testosterone at 56 days of age indicates that spermatids can still develop at such testosterone levels. In vivo experiments indicated that transient FSH suppression during the peripubertal period causes an increase in apoptosis of germ cells resulting from incomplete capacity of immature Sertoli cells to support the development of germ cells (Meachem et al, 2005). Therefore, it is reasonable to consider that immature Sertoli cells due to low levels of plasma FSH in lde/lde rats at 28 days of age could not support the development of early spermatocytes in the first round of spermatogenesis.
The development of male reproductive organs during fetal and perinatal periods is induced by testosterone secreted from fetal-type Leydig cells. Instead of the fetal-type Leydig cells, adult-type Leydig cells, as a primary source for testosterone in postnatal testis, differentiate from peritubular cells and proliferate rapidly in response to LH (Mendis-Handagama and Ariyaratne, 2001). In the lde/lde testes, although fetal-type Leydig cells were present at 21 days of age, the increases in numbers of 3β-HSD–positive adult-type Leydig cells and mature 11β-HSD–positive adult-type Leydig cells were markedly delayed. Therefore, the normal plasma testosterone level in lde/lde males at 21 days of age may be because of the considerable number of functional fetal-type Leydig cells, and the gradually increased but still low level of plasma testosterone in lde/lde males from 28 to 56 days of age was probably caused by decreased production of testicular testosterone because of decreased proliferation and delayed differentiation of adult-type Leydig cells.
Although plasma LH is critical for the establishment of a normal-size population of adult-type Leydig cells to produce physiological levels of testosterone (Mendis-Handagama and Ariyaratne, 2001; Ma et al, 2004), the initial differentiation of adult-type Leydig cells is independent of LH (Mendis-Handagama and Ariyaratne, 2001). In lde/lde males, early development of adult-type Leydig cells had already been affected under the conditions of normal plasma LH level at 21 days of age. Therefore, although significantly lower levels of plasma LH in lde/lde rats at 28 days of age may contribute to delayed maturation of adult-type Leydig cells, the primary causes of the defective development of adult-type Leydig cells in these animals may occur in the cells or testes. In addition, the almost normal levels of plasma LH in lde/lde males at 35 and 56 days of age may be inadequate to catch up to Leydig cell proliferation and differentiation with their normal levels. Although we could not confirm whether lde/lde males can produce sperm or not, the markedly low levels of plasma testosterone may not be adequate to induce male reproductive behavior.
Consistent with the lower levels of plasma FSH and LH at 28 days of age, the intensity of FSH immunostaining and the sizes of the cytoplasm of LH-positive cells seemed to be weaker and smaller, respectively, in lde/lde than in normal pituitary glands. Similar cytological alterations were observed in GH cells (Suzuki et al, 2007). Therefore, smaller cytoplasm may be a common feature of anterior cells of the lde/lde pituitary gland. It has been reported that rapid growths of testes after weaning are associated with the elevations in plasma FSH and LH levels, which are mediated by increased production of hypothalamic GnRH and increased frequency of pulsatile GnRH secretion during the prepubertal period (Dutlow et al, 1992; Ebling, 2005). Pathological lesions have been demonstrated in the hippocampus and amygdaloid body in lde/lde rats at 28 days of age (Suzuki et al, 2007). As these regions contain nuclear groups that influence GnRH release (Morrel and Montouris, 2004; Fawley et al, 2006), it is possible that the pathological alterations in these regions may affect the establishment of the secretion pattern and timing of GnRH during prepubertal period.
In humans, patients with epilepsy often have reproductive disorders, such as hypothalamic-pituitary axis disruption, and hypogonadotropic hypogonadism is a major underlying cause of sexual dysfunction in men with epilepsy (Morrel and Montouris, 2004; Fawley et al, 2006). Adult male rats with electroconvulsive shock seizure showed transient HGN characterized by decreased serum testosterone levels and lowered gonadal tissue weight (Edwards et al, 2000). At present, however, no suitable murine model for studying reproductive disorders accompanied by pediatric epilepsy has been reported. As lde/lde rats often experience epileptic seizures during the period when the hypothalamus-pituitary-testicular axis is established, lde/lde rats would be a useful model of reproductive disorder with pediatric epilepsy.
In summary, the reproductive phenotype of male lde/lde rats was characterized by early spermatocyte apoptosis in the initial round of spermatogenesis, delayed proliferation and differentiation of adult-type Leydig cells, transient lower levels of plasma FSH and LH, and continuous low plasma testosterone levels. Therefore, the normal lde allele may be expressed in male reproductive and endocrine organs and be related to the normal progression of testicular development and the initial round of spermatogenesis.
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Acknowledgments |
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Footnotes |
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