Ultrastructural evidence for phagocytosis of spermatozoa in the bovine rete testis and testicular straight tubules F.
Sinowatz, K.-H. Wrobel, S. Sinowatz and P. Kugler
Institutfür Anatomie der Universität Regensburg, Universitätsstraße 31, D-8400 Regensburg, West Germany
Summary. Ultrastructural examination showed that the epithelium of the bovine rete testis and the tubuli recti could phagocytose spermatozoa. Macrophages were regularly found in the basal parts of the epithelial cells and could be involved in the removal of degraded sperm material. Introduction
The rete testis occupies a comparatively extensive region in the mammalian testis. In the bull, as in many other mammalian species, it is situated in the central connective tissue core of the testis, the mediastinum testis. It consists of a series of anastomotic irregular spaces lined by a simple epithelium of varied height and appearance. The testicular straight tubules connect the semi¬ niferous tubules with the rete testis. The general morphology of bovine straight tubules and rete testis shows some remarkable specializations which have been discussed in detail by Wrobel, Sinowatz & Kugler (1978). Interest in these portions of the testis and especially in the rete was stimulated by the experiments of Voglmayr, Waites & Setchell (1966), Voglmayr, Larson & White (1970) and Setchell (1970, 1974) who have cannulated the rete testis of various mammals and collected rete testis fluid. Rete testis fluid has an electrolyte composition which is different from that of plasma, testicular lymph or fluid from seminiferous tubules. In addition rete testis fluid contains testosterone at levels which vary between the species (Cooper & Waites, 1974). The straight tubules and rete testis may therefore play a more significant role in reproduction than acting as a mere excurrent transport system for spermatozoa. The present study was performed to examine the ultrastructure of this area in the bull to see if any features could be related to function.
Materials and Methods The testes from 9 young bulls (approximately 1 5 years old) of proven fertility were used. The gonads were obtained immediately after slaughter and fixed by perfusion via the testicular artery (Wrobel et al., 1978) using the mixture described by Forssmann et al. (1977). Tissue from the mediastinum testis was removed after perfusion and post-fixed in 1% Os04 in phosphate buffer (Millonig, 1961) for 1 h. Following dehydration in a graded series of ethanol and immersion in propylene oxide the specimens were embedded in Epon-Araldite (Luft, 1973). Semi-thin sections (1 urn) were mounted on glass-slides and stained with azure II-methylene blue. Ultrathin sections were collected on unsupported copper grids and stained with uranyl acetate and lead citrate. They were examined in a Zeiss-EM 10 electron microscope.
Results of the electron micrographs provided ample evidence of phagocytosis of spermatozoa by Study the epithelium lining the distal portion of bovine straight tubules and parts of the rete testis (PI. 1, Fig. 1). Different stages of uptake and degradation of spermatozoa were frequently observed in many of the epithelial cells. In the initial step of phagocytosis the epithelial cells engulfed sperm cells by long pseudopodia-like processes (PI. 1, Fig. 2). These processes which protruded between the few microvilli of the apical cell surface contained no specific organelles. Fully ingested spermatozoa (PI. 1, Fig. 3; PI. 2, Figs 4 and 5) with only slightly damaged acrosomes were seen in the apical and supranuclear cytoplasm. However, sperm plasma membranes and acrosomal membranes were often still present and the acrosomal content was homogeneous and of normal density. Sperm heads in more advanced stages of degeneration (PI. 2, Figs 4 and 7) were restricted to the infranuclear and basal epithelial areas. These spermatozoa had lost their acrosomes and their nuclei were irregular in outline and ragged at the periphery. Besides sperm heads, the most obvious evidence of ingested spermatozoa was the presence in the cytoplasm of numerous vacuoles containing various types of cellular debris (PI. 2, Fig. 6). The mitochondrial sheath of the midpieces seemed to be broken down during the early stages of sperm degradation, but the fibrous sheath of the sperm tails seemed more resistant and was observed quite
frequently.
