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From the * Department for Reproductive Biology,
Leibniz-Institute for Zoo and Wildlife Research (IZW), Berlin, Germany; the
Institute of Veterinary Biochemistry, Freie
Universität Berlin, Germany; and the
Center for Species Survival, Department of
Reproductive Sciences, Smithsonian's National Zoological Park, Conservation
& Research Center, Front Royal, Virginia.
| Correspondence to: Katarina Jewgenow, Leibniz-Institute for Zoo and Wildlife Research (IZW), PF 601103, D-10252 Berlin, Germany (e-mail: Jewgenow{at}izw-berlin.de). |
| Received for publication August 27, 2008; accepted for publication January 27, 2009. |
Teratospermia (>60% morphologically abnormal sperm/ejaculate) is
associated with increased sperm output in the domestic cat. The objective of
this study was to determine whether increased sperm production in
teratospermic donors was associated with disturbances in germ cell apoptosis,
the usual mechanism for sperm cell elimination. Apoptosis was measured by
evaluating DNA fragmentation, expression of Caspase-3, and anti-apoptosis
repressor with caspase recruitment domain (ARC) in the testes of normospermic
compared with teratospermic cats. Testes (n = 6 males/group) were obtained by
bilateral castration and immediately fixed in Bouin solution. Results revealed
that greater than 97% of cells labeled as DNA fragmented were tubular
regardless of male type. Fewer (P < .05) apoptotic spermatogenic
cells per tubule (0.52 ± 0.11 cells/tubule,
± SEM) and per 100 Sertoli
cells (3.79 cells/100 Sertoli cells) were observed in teratospermic compared
with normospermic (1.25 ± 0.36 cells/tubule and 6.44 cells/100 Sertoli
cells) cats. Among the spermatogenic cells, fewer (P < .03)
spermatocytes were positively labeled in teratospermic (0.3 ±
0.07/tubule) compared with normospermic (0.83 ± 0.28/tubule)
counterparts. Neither donor type differed in Caspase-3 or ARC expression
activity. However, each factor was both cell- and stage-specific in
expression. Specifically, Caspase-3 was located in Sertoli cells,
A-spermatogonia, and round spermatids at stage V. The ARC was found in primary
spermatocytes at each stage of the spermatogenic cycle. These results
demonstrate that the high incidence of morphologically abnormal sperm in
teratospermic male cats is accompanied by a reduced elimination of defective
spermatogenic cells via apoptosis.
Key words: Teratozoospermia, felids, terminal deoxynucleotidyl transferase dUTP nick-end labeling, Caspase-3, antiapoptosis repressor with caspase recruitment domain
Cell degeneration in spermatogenesis occurs during the mitotic proliferation phase of spermatogonia and during meiosis (Blanco-Rodriguez, 2002). A meiotic index of 2.8 (based on number of round spermatids for each pachytene primary spermatocyte) suggests that the normal rate of cell loss during the 2 meiotic divisions in domestic cats is ca 30% (Franca and Godinho, 2003). In contrast, the teratospermic cat is known to lose only 15% of sperm cells while producing a meiotic index of ca 3.5 (Neubauer et al, 2004). This observation suggests that disturbances in germ cell depletion during spermatogenesis in the teratospermic cat could be contributing to increased sperm output at the expense of gamete quality. In many species, including felids, apoptosis occurs spontaneously throughout spermatogenesis as the normal physiological mechanism for eliminating abnormal germ cells to ensure quality control of gametes produced (Braun, 1998).
Incidence of apoptosis in testicular tissue has been examined in several species or taxa, including the mouse (Krishnamurthy et al, 1998; Marcon and Boissonneault, 2004), rat (Blanco-Rodriguez and Martinez-Garcia, 1996), stallion (Heninger et al, 2004), roe deer (Blottner and Schoen, 2005), nonhuman primate (Weinbauer et al, 2001), and domestic cat (Blanco-Rodriguez, 2002). Specific molecular, biochemical, and morphological alterations of cells during various apoptotic stages have been the basis for evaluative methods of understanding cell elimination (Van Cruchten and Van Den Broeck, 2002; Otsuki et al, 2003). Because sperm cell apoptosis is mediated through the Fas pathway (including the expression of transmembrane protein kinases Fas and FasL and the interacting cytosolic cascade of caspases), the detection of Caspase-3 activity is a useful indicator of apoptotic cell death (Tesarik et al, 2002). Apoptosis-induced cellular alterations also include internucleosomal DNA fragmentation. The detection of fragmented DNA by labeling free 3'-OH ends with the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) technique is the most common method for determining single cell apoptosis. This technique and the quantification of histone-associated DNA fragments by enzyme-linked immunosorbent assay (ELISA; Hingst and Blottner, 1995) are confounded by the risk of overestimation because of normal chromatin remodeling in spermatids. Specifically, it is possible that such DNA strand breaks are part of normative cellular differentiation rather than apoptosis (Marcon and Boissonneault, 2004; Laberge and Boissonneault, 2005). However, BAX inhibitor-1, granulysin, and apoptosis repressor with caspase recruitment domain (ARC) have been shown to be intimately involved in apoptosis regulation (Neuss et al, 2001) and, thus, are considered reliable markers for studying this phenomenon.
