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BOR - Papers in Press, published online ahead of print June 30, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.031062
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BIOLOGY OF REPRODUCTION 71, 1517–1524 (2004)
DOI: 10.1095/biolreprod.104.031062
© 2004 by the Society for the Study of Reproduction, Inc.

Testis

Quantity Rather Than Quality in Teratospermic Males: A Histomorphometric and Flow Cytometric Evaluation of Spermatogenesis in the Domestic Cat (Felis catus)1

Katrin Neubauer2,3, Katarina Jewgenow3, Steffen Blottner3, David E. Wildt4, and Budhan S. Pukazhenthi4

Institute for Zoo and Wildlife Research,3 D-10252 Berlin, Germany Smithsonian's National Zoological Park,4 Conservation and Research Center, Front Royal, Virginia 22630


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Teratozoospermia (ejaculation of <40% morphologically normal sperm) commonly occurs within the Felidae, including certain domestic cats, but the cellular and molecular mechanisms that give rise to this phenomenon remain unknown. This study quantified spermatogenesis to identify differential dysfunctions in teratospermic versus normospermic (>60% normal sperm/ejaculate) domestic cats. Sperm used were from electroejaculates and cauda epididymides. Testes from 10 normo- and 10 teratospermic males were obtained by castration and then evaluated by histomorphometry, flow cytometry, and testicular testosterone enzyme immunoassay. Some morphometric traits (tubular diameter, epithelium height, interstitial area, number of Leydig cells, and blood vessels per cross-section) as well as testicular testosterone concentrations were similar between groups, but testicular volume was greater in teratospermic males. Stage frequencies differed also between both cat populations, suggesting possible dysfunctions in spermiation. Quantification of cell populations in most frequent stages revealed more spermatogenic cells and fewer Sertoli cells per tubule cross-section as well as per tissue unit in teratospermic donors. Hence, the ratio of spermatogenic cells per Sertoli cell was elevated in the teratospermic cat. DNA flow cytometry confirmed higher total spermatogenic and meiotic transformations in teratospermic males. In summary, compared with normospermic counterparts, teratospermic cats have a higher sperm output achieved by more sperm-producing tissue, more germ cells per Sertoli cell, and reduced germ cell loss during spermatogenesis. Gains in sperm quantity are produced at the expense of sperm quality.

Sertoli cells, sperm, spermatid, spermatogenesis, testis, testosterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apart from the domestic cat (Felis catus), most members of the Felidae family (comprised of 37 species) are vulnerable, threatened, or endangered by extinction in nature. Teratozoospermia (ejaculation of <40% morphologically normal sperm) commonly occurs in ~70% of felid species/ subspecies (n = 28) studied to date, including endangered species such as the clouded leopard (Neofelis nebulosa) [1, 2], cheetah (Acinonyx jubatus) [1, 3, 4], and the Florida panther (Puma concolor coryi) [5]. The clouded leopard and cheetah consistently ejaculate >70% structurally abnormal spermatozoa, whereas the Florida panther produces an average of 94% pleiomorphisms, most of which have severely malformed acrosomes [6, 7]. Certain domestic cats also are teratospermic and represent a model for studying this reproductive disorder. Comparative studies of such males (including those from wild felid populations) have demonstrated an array of compromised sperm functions [2, 8–15]. Despite the fact that malformed sperm do not participate in fertilization [6], even normal-appearing sperm from teratospermic males have a reduced ability to capacitate, undergo the acrosome reaction, bind and penetrate the zona pellucida, and condense within the ooplasm [6]. There now is little doubt that populations of felids that produce high percentages of pleiomorphic sperm experience reduced fertility [7]. Teratozoospermia also impacts the efficiency of using assisted reproductive technologies for genetic management. For example, sperm from teratospermic donors are more susceptible to cold-induced acrosomal damage [14] during attempts to cryopreserve sperm for artificial insemination or in vitro fertilization.

