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From the * Department of Basic Veterinary
Sciences, The United Graduate School of Veterinary Sciences, Gifu University,
Gifu, Japan; the Laboratory of Veterinary
Physiology and the Department of Tissue
Physiology, Tokyo University of Agriculture and Technology, Tokyo, Japan; the
Department of Pathology, Sasaki Institute,
Tokyo, Japan; and the || School of Biological and
Molecular Science, Oxford Brookes University, Oxford, United Kingdom.
| Correspondence to: Dr Kazuyoshi Taya, Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, 3-5-8 Sai-wai-cho, Fuchu, Tokyo 183-8509, Japan (e-mail: taya{at}cc.tuat.ac.jp). |
| Received for publication February 21, 2002; accepted for publication June 6, 2002. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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C. In addition, we used the radioimmunoassay
(RIA) to measure immunoreactive (ir-)inhibin, FSH, luteinizing hormone (LH),
and testosterone. And finally, we used the proliferating cell nuclear antigen
(PCNA) and computer-assisted sperm motion analysis (CASA) methods to ascertain
how well spermatogenesis and sperm motility recover from the photoinhibition
caused by exposure to a short-day (SD; 10-hour light: 14-hour dark)
photoperiod. Animals were exposed to SD for 15 weeks, and then their testes
were checked carefully and found to be completely regressed. Thereafter, those
animals were transported to a long-day (LD; 14-hour light: 10-hour dark)
photoperiod. Sampling was carried out at weeks 0 (exposed SD 15 weeks), 1, 2,
4, 6, 8, and 10. Plasma FSH rapidly increased and reached peak levels 2 weeks
after transferral to the LD photoperiod and then declined to normal LD levels
at week 6. Circulating ir-inhibin, inhibin B, and inhibin pro-C rose to
normal LD levels by week 4. A highly significant inverse correlation was
observed between plasma FSH and inhibin B but not between FSH and either
ir-inhibin or inhibin pro-C. Plasma testosterone recovered to normal LD
levels within 1 week. Sperm motility parameters were low until week 2 and
recovered to normal LD levels from weeks 4 to 10. PCNA-labeled cells were
confined to the spermatogenic cells of the seminiferous tubules, though Leydig
and Sertoli cell nuclei were never stained for PCNA during the period studied.
The number of pachytene spermatocytes and the diameter of seminiferous tubules
increased in a time-dependent manner after transferral from SD to LD. In
conclusion, these results suggest that 1) secretion of inhibin B may be
stimulated by an early rise in FSH; 2) inhibin B suppresses FSH secretion from
weeks 2 to 10, after transferral to the LD photoperiod; and 3) testes
recrudescence is based on the increase in the number of sperm cells instead of
the increase in the number of Sertoli and Leydig cells of the male golden
hamster.
Key words: Photoperiod, proliferating cell nuclear antigen, sperm motion
It is well known that inhibin is a heterodimeric protein consisting of an
subunit and one of 2 ß subunits. Two related forms of inhibin,
inhibin A (/ßA) and inhibin B (/ßB), are secreted into
the circulation from the gonads and inhibit pituitary FSH secretion
(Ling et al, 1985;
Miyamoto et al, 1985;
Rivier et al, 1985; Robertson et al, 1985;
Vale et al, 1986). A recently
developed specific immunoassay has enabled the measurement of plasma levels of
dimeric inhibins and provided evidence that inhibin B is an important
physiological form of inhibins in male golden hamsters
(Jin et al, 2001b), as well as
in other males: male Gottingen miniature pigs
(Jin et al, 2001a), men
(Illingworth et al, 1996),
male monkeys (Foppiani et al,
1999; Ramaswamy et al,
2000), male chimpanzees (Kondo
et al, 2000), and male rats
(Sharpe et al, 1999). It has
also been suggested that in men, inhibin B regulates FSH secretion
(Anawalt et al, 1996;
Illingworth et al, 1996;
Nachtigall et al, 1996; Seminara et al, 1996).
