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Boehringer-Ingelheim Research and Development Center (K.T.B.), Ridgefield, Connecticut 06877; and Department of Pathology and Laboratory Medicine, Brown University (J.L., K.B.), Providence, Rhode Island 01912
Address all correspondence and requests for reprints to: Kim Boekelheide, Department of Pathology and Laboratory Medicine, Brown University, Box G-B518, Providence, Rhode Island 02912. E-mail: kim_boekelheide{at}brown.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Infertility affects approximately 15% of all couples, with half of these cases explained by male dysfunction (3, 4). Most male infertility is idiopathic and associated with decreased spermatozoon counts or motility; no adequate treatments exist for the vast majority of these patients. Although controversial (5), a number of reports have described a decrease in the sperm quality of men over the past five decades (6, 7). Due to the relatively short period of time for this trend to occur, this putative decrease in sperm quality has been ascribed to environmental rather than genetic factors (8).
In laboratory animals, a variety of environmental toxicants are known to produce infertility (9). Of these toxicants, 2,5-hexanedione (2,5-HD) is one intensively studied environmental toxicant that causes a long-lasting testicular atrophy and infertility in rats (10, 11). 2,5-HD is the active metabolite of n-hexane (12), and also the major metabolite found in the urine of humans exposed to n-hexane (13). Animal studies have identified testicular injury as a prominent feature of 2,5-HD exposure (10, 11, 12, 14).
When rats are exposed to 1% 2,5-HD in the drinking water for up to 5 weeks, a long-lasting testicular atrophy results (10, 11). At 2 weeks of exposure, Sertoli cell microtubule assembly kinetics are altered (9), followed by a reduction in seminiferous tubule fluid secretion by 3 weeks (15, 16). Histologically, by 4 weeks of 2,5-HD treatment, large basal vacuoles are observed, followed by a massive sloughing and loss of germ cells by 5 weeks of toxicant exposure (10, 11). This state of testicular atrophy is irreversible, because it continues more than 70 weeks after toxicant withdrawal (17). Although the 2,5-HD atrophic testis contains actively dividing spermatogenic stem cells and a residual type A spermatogonial population, more mature germ cells are absent (17, 18, 19). Due to the early biochemical and morphological alterations in Sertoli cells, this cell type is the presumptive target cell by which 2,5-HD exerts its injury.
Although various processes could contribute to the underlying mechanism of the 2,5-HD-induced irreversible testicular atrophy, previous studies in our laboratory have focused on stem cell factor (SCF; also known as c-kit ligand or mast cell growth factor) as a potential target. SCF is a germ cell growth factor produced by Sertoli cells that interacts with the c-kit receptor expressed on spermatogonia (20). This interaction between SCF and c-kit is essential for the progression of spermatogonia into more mature germ cells (21), a process blocked after 2,5-HD exposure (18, 19). In fact, blocking the interaction of SCF with c-kit in the testis using anti-c-kit antibodies results in an atrophic testis morphologically similar to the 2,5-HD atrophic testis (21). In a recent study, SCF messenger RNA (mRNA) expression was disrupted following 2,5-HD exposure, and exogenous administration of SCF enhanced spermatogonial proliferation in the 2,5-HD atrophic testis (22).
Two main forms of SCF are produced in the testis; however, one form can be released from the Sertoli cell membrane by protease cleavage (soluble SCF), whereas the other form (membrane SCF) is preferentially retained on the cellular membrane (20, 23). The form of SCF expressed is based on alternate mRNA splicing (24). Exon 6 of the SCF mRNA sequence codes for protease cleavage sites; alternate splicing produces mRNA lacking exon 6, thus coding for a form of SCF retained on the Sertoli cell membrane. Several lines of evidence suggest that preferential expression of membrane SCF is fundamentally important to spermatogenesis. This is obvious in Steel-Dickie (Sld) mutant mice that express only a soluble form of SCF, affecting germ cell differentiation and resulting in sterility (25). Even though the extracellular domain of SCF interacts with c-kit (26, 27), mutated forms of membrane SCF with altered cytoplasmic tails fail to support spermatogenesis (28). Also, Sertoli cells expressing membrane SCF as opposed to soluble SCF are more successful at promoting germ cell survival in vitro (29, 30). In the adult rat testis, SCF mRNA coding for membrane SCF is preferentially expressed; however, 2,5-HD treatment disrupts this expression and soluble SCF predominates in the 2,5-HD atrophic testis (22). Although this disruption in SCF mRNA expression correlated temporally with the onset and persistence of 2,5-HD-induced testicular atrophy, it is unknown whether altered SCF contributes to the underlying mechanism of this irreversible testicular injury.
