| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular article |
a Department of Anatomy, University of Bristol, School of Veterinary Science, Bristol BS2 8EJ, United Kingdom
b Institute of Veterinary, Animal, and Biomedical Sciences, Massey University, Palmerston North, New Zealand
ABSTRACT
Contractions of seminiferous tubules and epididymal duct walls promote spermiation and sperm transfer, and they are thought to be stimulated by the related peptides oxytocin and vasopressin. This study tested the hypothesis that if oxytocin and/or vasopressin play a physiological role in sperm shedding and transport, then local or circulating concentrations of these peptides would increase during puberty. Testes, epididymides, and trunk blood of sheep at stages during the first spermatogenic wave were collected, and radioimmunoassay measured significant increases in testicular and epididymal oxytocin during spermatogenesis. No changes were measured in circulating oxytocin or in local or circulating vasopressin. Localization and synthesis was investigated by immunohistochemistry and Western blot analysis employing antibodies recognizing epitopes of either oxytocin, oxytocin-associated neurophysin, vasopressin, or vasopressin-associated neurophysin. Marked expression of both oxytocin and its associated neurophysin in testicular Leydig and epididymal principal cells was seen, and weak neurophysin immunoreactivity was also identified in Sertoli cells. The intercellular distribution of oxytocin varied between regions of the epididymis, suggesting several roles for oxytocin. Vasopressin synthesis was not apparent in either tissue. These results confirm the presence and development of paracrine oxytocinergic systems in the ram testis and epididymis of ram during puberty while questioning the physiological importance of vasopressin.
epididymis, male reproductive tract, oxytocin, puberty, sperm motility and transport, testes, vasopressin
INTRODUCTION
The transfer of immotile sperm from the testis both to and through the epididymis relies on peristaltic-like contractions of the seminiferous tubules and the epididymal duct wall [1]. Contractions of seminiferous tubules and epididymal ducts are mediated by the smooth muscle-like cell layers surrounding them. To our knowledge, no evidence suggests neuronal innervation in the smooth muscle-type myoid cells of seminiferous tubules, and minimal innervation of the caput and corpus epididymis is seen [2]. Thus, other, nonneuronal factors must mediate contractility in these areas. Indeed, although the cauda epididymis does have considerable adrenergic innervation, disruption of the sympathetic supply from the mesenteric ganglion, while reducing its rate, does not abolish sperm transport [3].
The closely related neurohypophyseal peptides oxytocin and vasopressin have been implicated in stimulating contractile activity of the male reproductive tract, with each inducing dose-dependent seminiferous tubule contraction [4, 5]. Oxytocin also increases contraction of the epididymis both in vitro [6] and in vivo [7], as do higher doses of vasopressin [7]. Furthermore, oxytocin promotes spermiation and sperm transport [8], increases the volume of fluid and the number of sperm released from the cauda epididymis [9], and increases the volume of and the sperm concentration in the ejaculate [10, 11]. Vasopressin also increases the volume of seminal fluid and the concentration of spermatozoa in the ejaculate of the rabbit [11] but not the sheep [9]. Both peptides are released from the pituitary into the circulation during coitus [12, 13], and they are possibly involved in stimulating the increase in contractions of the reproductive tract that is seen at this time. Furthermore, treatment with a specific oxytocin antagonist significantly reduces basal fluid flow in the epididymis [9], suggesting that oxytocin may be involved in the regulation of basal contractility. In the testis, oxytocin does not affect basal contractility of seminiferous tubules, but it does increase specific contractile activity immediately before and at spermiation [14]. If oxytocin and/or vasopressin play a physiological role in the shedding and transport of sperm, then local or circulating concentrations of the peptides might be expected to increase with the onset of spermatogenesis.
Both oxytocin and vasopressin are present in the testes of several mammalian species [15, 16]. Oxytocin is produced locally by the Leydig cells [17, 18] and is under the control of LH and lipoproteins, but not testosterone [19]. Testicular vasopressin synthesis is less certain. Low levels of vasopressin mRNA are present in the rat testis, but its expression does not appear to be regulated [20]. In addition, although immunocytochemical staining has demonstrated its presence in the periphery of mouse seminiferous tubules [21], its localization has not been described in other species. In the epididymis, oxytocin has been identified by radioimmunoassay [22] and localized by immunocytochemistry to principal cells of the epithelium [22, 23]. The source of this oxytocin, however, is equivocal. Veermanchaneni and Amaan [24] suggest that in the sheep, oxytocin is not synthesized by principal cells but originates from the testis and is adsorbed from the lumenal fluid. However, neither efferent duct ligation nor castration removed immunoreactivity in the principal epithelium of the rat epididymis [23] suggesting that the testis is not the sole source of epididymal oxytocin, at least in this species. To our knowledge, there have been no reports of vasopressin in the epididymis.
