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Seasonal variations in the epididymis of the roe deer (Capreolus capreolus)
Animal Reproduction Science 111 (2009) 344–352
Short communication
Seasonal variations in the epididymis of the roe deer (Capreolus capreolus) Jennifer Sch¨on a,∗ , Steffen Blottner b a b
Freie Universit¨at Berlin, Institute of Veterinary Biochemistry, Oertzenweg 19b, 14163 Berlin, Germany Leibniz Institute for Zoo- and Wildlife Research (IZW), Berlin, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany
Received 29 November 2007; received in revised form 15 February 2008; accepted 13 March 2008 Available online 20 March 2008
Abstract The roe deer shows a distinct seasonal breeding pattern accompanied with significant changes in testicular structure and function during the annual cycle. It serves as a uniquely well-characterized ruminant model system to investigate the regulation of testicular activity. However, data regarding the seasonal variations taking place in the epididymis of the roe buck are not available. Therefore, this study provides a detailed morphological description of the roe buck’s epididymis (cell types and segments) and a qualitative as well as quantitative characterization of the seasonal changes in the different parts of the duct. For every second month of the complete seasonal cycle, five roe bucks were castrated (n = 30). Seasonal changes in the cellular composition of the epididymis were studied by computer aided image analysis of histological preparations. With regard to morphological criteria we defined 6 segments (S) within the epididymis (ductuli efferentes and S1–5) during the active period. S1–3 are located in the caput, 4 represents the corpus and 5 the cauda epididymidis. The epithelium consists of principal cells, basal cells, macrophages, lymphocytes and apical cells, except for the ductuli efferentes (cuboidal epithelium composed of ciliated and unciliated cells) and S5 (no apical cells). The quantification of the three functional compartments within the organ (lumina, epithelium and interstitial tissue) revealed distinct and region-specific seasonal changes in the cellular composition of caput, corpus and cauda epididymidis. As expected, the duct with its surrounding tissue expands towards rutting season. In the caput this enlargement of the duct is primarily caused by the growth of the epithelial compartment, whereas in the cauda it is predominantly attributed to the dilatation of the lumen, which is filled with testicular and epididymal fluid and spermatozoa towards the rut. This leads to distinct changes in the tissue composition of samples taken from the three main regions of the epididymis at different times of
∗
Corresponding author. Tel.: +49 30 838 62238; fax: +49 30 838 62584. E-mail address: [email protected] (J. Sch¨on).
0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.03.008
J. Sch¨on, S. Blottner / Animal Reproduction Science 111 (2009) 344–352
345
the year. This morphometric study provides the prerequisite for investigations of regulation mechanisms in epididymis function. © 2008 Elsevier B.V. All rights reserved. Keywords: Epididymis; Seasonal reproduction; Roe deer; Morphometry
1. Introduction Reproductive strategies of seasonal breeders are adaptations to annual changes in the environment and they minimize the animals’ energetic efforts for reproduction. Mature males show synchronized cycles of testicular growth and involution between breeding and non-breeding periods (Bronson and Heidemann, 1994). The roe deer is such a seasonal breeder and a uniquely well-characterized ruminant model system. It shows a short rutting season in summer (July/August) and completely arrested spermatogenesis in winter (Sempere et al., 1998; G¨oritz et al., 2003). The epididymis represents the main segment of the excurrent ducts in the reproductive tract which matures and stores spermatozoa under androgenic control (Robaire et al., 2006). It is the site where re-absorption of the testicular fluid and secretory activities occur, benefiting the maturational process of the spermatozoa (Kirchhoff et al., 1998; Cooper, 2007). It was reported that the morphology and function of the epididymis in seasonally reproducing animals change analogue to the testicular tissue (Calvo et al., 1997; Aguilera-Merlo et al., 2005). Basic data clarifying the seasonal variations in the cellular composition of the roe buck’s epididymis are not available. However, they are essential for the understanding of the reproductive physiology of the roe buck and especially for further studies of molecular regulation mechanisms in the epididymis. The aim of this study was to provide a detailed morphological picture of the roe buck’s epididymis (cell types and segments) and to describe the seasonal changes in the different parts qualitatively as well as quantitatively. 2. Materials and methods 2.1. Animals A total of 30 adult male roe bucks (Capreolus capreolus) were included between August 1998 and June 2004. Bucks were kept individually under semi-free ranging conditions in large outside enclosures. Animals were treated in accordance with the National Animal Welfare Legislation. All procedures were approved by the local Office for the Preservation of Environment and Nature, Eberswalde, Germany. For every second month of the complete seasonal cycle (February, April, June, August, October and December), five individual bucks were castrated as previously described (G¨oritz et al., 2003). 2.2. Tissue preparation Testis and epididymis were separated and samples were taken from different parts of the epididymis (caput, corpus and cauda). Samples were immersion-fixed in cold Bouin’s solution and embedded in paraffin.