However, the existence of negative feedback of inhibin B on FSH secretion
during photoperiod-induced testicular recrudescence in golden hamsters has
been disputed and is yet to be elucidated. Kirby et al
(1993) suggested that
testicular ir-inhibin secretion may not be directly related to circulating FSH
levels during recrudescence in the golden hamster.
The proliferating cell nuclear antigen (PCNA), a 36-kd acidic nuclear
protein that has been very highly conserved in the process of evolution, is a
cell proliferation marker currently drawing attention. PCNA is known to
function as a cofactor for DNA polymerase 
, with a biological half-life
of longer than 20 hours (Bravo and
Macdonald-Bravo, 1987). PCNA is required for both DNA replication
and DNA repair (Shivji et al,
1992; Xiong et al,
1992). It is synthesized primarily during the G1 phase of the cell
cycle and reaches its maximum levels during the S phase
(Hofstadter et al, 1995).
Given adequate fixation and tissue processing, the results of PCNA
immunohistochemistry directly reflect the proliferative status of the cells
and the fact that PCNA-labeled nuclei are observed during the G1 to the S
phase of the cell cycle (Morita et al,
1994). The localization of PCNA can be used to assess the
proliferative status of renewing spermatogonia and to analyze the
proliferative activity of the seminiferous epithelium of rodents and non-human
primates (Schlatt and Weinbauer,
1994) and men (Garrido et al,
1992). Thus, PCNA expression levels in the cell nuclei are
indicative of the proliferating activity of the renewing spermatogenic
cells.
Therefore, in the present study, we tested the hypothesis that triggering the secretion of inhibins and the resulting increased levels of inhibins subsequently affect the FSH during the photoperiod-induced testicular recrudescence in the male golden hamster. Furthermore, renewing spermatogenic cell proliferating activity was evaluated by PCNA. and sperm motility characteristics were also monitored by a computer-assisted sperm analysis system (CASA) to ascertain how well the spermatogenesis recovers from the photoinhibition caused by exposure to an SD photoperiod.
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Materials and Methods |
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Radioimmunoassays for FSH, LH, Testosterone, and ir-Inhibin
Plasma concentrations of FSH and LH were measured using National Institute
of Diabetes and Digestive and Kidney Diseases (NIDDK) radioimmunoassay (RIA)
kits for rat FSH and LH, as described previously
(Bast and Greenwald, 1974). The
antisera used were anti-rat FSH (S-11) and LH (S-10). Results were expressed
in terms of NIDDK rat FSH (RP-2) and LH (RP-2). The intra- and interassay
coefficients of variation were 4.4% and 14.6% for FSH and 6.7% and 8.9% for
LH, respectively.
Plasma concentrations of ir-inhibin were measured by a double-antibody RIA,
as described previously (Hamada et al,
1989). The antiserum used was raised in rabbits against bovine
inhibin (TNDH-1). Purified bovine 32-kd inhibin was used as the standard. The
assay system does not distinguish dimeric inhibin from subunit
monomer. The intra- and interassay coefficients of variation were 8.8% and
14.4%, respectively.
Plasma concentrations of testosterone were determined by a double-antibody RIA system using 125I-labeled radioligand, as described previously (Taya et al, 1985). Antiserum against testosterone was kindly supplied by Dr G. D. Niswender (Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colo) (Gay and Kerlan, 1978). The intra- and interassay coefficients of variation were 6.3% and 7.2%, respectively.
Enzyme-Linked Immunosorbent Assay
Plasma concentrations of inhibin B and inhibin pro-C were determined
using enzyme-linked immunosorbent assay (ELISA) kits (Serotec Ltd, Oxford,
United Kingdom). Inhibin A was not measured because we have previously
reported that plasma inhibin A is not detectable in male golden hamsters
(Jin et al, 2001b). We have
validated and reported these inhibin dimerspecific assays for male golden
hamsters (Jin et al,
2001b).