Oxytocin receptors are present in the Leydig and Sertoli cells of both human and primate testis [25, 26], and vasopressin receptors are present in the smooth muscle-like myoid cells of the seminiferous tubules [27]. This suggests that both vasopressin and oxytocin may stimulate tubule contraction via the vasopressin receptor, and that vasopressin is the more important physiologically. However, studies using specific agonists and antagonists of both peptides have revealed that the effects of oxytocin are probably mediated by a receptor that differs from the classical arginine vasopressin-V1
and uterine oxytocin receptor [5]. Receptors for both peptides have been identified in the pig epididymis [28], but only the localization of oxytocin receptors to the epithelial and stromal tissue of the primate epididymis has been described [25, 26].
To gain a solid basis on which to discuss the physiological importance of neurohypophyseal peptides in male reproduction, it is essential to clarify some of the anomalies that exist. The hypotheses that local concentrations of a physiologically important peptide will be maximal at the time of spermiation and that it is produced locally and in areas correlating with observed biological actions are central to our understanding. To our knowledge, this study describes for the first time testicular, epididymal, and circulating concentrations of oxytocin and vasopressin in the ram during the first wave of spermatogenesis. Furthermore, it augments previous studies by investigating the localization and production of both peptides in these tissues.
MATERIALS AND METHODS
Animals
Thirty-one Poll-Dorset cross rams between 2 and 7 mo of age were used in these experiments. Individuals were removed from the flock during the months May–October. The flock was grazing in pasture in the Bristol (United Kingdom) area. After removal, rams were housed under photoneutral conditions (12L:12D) for a maximum of 24 h, with free access to hay and water, before death. After death, which was induced by injection of 150 mg·kg-1 sodium pentobarbitol (Euthatol; Rhone Mérieux, Harlow, Essex), testes and epididymides were removed, weighed, and the equatorial circumference of the testes measured immediately. Tissue was then processed as described later. Investigations were conducted in accordance with the Home Office Animals (Scientific Procedures) Act 1986 of the United Kingdom.
Sperm Counts
One gram of tissue from the caput epididymis of each ram was removed and minced in 1 ml of 0.9% (w/v) NaCl according to the method described by Taylor et al. [29]. Sperm were counted in a hemocytometer, with the average count of 10 individual 0.1 µm3 divisions for three separate aliquots of each suspension being determined, giving a count variation of <5%.
Histological Determination of Stages of the Initial Spermatogenic Cycle
Samples of testicular tissue were fixed (24 h) in modified Bouin's solution [30], dehydrated, and embedded in paraffin wax. Thin sections (thickness, 5 µm) were cut and stained with hematoxylin and eosin. The stage of spermatogenesis in each ram was assessed according to the appearance of predetermined landmarks in the seminiferous tubules. Prepubertal stages (Pre) were determined as being those with only type A spermatogonia (n = 3). The appearance of pachytene spermatocytes was used to define the early pubertal stage (PS, n = 7), followed by the presence of early spermatids (ES, n = 6) and, subsequently, late spermatids (LS, n = 7) as the cytoplasm remodeled and moved away from the flagellum during spermatid elongation. Full spermatogenesis (FS) was assigned to those samples with residual bodies, indicating that spermiation had occurred (n = 8).
Oxytocin and Vasopressin Measurements
Testicular and epididymal tissue were frozen on dry ice within 5 min of death and stored at -80°C. Tissue samples were weighed and then extracted by homogenization in 10 ml·g-1(wet wt) acid extraction solution (1 mol·L-1 HCl containing 1% [w/v] NaCl, 5% [v/v] formic acid, and 15% trifluoroacetic acid) on ice. Samples were left overnight at 4°C, centrifuged at 4000 x g, and the supernatants further purified using C18 octadecyl silica cartridges (SepPak: Waters Corporation, Milford, MA) [31]. The oxytocin content of extracts was measured by a specific radioimmunoassay using antiserum 85/2 (cross-reacts < 0.001% with vasopressin, minimal level of detection of 2.5 pg·g-1 of tissue) [18]. The radioimmunoassay for vasopressin used the 84/1 antisera (cross-reacts < 1% with oxytocin , minimum limit of detection of 5 pg·g-1 of tissue) [32]. For each peptide, samples were assayed in one assay, with the intra-assay variations being 6.9% for oxytocin and 3.5% for vasopressin.