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2.3. Histology and morphometry Paraffin embedded samples were sectioned at approximately 3 m and stained with Hemalum–Eosin (HE). Slides were assessed using an Olympus microscope (BX41), camera (Olympus U-CMAD3) and the AnalySIS image-analyzing system (AnalySIS). 2.3.1. General morphology in the active reproduction period To measure the epithelial height and cilia length of the different segments, 10 pictures of straight perpendicularly cut epithelium sections were taken at 600× magnification. For the epithelium of segment 1 we had to employ a 400× magnification as the cells were too high in some parts to be fully displayed in one picture. Cilia were again measured using 600× magnification. In each picture epithelium and cilia were measured 3 times. 2.3.2. Qualitative and quantitative description of seasonal changes In three to four sections per animal (obtained from different parts of the caput, corpus and cauda) digital images employing a 400× magnification (35–70 per section) were taken to quantify the proportions of the tubular and interstitial compartment. The image series were generated by microscope scans across the broadest expansion of the histological sections. In these images (25.700 m2 /image) the total area of the interstitial and luminal compartment was measured. The area of the duct epithelium results from subtracting the interstitial and luminal area from the total picture area. In these picture series the epithelial height was also measured in epithelium parts, which were perpendicularly cut. We chose to only use perfectly fixed samples (no artefacts visible), which in most cases limited the number of measurable animals per month and sample type to 3–4. 2.4. Statistical analysis For each variable, seasonal means ± standard errors of means (S.E.M.) were calculated. The Kruskal–Wallis test (non-parametric ANOVA) was used to investigate the variation among monthly means for all parameters. Subsequently, Dunn’s multiple comparisons test was performed as pair wise post hoc test. Furthermore, each epididymal variable was summarised for the spermatogenic active (June + August) and the non-active season (December + February). Differences between these two periods were evaluated by the Mann–Whitney test. Relationships between the morphometric parameters were tested by calculation of the Spearman rank correlation including all single values. The analyses were performed using the GraphPad InStat software (version 3.0). 3. Results 3.1. General morphology of the roe buck’s epididymis in the active reproduction period (June) The epididymis of the roe buck can be divided into 6 parts: ductuli efferentes (DE) and segments 1–5 (S1–5). Segments 1–3 are located in the caput, segment 4 represents the corpus and segment 5 the cauda epididymidis.
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3.1.1. Caput epididymidis The DE show a low, cuboidal epithelium composed of ciliated and unciliated cells, basal cells and few lymphocytes. The segments 1–5 consist of principal cells, basal cells, macrophages, lymphocytes and apical cells. S1 is characterized by a tall, columnar epithelium, which has an irregular height. Therefore the duct shows a stellate lumen in this segment. S2 possesses a regular lumen and slightly more apical cells than S1. Principal cells in this area are characterized by a nucleus located near the base membrane and numerous vacuoles present in the apical part of the cytoplasm. In segment 3 the nuclei of principal cells are located noticeably more central in the cytoplasm and there are remarkably less vacuoles in this cell type. 3.1.2. Corpus epididymidis In segment 4 more basal cells were found than in any other region of the epididymis. Nuclei of the principle cells often appear invaginated and contain a diffuse euchromatic nucleoplasm. In some parts of all S4 samples under examination a high endocytotic activity was to be seen, making it difficult to measure cilia length in these areas. The measured cilia length therefore represents areas where less or no endocytosis occurs. 3.1.3. Cauda epididymidis Segment 5 presents a low but still columnar epithelium with wide lumina filled up with spermatozoa. Cilia become very short in this segment and there are no apical cells in the cell composition. Nuclei of principal cells show more heterochromatin than in the other segments and exhibit prominent nucleoli. Representative pictures of the different segments are shown in Fig. 1. The morphometric characteristics of the different segments of the epididymal epithelium are indicated in Table 1.
Fig. 1. Representative histological pictures of the different segments within the epididymis of the roe buck (DE: ductuli efferentes, S: segment). On the left side, conditions shortly before the rut (active spermatogenesis, June) and on the right side during non-active phase (no spermatogenesis, February). Magnification 200×, bars indicate 50 m. Hemalum/Eosin staining. In February S2 and S3 are indistinguishable, therefore the whole segment is referred to as S2.
