The epididymis, an androgen-dependent organ, plays a key role in the maturation and storage of spermatozoa (Bedford,1967). In fact, the epididymal epithelium is responsible for creating the ideal conditions that make spermatozoa fertilizing and motile via absorption, secretion, synthesis, and metabolic activity (Hinton,1990; Turner,1991).
Morphological characteristics of epididymis have been extensively described in the rat (Robaire and Hermo,1988; Serre and Robaire,1998), hamster (Calvo et al.,1997,1999), goat (Goyal and Williams,1991), cat (Hart and Schoning,1984), dog (Arrighi et al.,1993; Schimming and Vicentini,2001), monkey (Ramos and Dym,1977), and human (Vendrely,1981; Bedford,1994), among others.
Because of its morphological and functional complexity, the epididymis has been divided into four segments: initial, caput, corpus, and cauda. Early studies by Reid and Cleland (1957) described the histological characteristics of the different segments of rat epididymis. Several cell types have been also identified in the epididymal epithelium—principal, apical, basal, clear, halo, and narrow cells—whose abundance and distribution vary along the epididymis (Flickinger et al.,1978; Robaire and Hermo,1988; Arrighi et al.,1993).
Several authors described changes in the morphology of the epididymis as a result of aging or artificial photoperiod. Calvo et al. (1997) reported morphological and biochemical changes in the hamster epididymis induced by short light periods. Serre and Robaire (1998) and Calvo et al. (1999) demonstrated histological and ultrastructural variations during aging in the rat and hamster, respectively. Nevertheless, little is known about changes in the epididymis in relation to the natural photoperiod and their possible influence on sperm maturation.1
The viscacha (Lagostomus maximus maximus) is a nocturnal rodent that lives in burrows forming large colonies. Adult males exhibit an annual reproductive cycle. At least three periods have been well described. The period of maximum gonadal activity (summer and autumn) is followed by a brief gonadal regression period during the short days of winter (July and August) (Guzmán et al.,1980; Scardapane et al.,1983; Piezzi et al.,1984; Dominguez et al.,1987; Fuentes et al.,1993; Mohamed et al.,1994; Muñoz,1998). The reproductive system recovers gradually during spring (recovery period). The winter regression is characterized by a significant decrease in serum testosterone concentration and in the luteinizing hormone, follicle-stimulating hormone, and prolactin receptors at the testicular level (Fuentes et al.,1991,1993). The lowest levels of circulating testosterone are consistent with the presence of Leydig cells with evident signs of degeneration (Muñoz et al.,1997). Concomitantly, the reduced seminiferous tubules show epithelial disorganization and vacuolization, a lower number of germ cells, specifically spermatids, and cells in process of degeneration (Muñoz et al.,1998). Furthermore, Sertoli cells exhibit seasonal variations in nuclear and cytoplasmic features (Muñoz et al.,2001).
Considering the precedents described above, the main goals of this work was to describe seasonal variations in the morphology and cellular population of the epididymis during the gonadal periods of maximum and minimum activity and to compare these results with those recorded at the testicular level.
MATERIALS AND METHODS
Twelve male adult viscachas (Lagostomus maximus maximus) weighing 5–7 kg were captured in their habitat near San Luis, Argentina (33° 20′ south latitude, 760 m altitude) during periods of maximum reproductive activity (summer and autumn) and gonadal regression (winter).
In San Luis, summer days have 14 hr of light and average temperature of 25°C. In winter, the light phase is 10 hr and the average temperature is 10°C. The average rainfall is 206 mm in summer and 18 mm in winter.
Samples of Tissue
Viscachas were anesthetized with Nembutal (pentobarbital) and quickly decapitated. The left testis and epididymis were surgically removed and sliced into 2–3 mm cross-sectional slabs. The epididymis was cut into four segments, i.e., the initial, caput, corpus, and cauda segments. They were fixed in Bouin's fluid, embedded in paraffin, serially sectioned at 4–5 μm, stained with hematoxylin and eosin (H&E), and examined with the light microscope (Olympus SC 35). Small blocks (2 × 2 × 2 mm) of the right epididymis were obtained from the four segments and were fixed in glutaraldehyde-formaldehyde (Karnovsky,1965) in phosphate buffer. They were postfixed in cold 2% OsO4, dehydrated in acetone, and embedded in Spurr's resin. One micrometer thick sections were cut with a Porter-Blum ultramicrotome and dyed with toluidine blue (AT) for study under the light microscope. Concomitantly, testicular morphology was evaluated to confirm gonadal activity or regression.