Immunocytochemical Localization of Testicular and Epididymal Oxytocin and Vasopressin
Immunocytochemistry was performed on 5-µm sections of testes and epididymides using the oxytocin antiserum VA10 (donated by Dr. H. Gainer, NICHHD, Bethesda, MD) [33], which recognizes amidated oxytocin and C-terminally extended biosynthetic precursors, or the rabbit antivasopressin antiserum 84/2 (raised in the Department of Anatomy, University of Bristol, UK) [34], which recognizes mature vasopressin and not the extended forms. Both oxytocin and vasopressin are synthesized as precursor molecules, which are then cleaved to oxytocin and its associated neurophysin (NpI) or vasopressin, its associated peptide (NPII) and a glycopeptide. To demonstrate local synthesis, the presence of neurophysin epitopes were investigated using antibovine neurophysin I (anti-NpI) [35] and antibovine neurophysin II (anti-NpII) (both antisera donated by Dr. W.G. North, Dartmouth Medical College, Hanover, NH) [34]. Sections were dewaxed in xylene, rehydrated through graded alcohols, and washed in 0.05 mol·L-1 Tris/HCl with 0.15 mol·L-1 NaCl (pH, 7.6; Tris/HCl/NaCl) before preincubating with Tris/HCl/NaCl containing 3% (w/v) BSA and 0.5% (v/v) Triton X-100 for 1 h. Sections were then incubated overnight with a 1:100 dilution of primary antiserum in Tris/HCl/NaCl containing 3% (w/v) BSA and 0.5% (v/v) Triton X-100, at 4°C. Sections were then washed and incubated with a 1:100 dilution of peroxidase-conjugated swine anti-rabbit immunoglobulin (Dako Ltd, High Wycombe, UK) Tris/HCl/NaCl in 3% (w/v) BSA and 0.5% (v/v) Triton X-100, at room temperature for 2 h. Immunoreactive peptide was visualized by hydrogen peroxide (2 mg·ml-1) and diaminobenzidine (0.7 ng·ml-1) in 0.06 mmol·L-1 Tris-HCl (pH 7.5; Fast DAB; Sigma Chemical Co., Poole, UK). Control sections were incubated with normal rabbit serum in place of primary antiserum to indicate any nonspecific staining. Cross-reactivity of VA10 and 84/2 was determined by preadsorption of the antisera to either oxytocin-coupled, vasopressin-coupled, or vasotocin-coupled sephadex beads.
Western Blot Analysis for Oxytocin-Neurophysin and Vasopressin-Neurophysin
Results of immunocytochemical staining for NpI and NpII were confirmed using a sensitive Western blot procedure. Crude tissue extracts were prepared by homogenizing 0.5 g of tissue in 2 ml of PBS (154 mmol·L-1 NaCl, 1.9 mmol·L-1 NaH2PO4, 8.1 mmol·L-1 Na2HPO4 [pH 7.2]) containing "Complete" mini-protease inhibitor cocktail (Boehringer Mannheim UK, Lewes, East Sussex) at the manufacturer's recommended concentration. Homogenates were stored frozen until required. Equal concentrations (300 µg) of total protein were mixed with equivalent equal volumes of SDS reducing buffer (62.5 mmol·L-1 Tris-HCl [pH 6.8], 2% [w/v] SDS, 10% [w/v] glycerol, 100 mmol·L-1 dithiothreitol, and 0.05% [w/v] bromophenol blue) and incubated in a boiling water bath for 5 min before loading. Samples were then separated according to size by discontinuous SDS-PAGE [36] on 15% gels in running buffer (pH 8.3; 0.2 mol·L-1 Tris base, 1 mol·L-1glycine, 1% [w/v] SDS at 16.67 V·cm-1 for 90 min. Separated proteins were then electrotransfered onto polyvinylidene difluoride membrane (Boehringer Mannheim UK) at 200 mV for 90 min in transfer buffer (20 mmol·L-1 Tris base and 150 mmol·L-1 glycine [pH 8.3]). Blots were probed with either anti-NpI or anti-NpII at a 1:2000 dilution in PBS/0.02% (v/v) Tween-20 with 3% (w/v) dried milk for 16 h at 4°C with continuous agitation. Membranes were then washed with PBS/Tween and incubated with horseradish peroxidase-conjugated swine antirabbit immunoglobulin (1:4000 dilution in PBS/0.02% [v/v] Tween 20 with 3% [w/v] dried milk) for 1 h at room temperature. Detection of bound antibody was achieved by chemiluminescence (BM chemiluminescence [POD]; Boehringer Mannheim UK) and exposure of Hyperfilm (Amersham International, Amersham, UK). Positive controls were 100 ng of bovine neurophysin-I (BNpI) or -II (BNpII) and 300 µg of crude sheep pituitary extract, whereas 300 µg of smooth muscle crude extract was included as a negative control tissue. Control blots of all samples were incubated with normal rabbit immunoglobulin in place of the primary antibody.