To date, the mechanisms that give rise to teratozoospermia are still unknown [6]. A relationship between genetic homozygosity and increased numbers of malformed sperm has been proposed for the cheetah, a species well known for its lack of genetic variation [4, 16], and isolated populations of pumas and lions [5, 17]. Nonetheless, it remains possible that environmental factors (e.g., stress, inappropriate housing, diet, or other suboptimal management practices) [18, 19] may be important in regulating numbers of structurally normal sperm. Because previous studies have focused on qualitative sperm changes, there still is no conclusive evidence for the origin of the gamete malformation in felids.

For the first time, we designed a study to examine potential dysfunctions occurring during spermatogenesis in the teratospermic male. We especially were interested in an earlier observation that teratospermic male cats produce not only elevated numbers of abnormally shaped sperm, but also typically more total sperm per ejaculate while remaining fertile [8]. This higher sperm output by teratospermic compared with normospermic counterparts could be achieved by 1) a higher spermatogenic efficiency (expressed as the number of sperm produced daily per tissue unit), which is usually positively correlated with the Sertoli cell efficiency (number of germ cells supported by each Sertoli cell) [20, 21]; 2) more sperm-producing tissue; and/ or 3) insufficient selection of malformed germ cells during spermatogenesis. We hypothesized that, in the teratospermic cat, the ratio between spermatogenic and somatic cells is altered, leading to suboptimal conditions for germ cell development. This milieu is so unfavorable that especially Sertoli cells are unable to adequately support all sperm produced from the intensified output. As a consequence, sperm quantity is increased to the disadvantage of sperm quality in the teratospermic male. This impaired process also may have adverse implications for spermiation.

This project tested these hypotheses while examining how increased sperm production occurred. Studies were designed to take advantage of the availability of a well-characterized population of teratospermic domestic cats that were compared with normospermic controls. Because spermatogenesis in the teratospermic cat has never been investigated before, a particularly important objective of the present study was to do a detailed assessment of histomorphometric and flow-cytometric parameters as well as of testicular testosterone content, and thus to provide basic information that also will be valuable for future research.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

The comparative groups were normospermic (>60% structurally normal spermatozoa) versus teratospermic (<40% structurally normal spermatozoa) adult male cats. Age of animals ranged from 1 to 10 yr, with mean ages of 2.3 yr for normospermic and 2.5 yr for teratospermic cats. Testes from the former (n = 10 males) were obtained from local veterinary clinics and assigned to this group based on morphological assessment of spermatozoa recovered from the ductus deferens and cauda epididymides [22]. Testes from teratospermic males (n = 10) were obtained from a research colony housed at the National Institutes of Health (NIH) Animal Center, Poolesville, MD. These cats were confirmed to be teratospermic on the basis of at least three recent fertility assessments during the past 6 mo that included a detailed assessment of sperm morphology (postelectroejaculation [8]). Teratospermic phenotypes included acrosomal defects, head malformations, a bent midpiece, a bent or coiled flagellum, and persisting cytoplasmic droplets. A male was deemed teratospermic only if producing <40% structurally normal spermatozoa on all three occasions. These males were maintained in individual enclosures under a 12L:12D cycle and were provided dry cat food (Purina Cat Chow; Ralston-Purina Co., St. Louis, MO) and water ad libitum [8]. As with the normospermic controls, teratospermic males were subjected to castration under anesthesia and the testes recovered after castration. Because sperm production in the domestic cat is not influenced by season [23, 24], testes collection was not restricted to a specific period of the year. Nonetheless, all testes were recovered during the predominant mating season (March–April and September–October). All investigations were performed in accordance with the Guide for Care and Use of Laboratory Animals (NIH, 1996).