The objective of this study was to test the hypothesis that disturbed apoptosis plays a role in the pathogenesis of teratospermia in the domestic cat. Apoptosis in normo- compared with teratospermic domestic cats was determined by assessing the incidence of DNA fragmentation, expression of Caspase-3, and ARC in testicular tissue.
Materials and Methods
All chemical were obtained from Sigma Chemical Company (Sigma Chemie GmbH, Deisenhofen, Germany) unless otherwise stated.
Animals
The 2 donor types were normospermic (<40% structurally abnormal
spermatozoa) and teratospermic (>60% structurally abnormal spermatozoa)
adult male cats. Explicit data on seminal traits of these cats already have
been published (Neubauer et al,
2004)—specifically that the teratospermic cohort produces an
unusually high concentration of total sperm, including those with
malformations. Paired testes from randomly selected domestic cats (n = 6;
average age, 2.3 years) were obtained from local veterinary clinics. Only
testes that were comparable in size and sperm yield to those from fully mature
animals were used for determination of epididymal sperm quality. A
normospermic classification (Howard et al,
1990) was based on a detailed morphological assessment of
spermatozoa recovered after bilateral flushing of the ductus deferens and
cauda epididymidis (epididymal sperm motility = 68.3 ± 9.9%;
morphologically normal epididymal sperm = 61.0 ± 2.1%;
Table 1). Teratospermic males
were known members of a research colony housed at the National Institutes of
Health (NIH) Animal Center, Poolesville, Maryland. Animals in this colony were
maintained in individual enclosures under a 12:12 h light:dark cycle and
provided dry cat food (Purina Cat Chow; Ralston-Purina Co, St Louis, Missouri)
and water ad libitum. These conditions eliminate seasonal changes in sperm
quality and production, in contrast to moderate seasonal changes in testis
function described for free-ranging domestic cats
(Axner and Linde Forsberg,
2007; Blottner and Jewgenow,
2007). Sperm quality of teratospermic cats (n = 6; average age,
1.6 years) were confirmed on the basis of at least 3 recent semen evaluations
(at least 6 months before castration) after electro-ejaculation
(Neubauer et al, 2004). A male
was deemed to be teratospermic when greater than 60% of spermatozoa in the
ejaculate was structurally abnormal on all 3 occasions. In addition, detailed
morphological assessment of epididymal spermatozoa was performed after
castration (epididymal sperm motility = 71.1 ± 7.5%; morphologically
normal epididymal sperm = 5.8 ± 1.3%;
Table 1). Normospermic and
teratospermic cats were subjected to the same anesthesia/castration methods,
with normospermic testes recovered from March through April and teratospermic
males from March through October. All animal handling procedures were in
accordance with the National Research Council's Guide for Care and Use of
Laboratory Animals
(1996). The animal protocol
was approved by the Smithsonian's National Zoo. Furthermore, materials derived
from local veterinary clinics were surplus biomaterials; participating
veterinary clinics did not require additional review or approval.
|
Testes Tissue Preparation
Immediately postcastration, testes were weighed and prepared for further
analysis as previously described (Neubauer
et al, 2004). Briefly, each testis was separated from the
epididymis and decapsulated. Small pieces of testicular parenchyma (3–5
mm3) were prepared from the outer third of each testis avoiding
regions containing the rete testis. Tissue pieces for histology were fixed in
Bouin solution and embedded in paraffin. Subsequently, 3-µm testis sections
were prepared for TUNEL analysis and immunohistochemical detection of
Caspase-3 proteins and anti-apoptosis repressor with ARC.