Sections in paraffin (4–5 μm) stained with H&E and sections in plastic (1 μm) stained with toluidine blue were examined. Six animals from each period of the reproductive cycle were studied. For each animal, 35 sections for each epididymal segment were randomly chosen. Measurements were made with a micrometer ocular using an objective of 25× and 40× (total magnification, 250× and 400×, respectively). The following measurements were performed in paraffin sections: luminal diameter, epithelium height (excluding the stereocilia), lamina propria thickness, and abundance of each cellular type. Only transverse epididymal sections were included in this study. The cellular frequency was expressed as percentage of the total population. Since the preliminary histology of the viscacha epididymis proved similar to that reported for other species, we adopted the cell classifications according to the descriptions reported by Flickinger et al. (1978), Robaire and Hermo (1988), and Arrighi et al. (1993).
Means and standard errors were calculated for all data sets. Differences between groups were evaluated using one-way analysis of variance (ANOVA) or Kruskal-Wallis test (nonparametric ANOVA) followed by pairwise comparisons using a Tukey-Kramer or Dunn test, respectively. A P value of < 0.05 was accepted as statistically significant.
The epididymis of the viscacha is a single elongated and coiled tubule that lies in subsequent surface of the testis. The epididymis has been divided into four regions. They are the initial segment located in the upper pole of the testis, caput, corpus, and cauda in the lower pole. A fibromuscular capsule (Fig. 1) surrounds the last segment.
Variations in Epididymal Length During Reproductive Seasonal Cycle
In the period of maximum gonadal activity, epididymal length reached a value of 7.41 ± 0.063 cm. In the period of gonadal regression, the length was significantly reduced to 5.27 ± 0.075 cm (P < 0.05). The epididymal corpus was the segment that showed greatest changes in length (4.3 ± 0.15 in active epididymis vs. 3.25 ± 0.13 in regressed epididymis; P < 0.05).
Variations in Luminal Diameter, Epithelial Height, and Lamina Propria Thickness During Reproductive Seasonal Cycle
The analysis of these parameters in the four segments of the epididymis corresponding to both periods is shown in Table 1. In the period of maximum testicular activity, the luminal diameter increased in the cauda, being almost threefold higher than the rest of the organ. On the other hand, the height of the epithelium decreased throughout the epididymis, while the thickness of the lamina propria did not show significant variations in the three first segments, being higher in the cauda (Fig. 9a).
Table 1. Means values ± SEM of estructural parameters in each epididymal region and in each seasonal reproductive period
Values with significant difference in regression period. I, initial segment; II, caput; III, corpus; IV, cauda.
During the period of gonadal regression, the parameters appeared significantly modified with regard to the active period. The luminal diameters of the four segments were lower in comparison with the active period. The epithelial height of the initial segment, caput, and corpus showed nonsignificant differences, while in the cauda segment, an increase was observed compared to the active period. The thickness of the lamina propria reached higher values during gonadal regression, specifically in the epididymal cauda. Therefore, the quantitative analysis of these morphological parameters suggests that, in the viscacha, the most sensitive segment to seasonal changes is the epididymal cauda, showing changes in three studied parameters (compare Figs. 9a and 10a).
Variations in Relative Cellular Distribution in Epididymal Epithelium During Reproductive Cycle
Principal, basal, clear, narrow, and halo cells were quantified throughout the epididymis of viscachas sacrificed during the active and inactive gonadal periods. The results are shown in Figure 2. In both periods, the principal cells constituted the predominant epithelial population throughout the epididymal duct. In the regression period, the epididymal caput and cauda showed a lower percentage of these cells (Fig. 2A). The basal cells did not exhibit significant quantitative variations between the two periods. During the active period, this cell type showed a higher contribution to the epithelium only in the cauda segment (Fig. 2B). The frequency of narrow cells (present only in the initial segment) and clear cells (not found in the initial segment but are present in the other epididymis segments) was significantly lower compared to the other cellular types. During gonadal regression, the clear cells increased in the epididymal corpus, showing a significant decrease in cauda (Fig. 2C). Halo cells, located at the base of the epithelium, were found in all the studied segments and showed a higher frequency in the cauda during the active period (Fig. 2D).
Light Microscopy Studies
The epididymal duct of Lagostomus maximus maximus is composed of a pseudostratified columnar epithelium with extensive stereocilia. In the period of maximum gonadal activity (Fig. 3), the predominant cellular type was the principal cell. The nuclei of these principal cells (euchromatin with evident nucleoli) were located perpendicular to the lamina propria. The apical surface of the principal cells showed long and thin stereocilia. The basal cells were at the base of the epithelium, located between principal cells, and demonstrated oval nuclei with lax chromatin. Narrow cells, found exclusively in the initial segment of the epididymis, were scarce and characterized by elongated dense nuclei and poorly developed cytoplasm. The few halo cells showed round, moderately dense nuclei and clear cytoplasm without inclusions (classic halo). The apical cells were more abundant, exhibiting nuclei in the process of mitotic division. In the lumen, detached epithelial cells and scarce spermatic masses were observed. A thin lamina propria constituted mainly of smooth muscular fibers and fibroblasts were found under the epithelium.