Semen Collection and Analysis

Immediately before castration, teratospermic cats were subjected to a standardized electroejaculation protocol for collecting semen [25]. Briefly, males were anesthetized with an intramuscular injection of ketamine hydrochloride (Ketaset; Bristol Laboratories, Syracuse, NY; 3.5–4.0 mg/kg body weight) plus xylazine (Xyla-Ject; Phoenix Pharmaceuticals, Inc., St. Joseph, MO; 3.5–4.0 mg/kg body weight). Upon reaching a surgical plane of anesthesia, each male was electroejaculated using a 60-Hz sine-wave stimulator and a 1.0-cm-diameter rectal probe with three longitudinal electrodes (P.T. Electronics, Boring, OR). A total of 80 low-voltage (2–5 V) stimuli were delivered in three series over the course of 20 min. Each ejaculate was evaluated immediately for volume, sperm concentration, sperm motility percentage, and progressive sperm motility (i.e., speed of forward progress; scale, 0–5; 5 = best) by microscopic assessment before processing [8]. For morphological assessment, a 10-µl aliquot of the fresh semen was fixed in 0.3% (v/v) glutaraldehyde in PBS (pH 7.4; 320 mOsm) and evaluated (100 sperm/aliquot) by phase-contrast microscopy (1000x). There were no semen collections from the normospermic controls because these males were companion animals brought to local veterinary clinics for castration.

For evaluation of sperm morphology from the castrated biomaterials, the ductus deferens adjacent to the epididymis was flushed with 0.3% (v/ v) glutaraldehyde in PBS (pH 7.4; 320 mOsm), or a portion of the cauda epididymis was gently teased apart in a drop of 0.3% (v/v) glutaraldehyde in PBS (pH 7.4; 450 mOsm; the latter was used to help compensate for the increased osmotic strength of the cauda epididymal contents). As later demonstrated in the Results, there generally were no differences in traits for sperm recovered from the ejaculate versus the epididymis of teratospermic males.

Epididymal Sperm Recovery and Quality Assessment

For recovering epididymal sperm, the cauda epididymis was first carefully separated from the testis proper and associated blood vessels and transferred to a Petri dish. Each epididymis was minced while immersed in Hepes buffered Ham F10 medium (Gibco-BRL, Gaithersburg, MD) containing 5% fetal calf serum (FCS; HyClone Laboratories, Logan, UT) and adjusted to 450 mOsm. After a 15- to 20-min incubation at room temperature, 5 µl of sperm suspension was assessed for sperm motility and progressive status (as described earlier). Epididymal sperm morphology was determined by phase-contrast microscopy (1000x) on a sample obtained by mincing a 3-mm piece from the middle of the cauda epididymis directly into 0.3% (v/v) glutaraldehyde in PBS (450 mOsm).

Testes Tissue Preparation

Testes weights or volumes were measured with their ratio assumed as 1:1. This ratio was used recently in a quantitative characterization of spermatogenic efficiency in primates [26]. It is also warrantable from our own measurements of tissue density of entire cat testes (1 cm3 = 1.018 g, n = 24).

Thereafter, testes were immediately processed for histomorphometric and flow cytometric analysis of spermatogenesis. Briefly, each testis was dissected from the epididymis and decapsulated. Small pieces of testicular parenchyma (3–5 mm3) were prepared from the outer third of testis, avoiding regions containing the rete testis. The three parts of the rete testis were clearly identified [27] and not present in any of our studied sections. For subsequent flow cytometry and hormonal analysis, samples were frozen in liquid nitrogen. Tissue sections for histology were fixed in Bouin solution and embedded in paraffin. Subsequently, 2-µm testis sections were prepared and stained with standard Periodic Acid Schiff (PAS) and hematoxylin and eosin (HE) [28], respectively.

Histomorphometric Analysis of Spermatogenesis

Histomorphometric parameters were measured on HE-stained slides using a computer-assisted image analysis system (Carl Zeiss, Inc., Oberkochen, Germany) and a software package, analySIS 3.0 (Soft Imaging System GmbH, Muenster, Germany). For each cat, 100 circular or nearly circular tubules were randomly chosen to estimate minimal tubular diameter and seminiferous epithelium height. Each value for epithelium height was calculated as the average of four measurements/tubule taken at an angle of about 90°. Percentage of interstitial area on the total testis parenchyma was determined by labeling and measuring the intertubular compartment in a field of vision (at 400x). One field corresponded to two squares of 10 000 µm2; five fields/section and four sections/animal were chosen for the image series. Thus, a total of 400 000 µm2 was scored for each cat. For each population, the tubular compartment area was calculated by subtracting the interstitial area from the total area neglecting mediastinum testis and tunica albuginea. The area density of tubular compartment (Ad), which is proportional to volume density, was expressed as fraction of 1 (i.e., 100%). The total tubular compartment volume (portion of sperm-producing tissue) was computed by multiplying Ad by testis volume. Additionally, Leydig cell number and number of blood vessels were counted in every field used for interstitial area measurements.