Besides the lining epithelium involved in phagocytosis another type of cell was identified regularly in the epithelium of straight tubules and rete testis. These cells were always confined to the basal half of the epithelium and were never seen in more apical regions. They could be distinguished easily from normal epithelial cells by their dark cytoplasm, the heterochromasy of their nuclei and the irregularity of their outlines. These cells (PI. 2, Figs 6 and 7), presumably macrophages, exhibited considerable variation in size and shape and interdigitated with pseudopodia-like processes between the epithelial cells. No desmosomes could be seen joining them to neighbouring cells, another indication for their macrophagic nature. Their dark cytoplasm contained a comparatively high number of small vacuoles and many round electrondense granules. Discussion A considerable loss of spermatozoa during their passage along the male excurrent duct system has been reported for many vertebrate species (Roussel, Stallcup & Austin, 1967; Tingari & Lake, 1971; Crabo, Gustafsson, Nicander & Rao, 1971; Scheffels & Stolla, 1975; Hoffer, Hamilton & Fawcett, 1975). In the bull the number of spermatozoa obtained by exhaustive ejaculation techniques is less than half of the estimated number of spermatozoa produced by the seminiferous tubules (Amann & Almquist, 1962). Despite the large number of investi¬ gations, the mechanism of sperm elimination in the male genital tract is poorly understood. So far, the two main routes considered are either voidance in urine or résorption by epithelial cells of the epididymis (Zankl, 1970). Sperm counts from different levels of the epididymis have led to the suggestion that a selective removal of abnormal spermatozoa takes place in this organ, but morphological evidence of phagocytosis by cells of the ductuli efferentes or epididymis has rarely been reported (Crabo et ai, 1971; Tingari & Lake, 1971).
PLATE 1
Fig. 1. Epithelial cells of the tubulus rectus of the bull showing phagocytosed spermatozoa (arrows), 6000. Figs 2 and 3. Stages in the phagocytosis of spermatozoa by cells of the tubulus rectus. Fig. 2. In the early stages, the spermatozoon is engulfed by pseudopodia-like processes of the epithelial 16 000. cells, 10 000. Fig. 3. A fully ingested sperm cell,
The results presented here give ultrastructural evidence for phagocytotic ability of the epithelium of bovine testicular straight tubules and rete testis. All steps of ingestion of spermatozoa were seen but the mechanism of degradation and the fate of the degraded material is not clear. To some extent phagocytosed spermatozoa may be destroyed by intracellular enzymic activity. The phagocytosing epithelial cells give a strong histochemical reaction for leucine aminopeptidase (Wrobel et ai, 1978), but the number of lysosomes seen with the electron microscope in the epithelial cells is comparatively low. Acrosomal enzymes of ingested spermatozoa may contribute to the intracellular sperm digestion but it seems questionable whether the concentration of these hydrolytic enzymes is sufficient to dispose of such resistant structures as sperm nuclei or the coarse fibres of the sperm tails (Hoffer et /., 1975). The macrophages observed regularly in the basal parts of the epithelial cells could be involved in the removal of degraded sperm material. Macrophages have been described in several species as playing a significant role in the disposal of ingested spermatozoa under physiological conditions (Tingari & Lake, 1971). From the data presented it is obvious that spermatozoa can be phagocytosed by epithelial cells of the bovine testicular straight tubules and rete testis but further studies are required to clarify fully the physiological significance of these observations.
This
investigation was supported in part by a grant of the Deutsche Forschungs¬ gemeinschaft. We thank Dr C. G. Pierrepoint for his helpful review of the manuscript; Miss Fallenbacher for skilled technical help; and Mrs Kugler for secretarial assistance. References Amann, R.P. & Almquist, J.O. (1962) Reproductive
capacity of dairy bulls. VI. Effect of unilateral vasectomy and ejaculation frequency on sperm reserves; aspects of epididymal physiology. /. Reprod. Feri. 3, 260-268. Cooper, T.G. & Waites, G.M.H. (1974) Testosterone in rete testis fluid and blood of rams and rats. J.
Endocr. 62,619-629. Crabo, B., Gustafsson, B., Nicander, L. & Rao, A.R.
(1971) Subnormal testicular function in a bull concealed by phagocytosis of abnormal spermatozoa in the efferent ductules. J. Reprod. Fert. 26,393-396. Forssmann, W.G., Ito, S., Weihe, E., Aoki, ., Dym, M., & Fa wee«, D.W. (1977) An improved perfusion fixation method for the testis. Anat. Ree. 188,307314. Hoffer, P.A., Hamilton, D.W. & Fawcett, D.W. (1975) Phagocytosis of spermatozoa by the epithelial cells of the ductuli efferentes after epididymal obstruction in the rat. /. Reprod. Fert. 44, 1-9. Luft, J.H. (1973) Embedding media- old and new. In Advanced Techniques in Biological Electron Microscopy, pp. 1-34. Ed. J. K. Koehler, Springer, New York.
Millonig, G. (1961) Advantages of a phosphate buffer for Os04 solutions in fixation. /. appi. Physics. 32, 1637.