Tissue Processing
In each case, the right testis, including the epididymis, the seminal
vesicle, and the coagulating gland complex, was weighed, and sperm from the
right epididymis were used for sperm motion analyses. Testicular tissue
samples were immediately fixed in freshly prepared 4% paraformaldehyde (Sigma
Chemical Co, St Louis, Mo) in 0.05 M phosphate-buffered saline, pH 7.4, and
embedded in paraffin. The paraffin-embedded testicular tissues were serially
sectioned at 6-µm thickness and mounted onto poly-L-lysine (0.01% [wt/vol])
(Sigma) coated slide glasses (Dako Japan Co, Kyoto, Japan) for use in
immunohistochemistry.
Immunohistochemistry for PCNA
After being deparaffinized with xylene, the tissue sections were subjected
to antigen retrieval by autoclaving in 0.01 M sodium citrate buffer, pH 6.0,
at 121°C for 15 minutes. Sections were then incubated in 6%
H2O2 in methanol at room temperature for 1 hour,
followed by 0.5% casein-Tris saline (0.05 M Tris-HCl with 0.15 M NaCl, pH 7.6;
CTS) at 37°C for 1 hour, to quench nonspecific staining. Then, the tissue
sections were incubated at 37°C for 16 to 18 hours with a monoclonal
antibody raised against PCNA (Biomeda, Forster City, Calif) at a dilution of
1:200 in CTS. After incubation with the antibody, sections were treated with
0.25% (vol/vol) biotinylated goat anti-mouse secondary antibody (Elite ABC
kit, Vector Laboratories, Burlingame, Calif) in CTS at 37°C for 1 hour.
These sections were subsequently incubated with 2% (vol/vol) avidin-biotin
complex (Elite ABC kit) in CTS at 37°C for 30 minutes. The reaction
products were visualized by treatment with 0.025% (wt/vol)
3.3'-diaminobenzidine tetrachloride (DAB; Sigma) in 100 mM Tris-buffered
saline containing 0.01% H2O2 for 1 to 30 minutes.
Computer-Assisted Sperm Mobility Analyses
The sperm motility parameters were obtained using the C. IMAGING CASA
system. Sperm from the cauda epididymis were incubated at 37°C for 3
minutes in 0.01 M medium buffer, pH 7.2. The medium buffer was made up of 59.8
mg of HEPES (Dojindo, Kumamoto, Japan), 982 mg of medium 199 (Biocell, Carson,
Calif), 500 mg of bovine serum albumin (Sigma), and 220 mg of
NaHCO3 (Wako, Osaka, Japan) dissolved in 100 mL deionized water.
After the sperm were incubated in medium buffer at 37°C, an aliquot of
this solution was diluted 10- to 20- fold, and 10 µL was placed into the
microcell-HAC chamber, which has a depth of 50 µm (Conception Technologies,
San Diego, Calif). Analyses of motility characteristics were performed on at
least 200 cells for each sample. Sperm motion, as viewed on an Olympus
microscope (4x, pseudodark-field optics) with a stage warmer (37°C)
(MP-10DM; Kitazato Supply Co, Kitazato, Japan), was analyzed using the C.
IMAGING system. The C. IMAGING system settings were as follows: frames
analyzed, 15; framing rate, 30; maximum velocity, 1200 µm/s; threshold
velocity, 45 µm/s; minimum linearity for ALH (amplitude of lateral head
displacement), 3.5; pixel scale, 3.26 mm/pixel; maximum average number of
cells/field, 30; and cell size range, 350 to 1600 pixels. The following
characteristics were analyzed: percentage of motile spermatozoa, curvilinear
velocity (total distance traveled divided by total time the cell was tracked),
straight velocity (straight-line distance), mean ALH (deviation of the sperm
head from the mean trajectory), max ALH (the maximum amplitude of lateral head
displacement), linearity (ratio of the straight-line distance to the actual
tracked distance), and percentage of circular cells.
Histological Analysis
The PCNA-labeled germ cells were counted under the microscope (Nikon,
Tokyo, Japan). The diameters of 20 round tubules per animal were measured.
