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From the Department of Surgery * Division of
Urology and Neuroscience, UMD-New Jersey
Medical School Newark, New Jersey; and the
Veterans Affairs Medical Center, East Orange,
New Jersey.
| Correspondence to: Dr Hosea F. S. Huang, Department of Surgery Section of Urology, UMD-New Jersey Medical School, 185 S Orange Ave, Newark, NJ 07103 (e-mail: huanghf{at}umdnj.edu). |
| Received for publication September 30, 2002; accepted for publication November 16, 2002. |
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Abstract |
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Key words: Spinal cord injury, cAMP, sperm
The extent of SCI resulting from contusion is a function of the sheer force generated by the weight of a rod that drops from different heights (Kwo et al, 1989). By using this method, various degrees of SCI resembling those in SCI men can be induced in the rat. Spinal cordcontused (SCC) rats provide a unique animal model for evaluating the relationship between the extent of SCI and the integrity of peripheral functions. In this study, we examined the normalcy of spermatogenesis and sperm function in SCC rats with various degrees of SCI induced by weight drop from different heights.
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Materials and Methods |
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Experiments
Experiment 1—
Animals were subjected to a weight drop from a 12.5-, 25-, 50-, or 75-mm
height. Animals were killed at 5–7 days or at 14 weeks after injury by
decapitation to collect trunk blood for hormone measurement.
Experiment 2— Animals were subjected to a weight drop 1 (1x) or 3 times (3x) from 25- or 75-mm heights. Animals were hemicastrated 4 weeks after the injury and killed by decapitation 4 weeks later.
In both experiments, a portion of 1 testis was fixed in Bouin solution and processed for histology. The remaining tissues were stored at -80°C for further analysis.
Sperm Motility
The epididymides were dissected immediately from the testes after killing
and immersed in 4–5 mL of 37°C Kreb Ringer solution supplemented
with sodium pyruvate (1 mM), D-glucose (5.57 mM), sodium
bicarbonate (10 mM), HEPES (25 mM), and bovine serum albumin (2%). The distal
end of the caudal epididymis was punctured in 20–30 locations with a
19-G needle, and the sperm were flushed out with a gentle stream of buffer.
The sperm suspension was kept at 37°C for 10–15 minutes. A drop of
sperm suspension was then placed on a prewarmed hemocytometer and examined.
Sperm in 10–20 microscopic fields were videotaped, and sperm motility
was later determined. A sperm was considered "motile" if it did
not remain at the same location during the 5- to 10-second taping time.
Northern Blot Complementary DNA Hybridization
Detailed procedures for isolation of tissue RNA by the singlestep method
(Chomczynski and Sacchi, 1987),
enrichment of poly-A+ RNA by oligothymine dinucleotide cellulose
chromatography (Aviv and Leder,
1972), and Northern blot complementary DNA (cDNA) hybridization
(Sambrook et al, 1989) of
Sertoli and germ cell–specific transcripts have been previously reported
(Huang et al, 1999;
Ottenweller et al, 2000).
Hormone Measurement
Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone concentrations were determined by radioimmunoassay as previously described (Huang et al, 1991).
Immunostaining of Cyclic Adenosine Monophosphate Responsive Element
Modulator
Five-micron-thick sections of Bouin solution fixed testicular tissue were
deparaffinized in xylene, rehydrated in graded ethanol, and boiled in 0.01 M
citrate buffer (pH 6.0) for 10–15 minutes. After a phosphate-buffered
saline (PBS) wash (twice for 5 minutes each), the sections were incubated with
1.5% hydrogen peroxide for 10 minutes and washed twice in distilled water,
washed in 2 changes of PBS (5 minutes each), and blocked in 4% normal goat
serum in PBS containing 0.01% Tween 20 for 20 minutes. After 2 washes in PBS,
the sections were then incubated with polyclonal anti–cyclic adenosine
monophosphate (cAMP) responsive element modulator (CREM) antibody (1:300
dilution in blocking solution, Santa Cruz Biotechnology Inc, Santa Cruz,
Calif) at 4°C overnight. For the negative control, the CREM antibody was
incubated with a fivefold excess of purified antigen at 4°C overnight and
diluted to 1:300 in blocking solution before use. The sections were
subsequently washed in 2 changes of PBS, incubated with biotin-labeled
anti-rabbit immunoglobulin G (IgG; 1:2000, Sigma Chemical Co, St Louis, Mo)
for 30 minutes, and washed with PBS. The sections were then incubated with
Vectastain Elite ABC reagent (Vector Laboratories, Burlingame, Calif) for 30
minutes, washed in 2 changes of PBS, and visualized after adding stable
diaminobenzidine solution (Research Genetics, Huntsville, Ala). The slides
were dehydrated in graded ethanol solution, cleared in xylene, and
covered.