Recently, suppression of the pituitary-gonadal hormonal axis has been used to treat other states of irreversible testicular injury in the rat. This has been accomplished through treatments with GnRH antagonists, GnRH agonists, antiandrogens, and systemic androgens (31, 32, 33, 34). The overall aim of this study was to use GnRH agonist treatment as therapy for 2,5-HD-dependent testicular atrophy and to correlate testicular recovery with SCF expression, testing the hypothesis that decreased membrane SCF expression is responsible for the irreversible testicular injury that follows 2,5-HD exposure.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7 weeks
old, 175–200 g) were acclimated approximately 1 week before use. To
examine age-dependent changes in SCF mRNA, young animals ( 5 days
old or 21 days old) were used the day of arrival or allowed to age to
later time points. All chemicals and reagents were obtained from local
suppliers and were of the highest quality. Data were analyzed by a
single-factor ANOVA followed by the Fisher’s post hoc test. A
P value of < 0.05 was set as the level of
significance.
Leuprolide preparation
Leuprolide (Lupron Depot) was kindly provided by TAP
Pharmaceuticals Inc. (Deerfield, IL). Leuprolide injections (3 mg/ml)
were prepared by resuspending a vial of leuprolide (3.75 mg leuprolide
acetate, 0.65 mg purified gelatin, 33.1 mg DL-lactic and glycolic acids
copolymer, 6.6 mg D-mannitol) into 1.25 ml diluent (6.25 mg
sodium carboxymethylcellulose, 62.5 mg D-mannitol, 1.25 mg
polysorbate-80).
Experimental design
For the leuprolide experimental protocols (Fig. 1
), groups of 10 rats ( 200 g, 8
weeks old) were treated with 1% (vol/vol) 2,5-HD (Aldrich Chemical
Co., Milwaukee, WI) for either 23 or 42 days and then returned to
normal drinking water as previously described (11). For each of the two
protocols, immediately following the 2,5-HD exposure, animals were
divided into two groups; one group of five animals received three
sequential leuprolide injections (1.5 mg, 0.5 ml/animal, sc), whereas
the remaining group of five animals (2,5-HD-exposed controls) received
sterile filtered distilled water (0.5 ml/animal, sc) 23–24 days apart
(Fig. 1). This volume of leuprolide depot is predicted to release the
GnRH agonist for 3–4 weeks based on the surface area-to-volume ratio
for the human dose (34, 35, 36, 37). Twelve weeks after the last leuprolide
injection, the animals were killed by CO2 asphyxiation, and
each testis and epididymis was trimmed of surrounding adipose tissue
and individually weighed. For each animal, the left testis and
epididymis were processed for histopathology, and the right testis was
processed for RT-PCR analysis of membrane and soluble SCF mRNA. Body
weights for each animal were monitored throughout the study.
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Histopathological analysis
Following multiple puncture of the tunica albuginea with a
30-gauge needle, the left testis and left epididymis from each rat were
immersion fixed in 10% neutral buffered formalin. Testicular and
epididymal cross sections (1–2 mm thick) were embedded in glycol
methacrylate (Leica Historesin, Deerfield, IL), sectioned (2.5 µm),
and stained with periodic acid Schiff’s reagent and hematoxylin
(PAS/H) (38).
For the testicular cross-sections, the mean number and range of seminiferous tubules analyzed per rat in each group was: 2,5-HD 42-day, mean = 182, range 104–224; 2,5-HD 23-day, mean = 138, range 112–182; leuprolide + 2,5-HD 42-day, mean = 98, range 75–107; leuprolide + 2,5-HD 23-day, mean = 99, range 97–102. Each seminiferous tubule was classified as atrophic (no germ cells more mature than spermatogonia present), partially recovered (containing germ cells more mature than spermatogonia, but either missing or having less than a full complement of spermatocytes, round spermatids, or elongate spermatids), and recovered (containing a full complement of spermatocytes, round spermatids, and elongate spermatids); a full complement was defined as more than 10 cells of a specific type. In addition, the stage (grouped as either I-III, IV-VIII, or IX-XIV) of each recovered seminiferous tubule was determined using standard morphological criteria (39). The extent of calcification of seminiferous tubules was also noted; for counting purposes, calcified seminiferous tubules were included in the atrophic category. For photomicroscopy, a Zeiss Axiovert (Oberkochen, West Germany) mounted with a x20 objective and Tri-X pan film (Eastman Kodak, Rochester, NY) were used.
