BIOLOGY OF REPRODUCTION 68, 15541561 (2003) Published online before print 27 November 2002. DOI 10.1095/biolreprod.102.

010652

Testis Morphometry, Seminiferous Epithelium Cycle Length, and Daily Sperm Production in Domestic Cats (Felis catus)
Luiz R. Franc a and Christiane L. Godinho1
Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 31270-901
ABSTRACT
There is very little information regarding the testis structure and function in domestic cats, mainly data related to the cycle of seminiferous epithelium and sperm production. The testis weight in cats investigated in the present study was 1.2 g. Compared with most mammalian species investigated, the value of 0.08% found for testes mass related to the body mass (gonadosomatic index) in cats is very low. The tunica albuginea volume density (%) in these animals was relatively high and comprised about 19% of the testis. Seminiferous tubule and Leydig cell volume density (%) in cats were approximately 90% and 6%, respectively. The mean tubular diameter was 220 m, and 23 m of seminiferous tubule were found per testis and per gram of testis. The frequencies of the eight stages of the cycle, characterized according to the tubular morphology system, were as follows: stage 1, 24.9%; stage 2, 12.9%; stage 3, 7.7%; stage 4, 17.6%; stage 5, 7.2%; stage 6, 11.9%; stage 7, 6.8%; and stage 8, 11 %. The premeiotic and postmeiotic stage frequency was 46% and 37%, respectively. The duration of each cycle of seminiferous epithelium was 10.4 days and the total duration of spermatogenesis based on 4.5 cycles was 46.8 days. The number of round spermatids for each pachytene primary spermatocytes (meiotic index) was 2.8, meaning that signicant cell loss (30%) occurred during the two meiotic divisions. The total number of germ cells and the number of round spermatids per each Sertoli cell nucleolus at stage 1 of the cycle were 9.8 and 5.1, respectively. The Leydig cell volume was approximately 2000 m3 and the nucleus volume 260 m3. Both Leydig and Sertoli cell numbers per gram of testis in cats were approximately 30 million. The daily sperm production per gram of testis in cats (efciency of spermatogenesis) was approximately 16 million. To our knowledge, this is the rst investigation to perform a more detailed and comprehensive study of the testis structure and function in domestic cats. Also, this is the rst report in the literature showing Sertoli and Leydig cell number per gram of testis and the daily sperm production in any kind of feline species. In this regard, besides providing a background for comparative studies with other felids, the data obtained in the present work might be useful in future studies in which the domestic cat could be utilized as an appropriate receptor model for preservation of genetic stock from rare or endangered wild felines using the germ cell transplantation technique.

Leydig cells, Sertoli cells, spermatogenesis, testis

INTRODUCTION Although there are hundreds of millions of cats maintained by man as pets, companions, and entertainers, re1

Correspondence. FAX: 55 31 34992780; e-mail: lrfranca@icb.ufmg.br

Received: 31 August 2002. First decision: 24 September 2002. Accepted: 13 November 2002. 2003 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org

search on cat reproduction clearly lags behind that of more frequently used laboratory model species and other domestic animals [1]. The knowledge of male reproductive function in the domestic cat is very important because this species is useful for biomedical research [2, 3] and may become a valuable animal model for examining the physiology of reproduction of felines [3, 4]. For instance, in vitro studies showed that the cat is useful for studies of teratospermia and sperm capacitation [2, 3, 5]. Also, studies with domestic cats are particularly relevant mainly when it is considered that most wild felines are listed as threatened or endangered species [2, 3, 6]. Spermatogenesis is a cyclic and highly coordinated process in which spermatogonia differentiate into mature spermatozoa. This highly organized and complex process encompasses different cell associations called stages, which might be classied according to the changes in the shape of the spermatid nucleus, the occurrence of meiotic divisions, and the arrangement of spermatids within the germinal epithelium [7, 8]. Also, these stages can be identied based on the development of the acrosomic system and the morphology of developing spermatids [9, 10]. The sequence of events that occurs from the disappearance of a given cellular association to its reappearance in a given area of the seminiferous epithelium constitutes the cycle of seminiferous epithelium [9]. The time interval required for one complete series of cellular associations to appear at one point within the tubule is called duration of the cycle of the seminiferous epithelium [9]. The total duration of spermatogenesis, that takes approximately 4.5 cycles, lasts from 30 to 75 days in mammals [11]. Although strain or breed differences can be found in the literature among members of the same species [10, 11], the length of the spermatogenic cycle has been generally considered to be constant for a given species [12]. It is not established yet which genes regulate the duration of spermatogenesis; however, recent work has demonstrated that the spermatogenic cycle length is under the control of germ cell genotype [13]. Most quantitative investigations of spermatogenesis require identication of the stages of seminiferous epithelium cycle and the knowledge of the cycle length [8]. There are few reports in the literature concerning the testis structure, spermatogenic process, and testis morphometry in cats [14 19]. In these studies, the seminiferous epithelium cycle length and quantitative investigation of Sertoli and Leydig cells and daily sperm production were not investigated. In this regard, the objectives of the present study were to perform a more detailed and comprehensive histologic and morphometric investigation of the testis and to estimate the spermatogenic cycle length and daily sperm production in sexually mature cats.