In the regression period (Fig. 4), cells in the process of desquamation were observed. The principal cells, in regressed animals, showed thinner nuclei and grouped stereocilia. Anisocariosis was observed at the level of principal and basal cells. The apical cells were less frequent. Few classical halo cells were observed at the base of the epithelium. The number of narrow cells was reduced. Significant changes were not observed in lamina propria of the epididymal duct. No major change was seen in the epithelium of this segment during the gonadal regression period.
During the active period, the population of principal cells showed clear elongated nuclei, located at different levels (Fig. 5). The cytoplasm had numerous vacuoles and dense bodies. Scarce clear cells were observed. The apical cells exhibited lax round nuclei with peripheral heterochromatic clusters and evident nucleoli. Another type of halo cell was observed in this segment. Cells located at the base of epithelium and filled with cytoplasmic dense bodies were classified as mature halo cells. The basal cells showed similar characteristics to those found in the initial segment. The lamina propria showed morphological differences compared to the initial segment in the active period.
In the period of gonadal regression (Fig. 6a), the epididymal caput exhibited significant morphological changes with regards to the active period. The principal and basal cells both showed small nuclei with segmentation (Fig. 6b). Cytoplasmic vacuoles were not observed in the principal cells. These cells contain large, round or elongated nuclei located at the base of the epithelium and a clear cytoplasm. Obvious changes were not seen in the morphology of the lamina propria.
During the active period, the principal cells presented elongated and large nuclei with lax chromatin and one or more evident nucleoli (Fig. 7). Abundant dense bodies were present in the supranuclear region. A reduced number of clear cells and a higher frequency of mature halo cells were observed. The basal cells showed the same characteristic as in the previous segments. In the lumen, scarce spermatic and remainder cells were observed. The lamina propria was more developed than that of the caput during the active period.
In the period of regression (Fig. 8a), significant morphologic changes were observed. The principal cells exhibited elongated nuclei, with granular chromatin close to the nuclear envelope, and evident nucleoli. Giant vacuoles and numerous dense bodies with irregular shape and size were observed in this cellular type. Clear cells were found in this segment (Fig. 8b). The lamina propria did not present changes with respect to the epididymal corpus of the active period.
In the active period, the caudal segment was lined by a short epithelium and had a more extensive lumen than that observed in the anterior segments (Fig. 9a). A higher number of halo cells than in the rest of the organ was observed. In plastic-embedded sections, this epididymal segment showed the two types of halo cells, classical and mature, both located in the base of the epithelium. Compared to the other epididymal segments, a higher number of halo cells were observed in the cauda during the active period. The principal and basal cells did not show significant morphological changes. Abundant spermatozoids and early germinal cells constituted the luminal dense spermatic mass. A thicker lamina propria was found in this segment (Fig. 9b).
In the regression period (Fig. 10a), the cauda showed a strong reduction of the lumen and a significant increment of epithelial height compared to the active period. In this segment, principal and basal cells showed evident morphological modifications. Their nucleus showed patches of condensed chromatin in association with the nuclear envelope. A decrease in halo cells was observed compared to the period of gonadal activity. The lamina propria in this segment was more developed that in the active period.
Different environmental factors, such as ambient temperature, food availability, rainfall periods, and natural photoperiod may be considered important variables that control the reproductive processes in rodents (Lofts,1975). The viscacha is an animal normally not exposed to long periods of light (Llanos and Crespo,1952). Throughout the year, the viscacha emerge from their burrows approximately at the same hour, which in summer (active period) results in a greater period of light exposure due crepuscular delay.
Numerous morphological and biochemical studies have been carried out on epididymis of different experimental animals (Goyal,1985; Goyal and Williams,1991; Arrighi et al.,1993; Schimming and Vicentini,2001). Different authors (Cooper,1986; Hermo,1988; Hinton,1990; Amann et al.,1993; Haidl et al.,1996) have analyzed the structural characteristics and epididymal participation in the fertilizing capability of the spermatozoa. Additionally, morphological changes in the epididymis during aging have been investigated (Cran and Jones,1980; Serre and Robaire,1998; Calvo et al.,1999). Nevertheless, studies referring to cyclic variations in the epididymis in animals in the wild are scarce.
The objectives of the present work were to describe, for the first time, the morphological characteristics of the epididymis of the viscacha with relation to its annual reproductive cycle and natural photoperiod.
The results obtained from the morphological study of the viscacha epididymis show that this complex organ is clearly segmented. Each segment probably performs specific functions in the capacitation and maturation of the spermatozoa and shows a specific epithelial cytoarchitecture likely conditioned by the histophysiology of the preceding segment.