Column Chromatography of Testes Extracts
To confirm the authenticity of oxytocin and vasopressin immunoreactivity in the sheep testes extracts, the experiment of Kasson et al. [37] was repeated. Two separate extraction methods of sheep testicular tissue were used. Tissue was either homogenized on ice in 10 ml·g-1 (wet wt) of 100 mmol·L-1 acetic acid, followed by centrifugation at 4000 x g before separation by chromatography, or extracts were prepared as described for radioimunoassay, including purification using C18 octadecyl silica cartridges. Supernatants of either extracts (1-ml aliquots) or 125I-labeled oxytocin or vasopressin standard were applied separately to a 1- x 25-cm Sephadex-G25 column (Sigma, St. Louis, MO) and eluted with 0.1 mol·L-1 acetic acid at a flow rate of 0.8 ml·min-1. The column was washed with 10 column volumes between application of each sample. Ten-microliter aliquots of 1-ml fractions were assayed by specific radioimmunoassay as described.
Statistical Analysis
Data are given as mean values ± SEM. Significant differences between means of spermatogenic stage were tested by one-way ANOVA, followed by comparison of means by two-tailed t-test. Differences within means of spermatogenic stages were tested for by paired t-tests. Samples that contained values ± two standard deviations of the respective group mean were rejected and not included in the analysis.
RESULTS
Sperm Counts and Testis Circumference
Sperm count and testis circumference (Table 1) increased significantly (both P < 0.001, ANOVA) with progression of the spermatogenic cycle. No significant differences between left and right testis circumference of animals were seen at any stage. These data were used to confirm the classification of the spermatogenic stage in each animal.
|
Oxytocin and Vasopressin Concentrations
Testicular oxytocin concentrations increased significantly (ANOVA, P = 0.029) with spermatogenesis (Fig. 1A), with the most significant difference being between prepubertal and late spermatid stage (t-test; P = 0.002). Maximum concentrations of oxytocin occurred once there was evidence of spermiation, although increases between stages other than the prepubertal were not significant. Circulating oxytocin levels did not alter (ANOVA, P = 0.334; Fig. 1a). Neither testicular (ANOVA, P = 0.13) or circulatory (ANOVA, P = 0.99) vasopressin altered significantly with progression of spermatogenesis (Fig. 1b).
|
Oxytocin was detected in all three regions of the epididymis, although concentrations decreased with progression from caput to cauda (Fig. 2a) in all but prepubertal animals. Concentrations rose significantly (ANOVA, P = 0.021) in the caput epididymis during the onset of spermatogenesis, with significant differences found between prepubertal and PS (P = 0.007), ES (P = 0.001), LS (P = 0.028), and FS (P = 0.049). Changes in oxytocin concentrations between other stages were not significant. A similar trend was also measured in the corpus and cauda epididymis; however, changes associated with the onset of spermatogenesis in these regions were not significant. Paired t-test analysis of data for each spermatogenic stage showed significant concentration gradients from caput to cauda in all stages except the prepubertal and FS (Table 2). Vasopressin concentrations were greater than those for oxytocin but did not vary significantly (ANOVA, P = 0.993) with either spermatogenic stage or region of the epididymis (Fig. 2b).