Roussel, J.D., Stallcup, O.T. & Austin, C.R. (1967) Selective phagocytosis of spermatozoa in the epididymis of bulls, rabbits and monkeys. Fert. Steril. 18,509-516. Scheffels, W. & Stolla, R. (1975) Resorption von
Spermien im Nebenhoden des Ebers. Zentbl. Vet. Med. A 11, 302-312. Setchell, B.P. (1970) Fluid secretion in the testes of an Australian marsupial, Macropus eugenii. Comp. Biochem. Physiol. 36, 411-414. Setchell, B.P. (1974) Secretions of the testis and epididymis. J. Reprod. Fert. 37, 165-177. Tingari, M.D. & Lake, P.E. (1971) Ultrastructural evidence for résorption of spermatozoa and testicular fluids in the excurrent ducts of the testis of the domestic fowl, Gallus domesticus. J. Reprod. Fert. 31,373-381. Voglmayr, J.K., Waites, G.M.H. & Setchell,
B.P.
(1966)
Studies on spermatozoa and fluid collected directly from the testis of the conscious ram. Nature, Lond.
210,861-863.
PLATE 2
Fig. 4. Sperm heads during different stages of degradation in epithelial cells of the rete testis. The sperm cell in the basal cell area has lost its acrosome. 5000. Fig. 5. Spermatozoon with acrosome in the apical area of a rete testis cell, 10 000. Figs 6 and 7. Macrophages (MC) can be regularly observed in the basal parts of the cells which also contain sperm debris (arrow), 5000.
epithelial
L.H. & White, LG. (1970) Metabolism of spermatozoa and composition of fluid collected from the rete testis of living bulls. J. Reprod. Fert. 21, 449-460. Wrobel, K.H., Sinowatz, F. & Kugler, P; (1978) Zur funktioneilen Morphologie des Rete testis, der
Voglmayr, J.K., Larson,
Tubuli recti und der Terminalsegmente der Tubuli seminiferi des geschlechtsreifen Rindes. Zentbl. Vet. Med. C. Anat. Histol. Embryol. 7, 320-335. Zankl, H. (1970) Tierartliche Unterschiede der Spermienresorption im Nebenhoden. Zuchthygiene 5, 32-36. Received 16 October 1978
Sinowatz, K.-H. Wrobel, S. Sinowatz and P. Kugler
Institutfür Anatomie der Universität Regensburg, Universitätsstraße 31, D-8400 Regensburg, West Germany
Summary. Ultrastructural examination showed that the epithelium of the bovine rete testis and the tubuli recti could phagocytose spermatozoa. Macrophages were regularly found in the basal parts of the epithelial cells and could be involved in the removal of degraded sperm material. Introduction
The rete testis occupies a comparatively extensive region in the mammalian testis. In the bull, as in many other mammalian species, it is situated in the central connective tissue core of the testis, the mediastinum testis. It consists of a series of anastomotic irregular spaces lined by a simple epithelium of varied height and appearance. The testicular straight tubules connect the semi¬ niferous tubules with the rete testis. The general morphology of bovine straight tubules and rete testis shows some remarkable specializations which have been discussed in detail by Wrobel, Sinowatz & Kugler (1978). Interest in these portions of the testis and especially in the rete was stimulated by the experiments of Voglmayr, Waites & Setchell (1966), Voglmayr, Larson & White (1970) and Setchell (1970, 1974) who have cannulated the rete testis of various mammals and collected rete testis fluid. Rete testis fluid has an electrolyte composition which is different from that of plasma, testicular lymph or fluid from seminiferous tubules. In addition rete testis fluid contains testosterone at levels which vary between the species (Cooper & Waites, 1974). The straight tubules and rete testis may therefore play a more significant role in reproduction than acting as a mere excurrent transport system for spermatozoa. The present study was performed to examine the ultrastructure of this area in the bull to see if any features could be related to function.
Materials and Methods The testes from 9 young bulls (approximately 1 5 years old) of proven fertility were used. The gonads were obtained immediately after slaughter and fixed by perfusion via the testicular artery (Wrobel et al., 1978) using the mixture described by Forssmann et al. (1977). Tissue from the mediastinum testis was removed after perfusion and post-fixed in 1% Os04 in phosphate buffer (Millonig, 1961) for 1 h. Following dehydration in a graded series of ethanol and immersion in propylene oxide the specimens were embedded in Epon-Araldite (Luft, 1973). Semi-thin sections (1 urn) were mounted on glass-slides and stained with azure II-methylene blue. Ultrathin sections were collected on unsupported copper grids and stained with uranyl acetate and lead citrate. They were examined in a Zeiss-EM 10 electron microscope.