1554

TESTIS FUNCTION IN DOMESTIC CATS

1555

MATERIALS AND METHODS

Animals
Thirteen sexually mature domestic cats (Felis catus) were utilized in the present work. Additionally, 16 testes from sexually mature cats orchyectomized in veterinarian clinics were frozen at 20C to estimate the tunica albuginea and testis mediastinum relative weights. As sperm production in domestic cats appears unaffected by season [20, 21], the testis samples were not collected at any specic period of the year. Testes were xed by immersion (n 6) or were perfused xed (n 7) by gravityfed perfusion through the left ventricle with 0.9% saline and abdominal aorta with 45% buffered glutaraldehyde for 2530 min. Before surgery, all animals received i.m. injection of 0.4 ml of Rompum (Bayer, Leverkusen, Germany) 0.4 ml of Ketalar (Parke-Davis, Morris Plains, NJ) per kg of body weight. All surgical procedures were performed by a veterinarian and followed approved guidelines for the ethical treatment of animals.

seminiferous epithelium composition [7, 8]. This method provides eight stages of the seminiferous epithelium cycle. The relative stage frequencies were determined from the analysis of 200 seminiferous tubule cross sections per animal at 400 magnication. Both testes were analyzed for each animal. The histological sections utilized were those that presented better quality and more tubular cross sections. The duration of the spermatogenic cycle was estimated based on the stage frequencies and the most advanced germ cell type labeled at different periods postthymidine injections. The total duration of spermatogenesis took into account that approximately 4.5 cycles are necessary for this process to be completed, from type A1 spermatogonia to spermiation [24].

Cell Counts and Cell Numbers

All germ cell nuclei and Sertoli cell nucleoli present at stage 1 of the cycle were counted in 10 round or nearly round seminiferous tubule cross sections, chosen at random, for each animal. These counts were corrected for section thickness and nucleus or nucleolus diameter according to Abercrombie [25], as modied by Amann [26]. For this purpose, 10 nuclei or nucleoli diameters were measured for each cell type analyzed per animal. Cell ratios were obtained from the corrected counts obtained at stage 1. The total number of Sertoli cells was determined from the corrected counts of Sertoli cell nucleoli per seminiferous tubule cross section and the total length of seminiferous tubules according to Hochereau-de Reviers and Lincoln [27]. The daily sperm production (DSP) per testis and per gram of testis (spermatogenic efciency) were obtained according to the formula developed by Franc a [28] as follow: DSP total number of Sertoli cells per testis the ratio of round spermatids to Sertoli cells at stage 1 stage 1 relative frequency (%)/stage 1 duration (days). Individual volume of a Leydig cell was obtained from nucleus volume and the proportion between nucleus and cytoplasm. Because the Leydig cell nucleus in cats is spherical, its nucleus volume was obtained from the knowledge of the mean nuclear diameter. For this purpose, 30 nuclei showing evident nucleolus had their diameters measured for each animal. Leydig cell nuclear volume was expressed in m3 and obtained by the formula (4/3)R3, where R nuclear diameter/2. To calculate the proportion between nucleus and cytoplasm, a 441-point square lattice was placed over the sectioned material at 400 magnication. One thousand points over Leydig cells were counted for each animal. The number of Leydig cells per testis was estimated from the Leydig cell individual volume and the volume occupied by Leydig cells in the testis parenchyma.

Thymidine Injections and Tissue Preparation

To estimate the duration of seminiferous epithelium cycle, six animals received intratesticular injections of tritiated thymidine (thymidine [methyl-3H], specic activity 82.0 Ci/mmol; Amersham, Life Science, England). The injections of 150 Ci of tritiated thymidine were performed in three different regions (50 Ci in 0.5 ml) of each testis (near the epididymal head, body, and tail) using a hypodermic needle. Two animals were utilized for each time interval considered (1 h, 7 days, and 17 days) after thymidine injections. After xation by immersion, testes were trimmed out from the epididymis and weighed and cut longitudinally by hand with a razor blade. Tissue samples, measuring 13 mm in thickness, were taken near the site of thymidine injections. Testis fragments were routinely processed and embedded in plastic (glycol methacrylate). To perform autoradiographic analysis, unstained testis sections (3 m) were dipped in autoradiography emulsion (Kodak NTB-2; Eastman Kodak Company, Rochester, NY) at 45C. After drying for approximately 1 h at 25C, sections were placed in sealed black boxes and stored in a refrigerator at 4C for approximately 8 wk. Subsequently, testis sections were developed in Kodak D-19 solution at 15C [22] and stained with toluidine blue. Analyses of these sections were performed by light microscopy to detect the most advanced germ cell type labeled at different periods postthymidine injections. Cells were considered labeled when 45 or more grains were present over the nucleus in the presence of low-to-moderate background.

Statistical Analysis
All data are presented as the mean SEM. Analysis of correlation was made using the program STATISTICA for Windows (StatSoft, Inc., Tulsa, OK). The signicance level considered was P 0.05.