|
|
Location of Immunoreactive Oxytocin, Vasopressin, and Associated Neurophysins in the Ram Testis and Epididymis
The presence of both oxytocin (Fig. 3B) and vasopressin (Fig. 3C) was demonstrated by immunocytochemistry in the intertubular area of ram testes. No staining was seen in sections incubated with whole rabbit serum (Fig. 3A). Preadsorption of VA10 to oxytocin-coupled sephadex beads abolished staining, whereas vasopressin-coupled sephadex and vasotocin-coupled sephadex did not (results not shown). The 84/2 labeling was diminished by both vasopressin-coupled sephadex and oxytocin-coupled sephadex, but not by vasotocin-coupled sephadex (results not shown). Immunoreactive Np1 (Fig. 3D) was seen in corresponding areas to oxytocin, but no immunoreactivity was detected for the vasopressin-associated NpII (Fig. 3E). The NpI immunoreactivity was also seen in intratubular areas (Fig. 3D), with weak labeling of Sertoli cells.
|
Staining for oxytocin was found throughout the epididymis, although the location and density of staining in these cells varied between regions. In the initial segment, caput and corpus, staining was seen throughout discrete principal cells (Fig. 4, C–E). However, the intensity of this staining and the number of cells stained decreased from the initial segment to corpus. In the cauda, staining was localized to the apex of all principal cells in a supranuclear aspect (Fig. 4F). No reactivity was found in sections incubated with whole rabbit serum (Fig. 4A). Indeed, no immunoreactivity was detected for sections incubated with the antivasopressin antiserum 84/2 (Fig. 4H) or the vasopressin-associated NpII (Fig. 4I), whereas staining for the oxytocin-associated NpI was inconclusive, with staining observed in some, but not all, sections (Fig. 4G). This was investigated further by Western Blot analysis.
|
Identification of Oxytocin-Associated Neurophysin and Vasopressin-Associated Neurophysin by Western Blot Analysis
Immunoreactive-oxytocin associated neurophysin (NpI) and vasopressin-associated neurophysin (NpII) were present in crude pituitary extracts, indicating that the antisera cross-reacted with sheep NpI and NpII, respectively (Fig. 5, a and b). Both bands corresponded to those of purified BNpI and BNpII (Sigma) and were calculated to be of approximately 14 kDa and 10 kDa, respectively. Extracts of testes and epididymides probed with anti-NpI gave bands corresponding to those of the positive controls (BNpI and sheep pituitary), indicating the presence of this peptide in both tissues (Figs. 5a and 6a). Extracts of testes and epididymides probed with the anti-NpII did not yield immunoreactive bands of similar size to those of BNpII and pituitary extract (Figs. 5b and 6b). Negative control tissue (muscle) showed no immunoreactivity with either antiserum.
|
Presence of Nonauthentic Vasopressin in Ram Testes Extracts
Both crude acetic acid and octadecyl silica purified extracts of testicular tissue possessed major immunoreactive species that eluted in fractions before authentic [125I]vasopressin (Fig. 7). However, some authentic vasopressin was present in testicular tissue, as identified by a peak coeluting with that for [125I]vasopressin standard. In crude acetic acid extracts, the peak area of authentic vasopressin accounted for 2.5% of total immunoreactivity, whereas in the octadecyl silica purified extracts, this peak accounted for 25% of immunoreactivity. Two immunoreactive peaks of similar size were identified in acetic acid extractions assayed for the presence of oxytocin, the second of which coeluted with the authentic [125I]oxytocin standard. Octadecyl silica purified extracts possessed only one peak, which coeluted with authentic oxytocin.
|
DISCUSSION
In sheep, as in many other mammalian species [25, 26, 30, 38], Leydig cells appear to be the main source of testicular oxytocin. Concomitant NpI immunoreactivity provides evidence that oxytocin is synthesized here and is consistent with reports for the bull [38] and marmoset [26]. Western blot analysis of crude testicular extracts confirmed the presence of NpI in the testis, with an immunoreactive band that comigrated with BNpI and having an approximate size of 14 kDa, corresponding to that of the oxytocin-associated neurophysin in the sheep corpus luteum [39]. Weak immunoreactivity for NpI, but not for oxytocin, was also evident in Sertoli cells. The significance of this is uncertain, but both NpI and oxytocin immunoreactivity has been reported in bull [38] and marmoset [26] Sertoli cells as well as oxytocin mRNA expression in ram Sertoli cells [38]. Furthermore, in the rat [40] and sheep [24], evidence for low concentrations of oxytocin in the seminiferous tubule fluid has been found, with Sertoli cell secretion being the most likely source.