Results of the electron micrographs provided ample evidence of phagocytosis of spermatozoa by Study the epithelium lining the distal portion of bovine straight tubules and parts of the rete testis (PI. 1, Fig. 1). Different stages of uptake and degradation of spermatozoa were frequently observed in many of the epithelial cells. In the initial step of phagocytosis the epithelial cells engulfed sperm cells by long pseudopodia-like processes (PI. 1, Fig. 2). These processes which protruded between the few microvilli of the apical cell surface contained no specific organelles. Fully ingested spermatozoa (PI. 1, Fig. 3; PI. 2, Figs 4 and 5) with only slightly damaged acrosomes were seen in the apical and supranuclear cytoplasm. However, sperm plasma membranes and acrosomal membranes were often still present and the acrosomal content was homogeneous and of normal density. Sperm heads in more advanced stages of degeneration (PI. 2, Figs 4 and 7) were restricted to the infranuclear and basal epithelial areas. These spermatozoa had lost their acrosomes and their nuclei were irregular in outline and ragged at the periphery. Besides sperm heads, the most obvious evidence of ingested spermatozoa was the presence in the cytoplasm of numerous vacuoles containing various types of cellular debris (PI. 2, Fig. 6). The mitochondrial sheath of the midpieces seemed to be broken down during the early stages of sperm degradation, but the fibrous sheath of the sperm tails seemed more resistant and was observed quite
frequently.
Besides the lining epithelium involved in phagocytosis another type of cell was identified regularly in the epithelium of straight tubules and rete testis. These cells were always confined to the basal half of the epithelium and were never seen in more apical regions. They could be distinguished easily from normal epithelial cells by their dark cytoplasm, the heterochromasy of their nuclei and the irregularity of their outlines. These cells (PI. 2, Figs 6 and 7), presumably macrophages, exhibited considerable variation in size and shape and interdigitated with pseudopodia-like processes between the epithelial cells. No desmosomes could be seen joining them to neighbouring cells, another indication for their macrophagic nature. Their dark cytoplasm contained a comparatively high number of small vacuoles and many round electrondense granules. Discussion A considerable loss of spermatozoa during their passage along the male excurrent duct system has been reported for many vertebrate species (Roussel, Stallcup & Austin, 1967; Tingari & Lake, 1971; Crabo, Gustafsson, Nicander & Rao, 1971; Scheffels & Stolla, 1975; Hoffer, Hamilton & Fawcett, 1975). In the bull the number of spermatozoa obtained by exhaustive ejaculation techniques is less than half of the estimated number of spermatozoa produced by the seminiferous tubules (Amann & Almquist, 1962). Despite the large number of investi¬ gations, the mechanism of sperm elimination in the male genital tract is poorly understood. So far, the two main routes considered are either voidance in urine or résorption by epithelial cells of the epididymis (Zankl, 1970). Sperm counts from different levels of the epididymis have led to the suggestion that a selective removal of abnormal spermatozoa takes place in this organ, but morphological evidence of phagocytosis by cells of the ductuli efferentes or epididymis has rarely been reported (Crabo et ai, 1971; Tingari & Lake, 1971).
PLATE 1
Fig. 1. Epithelial cells of the tubulus rectus of the bull showing phagocytosed spermatozoa (arrows), 6000. Figs 2 and 3. Stages in the phagocytosis of spermatozoa by cells of the tubulus rectus. Fig. 2. In the early stages, the spermatozoon is engulfed by pseudopodia-like processes of the epithelial 16 000. cells, 10 000. Fig. 3. A fully ingested sperm cell,
The results presented here give ultrastructural evidence for phagocytotic ability of the epithelium of bovine testicular straight tubules and rete testis. All steps of ingestion of spermatozoa were seen but the mechanism of degradation and the fate of the degraded material is not clear. To some extent phagocytosed spermatozoa may be destroyed by intracellular enzymic activity. The phagocytosing epithelial cells give a strong histochemical reaction for leucine aminopeptidase (Wrobel et ai, 1978), but the number of lysosomes seen with the electron microscope in the epithelial cells is comparatively low. Acrosomal enzymes of ingested spermatozoa may contribute to the intracellular sperm digestion but it seems questionable whether the concentration of these hydrolytic enzymes is sufficient to dispose of such resistant structures as sperm nuclei or the coarse fibres of the sperm tails (Hoffer et /., 1975). The macrophages observed regularly in the basal parts of the epithelial cells could be involved in the removal of degraded sperm material. Macrophages have been described in several species as playing a significant role in the disposal of ingested spermatozoa under physiological conditions (Tingari & Lake, 1971). From the data presented it is obvious that spermatozoa can be phagocytosed by epithelial cells of the bovine testicular straight tubules and rete testis but further studies are required to clarify fully the physiological significance of these observations.
