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J Frayne and H D Nicholson


The aim of the present study was to determine whether LH stimulates oxytocin production by adult rat Leydig cells directly or indirectly via testosterone. Purified adult rat Leydig cells were cultured in the presence or absence of 0·1 ng/ml LH or 1, 10 or 100 ng/ml testosterone for 22 h. Culture medium was collected at 2-hourly intervals and assayed for oxytocin and testosterone. In the presence of LH, Leydig cells produced significantly higher levels of both testosterone (basal production 1·4± 0·13 ng, LH-stimulated 4·1 ±0·13 ng/106 cells per 2 h) and oxytocin (basal production 8·3± 1·2 pg, LH-stimulated 20·2± 1·3 pg/106 cells per 2 h). Testosterone also stimulated oxytocin secretion. However, the increase was smaller compared with that seen with LH and was not found to be dose-dependent. Furthermore, testosterone production was only significantly increased by LH during the first 10 h of the 22-h culture period whereas LH stimulated oxytocin production throughout the whole culture period.

To further determine the effect of LH on oxytocin production, cultures were performed in the presence of LH and/or 400 μm aminoglutethimide. In the presence of aminoglutethimide both the basal and LH-stimulated production of testosterone was significantly reduced. However, in the same cultures aminoglutethimide did not alter either the basal or LH-stimulated production of oxytocin.

These data show that LH does not act via testosterone to stimulate oxytocin production and therefore acts directly or by some alternative indirect mechanism.

In this study it was found that two other factors, cell density and lipoprotein, also influenced oxytocin production by isolated Leydig cells. Decreasing the density at which Leydig cells were cultured from 106 to 10 cells/well significantly increased both their basal and LH-stimulated production of oxytocin. Lipoproteins were also found to stimulate oxytocin production in a dose-dependent manner and to synergize with LH to further increase LH-stimulated oxytocin production. The results of this study show the production of oxytocin by Leydig cells to be regulated not only by the gonadotrophin LH but also by lipoproteins which are known to be present in interstitial fluid. These data add to the accumulating evidence that intratesticular oxytocin may be a paracrine factor.

Journal of Endocrinology (1994) 143, 325–332

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H D Nicholson, H M Greenfield and J Frayne


In the rat testis oxytocin has been localized to the Leydig cells, and these cells have been shown to produce oxytocin in vitro. The present study was performed to determine whether oxytocin is present in the interstitial fluid (IF) and seminiferous tubule fluid (TF) of the rat and whether concentrations of the peptide vary within the two compartments following germ cell destruction.

In order to destroy germ cells adult male rats were anaesthetized and their scrotal regions placed in a water bath at 43 °C for 20 min. Control animals were subjected to anaesthesia alone. Groups of 6 animals were killed 3, 7 and 21 days after heat treatment and their testes removed for histological examination or fluid extraction. IF and TF were separated and the oxytocin content of the fluids measured by radioimmunoassay.

Immunoreactive oxytocin was detected in both the IF (100 ±11 pg/ml) and TF (27± 4 pg/ml) of control rats and this immunoreactivity co-eluted with the authentic peptide following HPLC. Three days after heat treatment IF levels of oxytocin were significantly reduced but TF levels of the peptide were significantly increased. These changes were associated with a lack of pachytene spermatocytes in the histological sections. Seven and 21 days after heat treatment the levels of oxytocin in the IF and TF were not significantly different from control levels. Similar changes in IF and TF oxytocin levels were seen in a second experiment when pachytene spermatocytes were removed using the testicular toxicant methoxyacetic acid.

To investigate whether oxytocin passes from the IF into the TF, 3H-oxytocin was infused into the testicular arteries of both control and heat-treated rats and the rats killed at regular intervals over the next 24 h. In both groups of animals <5% of the 3H-oxytocin passed from the IF into the TF.

These data suggest that the seminiferous tubule as well as the Leydig cells may secrete oxytocin and that this secretion may be influenced by the presence of germ cells, particularly pachytene spermatocytes.

Journal of Endocrinology (1994) 143, 471–478

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H. D. Nicholson, R. T. S. Worley, H. M. Charlton and B. T. Pickering


Immunoreactive oxytocin is present in the testis and it has been shown that this hormone increases the contractility of seminiferous tubules. We have investigated the relationship between testicular oxytocin, tubular movements and the effects of LH and testosterone using, as a model, the hypogonadal (hpg/hpg) mouse, which is deficient in hypothalamic LH-releasing hormone (LHRH). Whilst both testicular oxytocin and seminiferous tubule movements, resembling those seen in the rat, can be found in normal adult mice, neither can be found in hypogonadal mice. After 2 weeks of treatment with LH (200 ng to 100 μg daily) low levels of testicular oxytocin and tubular movements were observed. Treatment with large doses of testosterone for 2–12 weeks led to higher concentrations of testicular oxytocin and tubular movements resembling those seen in the normal adult mouse. The results support the evidence that testicular oxytocin modulates seminiferous tubule movements. We suggest that testosterone may play a part in the accumulation of oxytocin in the testis.