PAS staining was applied to differentiate spermatogenic cells in the stages of the seminiferous epithelium cycle. Previous studies have characterized eight stages of feline spermatogenic cycle according to spermatid transformation, with emphasis on acrosomal development and specific composition and topography of cell generations in a seminiferous tubule cross-section [29, 30]. Stage I is defined by occurrence of new round spermatids, stage IV by spermiation, and stage VIII by meiotic division of spermatocytes. The relative stage frequencies were determined by analyzing 100 tubule cross-sections/cat, as practiced in comparable examinations [26, 31].

Stages IV and V (postspermiation stage) were selected for a detailed examination. These stages showed the highest frequencies and the most pronounced differences between the two cat populations. Eight (stage IV) or 10 (stage V) circular tubules/animal were chosen, and the Sertoli cells, spermatogonia, spermatocytes, and round and elongated spermatids (Fig. 1) were counted. Resulting cell numbers per cross-section also were expressed per Sertoli cell and converted to numbers per standardized tissue area of 1 mm2 as a parameter of spermatogenic efficiency. Sertoli and germ cell densities in 1 mm2 were calculated by multiplying respective cell number/tubule cross area by number of tubules/mm2. The number of tubules per 1 mm2 is given by

where AT = the mean tubular cross area (mm2).



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  		FIG. 1. Teratospermic domestic cat seminiferous tubule cross-section at (a) stage IV (spermiation) and (b) stage V (postspermiation) of the seminiferous epithelium cycle. Sc, Sertoli cells; sg, spermatogonia; spc, spermatocytes; r spt, round spermatids; e spt, elongated spermatids. Bars = 10 µm

Flow-Cytometric Analysis of Spermatogenesis

Cryopreserved testes samples were processed for flow cytometry as described earlier [32]. Briefly, testis cells were obtained by finely mincing 50 mg of decapsulated testicular tissue in 100 mM citric acid and 0.5% (v/v) Tween 20. Released nuclei were dispersed by gentle agitation for 20 min and then stained with 0.175% 4',6-diamidino-2-phenylindol (DAPI; Sigma, Deisenhofen, Germany) in 400 mM Na2HPO4. Flow cytometric analysis was performed in a PAS III flow cytometer (Partec GmbH, Muenster, Germany) with a mercury lamp using a wavelength of 360 nm for excitation and of 420 nm for emission. Resulting DNA histograms were analyzed by DPAC computer software to determine the proportions of cells in each peak. Contents of haploid (spermatids and spermatozoa), diploid (spermatogonia, preleptotene, and secondary spermatocytes and somatic cells), and tetraploid cells (all cells in the G2/M phase of cell cycle, mainly primary spermatocytes) were expressed as 1C, 2C, and 4C percentages, respectively. These proportions were used to characterize the total spermatogenic transformation (1C:2C cell ratio) and the meiotic transformation of primary spermatocytes to spermatids (1C:4C cell ratio).

Estimation of Testicular Testosterone by Enzyme Immunoassay

Testicular testosterone was measured for one testis in the pair by a double antibody enzyme immunoassay (EIA) as published earlier [32]. In brief, 200 mg of each testis was extracted with 1 ml methanol:water 70: 30 (v/v) for 30 min. The extract was diluted with assay buffer as required, and duplicates of 20 µl were analyzed. The assay used a polyclonal antibody raised in rabbits against testosterone-11-hemisuccinate-BSA, and the label was testosterone-3-carboxymethyl-oxime-horse radish peroxidase. The testosterone standard curve ranged from 0.4 pg to 50 pg/20 µl, and the cross-reactivity was 100% with testosterone, 10% with 5{alpha}-dihydrotestosterone, 2% with androstenedione, and <0.1% with estradiol and with progesterone. Results were given in nanogram testosterone per gram testis. The intra- and interassay coefficients of variation were 8.9% and 12.3%, respectively. One teratospermic male was excluded from this analysis due to insufficient amount of saved tissue.