Testis Morphometry
To perform light microscopic investigations, testis fragments were routinely processed and embedded in plastic (glycol methacrylate). Subsequently, sections of 3-m thickness were obtained and stained with toluidine blue. The tubular diameter and the height of seminiferous tubule epithelium were measured at 160 magnication using an ocular micrometer calibrated with a stage micrometer. At least 30 tubular proles that were round or nearly round were chosen randomly and measured for each animal. The epithelium height was obtained in the same tubules utilized to determine tubular diameter. The volume densities of various testicular tissue components were determined by light microscopy using a 441-intersection grid placed in the ocular of the light microscope. Fifteen elds chosen randomly (6615 points) were scored for each animal at 400 magnication. Artifacts were rarely seen and were not considered in the total number of points utilized to obtain volume densities. Points were classied as one of the following: seminiferous tubule, comprising tunica propria, epithelium, and lumen; Leydig cell; blood and lymphatic vessels; and connective tissue. The volume of each component of the testis was determined as the product of the volume density and testis volume. For subsequent morphometric calculations, the specic gravity of testis tissue was considered to be 1.0. To obtain a more precise measure of testis volume, the testis capsule plus mediastinum were excluded from the testis weight. The total length of seminiferous tubule (meters) was obtained by dividing seminiferous tubule volume by the squared radius of the tubule times the pi value [23].

RESULTS

Biometric Data and Testis Volume Density

Stages and the Length of the Seminiferous Epithelium Cycle

Stages of the cycle in cats were characterized based on the shape and location of spermatid nuclei, presence of meiotic divisions, and overall

The mean testis weight found for the adult cat was approximately 1.2 g, providing a gonadosomatic index (testes mass divided by body weight) of approximately 0.08% (Table 1). The mean weights observed for the left and right testes were very similar (P 0.05), and no signicant correlation was observed between testis weight and body weight (r 0.36). The mean percentage found for the tunica albuginea was approximately 19%. It was not possible to observe the testis mediastinum macroscopically; in this regard, its weight was not obtained. The volume density of seminiferous tubules and Leydig cells was approximately 90% and 6%, respectively (Table 1). This means that Leydig cells occupy 50% of the intertubular compartment. In this compartment, Leydig cells are organized in clusters and the lymphatic vessels, irregularly shaped, are randomly distributed. The mean tubular diameter and epithelium height were 223 and 81 m, respectively (Table 1). Based on the volume of the testis parenchyma (testis weight minus tunica albuginea weight) and the volume occupied by seminiferous tubules in the testis and the tubular diameter, approximately 23 m of seminiferous tubules were found per testis and testis gram (Table 1). It should be mentioned that the

1556

FRANC A AND GODINHO

TABLE 1. Biometric and morphometric data in sexually mature cats. Parameter (n 9) Body weight (kg) Testis weight (g)b Right testis Left testis Gonadosomatic index (%) Testis parenchyma volume density (%): Seminiferous tubule Tunica propria Seminiferous epithelium Lumen Intertubular compartment Leydig cell Connective tissue Blood vessels Lymphatic vessels Tubular diameter (m) Seminiferous epithelium height (m) Tubular length per gram of testis (meters) Total tubular length per testis (meters)
a b

Mean SEM 3.1 1.17 1.16 1.18 0.078 88.2 4.2 78 6 11.8 6.0 2.9 2.6 0.3 223 81 23.1 22.8 0.2a 0.07a 0.09 0.10 0.007a 1.2 0.2 1.5 0.7 1.2 0.6 0.9 0.8 0.1 5 3 3.5 3.8

testis parenchyma weight was almost 1.0 g in the animals investigated in the present work. In this regard, all values expressed per testis and per gram of testis were basically the same. The total length of seminiferous tubules per testis and the Leydig cell volume density showed signicant correlation (P 0.05) with testis weight (r 0.99 and r 0.80, respectively). However, no signicant correlation (P 0.05) was observed between tubular diameter and testis weight (r 0.11).
Stages of the Seminiferous Epithelium Cycle and Relative Stage Frequencies

n 13. Right testis plus left testis divided by two. n, Number of animals utilized.

The eight stages of the cycle in cats, characterized according to the tubular morphology system, are briey described in the caption of Figure 1. The mean percentage of each stage of the seminiferous epithelium cycle was as follows: stage 1, 24.9 1.9; stage 2, 12.9 1.0; stage 3, 7.7 1.1; stage 4, 17.6 1.7; stage 5, 7.2 1.4; stage 6, 11.9 0.9; stage 7, 6.8 1.3; and stage 8, 11.0 0.9. As can be observed, stage 1 was the most frequent and stage 7 presented the lowest frequency. The frequencies of

FIG. 1. Stages 18 of the seminiferous epithelium cycle based on the tubular morphology system. Stage 1 (a) shows pachytene primary spermatocytes (P) leptotene spermatocytes (L), round spermatids (R), and Sertoli cells (S). Stage 2 (b) presents type A spermatogonia (A), zygotene spermatocytes (Z), pachytene spermatocytes (P), and elongating spermatids (E). Stage 3 (c) contains type A spermatogonia (A), zygotene spermatocytes (Z), diplotene spermatocytes (D), elongate spermatids (E), and Sertoli cells (S). Stage 4 (d) shows predominantly cells in the pachytene phase of meiosis (P), meiotic gures (M), secondary spermatocytes (II), elongate spermatids (E), and Sertoli cells (S). Stage 5 (e) contains type A spermatogonia (A), pachytene spermatocytes (P), newly formed round spermatids (R), elongate spermatids (E), and Sertoli cells (S). Stage 6 (f) presents intermediate spermatogonia (In), pachytene spermatocytes (P), round spermatids (R), elongate spermatids (E), and Sertoli cells (S). Stage 7 (g) shows type B spermatogonia (B), pachytene spermatocytes (P), round spermatids (R), elongate spermatids (E), Sertoli cells (S), and residual bodies (Rb). Stage 8 (h) shows pachytene spermatocytes (P), round spermatids (R), elongate spermatids (E), Sertoli cells (S), and residual bodies (Rb). The bar present in all panels represents 20 m.

