Androgen receptors are only present in mesenchyme-derived dermal papilla cells of red deer (Cervus elaphus) neck follicles when raised androgens induce a mane in the breeding season.

Red deer stags produce an androgen-dependent mane of long hairs only in the breeding season; in the non-breeding season, when circulating androgen levels are low, the neck hair resembles the rest of the coat. This study was designed to determine whether androgen receptors are present in deer follicles throughout the year or only in the mane (neck) follicles when circulating testosterone levels are high in the breeding season. Although androgens regulate much human hair growth the mechanisms are not well understood; they are believed to act on the hair follicle epithelium via the mesenchyme-derived dermal papilla. The location of androgen receptors in the follicle was investigated by immunohistochemistry and androgen binding was measured biochemically in cultured dermal papilla cells derived from mane and flank follicles during the breeding season and from neck follicles during the non-breeding season. Immunohistochemistry of frozen skin sections using a polyclonal antibody to the androgen receptor localised nuclear staining only in the dermal papilla cells of mane follicles. Saturation analysis assays of 14 primary dermal papilla cell lines using [(3)H]-mibolerone demonstrated high-affinity, low-capacity androgen receptors were present only in mane (breeding season neck) cells; competition studies with other steroids confirmed the specificity of the receptors. Androgen receptors were not detectable in cells from either the breeding season flank nor the non-breeding season neck follicles. The unusual biological model offered by red deer of androgen-dependent hair being produced on the neck in the breeding, but not the non-breeding season, has allowed confirmation that androgen receptors are required in follicle dermal papilla cells for an androgen response; this concurs with previous human studies. In addition, the absence of receptors in the non-breeding season follicles demonstrates that receptors are not expressed unless the follicle is responding to androgens. Androgen receptors may be induced in mane follicles by seasonal changes in circulating hormone(s).

Ability to culture dermal papilla cells from red deer (Cervus elaphus) hair follicles with differing hormonal responses in vivo offers a new model for studying the control of hair follicle biology.

Red deer stags annually grow two distinct seasonal coats, a winter coat and a summer coat; in addition, they produce a mane during the breeding season when plasma testosterone levels are high, which is replaced by the short neck hairs of the summer coat when testosterone levels are low. As two very different hair types are produced from the same follicle under hormonal regulation, they offer an interesting model for studying the effects of hormones, particularly androgens, on mammalian hair growth. Since the dermal papilla of the hair follicle has a regulatory function and is probably the site of androgen action, we have investigated whether cells from the dermal papilla can be readily cultured from various types of red deer follicles; as the follicular connective tissue sheath may regenerate a new papilla in vivo, this was also examined. Individual dermal papillae and lower portions of the connective tissue sheath were microdissected from mane and flank follicles of red deer stags during the winter breeding season and from the summer coat during the nonbreeding season. Primary cultures were established from isolated dermal papillae, connective tissue sheath and dermal explants, subcultured and reestablished after freezing. Deer dermal papilla cells resembled sheep cells; they displayed a polygonal shape and irregular organisation, but did not form aggregates in contrast to human and rat vibrissa cells. Connective tissue sheath cell morphology was intermediate between that of dermal papilla cells and dermal fibroblasts. However, all three cell types derived during the breeding season grew at a much faster rate than the same cells derived during the nonbreeding season. Therefore, primary cell lines can be fairly readily derived from deer hair follicles. Since the red deer stag offers both androgen-dependent neck (mane) and control flank follicles in the breeding season, plus control nonbreeding season neck follicles, this means that stag follicular cells, particularly the dermal papilla cells, appear to offer a unique novel model system for the study of the hormonal regulation of hair growth.

Manipulating melatonin in red deer (Cervus elaphus): differences in the response to food restriction and lactation on the timing of the breeding season and prolactin-dependent pelage changes.