Tissue sections were incubated at 60°C for 1 hour, deparaffinized, and rehydrated via a 2-fold incubation in xylene for 15 minutes, immersion in decreasing concentrations of ethanol (2 x 98%, 96%, 80%, 70%, 50%; 2x distilled water, 5 min each) and a 5-minute wash in phosphate-buffered saline (PBS). Stained slides were microscopically assessed at x400 (Leica Microsystems, Bensheim, Germany). At least 3 sections (from different parts of the testis) per animal were randomly chosen for histological determination of apoptosis (see below).
In Situ Localization of Fragmented DNA (TUNEL Assay)
The detection of cells with fragmented DNA in testicular tissue sections
was performed using the DeadEnd Colorimetric Tunel System (Promega
Corporation, Madison, Wisconsin) according to manufacturer's instructions with
slight modifications for the analysis of apoptosis in paraffin-embedded tissue
sections. Briefly, for permeabilization, sections were incubated in
10-µg/mL proteinase K in PBS (10 minutes, room temperature), and the
Streptavidin HRP solution was diluted 1:1000 in PBS (rather than the
recommended dilution of 1:500).
Histomorphometry
Specific histomorphometrics were determined for both normo- and
teratospermic cats. For each cat, sections from 3 different parts of the
testis were chosen. Analyzing circular or nearly round tubules, the stage of
spermatogenesis was determined according to the cellular composition of the
seminiferous epithelium (Bohme and Pier,
1986, Neubauer et al,
2004). Tubules at the postspermiation stage (stage V) were
selected for a detailed examination of TUNEL-labeled cells and evaluated at
x400 magnification (10–30 cells/cat). The number of Sertoli cells,
spermatogonia, spermatocytes, spermatids, and spermatozoa labeled by the TUNEL
assay was counted, as well as the number of labeled fragmented (not
identifiable) cells. The overall number of Sertoli cells per tubule was
determined as described earlier by our laboratories
(Neubauer et al, 2004).
Immunohistochemistry
Caspase-3—
Caspase-3 expression was assessed by an affinity-purified rabbit
anti-human/mouse Caspase-3 reactive antibody (Cat. AF 835, Lot No. CFZ32;
R&D Systems, Wiesbaden, Germany). For exposure of antigenic epitopes,
slides were first boiled for 10 minutes in citrate buffer (pH 6.0), then
incubated in 3% H2O2-methanol for 15 minutes to block
endogenous peroxidases, and then incubated in 3% bovine serum albumin (BSA) in
PBS for 1 hour (37°C) to block nonspecific binding sites. Slides were
incubated overnight at 4°C in Caspase-3 antibody (0.05 µg/mL) followed
by a 30-minute incubation in HRP-conjugated secondary antibody
(DakoEnVision+system–HRP anti-rabbit antibody, code K4002; Dako
Diagnostika GmbH, Hamburg, Germany) at room temperature. Before adding each
antibody, slides were washed twice (5 minutes each) in PBS. Binding sites were
visualized with the DAKO chromogen system (code K3468; Dako), whereby slides
were developed for ca 0.5 to 2 minutes to avoid excessive background staining.
All slides were counterstained with hematoxylin. As a negative control, slides
were incubated with normal rabbit immunoglobulin fraction (code 0903; Dako),
rather than the primary antibody, and then processed (as above).
Anti-apoptosis Repressor With ARC— A rabbit polyclonal antibody generated against a peptide corresponding to amino acids 2 through 18 of human anti-apoptosis repressor with ARC (catalog PC486; anti-ARC 2-18; Calbiochem, an affiliate of Merck KGaA, Darmstadt, Germany) served as the primary antibody (2 µg/mL, incubated at 4°C overnight). Antigen recovery was performed by boiling the slides in Tris-HCl (pH 10) for 10 minutes. All other steps were as described above.
Western Blot
Snap-frozen testicular tissue samples from normospermic cats were utilized
for protein extractions. Samples were minced under liquid nitrogen in a mortar
and transferred to RIPA buffer (Sigma catalog R0278; on ice, 30 minutes) with
frequent vortexing to affect lysis. Insoluble constituents were pelleted by
repeated centrifugation at 12 000 x g for 15 minutes. Protein
quantification was performed with a reducing agent–compatible BCA
protein assay kit (Pr 23250; Pierce Chemical, distributed by Perbio Science
Deutschland GmbH, Bonn, Germany). Proteins (60 µg/lane) were separated by
sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Mini Protean
III; Bio-Rad, Munich, Germany) in a gel containing 10% acrylamide and
transferred to a polyvinylidene difluoride membrane (GE Healthcare, Munich,
Germany) with a semidry blotting methodology (1 hour, 40 mA/membrane). After
transfer, membranes were rinsed in double-distilled water and then immersed
for 1 hour (37°C) in blocking solution (ECL Advance Blocking Agent, GE
Healthcare). All antibodies were diluted in blocking solution. For Caspase-3
immunodetection, blots were incubated overnight at 4°C with the
Caspase-3-antiserum diluted 1:3000. After washing in PBS containing 0.1%
Tween-20 (PBS-T), specific binding was detected with a peroxidase-conjugated
goat anti-rabbit immunoglobulin G antibody (GE Healthcare) diluted 1:20 000
and visualized with the ECL plus detection reagent (GE Healthcare). ARC also
was detected with the above methods with an anti-ARC primary antibody diluted
1:5000.