Statistics
All data were analyzed to determine whether they were normally distributed.
The data then were evaluated using analyses of variance of treatment groups
(sham and SCC from different heights). When there was a significant treatment
effect (P < .05), Dunn tests were used to determine the
significance of the differences among groups and to correct for multiple
comparisons. In addition, regression analysis was used to determine whether
changes in parameters were related to the height of weight drop.
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Results |
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Organ Weights
The testis weight of SCC rats remained normal during the first week after
injury (experiment 1, data not shown). Fourteen weeks after cord contusion,
testis weights of SCC rats that were injured by a weight drop from the 50- and
75-mm heights were significantly lower than those of sham controls (sham: 1929
± 110, 50 mm: 1632 ± 92, 75 mm: 1781 ± 52 mg, P
< .05). Epididymis weights of these SCC rats were not different from those
of sham control rats. Prostate weight was decreased significantly in those SCC
rats injured by a weight drop from the 75-mm height (sham: 1154 ± 92,
75 mm: 894 ± 83, P < .05). The weights of the testis and
epididymis of SCC rats that were contused 1x or 3x by a weight
drop from the 25- or 75-mm height (experiment 2) were not different from those
of sham control rats at either time point (data not shown).
Spermatogenesis
Complete spermatogenesis, illustrated by the presence of mature spermatids
at the luminal edge of stage VII–VIII epithelium, was observed in all
SCC rats. Nevertheless, abnormal spermatogenesis was illustrated by the
underdevelopment of pachytene spermatocytes during the acute phase of the
injury (Figure 1G and
H). In addition, total
regression of the seminiferous epithelium was observed in some tubular cross
sections in some SCC rats 14 weeks after injury from a weight drop from the
75-mm height (data not shown).
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CREM Localization
In sham control rats, CREM was localized to young spermatids and maturing
spermatocytes; its presence in early spermatocytes or mature spermatids was
rare (Figure 1A,
D, and
E). Five to 7 days after cord
contusion, CREM distribution in spermatogenic cells was grossly abnormal. CREM
in young spermatids was decreased, and sometimes absent, in SCC rats injured
by a weight drop from different heights (Figure
1B and
C). Concomitantly, there was a
significant increase in CREM in young spermatocytes, including the
preleptotene spermatocytes and sometimes the spermatogonia in SCC rats (Figure
1F through
H). This phenomenon was least
pronounced in those rats injured by a weight drop from the 12.5-mm height and
most pronounced in those rats injured by a weight drop from the 75-mm height.
Such changes were no longer apparent 8 or 14 weeks after the injury (data not
shown).
Northern Blot
Fourteen weeks after the cord contusion, the levels of messenger RNA (mRNA)
for Sertoli cell–specific androgen binding protein (ABP) and the inhibin
subunit (1: 2.4 kilobase [kb]; 2: 1.3 kb) of SCC rats
were not different from those of sham control rats. Transferrin (Trf)
transcript levels, however, were significantly lower in SCC rats in the 25-
and 50-mm height groups (P < .05 and P < .01,
Figure 2A). On the other hand,
several germ cell–specific mRNAs exhibited various patterns of changes
among the SCC rats. The mRNA levels of nuclear transition protein-2 (TP-2) and
lactate dehydrogenase C (LDHC) were only significantly reduced in the 50-mm
height group (P < .05). In contrast, the levels of protamine-1
(Pm-1) mRNA were significantly lowered in SCC rats injured by weight drops
from the 25-, 50-, and 75-mm heights (P < .01,
Figure 2B), and this effect was
negatively correlated with the height of weight drop (r = -0.65,
P < .005). Hemiferrin (Hemif) mRNA levels in SCC rats were
comparable to those in sham controls. Linear regression analysis also revealed
a negative correlation between levels of CREM mRNAs and the height of the
weight drop (CREM1, 3.8 kb: r = -0.52, P = .018; CREM2,
2.3/1.6 kb: r = -0.70, P < .01). However, a significant
decrease in mRNA level was observed only in CREM2 mRNA of the 50- and 75-mm
height groups (P < .05, Figure
2C).