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Table 1 Morphometric characteristics of the different segments of the epididymal epithelium during fully active spermatogenesis in June (DE = ductuli efferentes, S = segment) Segment
Epithelial height
DE S1 high S1 low S2 S3 S4 S5
19.76 94.90 61.92 55.05 47.10 57.05 30.80
± ± ± ± ± ± ±
1.96 6.05 10.70 0.94 2.41 1.74 1.48
Cilia length 11.48 ± 0.63 18.99 ± 1.82 20.17 20.83 15.21 3.99
± ± ± ±
1.55 2.77 0.68 0.37
3.2. Morphological changes during the annual cycle 3.2.1. Qualitative evaluation Representative pictures of the seasonal changes in the different parts of the epididymis are shown in Fig. 1. Generally, the whole epithelium lining the DE and the epididymis appears more compact during non-active phases and the epithelial and luminal compartments seem to fade. In the caput, S2 and S3 are no longer distinguishable. Epithelial cells exhibit less cytoplasm and shorter cilia then during fully active sperm production. Towards highly active phases the lumen of the whole duct becomes wider. Most strikingly this can be seen in S5. In the active season the lumen of most cauda cross sections are filled up widely with spermatozoa, whereas in the inactive period spermatozoa are only sporadically observable in the cross sectioned lumina. The duct in S5 collapses during the inactive period and cross sections show a folded shape. 3.2.2. Quantitative description The proportion (in %) of the different functional compartments (lumen, epithelium, interstitial/connective tissue) within the epididymis revealed distinct variances depending on the time of year, in which the samples are taken. These seasonal changes in the composition of the three main epididymal parts are shown in Table 2. The combined data of spermatogenic inactive (December and February) and active (June and August) periods demonstrate the distinct differences between these stages (Fig. 2). This is also evident for the epithelium height (Fig. 3). 3.3. Correlations between morphometric data Relationships between the morphometric parameters are shown in Table 3. 4. Discussion The present study describes the histology of the roe buck’s epididymis qualitatively and quantitatively. The results demonstrate significant seasonal changes taking place in this organ throughout the year. In the bull, Goyal (1985) divided the epididymis into 6 segments excluding the ductuli efferentes. Segments 1–3 were located in the caput epididymis and S4 was found in the transition area between caput and corpus. Segment 5 represents the corpus and segment 6 the cauda epididymidis. Epididymis morphology of the roe buck is grossly similar to that of the bull. However, we were unable to distinguish 4 segments in the roe buck’s caput epididymidis. The seasonal
Table 2 Results of the morphometric measurements in the three main parts of the roe buck’s epididymis Epithelial height (m)a,**,A,*** 28.92b 45.93 67.24c,** 62.27c,* 48.44 42.95
± ± ± ± ± ±
2.91 12.82 12.13 4.00 9.56 5.29
Epithelial height (m)a,*,A,** Corpus February April June August October December
38.58b 60.55 63.76c,* 59.33 58.41 50.17
± ± ± ± ± ±
12.25 5.48 3.08 0.54 4.66 5.54
Epithelial height (m) Cauda February April June August October December
33.19 40.42 35.24 37.90 41.45 38.73
± ± ± ± ± ±
2.40 9.17 8.20 7.00 2.19 6.83
66.45b 37.25 25.66 22.32c,** 45.69 58.76
± ± ± ± ± ±
3.73 10.11 2.86 3.59 13.32 15.77
Interstitium (%)A,* 57.62 37.21 37.20 38.19 33.92 45.74
± ± ± ± ± ±
11.61 6.01 4.88 11.99 2.81 1.15
Interstitium (%)a,*,A,** 70.51b 48.20 55.20 37.28c,** 55.46 60.36
± ± ± ± ± ±
10.10 9.13 11.32 2.32 7.42 5.89
Lumina (%)a,* 7.31b 13.05 17.97 22.20c,** 15.34 16.80
± ± ± ± ± ±
Epithelium (%)a,**,A,***
0.90 1.40 2.13 3.77 8.47 13.65
Lumina (%)A,* 11.08 11.67 16.48 24.48 10.92 11.44
± ± ± ± ± ±
10.44 2.36 2.49 0.94 1.80 5.20
Lumina (%)a,*,A,** 5.94b 24.85 26.15 40.81c,** 19.89 13.89
± ± ± ± ± ±
4.81 9.13 9.49 9.35 0.35 0.89
26.25b 49.70 56.37c,* 56.28c,* 38.97 33.10
± ± ± ± ± ±
4.62 10.60 4.95 6.05 4.90 5.91
Epithelium (%)a,* 31.30b 51.12 46.32 37.34 55.16c,* 42.81
± ± ± ± ± ±
9.20 3.67 5.72 12.30 1.02 4.44
Epithelium (%) 21.73 26.95 18.65 22.92 24.65 25.75
± ± ± ± ± ±
5.88 15.91 10.67 7.45 7.09 6.08
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The proportions of the three compartments interstitial tissue, epithelium and lumen of the duct as well as the mean epithelial height (±S.E.M.). Indices indicate significances: a = significant differences in Kruskal–Wallis test; b to c = significant differences in Dunn’s multiple comparison test (b = reference point); A = significant in comparison non-breeding (December + February) to breeding (June + August) season evaluated by Mann–Whitney test. * Significant (p < 0.05). ** Very significant (p < 0.01). *** Highly significant (p < 0.001).
J. Sch¨on, S. Blottner / Animal Reproduction Science 111 (2009) 344–352
Caput February April June August October December
Interstitium (%)a,**,A,***
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Fig. 2. Graphic demonstration of the seasonal changes in tissue composition within the epididymis of the roe buck. Comparison of breeding (June + August) and non-breeding (December + February) period. A) caput, B) corpus, C) cauda epididymidis.