TESTIS FUNCTION IN DOMESTIC CATS

1557

tions are shown in Table 2 and Figure 3. Approximately 1 h after injection, the most advanced labeled germ cells were identied as preleptotene spermatocytes or cells in transition from preleptotene to leptotene. These cells were present at the beginning of stage 1 and were located in the basal compartment (Fig. 3a). The most advanced germ cell type labeled 7 days after thymidine injection was pachytene spermatocyte at the end of stage 5 (Fig. 3b). At the end of this stage, the elongated spermatid bundles were less evident and were moving toward the seminiferous tubule lumen. Also, at stage 5, intermediate spermatogonia were not present yet. Labeled meiotic gures from both rst and second meiotic divisions and secondary spermatocytes present at stage 4 were observed 17 days after thymidine injection (Fig. 3c). Labeled newly formed round spermatids were also eventually observed at late stage 4. Based on the most advanced labeled germ cell type observed at each time period investigated postthymidine injections (Figs. 3 and 4) and the stages frequencies, the mean duration of the seminiferous epithelium cycle was estimated to be 10.4 0.3 days (Table 2). The duration of various stages of the cycle was determined taking into account the cycle length and the percentage of occurrence of each stage (Fig. 4). The shortest stage was stage 7 (0.71 days), while the longest stage was stage 1 (2.59 days). Considering that approximately 4.5 cycles are necessary for the spermatogenic process to be completed, the total length of spermatogenesis was estimated as being 46.8 days. The approximate duration of the spermatogonial phase was 16.4 days. While the life span of primary spermatocytes and spermatids was 16.2 and 14.2 days, respectively, the meiotic divisions, corresponding to the duration of stage 4, lasted 1.83 days.
FIG. 2. Seminiferous tubule cross sections at stage 8 of the cycle. Observe missing generation of pachytene spermatocytes (a) and round spermatids (b). A, Type A spermatogonia; S, sertoli cells; P, pachytene primary spermatocytes; R, round spermatids; E, elongate spermatids; Rb, residual bodies. The bar present in both panels represents 16 m.

Testis Morphometry

premeiotic (stages 13), meiotic (stage 4), and postmeiotic (stages 58) stages were 45.5%, 17.6%, and 36.9%, respectively. It was not uncommon to nd abnormally shaped spermatids (data not shown) and missing generations of germ cells in the domestic cat, most noticeable of more advanced cell types (Fig. 2). Seminiferous tubules showing missing generations of germ cells were not utilized for stage frequencies and cell quantication.
Seminiferous Epithelium Cycle Length

The most advanced labeled germ cell type observed at different time periods investigated after thymidine injecTABLE 2. The length (days) of seminiferous epithelium cycle (X SEM).a

Leydig cell nuclear volume and cell size were approximately 260 and 2050 m3, respectively (Table 3). The number of Leydig and Sertoli cells per testis and per gram of testis were similar and around 30 million (Table 3). No signicant correlation (P 0.05) was observed between the Sertoli cell number per testis and testis weight and seminiferous tubule volume (r 0.67 and r 0.63, respectively). However, negative and signicant correlation (P 0.05) was found between Sertoli cell number per gram of testis and tubular diameter (r 0.89). Leydig cell number per testis showed signicant correlation (P 0.05) with testis weight (r 0.83). Although high (r 0.71), the correlation between Leydig cell number and seminiferous tubule volume was not signicant (P 0.05). The meiotic index, measured as the number of round spermatids produced per pachytene primary spermatocytes, was 2.8 0.3 (data not shown). This result shows that 30% of cell loss occurs during the two meiotic divisions. The

Time after injection 70 min 6.963 daysb 16.99 daysb

a

Most advanced germ cell type labeled P1/L P M/II/R

Stage of the cycle 1 5 4

Number of cycles traversed 0.703 1.631

Cycle length Cycle length based on based on labeling intermediate labeling in preleptotene/leptotene point 9.91 10.42 10.81

P1/L, Preleptotene/leptotene primary spermatocytes; P, pachytene primary spermatocytes; M, meiotic gures from rst and second meiotic division; II, secondary spermatocytes; R, newly formed round spermatids. b Total time after thymidine injection minus 70 min.

1558
FIG. 3. Most advanced labeled germ cell type found after different time periods following intratesticular injections of tritiated thymidine. One hour after injection, preleptotene/leptotene spermatocytes (arrows) at stage 1 (a). Seven days after injection, pachytene spermatocytes (arrows) at stage 5 (b). Seventeen days after injection, secondary spermatocytes and meiotic gures (arrows) at stage 4 (c). The bar present in all panels represents 11 m.