SCF RT-PCR
RT-PCR and quantification of the membrane/soluble SCF mRNA ratio
was conducted as described by Allard et al. (22). Briefly,
primers were designed corresponding to exon 2 (bp 199–223) and exon 7
(bp 896–874) of the SCF complementary DNA sequence reported by Martin
et al. (40). Because of differential splicing at exon 6,
this generates two PCR fragments; one of approximately 605 bp
corresponding to membrane SCF mRNA, and a second fragment of
approximately 698 bp corresponding to soluble SCF mRNA. The relative
ratio of membrane-to-soluble SCF mRNA expression was estimated by
quantifying the densities of PCR-generated DNA fragments using a
StrataScan-7000 with one-dimensional software (Cloning System;
Stratagene, La Jolla, CA). The densities of the PCR-generated DNA
fragments were within the linear portion of the curve, both for the
amount of product loaded on the gel and for the number of PCR
cycles.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). A small ( 9%) but statistically significant
decrease in body weight was observed in the leuprolide-treated animals
compared with their respective 23-day 2,5-HD-exposed controls (Table 1). In the 42-day 2,5-HD exposure
regimen, a similar difference in body weight between leuprolide-treated
animals and their respective vehicle-treated controls was observed;
however, this did not achieve statistical significance (Table 1).
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) when comparing the
2,5-HD-exposed animals with historical naïve control rats of
similar age (17-week-old rats, 1.52 g ± 0.02).
After 23 days of 2,5-HD exposure, leuprolide treatment markedly
increased testis weight by approximately 65% at the end of the 12-week
recovery period (Table 1). No weight differences in the epididymides
were observed between the 23-day 2,5-HD-treated control animals and
those that received leuprolide therapy. In animals exposed to 2,5-HD
for 42 days, no changes in testicular weights were observed with the
addition of leuprolide treatment. In 42-day 2,5-HD-exposed rats given
leuprolide, epididymides decreased in weight by approximately 22%
compared with their respective 2,5-HD-treated controls (Table 1). At
necropsy, several of the testes of rats from both leuprolide-treated
groups were noted to contain firm, white areas varying in size from
2–7 mm in diameter located centrally in the lower pole.
Histopathological analysis
For each rat, approximately 100 seminiferous tubule cross-sections
were analyzed and categorized as either atrophic, partially recovered,
or recovered. Using this approach, the comparison groups exposed to
2,5-HD alone for 23 or 42 days showed nearly complete testicular
atrophy (Fig. 2, A and B) and did not
differ from each other (Fig. 3). Both of
the groups of leuprolide-treated rats showed recovery of
spermatogenesis that was statistically significant (Fig. 3). However,
the two groups of leuprolide-treated rats differed from each other,
with the 23-day 2,5-HD-treated animals showing the most complete
recovery of spermatogenesis (Figs. 2C and 3). The leuprolide-treated
42-day 2,5-HD-treated animals had a higher proportion of atrophic
seminiferous tubules and fewer recovered seminiferous tubules compared
with the leuprolide-treated 23-day 2,5-HD-treated animals (Figs. 2D and 3). Interestingly, in the atrophic seminiferous tubules from
leuprolide-treated rats, occasional type A spermatogonia were seen
(data not shown), indicating that the failure of recovery was not a
result of elimination of the germ cell population.
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In some leuprolide-treated rats, isolated atrophic seminiferous tubules
or large groups of atrophic seminiferous tubules showed partial or
complete calcification (Fig. 4),
corresponding to the firm, white areas in the lower pole of
leuprolide-treated rat testes noted at necropsy. Calcification occurred
in two of five and three of five testicular cross-sections from the
leuprolide-treated rats that received 23 days and 42 days of 2,5-HD
exposure, respectively. No calcified seminiferous tubules were seen in
rats that received 2,5-HD alone.
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). However, in four of
five rats treated with leuprolide plus 2,5-HD (23 days), the ductal
lumens of the caput and cauda epididymides contained abundant,
normal-appearing spermatozoa (Fig. 5B).