variations in the epididymis somewhat mirror the up- and down-regulation of spermatogenesis taking place in the testicular tissue of the roe buck (Sch¨on et al., 2004). The whole epididymis increases its volume towards rut. This volume increase is not as striking in corpus as in caput and predominantly in cauda epididymidis (G¨oritz et al., 2003). Towards rutting season the caput epididymidis shows an increase of epithelial height and of the overall proportion of epithelium in the tissue composition, together with swelling lumina. The connective tissue surrounding the convoluted duct is displaced by the growing duct volume (epithelium and lumen). This is reflected by the decrease of interstitial tissue per area unit, indicating that no significant seasonal changes take place in this compartment. The alterations here are caused simply by the geometric changes within the organ originating from the growing epithelium and the enlargement of the duct’s lumen. In the caput, amongst other things, the re-absorption of testicular fluid occurs, whereas the corpus
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Fig. 3. Seasonal changes in the epithelium height. Comparison of breeding (June + August) and non-breeding (December + February) period. Significant differences as revealed by Mann–Whitney test are indicated by indices (**p < 0.01, ***p < 0.001).
seems to have more secretory functions. We observed a high exocytotic activity in this part of the duct during the rutting season. In the cauda epididymidis interestingly no growth of the epithelium takes place, even if the changes of tissue composition are very pronounced in this part of the organ. Towards the rut it is filled up with fluid and spermatozoa and the epithelium unfolds. The proportions of the interstitial and the luminal compartment are highly negatively correlated. Therefore, the expansion of the duct is compensated by the compression and displacement of interstitial material. Our results show, that substantial changes take place in the cellular composition of the epididymis throughout the year and that these changes are different in the different parts of the duct. These facts have to be taken into account when starting studies concerning endoor paracrine effectors or pathways and dealing with defined sample volumes or protein concentrations. Combining this morphological knowledge with localization studies and quantifications Table 3 Spearman rank correlations between morphometric data of the main parts of the roe buck’s epididymis (including all single values, independently from season) Pair of variables
Correlation coefficient
Significance (p)
Caput % Interstitium/% lumen % Interstitium/% epihelium Epithelium height/% epithelium
−0.8208 −0.9130 0.9117
<0.001 <0.001 <0.001
Corpus % Interstitium/% lumen % Interstitium/% epihelium Epithelium height/% epithelium
−0.3766 −0.7974 0.5173
0.092 <0.001 0.020
Cauda % Interstitium/% lumen % Interstitium/% epihelium Epithelium height/% epithelium
−0.7380 −0.4873 0.8182
<0.001 0.021 <0.001
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of receptors and effectors can lead to a more exact understanding of regulatory mechanisms in the reproductive tissues of seasonal reproducing animals. 5. Conclusions The results demonstrate distinct and region-specific changes in the cellular composition of caput, corpus and cauda epididymidis, in correspondence with the changes in gametogenesis throughout the year. This morphometric quantification of tissue components in roe deer epididymis provides necessary basic data for the investigation of specific effectors and biochemical processes in the regulation of seasonal changes in epididymis function. Acknowledgements This study was supported by grant of the “Deutsche Forschungsgemeinschaft” (DFG Scho 1231/1-1).The authors thank Katharina Topp and Johannes Ahlmann-Eltze for technical assistance. References Aguilera-Merlo, C., Munoz, E., Dominguez, S., Scardapane, L., Piezzi, R., 2005. Epididymis of viscacha (Lagostomus maximus maximus): morphological changes during the annual reproductive cycle. Anat. Rec. A. Discov. Mol. Cell. Evol. Biol. 282 (January), 83–92. Bronson, F.H., Heidemann, P.D., 1994. In: Knobil, E., Neill, J.D. (Eds.), Seasonal Regulation of Reproduction in Mammals. The Physiology of Reproduction. Raven Press, New York, pp. 541–583. Calvo, A., Bustos-Obregon, E., Pastor, L.M., 1997. Morphological and histochemical changes in the epididymis of hamsters (Mesocricetus auratus) subjected to short photoperiod. J. Anat. 191, 77–88. Cooper, T.G., 2007. Sperm maturation in the epididymis: a new look at an old problem. Asian J. Androl. 9, 533–539. G¨oritz, F., Quest, M., Wagener, A., Fassbender, M., Broich, A., Hildebrandt, T.B., Hofmann, R.R., Blottner, S., 2003. Seasonal timing of sperm production in roe deer: interrelationship between changes in ejaculate parameters, morphology and function of testis and accessory glands. Theriogenology 59, 1487–1502. Goyal, H.O., 1985. Morphology of the bovine epididymis. Am. J. Anat. 172, 155–172. Robaire, B., Hinton, B., Orgebin-Crist, M., 2006. The epididymis. In: Neil, J. (Ed.), Knobile and Neill’s Physiology of Reproduction, third ed. Elsevier, San Diego, pp. 1071–1148. Kirchhoff, C., Osterhoff, C., Pera, I., Schroter, S., 1998. Function of human epididymal proteins in sperm maturation. Andrologia 30, 225–232. Sch¨on, J., G¨oritz, F., Streich, J., Blottner, S., 2004. Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment. Anat. Embryol. 208, 151–159. Sempere, A.J., Mauget, R., Mauget, C., 1998. Reproductive physiology of roe deer. In: Andersen, R., Duncan, P., Linnel, J.D.C. (Eds.), The European Roe Deer: The Biology of Success. Scand Uni Press, Oslo, pp. 161–188.