FRANC A AND GODINHO

FIG. 4. Diagram showing the germ cell composition, the frequencies (%), and the duration in days for each stage of the seminiferous epithelium cycle. Also depicted is the most advanced germ cell type labeled at the eight stages of the cycle at the different time periods (1 h, 7 days, and 17 days) following tritiated thymidine injections. The roman numerals indicate the spermatogenic cycle. The space given to each stage is proportional to its frequency and duration. The letters within each column indicate germ cell types present at each stage of the cycle. A, Type A spermatogonia; In, intermediate spermatogonia; B, type B spermatogonia; Pl, preleptotene spermatocytes; L, leptotene; Z, zygotene; P, pachytene; D, diplotene; II, secondary spermatocytes; R, round spermatids; E, elongate spermatids.

TESTIS FUNCTION IN DOMESTIC CATS

TABLE 3. Leydig and Sertoli cell morphometry and daily sperm production.a Parameter (n 9) Nuclear diameter (m) Leydig cell volume (m3) Nuclear volume (m3) Cytoplasm volume (m3) Leydig cell number per gram of testis (106) Leydig cell number per testis (106) Sertoli cell number per gram of testis (106) Sertoli cell number per testis (106) Daily sperm production per gram of testis (106) Daily sperm production per testis (106)
a

1559

Mean SEM 7.9 2044 261 1783 30.0 29.1 32.0 31.6 15.7 15.8 0.1 149 14 159 2.9 3.8 3.9 4.1 1.6 2.5

n, number of animals utilized.

Sertoli cell efciency in cats, estimated from the total number of germ cells, and the number of round spermatids per Sertoli cell was 9.8 0.8 and 5.1 0.6, respectively (data not shown). Approximately eight primary spermatocytes at preleptotene/leptotene or pachytene phase were found per each type A spermatogonia present at stage 1 of the cycle. The daily sperm production per testis and per gram of testis in domestic cats was approximately 16 million (Table 3). The DSP per testis showed signicant correlation (P 0.05) with the total number of germ cells (r 0.84) and the number of round spermatids (r 0.84) per seminiferous tubule cross section at stage 1 of the cycle and also with the meiotic index (r 0.92). Although relatively high (r 0.79), DSP did not correlate (P 0.05) with the ratio of round spermatids per Sertoli cell. Also, no signicant correlation (P 0.05) was observed between the number of Leydig cells per testis and DSP (r 0.49).
DISCUSSION

To our knowledge, this is the rst study to perform a more comprehensive morphometric and functional investigation of the testis in the domestic cat or in any feline species. Compared with the mammalian species already investigated [29], the gonadosomatic index found for domestic cats is very low and similar to the value found for man, while the percentage occupied by the tunica albuginea in the testis is very high and also similar to the value found for men [30]. Seminiferous tubules comprise the main compartment of the testis and occupy from 70% to 90% of testis parenchyma in most mammals investigated [11, 31]. In this regard, compared with other mammalian species, the value found for this parameter in domestic cats is situated in the upper level. The value observed for tubular diameter in the present investigation is similar to that found in other reports for domestic cats [16] and is in the range cited for most mammals investigated (180350 m) [32, 33]. In general, 1015 m of seminiferous tubules are found per gram of testis parenchyma [11, 33]. However, probably due to the fact that the tubular diameter in domestic cats is not high and that almost 90% of seminiferous tubules are found in testis parenchyma, approximately 20 m of tubules per gram are observed in this species. Compared with most mammals investigated, Leydig cell size and volume density in domestic cats are not low [11, 34]. The organization of Leydig cells observed in the present work, where the lymphatic vessel is located near clusters of Leydig cells, is similar to the type II pattern according to Fawcett and collaborators [35]. Besides producing testosterone, Leydig cells secrete steroids and phero-

mones that are important for other reproductive functions such as sexual behavior and maintenance of sexual accessory gland function. For instance, it was recently demonstrated that estrogen is important for the male reproductive tract function (see review in Hess et al. [36]). It is also established in the literature that Leydig cell volume is correlated with the amount of smooth endoplasmic reticulum and with its capacity to secrete testosterone [37, 38]. However, it remains to be elucidated why very high variation in Leydig cell organization in the testis [34, 35] is observed in mammals. Also, there is very little knowledge regarding the reason why dramatic variation is observed for Leydig cell volume density (1% in rams to 35% in capybaras) and Leydig cell number per gram of testis (6 million in guinea pigs to 100 million in boars) [11, 34, 39, 40]. To our knowledge, there is only one report in the literature showing the stages of the seminiferous epithelium cycle in domestic cats [15]. In this report, eight stages were characterized according to the acrosomic system and stage frequencies were not determined. From the stage classication herein presented and from the germ cells present at each stage of the cycle, it seems that the criteria utilized by Bo hme and Pier [15] were merely an adaptation from the tubular morphology system. As mentioned for other species [4144], high variation of stage frequencies was observed among different animals investigated in the present work. It is suggested in the literature that stage frequencies grouped in premeiotic and postmeiotic phases of spermatogenesis might be phylogenetically determined among members of the same mammalian family [11, 41 44]. However, because this is the rst report in the literature regarding stage frequencies in felines, this aspect of spermatogenesis could not be comparatively analyzed. A recent study using the spermatogonial transplantation technique showed that the spermatogenic cycle length is under the control of the germ cell genotype [13]. From about 4000 mammalian species alive [45], the duration of spermatogenesis was estimated for only approximately 1% of them. To our knowledge, the spermatogenic cycle length was not determined for any feline species. The data available for the mammalian species investigated to date [46] show that the cycle length ranges from 9 to 12 days in approximately 50% of them. Predominantly, the value found for each spermatogenic cycle in these species is from 10 to 11 days. In this regard, the cycle length found for domestic cats is situated in the predominant range observed for other mammalian species investigated. It should be mentioned that, in mammals in general [11] and in cats in particular, each one of the three phases of spermatogenesis (spermatogonial, meiotic, and spermiogenic) lasts approximately one third of the entire process. Apoptosis occurs normally during specic steps of germ cell development [47, 48] and can be estimated comparing the ratio of germ cell numbers before and after a given developmental step [11, 49]. In mammals, only 2 or 3 of 10 spermatozoa are produced from differentiated type A1 spermatogonia and, as also observed in cats [17], the highest level of cell degeneration occurs during the spermatogonial proliferative phase and during meiosis [11]. In this regard, similar to what is found for most mammals [11, 48], the meiotic index observed for cats in the present work showed that 30% of cell loss occurred during the two meiotic divisions. We did not investigate the kinetics of spermatogonia in domestic cats. However, compared with other mammals investigated, such as stallions, donkeys, and rabbits [11, 46], the number of primary spermatocytes at pre-