In this study, we investigated the influence of food availability and lactation upon seasonality in red deer. This was examined by testing the hypothesis that advancing the timing of breeding and autumn moult using the hormone melatonin will be prevented when the food availability of lactating hinds is severely restricted. This hypothesis was rejected. Implanting 1 g of melatonin between June 22 and November 30 resulted in advances in the timing of the onset of ovarian activity and winter coat growth of 18 and 35 days, respectively. Whilst the onset of ovarian activity was unaffected by lactation and restricted food availability, these factors significantly delayed the winter coat growth by 20 days. The date of onset of winter primary fibre growth was negatively correlated to plasma concentrations of the hormone prolactin in July. We suggest that seasonal changes in the growth of primary hair fibres are modified by two mechanisms: the increasing duration of melatonin secretion, as day lengths decline, which depresses prolactin secretion, and low nutrition, which elevates prolactin secretion in lactating deer. To conclude, we have demonstrated that the sensitivity of red deer to photoperiodic influences is preserved in lactating animals at low levels of nutrition, and that the timing of the onset of the breeding season and winter coat growth differ detectably in their sensitivity to nutrition and lactation.

Evidence for a circannual rhythm of reproduction and prolactin secretion in a seasonally breeding macropodid marsupial, the Bennett's wallaby (Macropus rufogriseus rufogriseus).

Two groups of adult female wallabies were maintained in photoperiod-controlled rooms from June 1987 until August 1988. Group SSH was held on summer solstice photoperiods throughout the experiment; group SN was subjected to weekly stepwise simulated natural changes in photoperiod. Plasma melatonin concentrations reflected photoperiod with high concentrations during the dark phase in both groups. Group SN wallabies commenced oestrous cyclicity on 21 July (+/- 19 days, n = 6) entered reproductive quiescence on 14 February (+/- 10 days, n = 5) and recommenced cycling on 8 June (+/- 3 days, n = 4). Group SSH wallabies began cycling on 27 July (+/- 9 days, n = 7) at a time that was not significantly different from that of group SN. Three out of five of group SSH exhibited a spontaneous period of reproductive quiescence of between 59 and 70 days commencing between 3 December and 25 February. There was a highly significant difference between the transient plasma prolactin response to a dopamine antagonist during cycling and quiescent periods in both groups (P < 0.001) such that the response was increased during periods of quiescence. Our data support the hypothesis that prolactin is involved in the control of seasonal quiescence in the female Bennett's wallaby and demonstrate that spontaneous changes in reproductive state and prolactin can occur when animals are maintained on unchanging long photoperiods.

Hormones and hair growth: variations in androgen receptor content of dermal papilla cells cultured from human and red deer (Cervus elaphus) hair follicles.

Many hair follicles produce different types of hair in response to environmental changes or the mammals age, that are translated to the follicle by hormones. Androgens cause many changes, such as transforming vellus follicles producing insignificant hairs on the face to terminal beard ones at puberty or the reverse on the scalp. In male red deer the breeding season rise in androgens causes the annual production of a mane on the neck that is lost during the spring. Because the dermal papilla situated at the base of the hair follicle is important in determining the type of hair produced, androgens may act via the dermal papilla. Therefore, primary cell lines of dermal papilla cells from human and red deer follicles with different responses to androgens have been established. Specific saturable androgen receptors were present in all human papilla cells examined, with higher levels in cells from androgen-dependent follicles, e.g., beard than in control, non-balding scalp cells. In preliminary investigations of red deer, androgen receptors were only present in cells derived from mane follicles and were undetectable in flank or spring neck follicles. These similar results from both species support the hypothesis that androgens are acting on hair follicles via the dermal papilla. They also suggest that dermal papilla cells are potentially useful models for investigating the mechanism of androgen action because cultured cells appear to retain differences that relate to the androgen responsiveness of their parent follicle. The red deer seems particularly interesting in view of the much shorter hair-growth cycle than human scalp or beard follicles.

Gestation periods in the Père David's deer (Elaphurus davidianus): evidence for embryonic diapause or delayed development.

It has been suggested that the gestation length of the Père David's deer is around 280 days, which is significantly longer than any other deer species except the roe deer (approximately 300 days) which exhibits embryonic diapause. The present study was designed to determine whether embryonic diapause exists in the Père David's deer by accurately monitoring gestation length. There was no difference in gestation length (283-284 days) between animals mated early and later in the breeding season. Hence, although Père David's deer exhibit a longer gestation period than that predicted from maternal body weight, there is no evidence for seasonal control of implantation. Actual birth weight is as predicted from an interspecific comparison of ungulates. The data imply either that there is an obligate period of embryonic diapause, irrespective of season, or that postimplantation fetal growth rate is slow compared with that in other deer species.

Characterization of 2-[125I]iodomelatonin binding sites in the brain of a marsupial, Bennett's wallaby (Macropus rufogriseus rufogriseus).