Testicular oxytocin concentrations increased during the first spermatogenic cycle. No change in plasma oxytocin concentration occurred during this period, however, suggesting that testicular and not hypothalamic oxytocin is important. The most significant increase occurred with the appearance of pachytene spermatocytes at the onset of meiosis. It has been reported that whereas LH stimulates Leydig cell oxytocin secretion [18], the presence of oxytocin in interstitial fluid depends on the presence of pachytene spermatocytes [40]. Our data support this finding. Maximum concentrations of testicular oxytocin were measured once full spermatogenesis had occurred, which is consistent with seminiferous tubule contractility being maximally stimulated by oxytocin at the time of spermiation [14]. This increase in testicular oxytocin endorses the findings of a previous report for the pubertal regulation of oxytocin mRNA in the ram testis [41] and the suggestion that a local oxytocinergic system develops during puberty in the primate marmoset [26]. Indeed, in addition to seminiferous tubule contractility, which is mediated by smooth muscle-like myoid cells, oxytocin regulates the testosterone production of Leydig cells [42] and the conversion of testosterone to dihydrotestosterone by increasing testicular 5-reductase activity [43].
Obviously, all physiological effects of oxytocin must be mediated through an active receptor, which we have recently identified in the sheep testis (unpublished results). Receptor density is central to the amplitude of the response induced. In the marmoset, oxytocin immunoreactivity does not appear until spermatogenesis is underway, but the uterine-type oxytocin receptor is present before this in the juvenile [26], further illustrating the significant importance of the result presented here. However, changes in the density and regulation of an as-yet-uncharacterized receptor, as implicated in oxytocin-stimulated seminiferous tubule contractility [5], cannot be discounted.
In contrast to oxytocin, apparent immunoreactive vasopressin was found in Leydig cells, but no corresponding immunoreactive NpII was seen. Furthermore, NpII could not be detected by Western blot analysis of testicular extracts. Positive staining for vasopressin may result from cross-reactivity with oxytocin, as indicated by preadsorption controls, or from a nonauthentic immunoreactive species, as indicated by chromatographic separation. Measurements suggested high local concentrations, but chromatography showed that even after purification, only 25% of the measured value represented authentic vasopressin, which is comparable to those levels in plasma and is consistent with reports in the rat [37]. However, chromatography also suggested that a proportion of immunoreactive species in tissue extracts was authentic. This vasopressin is probably not synthesized in the testis. These findings are consistent with reports that vasopressin mRNA is, except in the rat, undetectable in mammalian species [44], and that authentic vasopressin is systemically derived. Failure to show any changes in the concentration of circulating or local vasopressin during puberty further indicate that this peptide probably does not have a significant role in spermatogenesis.
In the epididymis, oxytocin is thought to be involved in the increased contractility seen during ejaculation. An increase in systemic oxytocin occurs during coitus in the bull [12, 13] and in men [45], and increased sperm transport occurs after intravenous administration of oxytocin to rams [9]. Furthermore, this effect can be blocked by a specific oxytocin antagonist [9], so presumably, it is mediated by oxytocin receptors of the smooth muscle cells. Oxytocin receptors have been identified using binding studies in the pig epididymis [28] and localized to the epithelial and stromal regions in primates [25, 26] and specifically throughout the smooth muscle of the caput and cauda epididymis of the macaque [25]. Preliminary evidence indicates that oxytocin receptors are also present throughout the sheep epididymis (unpublished results). Along with an increase in epididymal contraction at ejaculation, there is a constant rhythmic contraction of the epididymis in the absence of stimulation [6] and a constant basal flow of sperm from the epididymis into the vas deferens [9, 46], both of which are inhibited by a specific oxytocin antagonist [9]. Local peptide is most likely involved in modulating this basal contractility. The significant increase in epididymal oxytocin concentration with the onset of spermatogenesis supports this hypothesis. Localization of oxytocin to the principal cells of the sheep epididymis and reduction in immunoreactivity with progression along the epididymis are consistent with previous reports for both sheep [24] and rat [23], and the significant concentration gradients measured by radioimmunoassay provide further support. Such distribution also suggests oxytocinergic roles other than contractility in the epididymis. Oxytocin may be involved in the androgen regulation of the epididymis. The concentration gradient of oxytocin measured in this study is very similar to the profile of 5-reductase type I expression [47] and activity [48]. The peptide increases the activity of epididymal 5-reductase and, hence, the conversion of testosterone to dihydrotestosterone, on which epididymal function depends [49]. The localization of oxytocin in the cauda epididymis was different with a perinuclear apical position and very granular appearance, similar to that of secretory granule staining seen in the pituitary. Oxytocin administration to the mouse by intratesticular injection reduces sperm motility in the vas deferens [50], whereas it suppresses human sperm motility in vitro [51]. Oxytocin may be secreted into the lumen of the cauda epididymis to help suppress sperm motility, thus ensuring that sperm do not enter the vas deferens prematurely. Immunoreactive NpI in all three regions of the epididymis indicated that oxytocin is synthesized in the principal cells of the epididymis. Although contradictory to previous reports for the sheep [22, 24], local synthesis is supported by data showing that oxytocin immunoreactivity in the rat epididymis is still present after castration or efferent duct ligation [23].