1560

FRANC A AND GODINHO

leptotene/leptotene found per each type A spermatogonia present at stage 1 suggests that at least ve generations of differentiated spermatogonia are present in this species. According to Wildt et al. [21], the normal cat ejaculate contains 2030% abnormally shaped sperm and it is not unusual to nd males showing more than 60% sperm pleiomorphisms, which characterizes animals considered as teratospermic. The present study did not aim to investigate these aspects of germ cell development. However, it was not uncommon to nd abnormally shaped spermatids in the present investigation. Also, it was not unusual to nd missing generations of germ cells. In humans, it is considered that missing generations of germ cells contribute to the low efciency of spermatogenesis [50]. Missing generations of germ cells are also a common nding after germ cell transplantation in mice [51, 52] and are probably related to the enhanced self-renewing stem cells carry out at the cost of producing differentiating spermatogonia. Missing generations that are most often seen at early time points after colonization begins and at the ends of colonies could also be related to the failure of stem cell division or stem cell degeneration [53]. Although a recent publication has suggested that glial cell line-derived neurotrophic factor is involved with the regulation of spermatogonial stem cell selfrenewal and differentiation [54], the biology of stem cell function remains largely unknown [55, 56]. In this regard, it remains to be investigated why, in some species, including the domestic cat, missing generations of germ cells are not a rare nding. It is currently accepted that the number of Sertoli cells established during testis development determines the rate of sperm production in sexually mature animals [57, 58]. This assumption is based on the fact that each Sertoli cell supports a limited number of germ cells in a species-specic manner [11, 59]. The literature shows that spermatogenic efciency, expressed as the number of sperm produced daily per gram of testis, is usually positively correlated with the number of germ cells supported by each Sertoli cell [11, 59, 60]. Other important aspects correlated with spermatogenic efciency are the volume density of seminiferous tubules, the length of spermatogenic cycle, the number of spermatogonial generations, the rate of germ cell loss during spermatogenesis, the number of Sertoli cells per gram of testis, and the size of Sertoli cells [11, 61]. Paradoxically, the higher the Sertoli cell size is, the lower its support capacity for germ cells [31]. Also, as found in the present work, higher numbers of Sertoli cells per gram of testis among individuals from the same species correlate negatively with tubular diameter and consequently with the number of germ cells per tubule cross section. In general, the number of Sertoli cells per gram of testis found for cats is situated in an intermediate level compared with the values found for most mammalian species investigated [11, 31]. However, Sertoli cell efciency in domestic cats is one of the lowest among mammals already investigated [11, 59, 60]. In spite of that and probably due to the high seminiferous tubule volume density and the fact that the cycle length is not long in domestic cats, spermatogenic efciency in this species is moderate. The signicant and positive correlation found in the present work between DSP and the number of germ cells per seminiferous tubule cross sections and the rate of germ cell loss during meiosis showed that these parameters are good predictors of spermatogenic efciency in domestic cats. Perhaps due to the sample size utilized in the present work,

the number of spermatids per Sertoli cell showed no signicant correlation with DSP. In conclusion, the results found in the present investigation will probably provide the baseline for comparative studies with other feline species. Also, these results might help future research in which the spermatogonial transplantation technique could be utilized as a tool to better understand testis function and to preserve the genetic stock from endangered feline species.
ACKNOWLEDGMENT
Technical help from Rubens Miranda is highly appreciated.