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In the second experiment, while levels of mRNA for ABP were only marginally
reduced in SCC rats injured by 1x weight drop from both the 25- and
75-mm heights (P < .06), Trf mRNA levels were significantly lower
in most groups of SCC rats 4 weeks after injury (P < .05,
Figure 3A). At 4 weeks
postinjury, the levels of inhibin 1 mRNA were not affected in those
rats injured by a weight drop from the 25-mm height, but they were
significantly decreased in the rats in the 75-mm height group (P <
.01). The level of inhibin 2 mRNA remained unaffected. As in the first
experiment, there were general decreases in the levels of mRNA for TP-2 and
Pm-1 in most SCC rats (P < .05 and P < .01,
Figure 3B); such effects were
not related to the height of weight drop. On the other hand, levels of mRNA
for hemif and LDHC were not significantly affected
(Figure 3B). While there was a
general decrease in the level of CREM mRNA transcripts, it was marginally
significant (P < .06).
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Sperm Motility
Fourteen weeks after the sham operation (experiment 1), more than 65% of
caudal epididymis sperm were motile in the control rats. Sperm motility of SCC
rats in the 12.5- and 25-mm weight-drop groups was not different from that of
sham control rats but was significantly lower in the SCC rats that were
injured by weight drops from the 50- and 75-mm heights (P < .05,
Figure 4A). In the second
experiment, sperm motility was reduced more than 50% in all SCC rats 4 weeks
after they were injured by 1x or 3x weight drops from both the 25-
and 75-mm heights (P < .01 and P < .05,
Figure 4B); however, this
decrease was not related to the height of weight drop. By the eighth week, the
sperm motility of rats injured by a 1x weight drop from the 25-mm height
had returned to approximately 60% of normal but remained below 50% in all
other groups (P < .01).
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Discussion |
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Previously, we reported a significant decrease in Trf mRNA levels (Huang et al, 1995) and changes in the response of Sertoli cell–specific transcripts to testosterone and/or FSH (Huang et al, 1999, 2003; Ottenweller et al, 2000) after spinal cord transection, which illustrated an alteration in Sertoli cell functions after SCI. While decreases in Sertoli cell transcript levels 4 weeks after cord contusion were consistent with abnormal Sertoli cell function in SCC rats, the return of normal levels of these transcripts 14 weeks postinjury indicated recovery of Sertoli cell functions in these rats. On the other hand, reduced levels of mRNA transcripts for germ cell–specific TP-2 and Pm-1 in SCC rats in both experiments were indicative of abnormal spermiogenesis. TP-2 and Pm-1 are spermatid-specific nuclear proteins that are involved in the condensation and maturation of spermatid nuclei, the processes essential for the final morphogenesis of sperm head (Bower et al, 1987; Kleene and Flynn, 1987). Abnormal expression of these genes could impair such processes and contribute to abnormal morphogenesis of the sperm head. Abnormal sperm morphology has been found in some studies of men with SCI (Hirsch et al, 1991). Decreases in these transcripts were not correlated with levels of serum hormones or Sertoli cell mRNA transcripts, suggesting that they were specific effects of SCI on spermiogenesis.
Recently, we reported changes in the response of testicular cell cAMP responsive genes to FSH and/or testosterone treatments after SCI (Huang et al, 2003), which suggest that altered cAMP signaling mediates some of the effects of SCI on spermatogenesis. Abnormal cellular distribution of CREM in testicular cells shortly after cord contusion was consistent with this thesis. Similar changes in CREM localization in spermatogenic cells have been observed in cord-transected rats given 3- or 5-cm testosterone capsule implants but not in sham control rats given identical regimens (Huang et al, unpublished data). This suggests that the changes in CREM localization resulted from the interaction between disruption of normal neural input to the testis and reduced testicular testosterone production. A similar mechanism might also exist for abnormal CREM in testicular cells in acute SCC rats, since these effects were more pronounced in those rats injured by a weight drop from 50- and 75-mm heights and had a lower testosterone production.
CREM is a nuclear transcription factor that, upon phosphorylation,
modulates the function of cAMP-responsive genes
(Habener et al, 1995;
Daniel and Habener, 1998). Because of differences in gene splicing and promoter usage, translation
products of this gene could either promote or repress the expression of
cAMP-dependent genes (Foulkes et al,
1992; Daniel and Habener,
1998). The CREMs ( and ß) expressed in spermatogonia
and early meiotic cells in prepubertal testes were of repressor isoforms
(Foulkes et al, 1992). On the
other hand, the CREMt transcript is transcribed in early spermatocytes and
translated in maturing spermatocytes and young spermatids and is a functional
switch for postmeiotic germ cell differentiation
(Sassone-Corsi, 1998). The
importance of CREM signaling in spermiogenesis and sperm production has been
demonstrated in clinical studies (Peri et
al, 1998; Steger et al,
1999). The precocious expression of CREM in early spermatocytes
and the lack of it in young spermatids during the acute phase after cord
contusion could indicate abnormal cAMP signaling. Such changes could disrupt
the differentiation of spermatogenic cells and could result in the formation
of sperm with abnormal morphology or function. In this regard, the promoter of
Pm-1 and LDHC genes contains the putative cAMP response element
(Ha et al, 1997;
Bonny et al, 1998) and
therefore could be affected by abnormal cAMP signaling. Premature translation
of Pm-1 in the transgenic mouse has been found to result in precocious
condensation of spermatid nuclei and arrest of spermatogenesis at the
spermatid stages (Lee et al,
1995).