Statistical Analysis

Mean comparisons were conducted using an unpaired Student t-test of a statistical program SPSS 9.0 (SPSS Inc., Chicago, IL). A two-tailed P value of <0.05 was considered significant. All data are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sperm-Quality Assessment

Sperm traits of normospermic and teratospermic cats are presented in Table 1. For the teratospermic donors, there was compatibility between electroejaculates and epididymal samples, both having sperm with similar motility traits and high proportions (~90%) of cellular pleiomorphisms. Epididymal samples from teratospermic donors were comprised of only 11.4% morphologically normal sperm versus 66.1% for normospermic counterparts (P < 0.001). Motility traits were comparable between donor types. The predominant abnormalities measured in ejaculated semen of teratospermic males were a persisting cytoplasmic droplet (62.4% ± 11.3% of all sperm with or without other defects) or a defective midpiece (52.3% ± 8.4%) or flagellum (19.7% ± 4.8%). Only a few head (4.4% ± 1.7%) and acrosomal (2.1% ± 0.5%) deformities were observed.


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  		TABLE 1. Sperm traits in normospermic versus teratospermic domestic cats (mean ± SEM)

Histomorphometric Analysis of Spermatogenesis

Histomorphometry revealed no differences in minimal tubular diameter, epithelium height, percentage of interstitial area, Leydig cell number, or number of blood vessels per square millimeter between the normospermic and teratospermic groups (Table 2). However, teratospermic males had about 35% bigger testis (P < 0.05) and a slightly greater portion of tubular compartment, resulting in ~40% more total tubule volume (P < 0.01; Table 3).


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  		TABLE 2. Histomorphometric data in normospermic versus teratosperm ic domestic cats (mean ± SEM)


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  		TABLE 3. Estimation of relative and total tubular volume for normospermic versus teratospermic domestic cats

For three stages, relative frequencies differed remarkably between cat populations (Table 4). In the normospermic group, the most commonly observed stage was postspermiation (V) followed by spermiation (IV). In contrast, teratospermic males were exactly the opposite with the most frequently observed stage being spermiation (IV) followed by postspermiation (V) (P < 0.01 for both stage frequencies). Additionally, stage I was more frequent (P < 0.01) in teratospermic compared with normospermic males.


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  		TABLE 4. Relative frequencies of stages of the seminiferous epithelium cycle in normospermic versus teratospermic domestic cats (mean ± SEM)

Within the postspermiation stage (stage V; Fig. 1), total number of germ cells/tubule cross-section differed between cat groups (N: 245.1 ± 13.6, versus T: 338.6 ± 11.9; P < 0.001). Differentiation of cell types revealed higher cell numbers per counted tubule for spermatogonia (P < 0.01), spermatocytes (P < 0.05), and particularly round spermatids (r spt; P < 0.001) in teratospermic (r spt, 184.3 ± 8.3) compared with normospermic (r spt, 117.6 ± 8.4) males. Interestingly, the total number of Sertoli cells was lower (P < 0.001) in teratospermic (13.7 ± 0.3) compared with normospermic (19.4 ± 1.1) donors (Fig. 2a). Consequently, the ratios of spermatogonia, spermatocytes, and round spermatids to Sertoli cells (r spt/Sc; N, 6.2 ± 0.4; T, 13.4 ± 0.5) were consistently greater in teratospermic cats (for all cell/cell ratios, P < 0.001) and these differences increased with proceeding germ cell development (Fig. 2b). Overall, the seminiferous tubules of the teratospermic male testis contained almost 100% more (P < 0.001) germ cells/Sertoli cell (24.7 ± 0.8) than tubules of normospermic counterparts (12.8 ± 0.8; Fig. 2b).