1. Specific high affinity binding of 2-[125I]iodomelatonin was detected in the brain of the pouch young of a marsupial, Bennett's wallaby. 2. Binding was rapid, stable, saturable and reversible. 3. Scatchard analysis indicated a single class of high affinity binding sites with an equilibrium dissociation constant (Kd) of 68 +/- 13 pM, a maximal number of binding sites (Bmax) of 0.7 +/- 0.1 fmol/mg protein and a Hill coefficient (nH) of 1.12 +/- 0.10. 4. Specific binding was inhibited by GTP (1 mM) indicating that the melatonin receptor is coupled to a guanine nucleotide binding protein, and by melatonin and closely related analogues with a potency order identical to that reported previously in the brain of eutherian mammals, birds and a reptile. 5. These studies suggest that the melatonin receptor is well-conserved through evolution.

Prostaglandin-induced secretion of oxytocin and prolactin in red (Cervus elaphus) and Pere David's (Elaphurus davidianus) deer hinds: evidence for oxytocin of luteal origin.

Peripheral plasma concentrations of oxytocin in female red deer during the luteal phase of the oestrous cycle (9.3 +/- 2.1 fmol/ml) exceeded those in the follicular phase (3.1 +/- 1.4) or during seasonal anoestrus (3.2 +/- 1.3). In both red and Père David's deer hinds during the mid-luteal phase of the cycle, systemic administration of a luteolytic dose of the prostaglandin F2 alpha analogue, cloprostenol, caused the concentration of oxytocin in the peripheral circulation to rise. Mean (+/- SEM) concentrations increased from 8.1 +/- 0.7 to 97 +/- 8 fmol/ml in red and from 6.2 +/- 0.7 to 153 +/- 30 fmol/ml in Père David's hinds within 5 min of treatment. During seasonal anoestrus oxytocin secretion in response to cloprostenol was reduced to less than 10% of that during the breeding season, in both species. Cloprostenol treatment raised circulating concentrations of prolactin in both species during the breeding season, and during anoestrus in red deer only. The concentration of oxytocin in a single corpus luteum removed at laparotomy from one red deer hind at the mid-luteal phase of the cycle was 66 nmol/g wet wt; identification was authenticated by HPLC. These results suggest that the corpus luteum secretes oxytocin in the Cervidae, as established previously in the Bovidae, and that luteal oxytocin secretion is stimulated by prostaglandin.

Efficacy of intermittent or continuous administration of GnRH in inducing ovulation in early and late seasonal anoestrus in the Père David's deer hind (Elaphurus davidianus).

Père David's deer hinds were treated with GnRH, administered as intermittent i.v. injections (2.0 micrograms/injection at 2-h intervals) for 4 days, or as a continuous s.c. infusion (1.0 micrograms/h) for 14 days. These treatments were given early (February-March) and late (May-June) in the period of seasonal anoestrus. The administration of repeated injections of GnRH increased mean LH concentrations from pretreatment values of 0.54 +/- 0.09 to 2.10 +/- 0.25 ng/ml over the first 8 h of treatment in early anoestrus, and from 0.62 +/- 0.11 to 2.73 +/- 0.49 ng/ml in late anoestrus. The mean amplitude of GnRH-induced LH episodes was greater (P less than 0.01) in late (4.03 +/- 0.28 ng/ml) than in early (3.12 +/- 0.26 ng/ml) anoestrus, but within each replicate (early or late anoestrus), neither mean LH episode amplitude nor mean plasma LH concentrations differed significantly between the four periods of intensive blood sampling. On the basis of their progesterone profiles, 6/12 hinds had ovulated in response to treatment with injections of GnRH (1/6 in early anoestrus and 5/6 in late anoestrus), and oestrus and a preovulatory LH surge were recorded in all of these animals. Oestrus and a preovulatory LH surge were also recorded in one other animal treated in early anoestrus in which progesterone concentrations remained low. The mean times of onset of oestrus (91.0 +/- 1.00 and 62.4 +/- 0.98 h) and of the preovulatory LH surge (85.8 +/- 3.76 and 59.4 +/- 0.25 h) both occurred significantly earlier (P less than 0.001) in animals treated in late anoestrus. Continuous infusion of GnRH to seasonally anoestrous hinds resulted in an increase in mean plasma LH concentrations, but this response did not differ significantly between early (2.15 +/- 0.28 ng/ml) and late (2.48 +/- 0.26 ng/ml) anoestrus. Ovulation, based on progesterone profiles, occurred in 2/7 hinds in early anoestrus and in 4/6 hinds in late anoestrus. Oestrus was detected in all except one of these hinds. The mean time of onset of oestrus occurred earlier in animals treated in late anoestrus (66.2 +/- 0.32 h and 46.7 +/- 0.67 h, P less than 0.01). The administration of GnRH, given either intermittently or continuously, will induce ovulation in a proportion of seasonally anoestrous Père David's deer. However, more animals ovulate in response to this treatment in late than in early anoestrus (75% compared with 23%).