|
Results
In Situ Localization of Fragmented DNA (TUNEL Assay)
Only a few cells per TUNEL-stained section were positive for DNA
fragmentation (Figure 1). Often
the TUNEL staining was characterized by a focal expression with unequal
distribution of labeled cells between tubules. Some tubules contained more
than 5 labeled cells, whereas many tubules were unlabeled, especially in
normospermic donors. Overall, fewer (P < .05) cells per tubule
were labeled in teratospermic (tubule n = 186; labeled cells n = 89; labeled
cells/tubule = 0.52) compared with normospermic (tubule n = 112; labeled cells
n = 164; labeled cells/tubule = 1.25) cats
(Table 2). Although there were
fewer Sertoli cells per tubule (Table
2), this was not the only reason for the decreased number of
apoptotic cells in terato-compared with normospermic males. In the context of
Sertoli cell density, teratospermic cats had fewer apoptotic cells (3.79/100
Sertoli cells) compared with normospermic counterparts (6.44/100 Sertoli
cells; Table 2).
|
More than 97% of labeled cells were identified as tubular in morphology. The frequency of TUNEL-positive cells among the various spermatogenic cell types revealed a significant difference only in apoptosis of spermatocytes. As indicated in Table 2, incidence of apoptotic spermatocytes was increased (P < .05) in normospermic compared with teratospermic donors, which was independent of stage of spermatogenesis. In contrast, the percentages of apoptotic spermatogonia or round spermatids were not different (P > .05) between the 2 donor types (Table 2).
Immunolabeling of Caspase-3 and ARC
Examples of the immunohistochemical localization of Caspase-3 and ARC are
presented in Figure 2.
Differences in the cell-specific expression of these apoptosis-related factors
were not detectable in normo- compared with teratospermic males. However, both
cell and stage-specific expression was found
(Table 3). The apoptotic
cascade enzyme Caspase-3 was located in cytoplasm and nuclei of Sertoli cells
and A-spermatogonia at every stage of the epithelial cycle. Additionally,
expression of Caspase-3 peptide was detected within nuclei of round spermatids
in stage V (Figure 2D). Round
spermatids were also slightly labeled by the caspase antibody at stage IV, but
no staining was found at stages I to III
(Figure 2B and C). At stage IV,
cytoplasmic remnants of elongated spermatids within the seminiferous tubular
lumen were strongly positive for Caspase-3
(Figure 2C). The ARC peptide
was found only in primary spermatocytes of each stage of spermatogenic cycle
(Figure 2E;
Table 3).
|
|
Western blot analyses of cat testis tissue revealed a detectable Caspase-3–positive signal at 17, 22, and 32 kd, with the most prominent band at 60 to 70 kd (Figure 3). The ARC immune reactivity was observed at 16 kd and 60 to 65 kd (Figure 3).
|
This study demonstrated, for the first time, a linkage between lower incidence of apoptosis during spermatogenesis and the incidence of teratospermia in the domestic cat. Immunohistochemistry and in situ nick-end labeling revealed the expression of factors known to be relevant to programmed cell death within testicular seminiferous epithelium of both the normo- and teratospermic male. Therefore, there was clear evidence of apoptotic mechanisms that no doubt contribute to elimination of spermatogenic cells. Caspase-3 is known to be involved in the 2 apoptotic pathways that converge on activating "downstream" caspases by regulating substrate cleavage and apoptotic cell death (Green, 1998). The first route is stimulated via mortality receptors on the cell surface (eg, Fas) that recruit adaptor proteins and eventually Caspase-8. In the second pathway, various cellular stressors trigger mitochondrial release of cytochrome c that is activated via Caspase-9. The first downstream caspase stimulated via both routes is Caspase-3 (Green, 1998), one of the target areas of our study. Additionally, anti-apoptosis repressor with ARC interacts with Caspase-2/8 and inhibits Caspase-8 enzyme activity. Therefore, expression of ARC is associated with a blocked death receptor–mediated pathway (Green, 1998).