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  		FIG. 2. Quantification of cell populations at stage V (postspermiation) in testes of the normospermic (N) and teratospermic (T) domestic cat. A) Number of cells per tubule cross-section, (B) ratio of spermatogenic cells/ Sertoli cell. Sc, Sertoli cells; sg, spermatogonia; spc, spermatocytes; r spt, round spermatids. Values are expressed as mean ± SEM. Superscripts indicate significant differences between normospermic and teratospermic cat populations (* P < 0.05; ** P < 0.01; *** P < 0.001)

Quantification of cell populations at spermiation (stage IV, Fig. 1) revealed results similar to those described before for postspermiation (data not shown). Here, in concordance with the round spermatid/Sertoli cell ratio in stage V, ratio of elongated spermatids to Sertoli cells also was about twice as high (P < 0.001) in teratospermic (10.3 ± 1.3) compared with normospermic (5.7 ± 0.8) cats. The resulting germ cell load of Sertoli cells amounted to 19.8 ± 1.9 and 28.0 ± 2.7 for normospermic and teratospermic males, respectively.

The ratio of round spermatids per pachytene spermatocyte (meiotic index) in stage V was higher in teratospermic (3.4 ± 0.1) compared with normospermic (2.6 ± 0.2) cats, indicating lower germ cell loss in teratospermic males (15% versus 35%) during the two meiotic divisions.

Numbers of round spermatids, elongated spermatids, and all germ cells per tissue area and volume unit represent parameters of spermatogenesis efficiency. These traits were notably higher in teratospermic compared with normospermic cats (Table 5). Potential sperm output, considering both spermatogenic efficiency of testis parenchyma (Table 5) and total testis volume (Table 3), was estimated for both groups. In a simplified model calculation, the higher values of teratospermic males for both traits (131.2% and 135.1%) resulted in an expected, elevated sperm production of 177% compared with normospermic cats.


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  		TABLE 5. Spermatogenic efficiency of normospermic versus teratospermic domestic cats: calculation of cells/mm2.a

Flow-Cytometric Analysis of Spermatogenesis

DNA flow cytometry revealed that, within tissue aliquots, the percentages of all cell fractions differed between the two cat populations (Table 6), leading to higher total spermatogenic transformation (P < 0.001) and meiotic transformation (P < 0.001) for the teratospermic group. Samples obtained from teratospermic donors contained a higher proportion of haploid cells (P < 0.001) than those from the normospermic cats. In contrast, percentages of diploid cells (P < 0.01), S-phase (P < 0.05), and tetraploid cells (P < 0.01) were lower in the teratospermic group.


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  		TABLE 6. Portions of haploid (1C), diploid (2C), S-phase, and tetraploid (4C) cells, total spermatogenic transformation (1C : 2C), and meiotic index (1C : 4C) in testis of normospermic versus teratospermic domestic cats as revealed by flow cytometry (mean ± SEM)

Testicular Testosterone

Testosterone concentration per gram of testis parenchyma tended to be lower in the teratospermic (893 ± 176 ng) compared with the normospermic (1239 ± 578 ng) group, but the difference was not significant. However, the average content per testis was at the same level (normospermic, 1.62 ± 0.43 µg/testis; teratospermic, 1.58 ± 0.22 µg/testis).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first study demonstrating a potential link between teratozoospermia and testicular dysfunction in felids based on detailed examinations of spermatogenesis in two distinct cat populations characterized by their sperm morphology. The most significant findings of our study were that higher sperm output in teratospermic cats was achieved at the expense of overall sperm quality and was associated with a germ cell overload of Sertoli cells.

Our results of morphology assessments of sperm in normo- versus teratospermic donors concurred with previous reports [2, 6, 8]. Although we noticed that in teratospermic males sperm concentration was higher and proportion of structurally normal sperm appeared even lower than in earlier reports. Predominant abnormalities, however, were identical [2, 6, 8]. The most striking finding in the teratospermic donors was the presence of a high proportion of sperm with a retained cytoplasmic droplet, which may have been indicative of impaired spermiation [33]. Our sperm morphology data also suggested that midpiece and flagellum angulations were secondary defects caused by the persisting droplet, as also proposed for the mouse [34]. By contrast, epididymal sperm isolated here from normospermic donors was better quality than described elsewhere [24], probably due to our use of a medium with an osmolality (450 mOsm) that more closely mimicked that within the epididymis (unpublished results). We also demonstrated that ejaculate and epididymal sperm traits in the teratospermic cat were similar, indicating that the latter can be used for spermatology studies when raw ejaculate is unavailable.