Short communication
Seasonal variations in the epididymis of the roe deer (Capreolus capreolus) Jennifer Sch¨on a,∗ , Steffen Blottner b a b
Freie Universit¨at Berlin, Institute of Veterinary Biochemistry, Oertzenweg 19b, 14163 Berlin, Germany Leibniz Institute for Zoo- and Wildlife Research (IZW), Berlin, Alfred-Kowalke-Straße 17, 10315 Berlin, Germany
Received 29 November 2007; received in revised form 15 February 2008; accepted 13 March 2008 Available online 20 March 2008
Abstract The roe deer shows a distinct seasonal breeding pattern accompanied with significant changes in testicular structure and function during the annual cycle. It serves as a uniquely well-characterized ruminant model system to investigate the regulation of testicular activity. However, data regarding the seasonal variations taking place in the epididymis of the roe buck are not available. Therefore, this study provides a detailed morphological description of the roe buck’s epididymis (cell types and segments) and a qualitative as well as quantitative characterization of the seasonal changes in the different parts of the duct. For every second month of the complete seasonal cycle, five roe bucks were castrated (n = 30). Seasonal changes in the cellular composition of the epididymis were studied by computer aided image analysis of histological preparations. With regard to morphological criteria we defined 6 segments (S) within the epididymis (ductuli efferentes and S1–5) during the active period. S1–3 are located in the caput, 4 represents the corpus and 5 the cauda epididymidis. The epithelium consists of principal cells, basal cells, macrophages, lymphocytes and apical cells, except for the ductuli efferentes (cuboidal epithelium composed of ciliated and unciliated cells) and S5 (no apical cells). The quantification of the three functional compartments within the organ (lumina, epithelium and interstitial tissue) revealed distinct and region-specific seasonal changes in the cellular composition of caput, corpus and cauda epididymidis. As expected, the duct with its surrounding tissue expands towards rutting season. In the caput this enlargement of the duct is primarily caused by the growth of the epithelial compartment, whereas in the cauda it is predominantly attributed to the dilatation of the lumen, which is filled with testicular and epididymal fluid and spermatozoa towards the rut. This leads to distinct changes in the tissue composition of samples taken from the three main regions of the epididymis at different times of
∗
Corresponding author. Tel.: +49 30 838 62238; fax: +49 30 838 62584. E-mail address: [email protected] (J. Sch¨on).
0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.03.008
J. Sch¨on, S. Blottner / Animal Reproduction Science 111 (2009) 344–352
345
the year. This morphometric study provides the prerequisite for investigations of regulation mechanisms in epididymis function. © 2008 Elsevier B.V. All rights reserved. Keywords: Epididymis; Seasonal reproduction; Roe deer; Morphometry
1. Introduction Reproductive strategies of seasonal breeders are adaptations to annual changes in the environment and they minimize the animals’ energetic efforts for reproduction. Mature males show synchronized cycles of testicular growth and involution between breeding and non-breeding periods (Bronson and Heidemann, 1994). The roe deer is such a seasonal breeder and a uniquely well-characterized ruminant model system. It shows a short rutting season in summer (July/August) and completely arrested spermatogenesis in winter (Sempere et al., 1998; G¨oritz et al., 2003). The epididymis represents the main segment of the excurrent ducts in the reproductive tract which matures and stores spermatozoa under androgenic control (Robaire et al., 2006). It is the site where re-absorption of the testicular fluid and secretory activities occur, benefiting the maturational process of the spermatozoa (Kirchhoff et al., 1998; Cooper, 2007). It was reported that the morphology and function of the epididymis in seasonally reproducing animals change analogue to the testicular tissue (Calvo et al., 1997; Aguilera-Merlo et al., 2005). Basic data clarifying the seasonal variations in the cellular composition of the roe buck’s epididymis are not available. However, they are essential for the understanding of the reproductive physiology of the roe buck and especially for further studies of molecular regulation mechanisms in the epididymis. The aim of this study was to provide a detailed morphological picture of the roe buck’s epididymis (cell types and segments) and to describe the seasonal changes in the different parts qualitatively as well as quantitatively. 2. Materials and methods 2.1. Animals A total of 30 adult male roe bucks (Capreolus capreolus) were included between August 1998 and June 2004. Bucks were kept individually under semi-free ranging conditions in large outside enclosures. Animals were treated in accordance with the National Animal Welfare Legislation. All procedures were approved by the local Office for the Preservation of Environment and Nature, Eberswalde, Germany. For every second month of the complete seasonal cycle (February, April, June, August, October and December), five individual bucks were castrated as previously described (G¨oritz et al., 2003). 2.2. Tissue preparation Testis and epididymis were separated and samples were taken from different parts of the epididymis (caput, corpus and cauda). Samples were immersion-fixed in cold Bouin’s solution and embedded in paraffin.
