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From the * Department of Physiology and the
Cooperative Reproductive Science Research Center, the
Department of Obstetrics and Gynecology, the
Department of Anatomy and Neurobiology and the
Neuroscience Institute, Morehouse School of Medicine, Atlanta, Georgia; and
the Department of Cell Biology and Physiology
and the Specialized Cooperative Center Program in Reproduction Research,
University of Pittsburgh, Pittsburgh, Pennsylvania.
| Correspondence to: Dr David R. Mann, Cooperative Reproductive Science Research Center, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA 30310 (e-mail: dmann{at}msm.edu). |
| Received for publication July 26, 2005; accepted for publication October 20, 2005. |
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Abstract |
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Key words: Spermatogenesis, seminiferous tubules, testis, cytomorphometry, micro array
Mammalian development is tightly regulated by cell proliferation as well as cell death (Ellis et al, 1991; Raff, 1992). Cell death that occurs during embryogenesis, metamorphosis, endocrine-dependent tissue atrophy, and normal tissue turnover is called programmed cell death or apoptosis (Ellis et al, 1991; Raff, 1992). When the testicular environment is not able to support spermatogenesis, the process of apoptosis decreases the level of proliferation of early germ cells (Lee et al, 1997). Apoptosis within germ cells is characterized by internucleosomal fragmentation of DNA, chromatin condensation, phagocytosis by Sertoli cells, and cell disintegration (Billig et al, 1995). It has been suggested that perhaps as high as 75% of potential mature spermatozoa are eliminated through the apoptotic pathways (Billig et al, 1995).
There are at least 2 major pathways for apoptosis, the extrinsic and intrinsic pathways (Sinha Hikim et al, 2003). The intrinsic pathway involves the release of cytochrome c from the mitochondria into the cytosol resulting in the activation of the initiator caspase 9 and the subsequent activation of the executioner caspases 3, 6, and 7. Caspases are cysteine proteases that mediate specific cleavage events in dying cells. Members of the Bcl-2 family of proteins play a major role governing this mitochondria-dependent pathway (Reed, 2000). The Fas-FasL system is involved in the extrinsic apoptotic pathway (Nagata and Golstein, 1995). Fas, a transmembrane receptor protein, and its ligand FasL belong, respectively, to the tumor necrosis factor (TNF) receptor and protein families (Watanabe-Fukunaga et al, 1992; Nagata and Golstein, 1995; Nagata, 1997). Binding of FasL to Fas results in the recruitment of Fas-associated death domain (FADD), and this complex then activates the initiator caspase, caspase 8. The intrinsic and extrinsic pathways converge on caspase 3 and other executioner caspases that drive the cleavage of various cellular substrates resulting in fragmentation of chromosomal DNA and subsequent formation of apoptotic bodies.
The objectives of the current study were to further characterize testicular morphology and spermatogenesis in the leptin-deficient mouse and assess the potential involvement of increased germ cell apoptosis in the processes that ultimately alter the fertility of this animal. Microarray technology was also employed to identify potential apoptosis-related genes whose expression levels within the testis are altered by the leptin deficiency.
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Materials and Methods |
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In Situ DNA 3' End Labeling of Apoptotic Cells
The Apop Tag apoptosis detection kit (Serological Co, Norcross, Ga) was
employed for labeling of DNA fragmentation. The in situ terminal
deoxynucleotidyl transferase (TdT) mediated by TUNEL method was used to
localize apoptotic cells in testis sections. Briefly, sections were washed
with phosphate-buffered saline (PBS) and pretreated with 20 µg/mL
proteinase K (25°C, 15 minutes). The sections were then incubated with TdT
reaction mixture in a humidified chamber (37°C, 60 minutes), washed with
PBS, and incubated with anti-digoxigenin antibody conjugated to a rhodamine
fluorescent marker in the humidified chamber (25°C, 30 minutes). Nuclei
were counterstained with 0.5 µg/mL 4', 6-diamino-2-phenylindole,
dihydrochloride (DAPI). For negative staining controls, the TdT reaction
mixture was omitted.
Hormone Assay
Serum samples were assayed for total leptin and testosterone using
commercially available enzyme-linked immunosorbent assay (ELISA) kits (Assay
Designs, Ann Arbor, Mich; Alpha Diagnostic International, San Antonio, Tex,
respectively). All samples were run in duplicate in the same assay. The
minimum detection limits for the leptin and testosterone assays were 5 and 10
pg/mL, respectively. The intra-assay coefficient of variations for leptin and
testosterone were 16% and 6%, respectively.
Histology and Histomorphometry of Testes
H&E-stained testis sections from 2 control and 2 ob/ob animals were
selected for detailed morphological analysis under light microscopy.