These results also show that the structure of the epididymis in this rodent changes during the seasonal reproductive cycle. Specifically, the morphometric analysis of luminal diameter, epithelial height, and thickening of lamina propria allowed us to confirm that these parameters present significant seasonal variation mainly in the cauda. During the short period of gonadal regression, the epididymal cauda shows a decrease in luminal diameter, a decrease in spermatozoa population, and an increase of lamina propria thickness. Calvo et al. (1997,1999) obtained similar results in the hamster subjected to artificial short photoperiods. These changes are likely due to a decrease in testosterone levels. These results permit us to postulate that the cauda is the epididymal segment most sensitive to the changes induced by the photoperiod.
In viscacha, a pseudostratified columnar epithelium with stereocilia covers the epididymal duct. This epithelium is constituted of diverse cell types: principal, basal, narrow, apical, clear, and halo cells, as in the rat and hamster (Robaire and Hermo,1988; Calvo et al.,1999). However, the distribution, frequency, and structural characteristics differ from those described in rat (Reid and Cleland,1957; Sun and Flickinger,1979; Robaire and Hermo,1988) and hamster (Flickinger et al.,1978). As occur in rat (Sun and Flickinger,1980), hamster (Flickinger et al.,1978), and mouse (Abou-Haila and Fain-Maurel,1984), in viscacha the narrow cells have been found only in the initial segment of the epididymis. This cell type may be a precursor of clear cells, as was postulated by Sun and Flickinger (1979).
Changes in epididymal epithelium were found throughout the annual reproductive cycle of the viscacha. During the period of gonadal regression, we observed the presence of giant vacuoles in the principal cells of the epididymal corpus. Vacuoles were wider than the width of several principal cells. Serre and Robaire (1998) found vacuoles with the same characteristics in the epididymal epithelium of aging rats. According to these authors, the presence of vacuoles that neither reach the epithelium nor touch the basement membrane suggests phagocytosis of the vacuolated principal cells by neighboring cells. It is possible that a temporary disruption of the endosomal-lysosomal cycling system may be caused by functional variations in principal cells during the seasonal reproductive cycle of the viscacha.
The halo cells are found in the two periods throughout the viscacha epididymis. They have been postulated to be lymphocytes or monocytes and are believed to play a role in the immunological barrier of the male reproductive duct (Wang and Holtein,1983). In the viscacha, a significant increment in the number of mature halo cells is observed at the base of the epithelium in epididymal cauda during the period of maximum gonadal activity. The epididymal lumen probably exhibits a greater content of abnormal or foreign material that induces an increase of the population of halo cells. In aged rats, it was demonstrated that a major recruitment of immune cells into the epithelium occurs in response to increased luminal content. This cellular type provides efficient protection from, and the prevention of autoimmune reaction against, spermatozoa (Serre and Robaire,1999). Moreover, during the period of regression, a significant reduction in the number of all the cellular populations corresponding to cauda and a decrease in luminal content occur.
Changes found in the epididymis during the gonadal regression period were correlated with observations obtained previously in our laboratory. In the period of gonadal regression, the lowest levels of circulating testosterone coincide with the presence of hypotrophic Leydig cells with evident signs of nuclear and cytoplasmic degeneration and a decreased testicular concentrations of LH, FSH, and PRL receptors (Fuentes et al.,1993; Muñoz et al.,1997). Likewise, we observed that testicular and epididymal weights vary along with the seasonal reproductive cycle. The highest values were observed during the period of maximum reproductive activity followed by a rapid and significant decrease in the period of regression (Fuentes et al.,1991). The seasonal changes of the epididymal epithelium together with specific changes in the periods of maximum activity and regression in testis would confirm epididymal dependence on levels of circulating testosterone. Therefore, as has been demonstrated in other rodents, the epididymis is an androgen-dependent organ (Reid and Cleland,1957; Eleftheriou and Lucas,1974). Nevertheless, we cannot discard the participation of local epididymal factors that might modulate this process.
In summary, our results show that the epididymis of the viscacha exhibits important seasonal morphological changes in synchrony with the external environment. The epididymal corpus and cauda are the segments most responsive to seasonal cyclical variations of the external environment, in agreement with Muñoz et al. (1997,1998,2001), who demonstrated similar changes in the testis of this rodent. Previous works of Fuentes et al. (1991,1993) reported seasonal variations in the testosterone serum levels and in the concentrations of testicular LH, FSH, and PRL receptors in viscacha. Testosterone levels were higher in the active period than in period of regression. As a result, we postulate that the epididymal morphology of viscacha is determined by seasonal variation of circulating testosterone.
The authors thank Mr. J. Arroyuelo and N. Perez for their technical participation.