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2.3. Histology and morphometry Paraffin embedded samples were sectioned at approximately 3 m and stained with Hemalum–Eosin (HE). Slides were assessed using an Olympus microscope (BX41), camera (Olympus U-CMAD3) and the AnalySIS image-analyzing system (AnalySIS). 2.3.1. General morphology in the active reproduction period To measure the epithelial height and cilia length of the different segments, 10 pictures of straight perpendicularly cut epithelium sections were taken at 600× magnification. For the epithelium of segment 1 we had to employ a 400× magnification as the cells were too high in some parts to be fully displayed in one picture. Cilia were again measured using 600× magnification. In each picture epithelium and cilia were measured 3 times. 2.3.2. Qualitative and quantitative description of seasonal changes In three to four sections per animal (obtained from different parts of the caput, corpus and cauda) digital images employing a 400× magnification (35–70 per section) were taken to quantify the proportions of the tubular and interstitial compartment. The image series were generated by microscope scans across the broadest expansion of the histological sections. In these images (25.700 m2 /image) the total area of the interstitial and luminal compartment was measured. The area of the duct epithelium results from subtracting the interstitial and luminal area from the total picture area. In these picture series the epithelial height was also measured in epithelium parts, which were perpendicularly cut. We chose to only use perfectly fixed samples (no artefacts visible), which in most cases limited the number of measurable animals per month and sample type to 3–4. 2.4. Statistical analysis For each variable, seasonal means ± standard errors of means (S.E.M.) were calculated. The Kruskal–Wallis test (non-parametric ANOVA) was used to investigate the variation among monthly means for all parameters. Subsequently, Dunn’s multiple comparisons test was performed as pair wise post hoc test. Furthermore, each epididymal variable was summarised for the spermatogenic active (June + August) and the non-active season (December + February). Differences between these two periods were evaluated by the Mann–Whitney test. Relationships between the morphometric parameters were tested by calculation of the Spearman rank correlation including all single values. The analyses were performed using the GraphPad InStat software (version 3.0). 3. Results 3.1. General morphology of the roe buck’s epididymis in the active reproduction period (June) The epididymis of the roe buck can be divided into 6 parts: ductuli efferentes (DE) and segments 1–5 (S1–5). Segments 1–3 are located in the caput, segment 4 represents the corpus and segment 5 the cauda epididymidis.
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3.1.1. Caput epididymidis The DE show a low, cuboidal epithelium composed of ciliated and unciliated cells, basal cells and few lymphocytes. The segments 1–5 consist of principal cells, basal cells, macrophages, lymphocytes and apical cells. S1 is characterized by a tall, columnar epithelium, which has an irregular height. Therefore the duct shows a stellate lumen in this segment. S2 possesses a regular lumen and slightly more apical cells than S1. Principal cells in this area are characterized by a nucleus located near the base membrane and numerous vacuoles present in the apical part of the cytoplasm. In segment 3 the nuclei of principal cells are located noticeably more central in the cytoplasm and there are remarkably less vacuoles in this cell type. 3.1.2. Corpus epididymidis In segment 4 more basal cells were found than in any other region of the epididymis. Nuclei of the principle cells often appear invaginated and contain a diffuse euchromatic nucleoplasm. In some parts of all S4 samples under examination a high endocytotic activity was to be seen, making it difficult to measure cilia length in these areas. The measured cilia length therefore represents areas where less or no endocytosis occurs. 3.1.3. Cauda epididymidis Segment 5 presents a low but still columnar epithelium with wide lumina filled up with spermatozoa. Cilia become very short in this segment and there are no apical cells in the cell composition. Nuclei of principal cells show more heterochromatin than in the other segments and exhibit prominent nucleoli. Representative pictures of the different segments are shown in Fig. 1. The morphometric characteristics of the different segments of the epididymal epithelium are indicated in Table 1.
Fig. 1. Representative histological pictures of the different segments within the epididymis of the roe buck (DE: ductuli efferentes, S: segment). On the left side, conditions shortly before the rut (active spermatogenesis, June) and on the right side during non-active phase (no spermatogenesis, February). Magnification 200×, bars indicate 50 m. Hemalum/Eosin staining. In February S2 and S3 are indistinguishable, therefore the whole segment is referred to as S2.