H&E-stained testicular sections from 6 ob/ob and 6 control mice were also
used to assess the effect of a leptin deficiency on seminiferous tubule area
(profile over the periphery of all the cross-sectioned tubules), number of
spermatocytes per cross-sectioned tubule, and percentage of cross-sectioned
tubules exhibiting sperm bundles (elongated spermatids and spermatozoa).
Imaging technology (ImagePro Plus Software, Media Cybernetics, Silver Spring,
Md) was employed for these measurements.
The number of TUNEL-positive germ cells was counted in 10 random fields from the testicular cross sections of 5 ob/ob and 5 control animals, and the average number of TUNEL-positive cells per tubule and the total number of TUNEL-positive cells for the 10 fields were calculated for both the ob/ob and control groups.
Microarray Sample Preparation and Hybridization
Total RNA from the testes of ob/ob (n = 3) and controls (n = 2) was
extracted with TRIzol Reagent (Life Technologies, Rockville, Md), cleaned
(RNAqueous kit, Ambion, Austin, Tex), and converted to double-stranded cDNA
(Invitrogen, Superscript Choice System, Carlsbad, Calif) using a T7-(dT)24
primer. The double-stranded cDNA was cleaned using phase Lock Gels (Eppendorf,
Westbury, NY), and an RNA transcript labeling kit (Enzo Diagnostics,
Farmingdale, NY) was used to synthesize cRNA. Biotin-labeled cRNA was cleaned
(GeneChip Sample Cleanup Module, Affymetrix Inc, Santa Clara, Calif) and
quantified spectrophotometrically. Next, 20 µg of the in vitro
transcription product was fragmented in fragmentation buffer at 94°C for
35 minutes. After fragmentation, 15 µg of the biotinylated cRNA was
hybridized to an Affymetrix Murine Genome U74AV2 GeneChip at 45°C for 16
hours, washed, stained with streptavidin phycoerythrin, and scanned according
to manufacturer's guidelines.
Microarray Data Processing
Data analysis was performed by Affymetrix Microarray Suite (MAS) 5.0
software. The microarray suite references the experimental file to select an
analysis algorithm for a cell intensity file that generates a gene chip file.
Single array analysis was used to build the databases of gene expression
profiles. Affymetrix GCOS software was used to normalize and analyze the data.
Detection P value (set at P < .05) was used to
statistically determine whether a transcript is expressed on the chip. The
software generated a present (P), marginal (M), or absent (A) call for each
transcript based on the P value. To obtain differentially expressed
genes for each condition, Affymetrix gene chip software was used to compare
each of the ob/ob testes arrays to that of the control arrays. Absolute calls
(present, marginal, and absent) and the average difference (RNA abundance) for
each gene were then imported into Genespring software (Silicon Genetics,
Redwood City, Calif) for further analysis. By combining the fold change and
the present calls derived from the comparisons, we obtained a list for each
condition. Differential expression was calculated as the increase between the
2 conditions (ie, ob/ob testes versus controls). A gene was considered
differentially expressed when the standard deviation of the signal increase or
decrease was significantly smaller than the absolute change in average
difference and the calculated confidence level of a gene was set greater than
95% (P < .05 based on unpaired t test). A general view of
the effect of the leptin deficiency on gene expression in the testes was
obtained by Self Organizing Map (SOM) cluster analysis using Genespring
software (Silicon Genetics) on replicate samples. Selected clusters were
examined for biological function and pathway analysis using Affymetrix Netfix
Analysis Center
(http://www.affymetrix.com).
Netfix detailed and annotated individual probe sets based on biological and
molecular function or cellular localization using the Gene Ontology public
database.
Statistical Analysis
One-way analysis of variance (ANOVA) was used to compare the effects of a
leptin deficiency on body and organ weights, serum leptin and testosterone
concentrations, seminiferous tubule area, number of spermatocytes, percent
tubules with sperm bundles, total number of TUNEL-positive germ cells, and the
number of apoptotic germ cells/tubule between testes from ob/ob and control
animals. P values less than .05 were considered as significant for
the differences observed between the ob/ob and control animals. Data are
presented as the mean ± SEM.
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Results |
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Histology and Histomorphometry of Testes
Light microscopy of the testes of ob/ob mice suggested that spermatogenesis
was impaired. There was evidence of increased germ cell degeneration and
condensation of germ cell nuclei (Figure
1; compare Panels B and C with Panel A). Sertoli cell
vacuolization was also observed in leptin-deficient mice, and some of the
Leydig cells of the ob/ob testes had an abnormal fibroblast-like appearance.
The effect of the leptin deficiency on testicular morphology was not
universal. While some of the tubules of ob/ob animals appeared to be comprised
of only Sertoli cells and without a clear lumen, there were regions in which
tubular morphology and spermatogenesis appeared normal
(Figure 1, Panel D).