Gross histomorphometric traits were similar in both cat populations and within the range of previous reports [30, 35–38], indicating a normal testis structure in both groups. However, highly significant was the finding that the teratospermic cat has larger testes, eventually leading to ~40% increase in total tubule volume. Thus, one reason for the higher sperm output in these males seems to be more sperm-producing tissue.

Stages of the seminiferous epithelium cycle were characterized according to Pier [30]. A similar system was recently presented by Franca and Godinho [35]. Relative stage frequencies obtained were within the range of earlier published values for the cat [30, 35]. However, frequency of the spermiation stage was higher in the teratospermic than normospermic male, apparently also reflected by an opposing observation for the postspermiation stage. Because a higher frequency corresponds to a longer duration of a stage, the remarkable shift of stage frequencies for the teratospermic males indicated prolonged (and/or delayed) spermiation. Spermatogenesis is a genetically determined process [39], which normally is rigidly timed. Its species-specific duration with all successive steps has been found to be invariant, meaning that spermatogenic cycle length [40] and stage frequencies [41, 42] cannot easily be influenced. However, other authors have shown that drugs can alter the frequency of particular stages of spermatogenesis [43–45]. Spermiation, as the final step in this process, is most vulnerable to perturbations, such as inadequate hormonal support or stress exposure (e.g., heat, chemicals, or vitamin A-deficiency) [20, 33, 46]. There also are recent reports of genetically determined disturbances, including an overexpression or absence of expression of particular genes in testicular cells. For example, the overexpression of {gamma}-tubulin disrupts the Sertoli cell microtubule network, causing impaired spermatid release [47]. Normally, such unliberated cells are phagocytosed by Sertoli cells [20, 33]. In the case of the teratospermic cat, we suspect that the high proportion of sperm with a persisting droplet indicates defective cytoplasmic elimination and phagocytosis of retained spermatids. This and prolonged spermiation process might be caused by insufficient functional competence of Sertoli cells.

The total number of Sertoli cells per tubule cross-section in teratospermic males was higher than described for another cat population [37], but was significantly less than in the normospermic controls. It is well documented that each Sertoli cell has a species-specific, limited capacity for supporting a certain number of germ cells through development into spermatozoa [20, 48]. For the rat [49, 50] and human [51], there is a strong positive correlation between Sertoli cell number and sperm production as well as testicular weight in adulthood. By contrast, our quantification of cell populations at the spermiation stage revealed that teratospermic males produced more elongated spermatids with fewer Sertoli cells, resulting in an elongated spermatids: Sertoli cell ratio almost twice that of normospermic counterparts. The disparity became even more pronounced with the observation of a round spermatids:Sertoli cell ratio in teratospermic males at the postspermiation stage that was more than twice as high as in normospermic donors. Thus, agreeing with Franca and Godinho [35], Sertoli cell efficiency in the normospermic domestic cat appeared rather low. However, for the teratospermic male, this value was one of the highest measured in any mammal to date [20, 21]. Additionally, teratospermic cats produced more round spermatids (stage V) and accordingly more elongated spermatids (stage IV) per square millimeter. Combining these observations, we concluded that the teratospermic domestic cat has an intensified spermatogenesis per Sertoli cell and per tissue unit and a higher total tubule volume than the normospermic donor. The simplified calculation of ~77% higher sperm production in the teratospermic male corresponded to the high sperm concentration and total sperm count in these samples and was in remarkable concordance with the ~70% higher total sperm number per ejaculate already noted in teratospermic cats from this same colony [8].

Obviously, the teratospermic cat has high sperm output, but at the expense of sperm quality. Because, in a teratospermic male, fewer Sertoli cells had to support more germ cells (observed in both of the examined stages), they might be simply overtaxed. Sertoli cells play a crucial role in complex spermatogenic processes, mediating endocrine signals by autocrine and paracrine factors and exerting extensive communications with germ cells [52]. Sertoli cells also produce key regulator molecules affecting testicular germ cell proliferation and differentiation as well as Sertoli and Leydig cell functions [53, 54]. An overtaxation of Sertoli cells and an inability to provide all necessary regulatory factors could provoke a suboptimal milieu for normal germ cell development. It also is possible that Sertoli cells are dysfunctional in teratospermic males, which causes impaired spermatogenesis [48]. For example, the loss of adhesion molecules at Sertoli-spermatid junctions in the mouse leads to severe sperm malformations and infertility [55].