Statistics
A one-way analysis of variance was performed. Significance was determined
by the Duncan multiple range test (Steel
and Torrie, 1960). Correlation analysis between inhibins and FSH
was performed using the Pearson method. All data are presented as the mean
plus or minus the standard error of the mean. Differences were considered
significant when P was less than .05.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Changes in Plasma Concentrations of FSH, LH, ir-Inhibin, Inhibin B,
Inhibin pro-C, and Testosterone
The changes in plasma concentrations of reproductive hormones are shown in
Figure 2. Rapid increases in
concentrations of plasma FSH occurred at 1 week after transferral from an SD
to an LD photoperiod and reached peak levels (22 ng/mL) (P < .01)
at week 2, followed by a rapid decline to normal LD levels (1.5-3 ng/mL) at
week 6 (Figure 2A). In
contrast, basal concentrations of plasma LH steadily increased after
transferral from the SD to the LD photoperiod and were significantly high at
weeks 4 and 10 compared with week 0, although these levels were not
significantly different from those in the control group
(Figure 2B). Plasma
concentrations of testosterone were significantly (P < .05) low at
week 0 in the treatment group compared with those in the control group. These
concentrations then gradually increased and reached peak levels at week 4
before declining to normal LD levels
(Figure 2C). Plasma
concentrations of ir-inhibin (Figure
2D) and inhibin B (Figure
2E) began to increase at week 2 and reached normal LD levels at
week 4. Plasma concentrations of inhibin pro-C were significantly
(P < .05) low at week 0 in the treatment group compared with those
in the control group, but they then began to increase at week 1 and remained
at higher levels than those in the control animals from weeks 2 to 10
(Figure 2F). Plasma FSH and
inhibin B concentrations were inversely correlated (r = -0.56,
P < .01) during the period from weeks 2 to 10
(Figure 3). However, neither
ir-inhibin nor inhibin pro-C was correlated with plasma FSH (data not
shown).
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Changes in Sperm Motility Parameters
No sperm were available in the cauda epididymis for motile parameter
analysis until week 2, but 1 of the 5 animals had some sperm at week 2 (data
not shown). The percentage of motile spermatozoa was abruptly increased at
week 4 and reached normal LD levels at week 6
(Figure 4A). At week 10, the
percentage of motile sperm was reduced slightly but significantly (P
< .05) in the treatment group compared with that in the control group. The
ALH mean (Figure 4B) and ALH
max (Figure 4C) in the
treatment group increased abruptly at week 4, although these levels were still
significantly low compared with those in the control groups; however, this was
followed by further increases to the control levels by week 6. Linear index
(Figure 4D) and straight
velocity (Figure 4E) were still
significantly (P < .05) low in the treatment group at weeks 4 and
6 compared with those in the control animals. These parameters recovered to
those of the control levels at week 8. Curvilinear velocity
(Figure 4F) abruptly recovered
to normal LD levels at week 4.
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Immunohistochemistry for PCNA
In order to better visualize cell types, the sections were stained with
hematoxylin and eosin at weeks 0, 2, 4, 6, and 8 in the treatment group
(Figure 5A, B, C, D, and E,
respectively) as well as in the control group
(Figure 5F). At weeks 0 and 2,
the seminiferous tubules contained primarily Sertoli cells and spermatogonia,
but spermatocytes and round spermatids were also occasionally seen
(Figure 5A and B). The testes
of animals in the treatment group at weeks 0 and 2 showed dilated seminiferous
tubules with small-sized spermatogenic cells, Sertoli cells, and Leydig cells.
However, there were no mature spermatids contained within the seminiferous
tubules. In early recrudescence, there was an increase in the number of germ
cells in the basal compartment. The increase in the cell volume of Sertoli and
Leydig cells, as well as the number of germ cells in the seminiferous tubules,
appeared to be time-dependent during weeks 4, 6, and 8
(Figure 5C, D, and E,
respectively). The testes in animals exposed to the SD photoperiod recovered
to normal LD appearance at 10 weeks.