To date, investigations of this biological process have been performed mainly in laboratory animals (for review, see Blanco-Rodriguez, 1998). Our study revealed that these mechanisms also appeared conserved in a carnivore species (ie, the domestic cat) and, more importantly, varied between 2 populations of animals that differed in sperm quality (both in morphology and function; Long et al, 1996; Pukazhenthi et al, 1996, 1999, 2001). Thus, our results suggest that in addition to reduced gene diversity (Pukazhenthi et al, 2006), lower incidence of apoptosis also appears to be a significant contributor to the condition of teratospermia in the cat.
With the TUNEL method, the incidence of apoptotic cells was shown to be a rare event in cat testicular tissue, with only about 1 cell per tubule exhibiting positive staining (ie, DNA nicks). Apoptosis is known to be a rapid event, with cells being eliminated quickly (Brinkworth et al, 1997), which could mean that only a few dying cells could be detected at any given moment of sampling. Siemieniuch (2008) also found that only a few cells were labeled by TUNEL in testis sections of domestic cats with slight age relatedness. They described that the incidence of TUNEL-labeled testicular cells is increased before onset of spermatogenesis. In sexually mature males (comparable to our domestic cats), the programmed cell death did not increase with age and was not influenced by season (Siemieniuch, 2008).
In contrast to the rare event of DNA strand breaks, our observations revealed a more generalized articulation pattern of Caspase-3 and ARC that was cell- and spermatogenic cycle–specific, as well as independent of the more focused expression of TUNEL-positive cells. Caspase-3 was found in the cytoplasm of all A-spermatogonia and within nuclei of secondary spermatocytes. This finding was consistent with those of Rodriguez et al (1997), who indicated that apoptotic cell death largely occurs in spermatogonia. We suspect that the antibody labeled the inactive proenzyme of feline Caspase-3, indicating a specific potency of these spermatogenic cells for apoptotic cell death.
Caspases are present in most cells, residing in the cytosol as a
single-chain proenzyme. During apoptotic cell death, caspases convert to
functionally active proteases by a proteolytic cleavage that divides the chain
into large and small subunits. A second cleavage removes the N-terminal
domain, and a 17- and 12-k subunit for Caspase-3 assembles into a tetramer
with 2 active sites. The antibody used in this study was directed against a
peptide sequence from the human 17-kd Caspase-3 subunit, thereby making it
unfeasible to identify the unconjugated 12-kd domain. But Western blot
analysis of feline testis protein lysates allowed recognizing the proenzyme
(32 kd), the large subdomain of the proenzyme (23 kd), and the 17-kd subunit.
We believe that the prominent protein band evident in
Figure 3 is the active tetramer
comprising the 2 subunits of the 17- and 12-kd (
70-kd) proteins.
The most prominent cellular localization of Caspase-3 was found within the cytoplasmic remnants of elongated spermatozoa in sections of both normo- and teratospermic (Figure 2C; Table 3) testicular tissue. Cells that fail to be released into the lumen because of defective apoptosis are phagocytosed by Sertoli cells (Russell, 1993; Guraya, 1995). It is well established that one of the most prominent malformations in the teratospermic cat ejaculate is a high proportion of spermatozoa with a retained cytoplasmic droplet (Howard et al, 1990). Thus, clearly, cytoplasmic elimination and phagocytosis of retained spermatids are compromised in the teratospermic cat. These anomalies and overall prolonged spermiation might be caused by deranged Sertoli cell function, as we have suggested previously (Neubauer et al, 2004).
Expression of ARC exclusively within the cytoplasm of pachytene spermatocytes suggested that the death receptor pathway was mediated via Caspase 8 that targeted specifically the primary spermatocytes. Perhaps apoptosis in testicular tissue is either inhibited or regulated expressly by the presence of ARC in these spermatogenic cells. Such an assertion would agree with the idea of a stage-specific intra- and extracellular control of apoptosis in spermatogenesis (Blanco-Rodriguez, 1998). Proper stability between cell death and survival-promoting proteins is essential to sustaining an appropriate ratio of maturing germ cells to Sertoli cells (Jegou et al, 1993). Our TUNEL labeling also revealed that spermatocytes were the most frequent (>55%) target for cell death, with a significantly increased elevation in apoptosis for normo- compared with teratospermic males. In other species, apoptotic cell death has been found also in all stages of the spermatogenic process, with the majority consisting of spermatogonia (rat, Blanco-Rodriguez and Martinez-Garcia, 1996; mouse, Rodriguez et al, 1997; Krishnamurthy et al, 1998), primary spermatocytes (common marmoset, Weinbauer et al, 2001), or both. Thus, despite these species-specific variations, the elimination of spermatogenic cells occurs mostly before the first meiotic division.