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). The membrane SCF and soluble
SCF specificity of these two bands was confirmed by two separate
restriction enzyme digestions (SspI and EcoRI),
each of which cut the fragments into smaller lengths of the predicted
size (data not shown). As shown in Fig. 6A, early in life (5–7 days
old) SCF mRNA in the testis is primarily of the soluble form. However,
a dramatic change in expression occurs between 7 and 11 days of life,
resulting in a greater expression of membrane SCF compared with soluble
SCF mRNA (Fig. 7).
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), a decrease in the ratio
of membrane SCF to soluble SCF mRNA was observed. When quantitated by
densitometry, a statistically significant decrease in the percent
membrane SCF was observed by 5 weeks of 2,5-HD treatment, which
persisted long after toxicant withdrawal (Fig. 7).
SCF expression after leuprolide therapy
To assess the relationship between the form of SCF mRNA expressed
and testicular atrophy, RT-PCR was performed on RNA extracted from the
testes of rats exposed to 2,5-HD with and without leuprolide therapy.
In the absence of leuprolide treatment, the proportion of membrane SCF
expressed (Fig. 8) depended on the length
of 2,5-HD exposure (23 days vs. 42 days), even though both
groups of exposed rats had nearly complete testicular atrophy (Fig. 2).
In both 23-day and 42-day 2,5-HD- exposed rats, leuprolide treatment
caused a significant increase in the proportion of membrane SCF
expressed (Fig. 8). However, the proportion of membrane SCF in the
42-day 2,5-HD-exposed rats treated with leuprolide was the same as that
of rats exposed to 2,5-HD for 23 days that did not receive leuprolide
(Fig. 8), despite a significant difference in their extent of recovery
as assessed by histopathology (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leuprolide, given as an extended release depot or daily injections over a long period of time, produces a transient increase, followed by a sustained suppression, in testosterone, LH, and FSH in the blood (35, 41, 42). Systemic suppression of LH and FSH allows recovery from irreversible testicular atrophy produced by a number of agents (31, 32, 33, 34). Interestingly, testicular atrophy is often accompanied by an elevation in serum LH and FSH (17); presumably, altered hemodynamics reduce the levels of testosterone leaving the atrophic testis, decreasing the negative feedback on LH and FSH secretion (43). However, the beneficial effect of leuprolide is unlikely to be simply a consequence of suppressing serum gonadotropins, because hormonal treatments that differ in their ability to rescue the injured testis (testosterone alone vs. testosterone plus estrogen) suppress gonadotropin levels to the same extent (44).
Leuprolide could act directly to stimulate germ cell maturation, division, or survival, although this is unlikely, because GnRH receptors have not been found on germ cells or associated with any cells of the seminiferous tubules (45). Alternatively, leuprolide could act indirectly to modulate the growth factor milieu of the seminiferous epithelium. For example, GnRH receptors have been identified on rat Leydig cells (46), and systemically administered leuprolide could alter the germ cell environment by stimulating Leydig cells to produce paracrine factors beneficial to spermatogenesis. Or by decreasing intratesticular testosterone concentrations, leuprolide could affect the production of Sertoli cell-derived growth factors, such as androgen binding protein, which are known to be influenced by testosterone interacting with androgen receptors in the Sertoli cell (47). Similarly, the expression of SCF, a Sertoli cell-derived growth factor, could be affected by leuprolide-induced alterations in the hormonal milieu of the seminiferous epithelium. Although testosterone itself does not increase SCF secretion by Sertoli cells in culture (48) or alter the activity of the human SCF proximal promoter (49), other multifactorial effects of leuprolide-induced suppression of the hypothalamic-pituitary-gonadal axis could alter the expression pattern of this growth factor.
A dramatic switch from preferential expression of soluble SCF to
membrane SCF occurred during reproductive maturation in the rat (Figs. 6 and 7), as previously described in mice (23). This alteration in
expression occurred 7–10 days postnatally in association with the
initiation of the first wave of spermatogonial proliferation in the rat
(50), and it is likely that membrane SCF is more important than soluble
SCF with regard to spermatogenesis in the adult (29, 30). The
preferential expression of membrane SCF associated with the onset of
spermatogenesis was altered to primarily soluble SCF in the atrophic
testis. If the reduction in membrane SCF mRNA expression is critical to
2,5-HD-induced testicular injury, enhanced germ cell survival and
proliferation would be expected if this deficit could be corrected.