REFERENCES
1. Concanon PW. Preface and dedication. J Reprod Fertil Suppl 1989; 39:viiviii. 2. Goodrowe KL, Howard JG, Schmidt PM, Wildt DE. Reproductive biology of the domestic cat with special reference to endocrinology, sperm function and in-vitro fertilization. J Reprod Fertil Suppl 1989; 39:7390. 3. Pukazhenthi BS, Wildt DE, Howard JG. The phenomenon and signicance of teratospermia in felids. J Reprod Fertil Suppl 2001; 57: 423433. 4. Wildt DE, Schiewe MC, Schmidt PM, Goodrowe KL, Howard JG, Phillips LG, OBrien SJ, Bush M. Developing animal model systems for embryo technologies in rare and endangered wildlife. Theriogenology 1986; 25:3351. 5. Pukazhenthi B, Pelican K, Wildt D, Howard JG. Sensitivity of domestic cat (Felis catus) sperm from normospermic versus teratospermic donors to cold-induced acrosomal damage. Biol Reprod 1999; 61: 135141. 6. CITES. Convention on International Trade in Endangered Species of Wild Flora and Fauna, part of the Endangered Species Act (PL 93-205, 93rd Congress) and in 50 append. Code Fed Reg 1973; part 23. 7. Courot M, Hochereau-de Reviers MT, Ortavant R. Spermatogenesis. In: Johnson AD, Gomes WR, Vandemark NL (eds.), The Testis, 1st ed. New York: Academic Press; 1970: 339432. 8. Berndtson WE. Methods for quantifying mammalian spermatogenesis: a review. J Anim Sci 1977; 44:818883. 9. Leblond CP, Clermont Y. Denition of the stages of the cycle of the seminiferous epithelium in the rat. Ann N Y Acad Sci 1952; 55:548 584. 10. Russell LD, Ettlin RA, Sinha-Hikim AP, Clegg ED. Histological and Histopathological Evaluation of the Testis. Vienna, IL: Cache River Press; 1990. 11. Franc a LR, Russell LD. The testis of domestic animals. In: Mart nez F, Regadera J (eds.), Male Reproduction. A Multidisciplinary Overview, 1st ed. Madrid: Churchill Livingstone; 1998: 197219. 12. Clermont Y. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renew. Physiol Rev 1972; 52: 198236. 13. Franc a LR, Ogawa T, Avarbock MR, Brinster RL, Russell LD. Germ cell genotype controls cell cycle during spermatogenesis in the rat. Biol Reprod 1998; 59:13711377. 14. Elcock LH, Schoning P. Age-related changes in the cat testis and epididymis. Am J Vet Res 1984; 45:23802384. 15. Bo hme G, Pier C. Ordnungkriterien fu r den Zyklus des Epithels der Tubuli seminiferi, nach Untersuchungen beim Kater (Felis catus). Berl Mu nch Tiera rztl Wschr 1986; 99:232236. 16. Sa nchez B, Pizarro M, Garcia P, Flores JM. Postnatal development of seminiferous tubules in the cat. J Reprod Fertil Suppl 1993; 47:343 348. 17. Sa nchez B, Pizarro M, Garcia P, Flores JM. Histological study of Leydig cells in the cat from birth to sexual maturity. J Reprod Fertil Suppl 1993; 47:349353. 18. Blanco-Rodriguez J. DNA replication and germ cell apoptosis during spermatogenesis in the cat. J Androl 2002; 23:484490. 19. Nie R, Zhou Q, Jassim E, Saunders PTK, Hess RA. Differential expression of estrogen receptors and in the reproductive tracts of adult male dogs and cats. Biol Reprod 2002; 66:11611168. 20. Spindler RE, Wildt DE. Circannual variations in intraovarian oocyte but not epididymal sperm quality in the domestic cat. Biol Reprod 1999; 61:188194.

TESTIS FUNCTION IN DOMESTIC CATS

21. Wildt DE, Brown JL, Swanson WF. Cats. In: Knobil E, Neill J (eds.), Encyclopedia of Reproduction, vol. 1. New York: Academic Press; 1999: 497510. 22. Bundy DC. Photographic emulsions and processing. In: Stumpf WE, Solomon HF (eds.), Autoradiography and Correlative Imaging, 1st ed. San Diego, CA: Academic Press; 1995: 4957. 23. Johnson L, Neaves WB. Age-related changes in the Leydig cell population, seminiferous tubules and sperm production in stallions. Biol Reprod 1981; 24:703712. 24. Amann RP, Schanbacher BD. Physiology of male reproduction. J Anim Sci 1983; 57(suppl 2):380403. 25. Abercrombie M. Estimation of nuclear populations from microtome sections. Anat Record 1946; 94:238248. 26. Amann RP. Reproductive capacity of dairy bull. III. The effect of ejaculation frequency, unilateral vasectomy and age on spermatogenesis. Am J Anat 1962; 110:4967. 27. Hochereau-de Reviers MT, Lincoln GA. Seasonal variation in the histology of the testis of the red deer, Cervus elephus. J Reprod Fertil Suppl 1978; 54:209213. 28. Franc a LR. Daily sperm production in Piau boars estimated from Sertoli cell population and Sertoli cell index. In: Proceedings of the 12th International Congress on Animal Reproduction and Articial Insemination; 1992; The Hague; 4:17161718. 29. Kenagy GJ, Trombulak SC. Size and function of mammalian testes in relation to body size. J Mammol 1986; 67:122. 30. Johnson L, Petty CS, Neaves WB. A comparative study of daily sperm production and testicular composition in humans and rats. Biol Reprod 1980; 22:12331243. 31. Russell LD, Ren HP, Sinha-Hikim I, Schulze W, Sinha-Hikim AP. A comparative study in twelve mammalian species of volume densities, volumes, and numerical densities of selected testis components, emphasizing those related to the Sertoli cell. Am J Anat 1990; l88:2l 30. 32. Roosen-Runge EC. The process of spermatogenesis in animals. Cambridge: University Press; 1977. 33. Setchell BP, Maddocks S, Brooks DE. Anatomy, vasculature, innervation, and uids of the male reproductive tract. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994: 10631175. 34. Russell LD. Mammalian Leydig cell structure. In: Payne AH, Hardy MP, Russell LD (eds.), The Leydig Cell. Vienna, IL: Cache River Press; 1996: 4396. 35. Fawcett DW, Neaves WB, Flores MN. Comparative observations on intertubular lymphatic and the organization of the interstitial tissue of the mammalian testis. Biol Reprod 1973; 9:500532. 36. Hess RA, Bunick D, Bahr J. Oestrogen, its receptors and function in the male reproductive tracta review. Mol Cell Endocrinol 2001; 178:2938. 37. Ewing LL, Zirkin BR, Cochran RC, Kromann N, Peters C, RuizBravo N. Testosterone secretion by rat, rabbit, guinea pig, dog, and hamster testes perfused in vitro: correlation with Leydig cell mass. Endocrinology 1979; 105:11351142. 38. Zirkin BR, Ewing LL, Kromann N, Cochran RC. Testosterone secretion by rat, rabbit, guinea pig, dog, and hamster testes perfused in vitro: correlation with Leydig cell ultrastructure. Endocrinology 1980; 107:18671874. 39. Paula TAR. Histometrical and functional analyses of the testis in adult capybaras (Hydrochoerus hydrochaeris) [in Portuguese]. Belo Horizonte, Brazil: Federal University of Minas Gerais; 1999. Dissertation. 40. Franc a LR, Silva VA Jr, Chiarini-Garcia H, Garcia SK, Debeljuk L. Cell proliferation and hormonal changes during postnatal development of the testis in the pig. Biol Reprod 2000; 63:16291636. 41. Franc a LR, Cardoso FM. Duration of spermatogenesis and sperm tran-