Why such high numbers of predominantly malformed sperm reach the epididymis and eventually the ejaculate in the cat is unknown. During normal spermatogenesis, overproliferation is avoided by selective apoptosis to adjust germ cell numbers to the supportive capacity of the Sertoli cells [56]. Recent findings in the human suggest that ejaculated, structurally aberrant spermatozoa also experiencing irregular biochemical functions or nuclear DNA damage (all described in the teratospermic cat [6]) fail to be eliminated due to an abortive apoptotic mechanism [56]. Testicular germ cell apoptosis is known to occur continuously throughout the life of a male at certain stages of the spermatogenic cycle [56–58]. In the cat, Franca and Godinho [35] observed a 30% cell loss during the two meiotic divisions. Indeed, from examining ratios of round spermatids per pachytene spermatocyte, we found a 35% cell loss in normospermic, but only a 15% reduction in the teratospermic male. DNA flow cytometry confirmed histomorphometric results by revealing the same alterations of cell ratios between the two cat populations. Most importantly, both histomorphometry and flow cytometry indicated that the teratospermic cat produced about 30% more spermatids per pachytene spermatocyte than normospermic males, an amount that matched the average percentage of spermatozoa normally eliminated during the meiotic divisions [35, 59]. Therefore, we propose that, in addition to compromised phagocytosis, disturbed germ cell apoptosis during spermatogenesis also may be contributing to teratozoospermia. Preliminary cDNA microarray analysis of testis from these two cat populations has revealed a difference in expression of multiple genes, some of which are involved in regulating apoptosis [60].

In contrast with sperm production, there were no differences in testicular testosterone content between the two sperm-donor types. To our knowledge, intratesticular testosterone has not been measured in felids. Howard et al. [8] earlier reported that mean serum testosterone was lower in teratospermic than normospermic domestic cats. In contrast, Brown et al. [61] compared ejaculate characteristics, testosterone secretion, and gonadotropin receptor concentrations in two free-living populations of lions (an outbred normospermic group versus an inbred teratospermic group) and found no link between peripheral testosterone concentration and proportions of morphologically abnormal sperm. Our results here would suggest that testosterone is playing no discernible role in contributing to teratozoospermia. However, due to the dynamics of testosterone secretion and the great individual variations, a more detailed assessment may be worthwhile.

In summary, our findings indicate that, compared with its normospermic counterpart, the teratospermic cat has a higher sperm output that is achieved by more sperm-producing tissue, more germ cells per Sertoli cell, and reduced germ cell loss due to compromised phagocytosis and apoptosis during spermatogenesis. An increased proportion of elongated spermatids at the spermiation stage, a higher frequency of this stage, and a high percentage of sperm with a persisting cytoplasmic droplet suggest an impaired spermiation process in the teratospermic male. Gains in sperm quantity are produced at the expense of sperm quality. These results provide a basis for further exploring the causes for mutated testicular function and will eventually lead to understanding the etiology of teratozoospermia.


   ACKNOWLEDGMENTS
 
The authors thank Melody Roelke and the staff of local veterinary clinics for providing access to source material. The technical assistance from Ms. Lena May Bush, Ms. Doris Fichte, Ms. Christiane Franz, and Ms. Dagmar Viertel is appreciated.


   FOOTNOTES
 
1 Supported by the Deutsche Forschungsgemeinschaft (grant Je 163/4-1), the National Institutes of Health (K01 RR00135), and Friends of the National Zoo. Back

2 Correspondence: Katrin Neubauer, Institute for Zoo and Wildlife Research, Postfach 601103, D-10252 Berlin, Germany. FAX: ++49 30 5126104; neubauer{at}izw-berlin.de Back

Received: 19 April 2004.

First decision: 10 May 2004.

Accepted: 24 June 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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