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Germ cells from the testes were positively stained for PCNA at weeks 0, 2, 4, 6, and 8 in the treatment group (Figure 5M, N, O, P, and Q, respectively) and in the control group (Figure 5R) in the male golden hamsters. PCNA was positively stained in early spermatogenic cells but not in the nuclei of Sertoli and Leydig cells at weeks 0 and 2 (Figure 5M through R). The increase in the size of immunopositive cells and in the number of spermatogenic cells was time-dependent. Relatively large nuclei of spermatogenic cells were positively stained with anti-PCNA antibody in the testes of the LD controls (Figure 5R). Sections incubated with normal mouse plasma instead of primary antibody did not show any immunopositive staining at weeks 0, 2, 4, 6, and 8 in either the treatment group (Figure 5G, H, I, J, and K, respectively) or the control group (Figure 5L).
Testes Histological Analysis
The numbers of pachytene spermatocytes and the diameter measurements of
seminiferous tubules are shown in the Table. Both sets of numbers increased in
a time-dependent manner during testicular recrudescence. The number of
pachytene spermatocytes increased to normal LD levels at week 4 and reached
peak levels at week 8. The diameter of seminiferous tubules also increased but
still did not recover to normal LD levels at week 4; however, this was
followed by a further increase to supracontrol levels at weeks 6 and 8.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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C—and
indirectly indicates that inhibin B affects FSH secretion during testicular
recrudescence in the golden hamster. Furthermore, the results of PCNA together
with the histological analysis suggest that testicular recrudescence is based
on the increase in the number of spermatogenic cells rather than the increase
in the number of Sertoli and Leydig cells in the testes of the male golden
hamster. The present study shows that plasma FSH is rapidly elevated after the
transfer of animals from an SD to an LD photoperiod and that it then begins to
decline at a time when plasma inhibin B is elevated, suggesting that inhibin B
is the major regulator of plasma FSH concentrations during this time. This
finding is also reflected in a strong inverse correlation between these 2
hormones. The present study also demonstrates that gradual increases in plasma
LH and testosterone occurring at the later stage of the LD photoperiod restore
spermatogenesis, which has been adversely affected during exposure to the SD
photoperiod.
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Previous studies have suggested that in men, inhibin B regulates FSH
secretion (Anawalt et al, 1996;
Illingworth et al, 1996;
Nachtigall et al, 1996; Seminara et al, 1996). We have
also shown in our previous studies that inhibin B is the major circulating
form of dimeric inhibins in the male golden hamster
(Jin et al, 2001b) and in the
Gottingen miniature pig (Jin et al,
2001a). Others have shown inhibin B as the major form of inhibins
in other species as well, including male monkeys
(Plant et al, 1997;
Foppiani et al, 1999; Ramaswamy et al, 2000), male
rats (Woodruff et al, 1996), and male chimpanzees (Kondo et al,
2000). It has been reported that inhibin B may have a
physiological role in the development of spermatogenesis in the testes of
adult male animals (Foppiani et al,
1999; Ramaswamy et al,
2000), though equine fetal testes
(Tanaka et al, 2002) and ovine
adult testes (McNeilly et al,
2002) secrete a large amount of inhibin A. In line with these
findings, it is quite possible that there is inhibin B-FSH negative feedback
during testicular recrudescence in the male golden hamster. Kirby et al
(1993) did not observe any
correlation between inhibin and FSH levels during testicular
recrudescence. We did not measure plasma inhibin A levels in the present study
because we have shown in a previous study that plasma inhibin A is
undetectable in the male golden hamster
(Jin et al, 2001b).