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Table 1 Morphometric characteristics of the different segments of the epididymal epithelium during fully active spermatogenesis in June (DE = ductuli efferentes, S = segment) Segment
Epithelial height
DE S1 high S1 low S2 S3 S4 S5
19.76 94.90 61.92 55.05 47.10 57.05 30.80
± ± ± ± ± ± ±
1.96 6.05 10.70 0.94 2.41 1.74 1.48
Cilia length 11.48 ± 0.63 18.99 ± 1.82 20.17 20.83 15.21 3.99
± ± ± ±
1.55 2.77 0.68 0.37
3.2. Morphological changes during the annual cycle 3.2.1. Qualitative evaluation Representative pictures of the seasonal changes in the different parts of the epididymis are shown in Fig. 1. Generally, the whole epithelium lining the DE and the epididymis appears more compact during non-active phases and the epithelial and luminal compartments seem to fade. In the caput, S2 and S3 are no longer distinguishable. Epithelial cells exhibit less cytoplasm and shorter cilia then during fully active sperm production. Towards highly active phases the lumen of the whole duct becomes wider. Most strikingly this can be seen in S5. In the active season the lumen of most cauda cross sections are filled up widely with spermatozoa, whereas in the inactive period spermatozoa are only sporadically observable in the cross sectioned lumina. The duct in S5 collapses during the inactive period and cross sections show a folded shape. 3.2.2. Quantitative description The proportion (in %) of the different functional compartments (lumen, epithelium, interstitial/connective tissue) within the epididymis revealed distinct variances depending on the time of year, in which the samples are taken. These seasonal changes in the composition of the three main epididymal parts are shown in Table 2. The combined data of spermatogenic inactive (December and February) and active (June and August) periods demonstrate the distinct differences between these stages (Fig. 2). This is also evident for the epithelium height (Fig. 3). 3.3. Correlations between morphometric data Relationships between the morphometric parameters are shown in Table 3. 4. Discussion The present study describes the histology of the roe buck’s epididymis qualitatively and quantitatively. The results demonstrate significant seasonal changes taking place in this organ throughout the year. In the bull, Goyal (1985) divided the epididymis into 6 segments excluding the ductuli efferentes. Segments 1–3 were located in the caput epididymis and S4 was found in the transition area between caput and corpus. Segment 5 represents the corpus and segment 6 the cauda epididymidis. Epididymis morphology of the roe buck is grossly similar to that of the bull. However, we were unable to distinguish 4 segments in the roe buck’s caput epididymidis. The seasonal
Table 2 Results of the morphometric measurements in the three main parts of the roe buck’s epididymis Epithelial height (m)a,**,A,*** 28.92b 45.93 67.24c,** 62.27c,* 48.44 42.95
± ± ± ± ± ±
2.91 12.82 12.13 4.00 9.56 5.29
Epithelial height (m)a,*,A,** Corpus February April June August October December
38.58b 60.55 63.76c,* 59.33 58.41 50.17
± ± ± ± ± ±
12.25 5.48 3.08 0.54 4.66 5.54
Epithelial height (m) Cauda February April June August October December
33.19 40.42 35.24 37.90 41.45 38.73
± ± ± ± ± ±
2.40 9.17 8.20 7.00 2.19 6.83
66.45b 37.25 25.66 22.32c,** 45.69 58.76
± ± ± ± ± ±
3.73 10.11 2.86 3.59 13.32 15.77
Interstitium (%)A,* 57.62 37.21 37.20 38.19 33.92 45.74
± ± ± ± ± ±
11.61 6.01 4.88 11.99 2.81 1.15
Interstitium (%)a,*,A,** 70.51b 48.20 55.20 37.28c,** 55.46 60.36
± ± ± ± ± ±
10.10 9.13 11.32 2.32 7.42 5.89
Lumina (%)a,* 7.31b 13.05 17.97 22.20c,** 15.34 16.80
± ± ± ± ± ±
Epithelium (%)a,**,A,***
0.90 1.40 2.13 3.77 8.47 13.65
Lumina (%)A,* 11.08 11.67 16.48 24.48 10.92 11.44
± ± ± ± ± ±
10.44 2.36 2.49 0.94 1.80 5.20
Lumina (%)a,*,A,** 5.94b 24.85 26.15 40.81c,** 19.89 13.89
± ± ± ± ± ±
4.81 9.13 9.49 9.35 0.35 0.89
26.25b 49.70 56.37c,* 56.28c,* 38.97 33.10
± ± ± ± ± ±
4.62 10.60 4.95 6.05 4.90 5.91
Epithelium (%)a,* 31.30b 51.12 46.32 37.34 55.16c,* 42.81
± ± ± ± ± ±
9.20 3.67 5.72 12.30 1.02 4.44
Epithelium (%) 21.73 26.95 18.65 22.92 24.65 25.75
± ± ± ± ± ±
5.88 15.91 10.67 7.45 7.09 6.08
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The proportions of the three compartments interstitial tissue, epithelium and lumen of the duct as well as the mean epithelial height (±S.E.M.). Indices indicate significances: a = significant differences in Kruskal–Wallis test; b to c = significant differences in Dunn’s multiple comparison test (b = reference point); A = significant in comparison non-breeding (December + February) to breeding (June + August) season evaluated by Mann–Whitney test. * Significant (p < 0.05). ** Very significant (p < 0.01). *** Highly significant (p < 0.001).
J. Sch¨on, S. Blottner / Animal Reproduction Science 111 (2009) 344–352
Caput February April June August October December
Interstitium (%)a,**,A,***
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Fig. 2. Graphic demonstration of the seasonal changes in tissue composition within the epididymis of the roe buck. Comparison of breeding (June + August) and non-breeding (December + February) period. A) caput, B) corpus, C) cauda epididymidis.