This
investigation was supported in part by a grant of the Deutsche Forschungs¬ gemeinschaft. We thank Dr C. G. Pierrepoint for his helpful review of the manuscript; Miss Fallenbacher for skilled technical help; and Mrs Kugler for secretarial assistance. References Amann, R.P. & Almquist, J.O. (1962) Reproductive
capacity of dairy bulls. VI. Effect of unilateral vasectomy and ejaculation frequency on sperm reserves; aspects of epididymal physiology. /. Reprod. Feri. 3, 260-268. Cooper, T.G. & Waites, G.M.H. (1974) Testosterone in rete testis fluid and blood of rams and rats. J.
Endocr. 62,619-629. Crabo, B., Gustafsson, B., Nicander, L. & Rao, A.R.
(1971) Subnormal testicular function in a bull concealed by phagocytosis of abnormal spermatozoa in the efferent ductules. J. Reprod. Fert. 26,393-396. Forssmann, W.G., Ito, S., Weihe, E., Aoki, ., Dym, M., & Fa wee«, D.W. (1977) An improved perfusion fixation method for the testis. Anat. Ree. 188,307314. Hoffer, P.A., Hamilton, D.W. & Fawcett, D.W. (1975) Phagocytosis of spermatozoa by the epithelial cells of the ductuli efferentes after epididymal obstruction in the rat. /. Reprod. Fert. 44, 1-9. Luft, J.H. (1973) Embedding media- old and new. In Advanced Techniques in Biological Electron Microscopy, pp. 1-34. Ed. J. K. Koehler, Springer, New York.
Millonig, G. (1961) Advantages of a phosphate buffer for Os04 solutions in fixation. /. appi. Physics. 32, 1637.
Roussel, J.D., Stallcup, O.T. & Austin, C.R. (1967) Selective phagocytosis of spermatozoa in the epididymis of bulls, rabbits and monkeys. Fert. Steril. 18,509-516. Scheffels, W. & Stolla, R. (1975) Resorption von
Spermien im Nebenhoden des Ebers. Zentbl. Vet. Med. A 11, 302-312. Setchell, B.P. (1970) Fluid secretion in the testes of an Australian marsupial, Macropus eugenii. Comp. Biochem. Physiol. 36, 411-414. Setchell, B.P. (1974) Secretions of the testis and epididymis. J. Reprod. Fert. 37, 165-177. Tingari, M.D. & Lake, P.E. (1971) Ultrastructural evidence for résorption of spermatozoa and testicular fluids in the excurrent ducts of the testis of the domestic fowl, Gallus domesticus. J. Reprod. Fert. 31,373-381. Voglmayr, J.K., Waites, G.M.H. & Setchell,
B.P.
(1966)
Studies on spermatozoa and fluid collected directly from the testis of the conscious ram. Nature, Lond.
210,861-863.
PLATE 2
Fig. 4. Sperm heads during different stages of degradation in epithelial cells of the rete testis. The sperm cell in the basal cell area has lost its acrosome. 5000. Fig. 5. Spermatozoon with acrosome in the apical area of a rete testis cell, 10 000. Figs 6 and 7. Macrophages (MC) can be regularly observed in the basal parts of the cells which also contain sperm debris (arrow), 5000.
epithelial
L.H. & White, LG. (1970) Metabolism of spermatozoa and composition of fluid collected from the rete testis of living bulls. J. Reprod. Fert. 21, 449-460. Wrobel, K.H., Sinowatz, F. & Kugler, P; (1978) Zur funktioneilen Morphologie des Rete testis, der
Voglmayr, J.K., Larson,
Tubuli recti und der Terminalsegmente der Tubuli seminiferi des geschlechtsreifen Rindes. Zentbl. Vet. Med. C. Anat. Histol. Embryol. 7, 320-335. Zankl, H. (1970) Tierartliche Unterschiede der Spermienresorption im Nebenhoden. Zuchthygiene 5, 32-36. Received 16 October 1978