Persistently reduced sperm motility after a prolonged recovery period in cord-contused rats injured by weight drops from the 50- and 75-mm heights resembled that which occurred in SCI men after they had suffered various degrees of SCI (Perkash et al, 1985; Brackett et al, 1994). Normal sperm motility observed in rats 14 weeks after they were injured by a weight drop from the 25-mm height may reflect recovery of sperm function, since sperm motility was significantly decreased 4 weeks after the rats were injured by a 1x weight drop from the 25-mm height. Of note, the sperm motility in rats injured by 3x weight drops from the 25-mm height was comparable to that of rats injured by a 1x weight drop from the 75-mm height at both time points, and sperm motility did not recover 8 weeks after the injury. This observation illustrates that cord injured by repeated minor contusions could impair sperm motility (and perhaps other sperm functions) as much as does a single contusion with greater force. Together, the current results demonstrate, first, a relationship between impaired sperm function and the extent of SCI and, second, that impaired sperm motility resulting from minor SCI is reversible, whereas that from more severe SCI might be permanent. Impaired sperm maturation due to abnormal epididymal function (Billups et al, 1990) might contribute to reduced sperm motility after SCI. However, persistent decreases in the expression of spermatid-specific proteins suggest that abnormal spermiogenesis could also contribute to defects in sperm function after SCI. These findings offer alternative mechanisms, in addition to the abnormal seminal fluid property (Brackett et al, 2000), for reduced sperm motility in SCI men (Ohl et al, 1999).
In summary, current results demonstrate persistent spermatogenesis in SCC rats with varying degrees of cord injury. However, abnormal CREM distribution in spermatogenic cells and decreased levels of germ cell–specific transcript denote abnormalities in spermatogenic differentiation. These effects were associated with a decrease in sperm motility that correlated with the extent of SCI and the time postinjury. Further understanding of the causes and mechanisms underlying such effects will form the basis for therapeutic interventions to preserve sperm function after SCI and could be beneficial to the reproductive health of SCI men.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Billups KL, Tillman SL, Chang TS. Reduction of epididymal sperm motility after ligation of the inferior mesenteric plexus in the rat. Fertil Steril.1990; 35:1076 –1082.
Bonny C, Cooker LA, Goldberg E. Deoxyribonucleic acid–protein
interactions and expression of the human testis-specific lactate dehydrogenase
promoter: transcription factor Sp1 plays a major role. Biol
Reprod. 1998;58:754
–759.
Bower PA, Yelick PC, Hecht NB. Both P1 and P2 protamine genes are expressed in hamster, mouse and rat. Biol Reprod.1987; 37:479 –488.[Abstract]
Brackett NL, Lynne CM, Aballa TC, Ferrell SM. Sperm motility from the vas deferens of spinal cord injured men is higher than from the ejaculate. J Urol. 2000;164:712 –715.[Medline]
Brackett NL, Lynne CM, Weizman MS, Bloch WE, Abae M. Endocrine profiles and semen quality of spinal cord injured men. J Urol. 1994; 151:114 –119.[Medline]
Chomczynski P, Sacchi N. Single step method of mRNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156 –159.[Medline]
Chow S-H, Giglio W, Anesetti R, Ottenweller JE, Pogach LM, Huang HFS. The effects of testicular denervation on spermatogenesis in the Sprague Dawley rat. Neuroendocrinology.2000; 72:37 –45.[Medline]
Daniel PB, Habener JF. Cyclic alternative axon splicing of
transcription factor cyclic adenosine monophosphate response element binding
protein (CREB) messenger ribonucleic acid during rat spermatogenesis.
Endocrinology.1998; 139:3721
–3729.