1561

42. 43. 44. 45. 46.

47. 48. 49.

50.

51. 52. 53. 54.

55. 56. 57.

58.

59. 60. 61.

sit time through the epididymis in the Piau boar. Tissue Cell 1998; 30:573582. Franc a LR, Becker-Silva SC, Chiarini-Garcia H. The length of the cycle of seminiferous epithelium in goats (Capra hircus). Tissue Cell 1999; 31:274280. Paula TAR, Chiarini-Garcia H, Franc a LR. Seminiferous epithelium cycle and its duration in capybaras (Hydrochoerus hydrochaeris). Tissue Cell 1999; 31:327334. Neves ES, Chiarini-Garcia H, Franc a LR. Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules. Biol Reprod 2002; 67:247255. Nowak RM. Walkers Mammals of the World, vols. I and II, 6th ed. Baltimore: The Johns Hopkins University Press; 1999. Neves ES. Comparative study of the testis structure and spermatogenic process in donkeys (Equus asinus) and mules (Equus mulus mulus) [in Portuguese]. Belo Horizonte, Brazil: Federal University of Minas Gerais; 2001. Dissertation. Blanco-Rodr guez J. A matter of death and life: the signicance of germ cell death during spermatogenesis. Int J Androl 1998; 21:236 248. Roosen-Runge EC. Germinal cell loss in normal metazoan spermatogenesis. J Reprod Fertil 1973; 35:339348. Matta SLP, Vilela DAR, Godinho HP, Franc a LR. The goitrogen 6-npropyl-thiouracil (PTU) given during testis development increases Sertoli and germ cell numbers per cyst in sh: the tilapia (Oreochromis niloticus) model. Endocrinology 2002; 143:970978. Johnson L, Chaturvedi PK, Willians JD. Missing generations of spermatocytes and spermatids in seminiferous epithelium contribute to low efciency of spermatogenesis in humans. Biol Reprod 1992; 47:1091 1098. Russell LD, Franc a LR, Brinster RL. Ultrastructural observations of spermatogenesis in mice resulting from transplantation of mouse spermatogonia. J Androl 1996; 17:603614. Parreira GG, Ogawa T, Avarbock MR, Franc a LR, Brinster RL, Russell LD. Development of germ cell transplants in mice. Biol Reprod 1998; 59:13601370. Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999; 60:14291436. Meng X, Lindadl M, Hyvo nen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M, Pichel JG, Westphal H, Saarma M, Sariola H. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287: 14891493. De Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Androl 2000; 21:776798. Spradling A, Drummond-Barbosa D, Kai T. Stem cells nd their niche. Nature 2001; 414:98104. Orth JM, Gunsalus GL, Lampert AA. Evidence from Sertoli cell-depleted rats indicates that spermatid number in adults depends on numbers of Sertoli cells produced during perinatal development. Endocrinology 1988; 122:787794. Hess RA, Cooke PS, Bunick D, Kirby JD. Adult testicular enlargement induced by neonatal hypothyroidism is accompanied by increased Sertoli cell and germ cell number. Endocrinology 1993; 132: 26072613. Russell LD, Peterson RN. Determination of the elongate spermatidSertoli cell ratio in various mammals. J Reprod Fertil 1984; 70:635 664. Sharpe RM. Regulation of spermatogenesis. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction. New York: Raven Press; 1994: 13631434. Johnson L. Spermatogenesis. In: Cupps PT (ed.), Reproduction in Domestic Animals, 3rd ed. New York: Academic Press, 1991: 173219.