In our present study, abrupt increases in plasma FSH levels preceded changes in plasma LH and testosterone after the animals were transferred from the SD to the LD photoperiod. Plasma FSH levels continued to rise through week 2 after transferral from the SD to the LD photo-period, while there were gradual increases in basal plasma LH and testosterone. This result corroborates previous studies (Milette et al, 1988; Donham et al, 1994), which have reported that elevated FSH levels preceded spontaneous recrudescence of the testes in male golden hamsters. It also has been suggested that a rapid increase in plasma FSH after photostimulation is the primary signal for initiating testicular development in Djungarian hamsters (Milette et al, 1988). It was reported that the administration of exogenous FSH causes the regrowth of testes and complete spermatogenesis in photoinhibited Djungarian hamsters (Niklowitz et al, 1989). It is reasonable to suggest that FSH is important for seminiferous tubular development as well as for inhibin production by testes. On the other hand, gradual increases in basal plasma LH and testosterone levels were also observed in the present study. It is possible that the early elevation of FSH levels stimulates the induction of LH receptors in Leydig cells, thereby increasing the responsiveness of Leydig cells to endogenous and exogenous LH and thus stimulating testosterone production (Parvinen et al, 1984; Verhoeven and Cailleau, 1985). Niklowitz et al (1989) also reported that FSH alone initiated complete spermatogenesis, whereas LH alone induced full redifferentiation of Leydig cell function, resulting in increased testosterone production in the photoinhibited and hypophysectomized Djungarian hamsters. However, plasma inhibin concentrations continued to rise until week 4, during which time plasma FSH had fallen to low levels. Inhibin B production was apparently stimulated by an early rise in FSH, thus resulting in circulating levels sufficient to suppress FSH release. This finding shows further that there is a dynamic change in hormonal interrelationships.
The present study evaluated the localization of PCNA in the cell nuclei in order to identify cellular proliferating activity occurring during testicular recrudescence. Immunolocalization of PCNA has been widely used as a method for the detection of proliferating cells in tumors (Wada et al, 1993), developing tissues (Casasco et al, 1993), and testes (Schlatt and Weinbauer, 1994). In the present study, we have identified PCNA as being localized to the nuclei of early spermatogenic cells; in addition, we have found that the number and size of these immunopositive spermatogenic cells appear to increase in a time-dependent manner. In contrast, nuclei of both Leydig and Sertoli cells were not stained for PCNA. This result agrees with previous findings that both Leydig cells (Hikim et al, 1988; Sinha Hikim et al, 1993) and Sertoli cells (Hikim et al, 1988) increased in volume but not in number during testicular recrudescence in the male golden hamster. In line with these results, Liang et al (2001) reported that, in male monkeys, PCNA-immunopositive spermatogenic cells were increased in an age-dependent manner and that the positive staining for PCNA in Sertoli cell nuclei was observed in the testes of animals from the neonatal to the pubertal stage but not in adult animals. Together with the present histological analysis (shown in the Table), it is likely that the abrupt increase in plasma FSH and the steady increase in plasma LH, which occurred at an early stage of testicular recrudescence in the golden hamster, were responsible for the increased volume of the Sertoli and Leydig cells. This rise in plasma FSH and LH levels possibly increases Leydig cell capacity to produce an increased amount of inhibins, because Leydig cells are the major source of inhibin B in the testes of the golden hamster (Jin et al, 2001b).
Although photoperiod-induced testicular recrudescence appears to begin at an early stage in the golden hamster, we could not detect any sperm until 2 weeks after transferral to the LD photoperiod. This suggests that the first wave of spermatogenesis apparently requires approximately 1 to 2 weeks after the transfer of animals from the SD to the LD photoperiod. It should be noted, however, that although sperm were detected in an animal in the treatment group at week 2, the profiles of sperm motility parameters were very low (data not shown). It should also be noted that in other animals, although sperm were present in the cauda epididymis after 4 weeks of normal LD exposure, in general, none of the parameters returned to normal values until about weeks 6 to 8.
In conclusion, the present results demonstrate the existence of a negative feedback of inhibin B on FSH secretion in male golden hamsters after transferral from an SD to an LD photoperiod. This feedback appears to begin functioning at an early stage of photoperiod-induced spontaneous testicular recrudescence. Plasma FSH as a primary signal, in combination with other reproductive hormones such as inhibin B, is likely to be the stimulus for the proliferation of various cells, leading to the reestablishment of the spermatogenic process.
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Acknowledgments |
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Footnotes |
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