Occasionally, multinuclear giant cells were encountered in the center of the
tubule. Intertubular space of the ob/ob testis appeared narrower and fewer
Leydig cells were found.
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Discussion |
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The reduced germ cell numbers and the absence of mature spermatozoa in many seminiferous tubules suggest that the level of germ cell apoptosis might be increased as a consequence of the leptin deficit. This theory was confirmed in the current study using the in situ TUNEL assay for apoptotic activity. We were also able to identify with microarray technology 9 proapoptotic-related genes within the testes of leptin-deficient animals whose expression levels were significantly higher than in control testes. These genes and their protein products are components of both the intrinsic and extrinsic apoptotic pathway, suggesting that both pathways of programmed cell death are accelerated within germ cells in the presence of a leptin deficiency.
To the best of our knowledge, the present study is the first to assess the impact of a leptin deficiency on apoptotic activity within germ cells of the testis. The demonstration that a leptin deficiency is associated with a greater than threefold increase in apoptosis within germ cells (particularly pachytene spermatocytes) and impaired sperm production supports the previous suggestion that, in general, the loss of germ cells in the testes occurs primarily through programmed cell death (Bartke, 1995). The induction of a gonadotropin and testosterone deficit in rats with GnRH antagonist treatment is associated with increased apoptosis within preleptotene and pachytene spermatocytes and spermatids (Sinha Hikim et al, 1995), and pachytene spermatocytes, dividing spermatocytes, and early round spermatids are especially susceptible to heat-induced apoptosis (Lue et al, 1999). Thus, the data from the current study extend previous findings to show that the impaired spermatogenic process in the leptin-deficient mouse is associated with a quantifiable elevation in germ cell apoptosis, and are consistent with the previous observations that early germ cell stages are especially vulnerable to increased programmed cell death during adverse conditions.
The present study is also the first to attempt to identify (via microarray) apoptosis-related genes whose expression levels within the testis are altered by a leptin deficiency. Of the 9 genes identified, 3 are components of the extrinsic apoptotic pathway and 5 are from the intrinsic apoptotic pathway. The final gene identified encodes for caspase-7, one of the executioner genes upon which the intrinsic and extrinsic pathways converge in the apoptotic cascade.
One of the genes associated with the extrinsic pathway that was highly expressed (increased 5.1-fold over control levels) in the testis of the ob/ob mouse encodes for the peptidoglycan recognition protein (PGRP). PGRPs form stable complexes with heat shock protein 70 (Hsp70) that is synthesized during the meiotic phase of spermatogenesis and is abundantly expressed in pachytene spermatocytes (Eddy, 1994; Dziarski 2004; Sashchenko et al, 2004). Neither the PGRPs nor Hsp70 is cytotoxic alone, but when complexed together they induce apoptotic cell death in several tumor cell lines (Sashchenko et al, 2004).
Expression levels of the 2 genes that encode for TNFR1 and 5 were also
increased above control values in the testes of leptin-deficient mice. TNFR1
is known to be involved in the extrinsic cell death pathway
(Baker and Reddy, 1998). Its
ligand, TNF-
, binds to TNFR1 activating caspases. Murine pachytene
spermatocytes and round spermatids express TNF- mRNA, and the latter
are also capable of secreting TNF- bioactivity in vitro
(De et al, 1993). Since our
gene array study was performed using whole testes it is not possible to
ascertain the exact cell types overexpressing TNF receptors.
Expression levels for 2 genes that code for 2 components (granzyme A and B) of the intrinsic apoptotic pathway were also elevated in the testes of ob/ob mice. Granzymes are serine proteases that serve as effector molecules for cytotoxic T lymphocytes and natural killer cells (Yamada et al, 2003). The combined action of these molecules is known to initiate apoptosis of target cells. Granzyme A mediates glucocorticoid-induced apoptosis in 697 leukemia cells by increasing caspase-3 activity, perhaps upstream of Bcl-2 signaling (Yamada et al, 2003). Granzyme B appears to trigger apoptosis by directing the proapoptotic molecule, Bid, to the mitochondrial membranes facilitating cytochrome c release (Ida et al, 2003). The present study appears to be the first to report granzyme expression in the murine testis.
Sphingosine-1-phosphate lyase (SPL), an enzyme that catalyzes the cleavage of intracellular second messenger sphingosine-1-phosphate (Maceyka et al, 2002), causes ceramide accumulation and/or sphingosine phosphate depletion leading to sustained cytochrome release and increased apoptosis in HEK293 cells (Reiss et al, 2004). During male germ cell apoptosis, ceramide levels increase before appearance of caspase-3 activation and DNA fragmentation, and germ cell death can be inhibited by exogenous administration of sphingosine phosphate to the cultured human seminiferous tubules (Suomalainen et al, 2003). In the present study, expression of the gene encoding SPL (another component of the intrinsic apoptotic pathway) was elevated in the testes of ob/ob mice, suggesting that SPL may play a role in mediating the enhanced level of germ cell apoptosis in leptin-deficient animals.