Melatonin implants prevent the onset of seasonal quiescence and suppress the release of prolactin in response to a dopamine antagonist in the Bennett's wallaby (Macropus rufogriseus rufogriseus).

Three groups of adult female wallabies were maintained out of doors under conditions of natural photoperiod and temperature from late December to mid-August. One group (M1; N = 6) received Silastic elastomer melatonin implants on 14 December, a second group (M2; N = 5) were given implants on 16 February and a third group (C; N = 7) were unimplanted controls. Group C animals had all ceased cycling by 15 March and the subsequent breeding season commenced on 5 July +/- 6.9 days. Group M1 wallabies continued to cycle throughout the experimental period and did not exhibit ovarian quiescence. In Group M2, 2/5 animals continued to undergo repeated oestrous cycles and 3/5 ceased cycling between 14 December and 27 January and began again after the insertion of melatonin implants on 16 February. The prolactin response 30 min after s.c. administration of the dopamine antagonist domperidone was determined approximately every 4 weeks. In Group C, peak responses were high during the period of seasonal quiescence (January-June; mean range 14.2-19.6 ng/ml) and fell significantly (P less than 0.02) at the beginning of the breeding season in early July to 7.4 +/- 3.1 ng/ml. In Group M1, prolactin levels remained low (2.8-8.2 ng/ml) throughout the course of the experiment while in Group M2, response to domperidone fell following the insertion of the implants and subsequently remained at levels similar to those in Group M1. Our data support the hypothesis that photoperiod-induced changes in the secretion of melatonin after the winter solstice drive this species into seasonal quiescence by influencing the dopaminergic control of prolactin secretion.

Roles of prolactin and the uterus in the control of luteal regression in the Bennett's wallaby (Macropus rufogriseus rufogriseus).

The factors responsible for controlling luteal regression in macropodid marsupials are unclear. Experiments were designed to examine the role of the uterus and the pre-partum prolactin pulse in this process. In experiment 1, two groups of adult female Bennett's wallabies were hysterectomized (n = 3) or sham-hysterectomized (n = 3) on Day 16 of a non-pregnant oestrous cycle and daily peripheral blood samples were collected between Days 10-37. In both groups, circulating progesterone concentrations declined to below 0.96 nmol L-1 at luteolysis on Days 28-33. Circulating prolactin concentrations remained low over the period of luteal regression. In experiment 2, two groups of animals, which were maintained in the presence of a fertile male and synchronized by the removal of pouch young (RPY), were treated on Day 16 after RPY with a single injection of long-acting bromocriptine (Parlodel LA, Sandoz Ltd, Basle, Switzerland; 50 mg per animal n = 5) or vehicle (n = 4). Vehicle-treated animals gave birth on Day 28 (n = 3) or Day 29 (n = 1) with a significant elevation of prolactin on Day 28. In contrast, 4 of the 5 bromocriptine-treated animals failed to give birth and one gave birth on Day 29. No comparable prolactin pulse was detected in any of them. Circulating progesterone concentrations declined later in the bromocriptine-treated group than in the controls. These data suggest that different mechanisms lead to luteolysis in pregnant and non-pregnant animals; in pregnancy the luteolytic signal is prolactin, whereas in the non-pregnant cycle another signal of non-uterine origin is involved.

Effect of exogenous prolactin and bromocriptine on seasonal reproductive quiescence in the Bennett's wallaby (Macropus rufogriseus rufogriseus).