Of particular interest was the functional difference in cell elimination
effectiveness between the normo- and teratospermic cat. It is well established
in normal males of this species that approximately one-third of cells are lost
during transition from pachytene spermatocyte to round spermatid stage
(Franca and Godinho, 2003;
Neubauer et al, 2004). In
contrast, only 15% of cells are eliminated in teratospermic counterparts
(Neubauer et al, 2004).
Multiapproach comparative analysis of testicular tissue in this study revealed
only about half the number of apoptotic cells (per 100 Sertoli cells) in
terato- (3.79) compared with normospermic (6.44) individuals. The latter value
for "normal" male cats was in the range of this same ratio for the
few other species that have been studied, including the human (5–10
apoptotic cells; Oldereid et al,
2001), rat (7.5 in stage XIV;
Blanco-Rodriguez, 1998), and
stallion (15.5; Heninger et al,
2004). The unusually low ratio in teratospermic male cats
suggested that compromised apoptosis likely contributed to the peculiarly high
sperm concentration routinely detected in males with this condition
(Neubauer et al, 2004).
However, the reduced elimination of defective sperm alone cannot explain the
almost 10-fold increase in sperm production. We suggest an overlay of another,
compensatory effect, which partly ensures the fertility of affected males.
Interestingly, the absolute number of morphologically intact sperm in
ejaculates of both normo- and teratospermic males is identical (10
x 106 of intact and motile sperm per ejaculate;
Neubauer et al, 2004). Thus,
it is very likely, that 10 million intact sperm present a kind of threshold
for fertility in domestic cats. In the case of a teratospermic male, reduced
apoptosis is accompanied by an increase in defective sperm within the
ejaculate, which is later compensated by higher sperm production.
It is noteworthy that teratospermia appears more prevalent in the domestic cat than once believed (Axner and Linde Forsberg, 2007). Also, different types of this condition are likely. One is "occasional," with some cats periodically producing increased numbers of malformed sperm that appear partly regulated by seasonality (Blottner and Jewgenow, 2007) or perhaps to sexual abstinence, nutrition, or health factors. This type of teratospermia is usually accompanied by a reduced sperm production, thus reduced fertility. However, others males (including the ones targeted in this study) consistently and always ejaculate high concentrations of sperm pleiomorphisms regardless of environmental factors. Detailed studies of these spermatozoa have revealed that even morphologically normal cells are compromised in the ability to capacitate, undergo the acrosome reaction, survive cryopreservation, proceed with tyrosine phosphorylation of sperm proteins, or penetrate the zona pellucida (for review, see Pukazhenthi et al, 2006). Teratospermia appears to have a genetic basis related to increasing loss of heterozygosity. Furthermore, this form appears with some commonality in various wild felid populations living in fragmented or isolated habitats or in poorly managed zoos, again likely directly related to increasing levels of homozygosity (Wildt et al, 1983; O'Brien, 1994). Although the database on the influences and etiology of teratospermia in felids is growing, the effect of this condition on actual fertility remains difficult to assess. Teratospermic male felids can (and do) sire offspring, although it is clear that malformed spermatozoa do not participate in fertilization (Pukazhenthi et al, 2001). Therefore, the overall effect of teratospermia on reproductive fitness remains ambiguous. And, perhaps such felids are undergoing compensatory adaptations, including developing an exquisitely efficient ability to remain reproductively successful in the presence of few normative ejaculated sperm. Regardless, it is clear that the cat is an excellent model for studying testis functions, including malformed sperm production, and the influence and fate of such cells. Although of basic research interest, this area of investigation will continue to be relevant to understanding the potential effect of teratospermia in wild felid species and populations that are growingly rare because of persistent and relentless environmental change.
Acknowledgments
The authors thank Ms Christiane Franz, Ms Katharina Topp, and Ms Sigrid Holz for technical assistance and Drs Michael Cranfield and Brent Whitaker (Maryland Line Animal Rescue) for providing domestic cat testes.
Footnotes
Supported by Deutsche Forschungsgemeinschaft DFG Je 163/9-1 and Scho 1231/2-1 and the Friends of the National Zoo.
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