Expression of the growth arrest and DNA damage 45 (GADD45) gene was also increased above control levels in the testes of ob/ob mice, suggesting that GADD45 may participate in the apoptosis and/or DNA repair occurring in the germ cells of leptin-deficient mice. Exposure to ionizing radiation induces the transcription of GADD 45, which inhibits proliferation and stimulates DNA excision repair in mammalian cells (Fornace et al, 1989; Hollander et al, 1993). Phorbol ester treated MCF-7 breast cancer cells expressed GADD45 before the onset of apoptosis (De Vente et al, 1995). The expression of GADD45 in brain regions of rats following excitotoxic lesion correlated with DNA fragmentation as detected by TUNEL staining (Hughes et al, 1996).
We also found increased levels of expression of genes coding for caspase 7 and 9 in the testes of leptin-deficient mice. Caspase 9 is an initiator of activation of the caspases that act as executioners of apoptotic processes (Johnson and Bridgham, 2002). Caspase 7 is one of the executioners of apoptosis that is thought to play a role in ovarian follicular atresia (Matikainen et al, 2001; Johnson and Bridgham, 2002). Our findings in the current study of increased germ cell apoptosis in conjunction with elevated gene expression levels for caspase 7 and 9 suggest that these genes are integral components of the cascade that results in increased germ cell death within the testis of mice with a leptin deficit.
The expression of 3 antiapoptotic genes was also up-regulated in the testis of leptin deficient mice. Among these were proviral integration site 2 (Pim-2), microphthalmia-associated transcription factor (Mitf), and baculoviral IAP repeat containing 4 (BIRC4). Pim-2 is a member of a small family of oncogenic serine/threonine kinases and provides long-term resistance to a variety of apoptotic stimuli (Fox et al, 2003), and it has been shown previously to be expressed in the spermatocytes and interstitial tissue of normal human testes (Baytel et al, 1998). Mitf belongs to a family of transcription factors and is expressed in spermatogonia, spermatocytes, and round spermatids of mouse testis (Hodgkinson et al, 1993; Saito et al, 2003). The physiological significance of Mitf expression in male germ cells is not known. BIRC4 belongs to the baculovirus IAP repeat-containing protein family known as the inhibitors of apoptosis (Wang et al, 2004). The reasons why these antiapoptotic genes are up-regulated in the testes of ob/ob mice that show elevated apoptosis are unknown, but these genes may be attempting to serve as protectors of germ cells during accelerated programmed cell death.
A question that remains unresolved is the mechanism by which the leptin deficit adversely alters germ cell production. Gonadotropins are known to be antiapoptotic agents (Tapanainen et al, 1993). It appears appropriate, therefore, to suggest that the gonadotropin deprivation may be responsible for the increased germ cell loss in the leptin-deficient model. Alternatively, the leptin deficiency may directly alter the spermatogenic process since testicular tissue expresses leptin receptors (El-Hefnawy et al, 2000; Caprio et al, 2003), and leptin can directly alter testicular function in vitro (Tera-Sempere et al, 1999; Giovambattista et al, 2003).
Leydig cells were morphologically abnormal and fewer and seminal vesicles were smaller in ob/ob animals. This is suggestive of under-androgenization, but surprisingly total serum testosterone concentrations were in the normal range. This finding is in agreement with an earlier study from our laboratory (Bhat et al, 2003). The reasons for these seemingly conflicting data are unclear. The normal total testosterone levels in leptin-deficient mice were unexpected because these animals have reduced circulating gonadotropin levels and impaired GnRH secretion (Swerdloff et al, 1976; Batt et al, 1982). This could be due to the fact that testosterone is secreted in a pulsatile and circadian pattern and that by only measuring the hormone at 1 time point, we may have missed a significant reduction at another time point that resulted in a reduced 24-hour secretory rate. Another possible explanation is that the leptin-deficient mouse produces a binding protein that lowers free biologically active testosterone while reducing the clearance rate of the androgen. Related to this issue, we have found a similar phenomenon in the ob/ob female mouse in which total serum estradiol and progesterone levels are actually elevated above control levels but uterine weights are subnormal (Olatinwo et al, unpublished data). In any case, this is an interesting issue that deserves further study.
In conclusion, we have identified a group of genes via microarray technology that may play a prominent role in mediating the increased germ cell apoptosis and impaired sperm production exhibited by leptin-deficient mice. These genes are components of both the extrinsic and intrinsic pathway of programmed cell death. Future studies will be needed to determine whether the effects of the leptin deficiency on the spermatogenic process are a consequence of the hypogonadotropic status of these animals or whether leptin can directly modulate spermatogenesis at the level of the testis.
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Footnotes |
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