The lack of a change in vasopressin concentration with puberty and the absence of any evidence to support local synthesis in the sheep epididymis do not support a role in any paracrine or juxtacrine role in the epididymis. These results are consistent with the inability to induce epididymal contractility at physiological concentrations [9].
In conclusion, correlation of increasing oxytocin concentrations in the testis and epididymis with progression of spermatogenesis, and its synthesis in these tissues, confirms the presence of an oxytocinergic paracrine system in sheep. In contrast, the lack of association of vasopressin concentrations with spermatogenesis and failure to localize the peptide or associated neurophysin in the reproductive tract raises significant doubt regarding its physiological significance in sheep.
|
ACKNOWLEDGMENTS
We are grateful to Drs. H. Gainer and W. North for their kind gifts of the VA10 antiserum and neurophysin antisera, respectively, and to Nicola Latham and Karen Greenwood for their assistance in care of the sheep.
FOOTNOTES
First decision: 20 December 1999.
1 Supported by grant 7/S06866 from the Biotechnology and Biological Sciences Research Council. 
2 Correspondence: Stephen J. Assinder, Department of Anatomy, University of Bristol, School of Veterinary Science, Southwell Street, Bristol BS2 8EJ, UK. FAX: 0044 117 9254794; s.j.assinder{at}bristol.ac.uk
Accepted: March 14, 2000.
Received: November 16, 1999.
REFERENCES
-Reductase activity increased by oxytocin in the rat testis. In: Bartke A (ed.), Function of Somatic Cells in the Testis. New York: Springer-Verlag; 1994: 278–285. reductase type 1 along the rat epididymis during postnatal development. Endocrinology 1991; 134:2298–2306.[Abstract]
4-5-reductase and 3-hydroxysteroid dehydrogenase. Endocrinology 1981; 109:1225–1233.This article has been cited by other articles:
|
|
 |
|
  C. E. Marinho, S. M. Almeida-Santos, S. M. Carneiro, S. C. Yamasaki, and P. F. Silveira Peptidase activities in Crotalus durissus terrificus semen Reproduction, December 1, 2008; 136(6): 767 - 776. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mewe, I. Wulfsen, R. Middendorff, and C. K Bauer Differential modulation of bovine epididymal activity by oxytocin and noradrenaline Reproduction, September 1, 2007; 134(3): 493 - 501. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Thackare, H. D. Nicholson, and K. Whittington Oxytocin--its role in male reproduction and new potential therapeutic uses Hum. Reprod. Update, July 1, 2006; 12(4): 437 - 448. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Filippi, A. Morelli, L. Vignozzi, G. B. Vannelli, M. Marini, P. Ferruzzi, R. Mancina, C. Crescioli, N. Mondaini, G. Forti, et al. Oxytocin Mediates the Estrogen-Dependent Contractile Activity of Endothelin-1 in Human and Rabbit Epididymis Endocrinology, August 1, 2005; 146(8): 3506 - 3517. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X H Zhang, S Filippi, L Vignozzi, A Morelli, R Mancina, M Luconi, S Donati, M Marini, G B Vannelli, G Forti, et al. Identification, localization and functional in vitro and in vivo activity of oxytocin receptor in the rat penis J. Endocrinol., March 1, 2005; 184(3): 567 - 576. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