J. Endocr. (1986) 110, 159–167

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H. D. Nicholson, A. J. Smith, S. D. Birkett, P. A. Denning-Kendall and B. T. Pickering


Vasopressin (VP)-like immunoreactivity (IR) has been located in the testes of several species of mammal. There is evidence that most of this IR in the rat does not represent authentic arginine vasopressin (AVP) and that a second AVP-like peptide may exist. We have studied testis samples from the pig, which produces lysine vasopressin (LVP) in its pituitary, and have found both LVP- and AVP-like IR.

High-performance liquid chromatography (HPLC) of testis extracts showed two peaks of VP-IR. The first peak co-eluted with authentic LVP and was recognized only by antisera which cross-reacted with LVP. The second peak co-eluted with authentic AVP and was recognized by antisera raised against AVP. Both VP-like peptides bound to a neurophysin affinity column and the HPLC elution profiles of the bound peptides were similar to those of the authentic hormones. When the LVP-like material was oxidized with performic acid, a peak of IR running in the same position as oxidized authentic LVP on HPLC was produced. Similarly, the performic acid-oxidized AVP-like material co-eluted with oxidized authentic AVP.

The presence of both LVP- and AVP-like peptides in the pig testis may mean that more than one gene is involved. A second VP-like gene could also explain the anomalies of VP-IR in other species.

J. Endocr. (1988) 117, 441–446

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H-L Ang, R Ivell, N Walther, H Nicholson, H Ungefroren, M Millar, D Carter and D Murphy


The bovine oxytocin gene has been expressed in the testes of two independent transgenic mouse lines. Hybridization and RNase protection analysis showed that the oxytocin transgene was transcribed from the normal functional promoter in the Sertoli cells of the seminiferous tubules in a developmentally regulated manner. Immunohistochemistry indicated that both oxytocin and neurophysin epitopes were expressed together in the Sertoli cells at stages I–V and X–XII of the cycle of the seminiferous epithelium. Furthermore, analysis with high- performance liquid chromatography showed that there was a tenfold increase in the amount of amidated oxytocin present in testicular extracts from the transgenic mice. However, there appeared to be no detectable effect of this overproduction of hormone on testicular morphology or fertility parameters. A significant decrease by 50% was detected only in the levels of intratesticular testosterone and dihydrotestosterone. The results point to a local paracrine role for oxytocin in the modulation of Leydig cell function.

Journal of Endocrinology (1994) 140, 53–62

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H. D. Nicholson, R. T. S. Worley, S. E. F. Guldenaar and B. T. Pickering


An oxytocin-like peptide is present in the interstitial cells of the testis, and testicular concentrations of oxytocin have been shown to increase seminiferous tubule movements in vitro. We have used the drug ethan-1,2-dimethanesulphonate (EDS), which depletes the Leydig cell population of the adult rat testis, to examine further the relationships between the Leydig cell, testicular oxytocin and tubular movements. Adult rats were injected i.p. with a single dose of EDS (75 mg/kg) or of vehicle (25% dimethyl sulphoxide). Histological study 3 and 10 days after treatment with EDS showed a reduction in the number of interstitial cells, and levels of oxytocin immunoreactivity were undetectable by radioimmunoassay. Immunostaining revealed very few oxytocin-reactive cells. Spontaneous contractile activity of the seminiferous tubules in vitro was also dramatically reduced, but could be restored by the addition of oxytocin to the medium. Four weeks after EDS treatment, the interstitial cells were similar to those in the control animals both in number and in immunostaining; immunoassayable oxytocin was present and tubular movements were normal. The EDS effect, seen at 3 and 10 days, was not altered by daily treatment with testosterone. However, repopulation of the testes with oxytocin-immunoreactive cells was not seen until 6 weeks in the testosterone-treated animals.

We suggest that the Leydig cells are the main source of oxytocin immunoreactivity in the testis and that this oxytocin is involved in modulating seminiferous tubule movements and the resultant sperm transport. The results also imply that testosterone does not play a major role in controlling tubular activity in the mature rat.

J. Endocr. (1987) 112, 311–316

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H. D. Nicholson, S. E. F. Guldenaar, G. J. Boer and B. T. Pickering


The long-term effects of oxytocin administration on the testis were studied using intratesticular implants. Adult male rats had an Accurel device containing 20 μg oxytocin (releasing approximately 200 ng/day) implanted into the parenchyma of each testis; control animals received empty devices. The animals were killed at weekly intervals for 4 weeks. Some animals were perfused and the testes processed for light and electron microscopy. Blood was collected from the remaining animals for the measurement of testosterone, dihydrotestosterone, LH, FSH and oxytocin; epididymal sperm counts were measured and the testes were extracted and radioimmunoassayed for testosterone, dihydrotestosterone and oxytocin.

Long-term administration of oxytocin resulted in a significant reduction in testicular and plasma testosterone levels throughout the 4-week period examined and, after 14 days of treatment, lipid droplets were seen in the Leydig cells of treated but not control animals. Concentrations of dihydrotestosterone in the plasma and testes of the oxytocin-treated animals, however, were significantly elevated after 7 and 14 days and at no time fell below control values. Plasma FSH levels were also lower in the oxytocin-treated animals. Intratesticular oxytocin treatment did not affect LH or oxytocin concentrations in the plasma, epididymal sperm counts or the number of Leydig cells in the testis. Empty Accurel devices had no effect on testicular morphology.

This study provides the first evidence that oxytocin in vivo can modify steroidogenesis in the testis.

Journal of Endocrinology (1991) 130, 231–238