variations in the epididymis somewhat mirror the up- and down-regulation of spermatogenesis taking place in the testicular tissue of the roe buck (Sch¨on et al., 2004). The whole epididymis increases its volume towards rut. This volume increase is not as striking in corpus as in caput and predominantly in cauda epididymidis (G¨oritz et al., 2003). Towards rutting season the caput epididymidis shows an increase of epithelial height and of the overall proportion of epithelium in the tissue composition, together with swelling lumina. The connective tissue surrounding the convoluted duct is displaced by the growing duct volume (epithelium and lumen). This is reflected by the decrease of interstitial tissue per area unit, indicating that no significant seasonal changes take place in this compartment. The alterations here are caused simply by the geometric changes within the organ originating from the growing epithelium and the enlargement of the duct’s lumen. In the caput, amongst other things, the re-absorption of testicular fluid occurs, whereas the corpus
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Fig. 3. Seasonal changes in the epithelium height. Comparison of breeding (June + August) and non-breeding (December + February) period. Significant differences as revealed by Mann–Whitney test are indicated by indices (**p < 0.01, ***p < 0.001).
seems to have more secretory functions. We observed a high exocytotic activity in this part of the duct during the rutting season. In the cauda epididymidis interestingly no growth of the epithelium takes place, even if the changes of tissue composition are very pronounced in this part of the organ. Towards the rut it is filled up with fluid and spermatozoa and the epithelium unfolds. The proportions of the interstitial and the luminal compartment are highly negatively correlated. Therefore, the expansion of the duct is compensated by the compression and displacement of interstitial material. Our results show, that substantial changes take place in the cellular composition of the epididymis throughout the year and that these changes are different in the different parts of the duct. These facts have to be taken into account when starting studies concerning endoor paracrine effectors or pathways and dealing with defined sample volumes or protein concentrations. Combining this morphological knowledge with localization studies and quantifications Table 3 Spearman rank correlations between morphometric data of the main parts of the roe buck’s epididymis (including all single values, independently from season) Pair of variables
Correlation coefficient
Significance (p)
Caput % Interstitium/% lumen % Interstitium/% epihelium Epithelium height/% epithelium
−0.8208 −0.9130 0.9117
<0.001 <0.001 <0.001
Corpus % Interstitium/% lumen % Interstitium/% epihelium Epithelium height/% epithelium
−0.3766 −0.7974 0.5173
0.092 <0.001 0.020
Cauda % Interstitium/% lumen % Interstitium/% epihelium Epithelium height/% epithelium
−0.7380 −0.4873 0.8182
<0.001 0.021 <0.001
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of receptors and effectors can lead to a more exact understanding of regulatory mechanisms in the reproductive tissues of seasonal reproducing animals. 5. Conclusions The results demonstrate distinct and region-specific changes in the cellular composition of caput, corpus and cauda epididymidis, in correspondence with the changes in gametogenesis throughout the year. This morphometric quantification of tissue components in roe deer epididymis provides necessary basic data for the investigation of specific effectors and biochemical processes in the regulation of seasonal changes in epididymis function. Acknowledgements This study was supported by grant of the “Deutsche Forschungsgemeinschaft” (DFG Scho 1231/1-1).The authors thank Katharina Topp and Johannes Ahlmann-Eltze for technical assistance. References Aguilera-Merlo, C., Munoz, E., Dominguez, S., Scardapane, L., Piezzi, R., 2005. Epididymis of viscacha (Lagostomus maximus maximus): morphological changes during the annual reproductive cycle. Anat. Rec. A. Discov. Mol. Cell. Evol. Biol. 282 (January), 83–92. Bronson, F.H., Heidemann, P.D., 1994. In: Knobil, E., Neill, J.D. (Eds.), Seasonal Regulation of Reproduction in Mammals. The Physiology of Reproduction. Raven Press, New York, pp. 541–583. Calvo, A., Bustos-Obregon, E., Pastor, L.M., 1997. Morphological and histochemical changes in the epididymis of hamsters (Mesocricetus auratus) subjected to short photoperiod. J. Anat. 191, 77–88. Cooper, T.G., 2007. Sperm maturation in the epididymis: a new look at an old problem. Asian J. Androl. 9, 533–539. G¨oritz, F., Quest, M., Wagener, A., Fassbender, M., Broich, A., Hildebrandt, T.B., Hofmann, R.R., Blottner, S., 2003. Seasonal timing of sperm production in roe deer: interrelationship between changes in ejaculate parameters, morphology and function of testis and accessory glands. Theriogenology 59, 1487–1502. Goyal, H.O., 1985. Morphology of the bovine epididymis. Am. J. Anat. 172, 155–172. Robaire, B., Hinton, B., Orgebin-Crist, M., 2006. The epididymis. In: Neil, J. (Ed.), Knobile and Neill’s Physiology of Reproduction, third ed. Elsevier, San Diego, pp. 1071–1148. Kirchhoff, C., Osterhoff, C., Pera, I., Schroter, S., 1998. Function of human epididymal proteins in sperm maturation. Andrologia 30, 225–232. Sch¨on, J., G¨oritz, F., Streich, J., Blottner, S., 2004. Histological organization of roe deer testis throughout the seasonal cycle: variable and constant components of tubular and interstitial compartment. Anat. Embryol. 208, 151–159. Sempere, A.J., Mauget, R., Mauget, C., 1998. Reproductive physiology of roe deer. In: Andersen, R., Duncan, P., Linnel, J.D.C. (Eds.), The European Roe Deer: The Biology of Success. Scand Uni Press, Oslo, pp. 161–188.