Two experiments were performed to investigate whether prolactin blocks the reactivation of the corpus luteum during seasonal reproductive quiescence in the Bennett's wallaby. In the first experiment three groups of non-lactating females (groups A C) were subjected in mid-April to photoperiods corresponding to those of the summer solstice from day 0 to day 13 of the experiment. Between days 14 and 52, photoperiods were reduced to correspond to those of the winter solstice (groups B and C). Animals in group A were maintained on the long photoperiods. Two milligrams ovine prolactin (groups A and C) or vehicle (group B) were administered on the mornings of days 14-22. In group A, no animal showed evidence of an active corpus luteum based on increased plasma progesterone levels. All animals in groups B and C exhibited reactivation of the corpus luteum. In group C, reactivation was significantly (P less than 0.01) delayed by a mean of 6.3 days. In the second experiment, two groups of nonlactating female wallabies in 'seasonal quiescence' were injected daily for 7 days with either 60 mg of the dopamine agonist bromocriptine or vehicle. The corpora lutea did not reactivate in either group. We conclude that exogenous prolactin is able to block the effect of short photoperiods in reactivating the quiescent corpus luteum during seasonal quiescence. However, the absence of an effect of bromocriptine suggests that if prolactin is the endogenous hormone responsible for maintaining seasonal quiescence it may not be under dopaminergic control at this time of year.

Melatonin in the plasma of growing sheep subjected to short and skeleton long photoperiods.

The levels of melatonin in plasma were measured at hourly intervals for 24 h in 8 sheep, 4 under 8L:16D (short day) and 4 under 7L:10D:1L:6D (skeleton long day) after 38 days of exposure. Mean concentrations did not differ significantly between treatments (52 pg/ml under short days; 91 pg/ml for skeleton long days), but the levels were more stable during 24 h in the SD treatment. Under skeleton long days there were 3 peaks during the 10D scotophase, with low levels during the 6D scotophase and the 7L photophase.

Effect of pinealectomy on the plasma concentrations of prolactin, cortisol and testosterone in sheep in short and skeleton long photoperiods.

Two experiments were carried out to investigate the effects of pinealectomy on the responses of prolactin, cortisol and testosterone to skeleton long photoperiods (7 h light: 10 h darkness: 1 h light: 6 h darkness; 7L: 10D: 1L: 6D) compared with short photoperiods (8L: 16D) in lambs. The first experiment included 23 female Suffolk cross sheep aged 10 months, of which six were pinealectomized. The skeleton long photoperiod significantly increased plasma levels of prolactin but this was blocked by pinealectomy; there was a peak around dusk and a trough around dawn and at the time of the 1-h period of light. There was no effect of either photoperiod or pinealectomy on plasma levels of cortisol. Testosterone was not measured in this experiment. In the second experiment there were 12 intact males and 11 castrated males aged 3 months; six of the lambs in each group were pinealectomized. Prolactin was again greatly stimulated by skeleton long photoperiods and the effect was blocked by pinealectomy; there was a trough in plasma prolactin at dawn in all groups. In addition, castration increased prolactin levels on two of the four sampling days. Plasma cortisol concentrations were significantly lower under skeleton long photoperiods and this was also blocked by pinealectomy; there was no effect of castration. Testosterone was much higher in intact males. After 10 weeks of exposure, skeleton long photoperiods produced significantly lower concentrations than short photoperiods in the intact ram with pineal glands but not in those which were pinealectomized.

The effect of short and skeleton long photoperiods on the plasma concentrations of prolactin and cortisol in sheep.

Suffolk-cross lambs, aged eight weeks, were exposed to either short photoperiods (8L : 16D, SP; 7 castrated males and 6 females) or skeleton long photoperiods (7L : 10D : 1L : 6D, SLP; 6 castrated males and 7 females). They were fed individually on a complete pelleted diet at 70 g/kg live weight 0.75/day. Blood samples were taken monthly by jugular puncture for three months and, after 39 days on experiment, jugular catheters were inserted for frequent sampling over a 24 h period. The samples were assayed for prolactin using radioimmunoassay and for cortisol by competitive protein binding. SLP caused a large and significant increase in plasma prolactin throughout the experiment, with no effect on sex. During the 24 h period there were consistent peaks of prolactin at the start of both dark phases under SLP, with lower levels around the start of both light phases; under SP, prolactin tended to be higher in the middle of the light phase and the middle of the dark phase than at other times. Considerable hourly fluctuations were observed in cortisol levels but an obvious 24 h rhythm was not. Mean levels were, however, significantly higher under SP (38.3 nmol/l) than under SLP (26.8 nmol/l).