Continuous infusion for 2 weeks of recombinant soluble SCF into the
2,5-HD-atrophic testes promoted recovery (22); however this beneficial
effect was modest. To date, technical limitations have prevented
examination of the effect of added membrane SCF on germ cell
proliferation and survival in the atrophic testis.
Leuprolide therapy increased the relative expression of the membrane
SCF vs. soluble SCF mRNA in 2,5-HD-exposed rats and resulted
in recovery from testicular atrophy; that is, preferential expression
of membrane SCF correlated with spermatogenesis, whereas, expression of
soluble SCF was related to a lack of spermatogenesis. Leuprolide
therapy was more efficient at stimulating spermatogenesis in rats
exposed to 2,5-HD for 23 days as opposed to 42 days. This is not
surprising, given the dose-response relationship between the length of
2,5-HD exposure and the extent of testicular atrophy that follows (11).
Certainly, both 23 and 42 days of toxicant exposure resulted in
testicular atrophy (Figs. 2 and 3). However, testicular weights from
the 42-day exposure were less than those from the 23-day exposure
(Table 1), and one animal in the 23-day exposure group had minimal
recovery of spermatogenesis. In any case, a 23-day exposure to 2,5-HD,
compared with a 42-day exposure, resulted in less testicular injury and
an atrophic testis more responsive to leuprolide-induced recovery.
Interestingly, the proportion of membrane SCF expressed was
significantly greater with the shorter 2,5-HD exposure consistent with
the greater recovery potential of these atrophic testes.
The relationship between the ratio of membrane/soluble SCF expression and the amount of spermatogenesis was not completely predictive as demonstrated by the similar proportion of membrane SCF but nearly universal testicular atrophy in the rats that received 23 days of 2,5-HD alone compared with rats that received 42 days of 2,5-HD plus leuprolide and had significant recovery of spermatogenesis. This comparison is complicated by the different analytical approaches, because SCF RT-PCR used mRNA extracted from the whole testis, whereas recovery of spermatogenesis was assessed histopathologically by examining individual seminiferous tubules. Membrane SCF may be a marker of both the potential to recover and the extent of recovery. Whether the link between membrane SCF expression and spermatogenesis is causative or merely correlative requires additional experimentation.
Another variable in the two exposure protocols (23 vs. 42
days of 2,5-HD exposure) involved the timing of leuprolide therapy
relative to the onset of atrophy. A temporally well-defined sequence of
events is involved in 2,5-HD-induced testicular injury, irrespective of
the length of toxicant exposure (11). When rats are exposed to 2,5-HD,
testicular microtubule assembly is altered by 2 weeks, seminiferous
tubule fluid secretion is decreased by 3 weeks, and basal vacuolization
and massive germ cell apoptosis is observed between 4 and 6 weeks
(9, 10, 11, 16). Because leuprolide therapy immediately followed toxicant
exposure (see Fig. 1), its timing varied with respect to the onset of
atrophy in the two exposure paradigms. Future experiments will be
designed to determine whether the timeframe of leuprolide treatment is
crucial to the extent of recovery from testicular atrophy.
The current study describes an otherwise irreversible model of testicular atrophy that can be significantly corrected by extended GnRH-agonist therapy. The underlying mechanism of this response is unknown but may be mediated through alterations in hormones of the gonadal-pituitary axis and/or intratesticular growth factors such as SCF. Future studies focused on these mechanisms can provide further insight into the treatment of testicular injuries and infertility.
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Acknowledgments |
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Footnotes |
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Received July 21, 1997.
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H. Hakovirta, W. Yan, M. Kaleva, F. Zhang, K. Vänttinen, P. L. Morris, M. Söder, M. Parvinen, and J. Toppari Function of Stem Cell Factor as a Survival Factor of Spermatogonia and Localization of Messenger Ribonucleic Acid in the Rat Seminiferous Epithelium Endocrinology, March 1, 1999; 140(3): 1492 - 1498. [Abstract] [Full Text]
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W. Yan, J. Linderborg, J. Suominen, and J. Toppari Stage-Specific Regulation of Stem Cell Factor Gene Expression in the Rat Seminiferous Epithelium Endocrinology, March 1, 1999; 140(3): 1499 - 1504. [Abstract] [Full Text]
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