Foulkes NS, Mellstrom B, Benusiglio E, Sassone-Corsi P. Developmental switch of CREM function during spermatogenesis: from antagonist to activator. Nature.1992; 355:80 –84.[Medline]
Ha H, van Wijne AJ, Hecht NB. Tissue-specific protein–DNA interactions of the mouse protamine 2 gene promoter. J Cell Biochem. 1997;64:94 –105.[Medline]
Habener JF, Miller CP, Vallejo M. cAMP-dependent regulation of gene transcription by cAMP response element-binding protein and cAMP response element modulator. Vitam Horm.1995; 51:1 –57.[Medline]
Hirsch IH, McCue P, Allen J, Lee J, Staas WE. Quantitative testicular biopsy in spinal cord injured men: comparison to fertile controls. J Urol. 1991;146:337 –341.[Medline]
Huang HFS, Li M-T, Giglio W, Anesetti R, Ottenweller JE, Pogach P.
The detrimental effects of spinal cord injury on spermatogenesis in the rat is
partially reversed by testosterone, but enhanced by folliclestimulating
hormone. Endocrinology.1999; 140:1349
–1355.
Huang HFS, Li MT, Giglio W, Anesetti R, Pogach LM, Ottenweller JE. Alteration of cAMP signaling in rat testicular cells after spinal cord injury. J Spinal Cord Med. 2003. In press.
Huang HFS, Linsenmeyer TA, Anesetti R, Giglio W, Ottenweller JE,
Pogach L. Suppression and recovery of spermatogenesis following spinal cord
injury in the rat. J Androl.1998; 19:72
–80.
Huang HFS, Linsenmeyer TA, Li MT, Giglio W, Anesetti R, von Hagen
J, Ottenweller JE, Pogach L. Acute effects of spinal cord injury on the
pituitary-testicular hormone axis and Sertoli cell functions: a time course
study. J Androl.1995; 16:148
–157.
Huang HFS, Pogach L, Nathan E, Giglio W, Seebode JJ. Synergistic effects of FSH and testosterone upon the maintenance of spermiogenesis in hypophysectomized rats. Relationship with the androgen binding protein status. Endocrinology.1991; 128:3152 –3161.[Abstract]
Kleene KC, Flynn JF. Characterization of a cDNA clone encoding a
basic protein, TP2, involved in chromatin condensation during spermatogenesis
in the mouse. J Biol Chem.1987; 262:17272
–17277.
Kwo S, Young W, De Crescito V. Spinal cord sodium, potassium, calcium, and water concentration changes in rats after graded contusion injury. J Neurotrauma.1989; 6:13 –24.[Medline]
Lee K, Haugen HS, Clegg CH, Braun RE. Premature translation of
protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid
differentiation in mice. Proc Natl Acad Sci U S A.1995; 92:12451
–12455.
Linsenmeyer TA, Ottenweller JE, D'Imperio JS, Pogach LM, Huang HFS. Epididymal sperm transport in normal and recent spinal cord injured Sprague Dawley rats. J Spinal Cord Med.1999; 22:102 –106.[Medline]
Linsenmeyer TA, Perkash I. Infertility in men with spinal cord injury. Arch Phys Med Rehab.1991; 72:747 –754.[Medline]
Linsenmeyer TA, Pogach LM, Ottenweller JE, Huang HFS. Spermatogenesis and the pituitary testicular hormone axis in rats during the acute phase of spinal cord injury. J Urol.1994; 152:1302 –1307.[Medline]
Ohl DA, Menge AC, Jarow JP. Seminal vesicle aspiration in spinal cord injured men: insight into poor sperm motility. J Urol. 1999;162:2048 –2051.[Medline]
Ottenweller JE, Li M-T, Giglio W, Anesetti R, Pogach LM, Huang HFS.
Alteration of the follicle-stimulating hormone and testosterone regulation of
messenger ribonucleic acid for Sertoli cell proteins in the rat during the
acute phase of spinal cord injury. Biol Reprod.2000; 63:730
–735.
Peri A, Krausz C, Cioppi F, Granchi S, Forti G, Francavilla S,
Serio M. Cyclic adenosine 3', 5'-monophosphate–responsive
element modulator gene expression in germ cells of normo- and oligoazoospermic
men. J Clin Endocrinol Metab.1998; 83:3722
–3726.
Perkash I, Martin DE, Warner H, Blank MS, Collins DC. Reproductive biology of paraplegics: results of semen collection, testicular biopsy and serum hormones. J Urol.1985; 134:285 –288.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Press; 1989.
Sassone-Corsi P. CREM: a master-switch governing male germ cell differentiation and apoptosis. Cell Dev Biol.1998; 9:475 –482.
Steger K, Klonisch T, Gavenis K, et al. Round spermatids show
normal testis-specific H1t but reduced cAMP-responsive element modulator and
transition protein expression in men with round spermatid maturation arrest.
J Androl. 1999;20:747
–754.
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