Depressed pituitary prolactin mRNA, prolactin synthesis, and prolactin storage after light-deprivation in female hamsters is not due to loss of estrous cyclicity alone

Pituitary prolactin (PRL) cell activity (i.e. PRL messenger ribonucleic acid [mRNA] levels, PRL synthesis, and radioimmunoassayable [RIA]-PRL), and serum RIA-PRL were measured in female golden Syrian hamsters that were (1) light-deprived and then ovariectomized before loss of estrous cyclicity, (2) light-deprived but not yet acyclic, and (3) light-deprived and ovariectomized simultaneously. The results indicate that light-deprivation can decrease PRL cell activity in ovariectomized hamsters but not in animals that continue to cycle. Thus, estrous cyclicity can be said to largely protect PRL cell activity from depressions due to light deprivation. After acyclicity/ovariectomy, however, PRL cell activity is no longer protected and light-deprivation leads to large depressions in PRL mRNA levels, PRL synthesis, and RIA-PRL beyond that caused by acyclicity/ovariectomy alone. As seen in previous studies of total light-deprivation in nonovariectomized female hamsters, we found that removing the pineal gland in conjunction with light-deprivation in ovariectomized hamsters can completely, partially, or fail to restore various measures of PRL cell activity.     

Effects of light deprivation on prolactin cells in golden hamsters: an immunoelectron microscopic study.

In the golden hamster light deprivation has been shown to induce gonadal regression and reduction of pituitary and plasma levels of prolactin (PRL). In the present study we examined changes in morphology and population ratios of three types of PRL cells 8 weeks after light deprivation, by means of blinding or exposure of hamsters to continuous darkness. In the pituitary of intact hamsters of either sex, which were entrained to a 14-h light: 10-h dark cycle, Type C cells with large secretory granules were the most numerous and Type A with smaller granules the least. After light deprivation the pituitary was found to contain remarkably atrophic PRL cells and showed a profound change in population ratio of PRL cell types, i.e., Type A cells prevailed over the other two types. Pituitary glands from light-deprived and concurrently pinealectomized hamsters exhibited structures and a population ratio of three types of PRL cells similar to those from intact animals. It is suggested that small-granule-containing PRL cells represent an inactive stage of PRL cells, whereas medium- and large-granule-containing cells are functionally active cells. The atrophy of PRL cells can account for the decreased pituitary level of PRL in light-deprived hamsters reported previously.     

Melatonin-pineal relationships in female golden hamsters.

Light deprivation by blinding in female hamsters was followed by a regression of the reproductive organs, an elevation of pituitary LH concentration and a depression of pituitary prolactin levels. Pinealectomy negated almost completely the effects of light deprivation on the neuroendocrine-reproductive axis. Weekly subcutaneous implants of a melatonin:beeswax pellet completely prevented the pineal gland from inhibiting reproductive physiology in blinded hamsters. The findings suggest that melatonin is not pineal antigonadotrophic factor in female golden hamsters. Melatonin implanted hamsters also had higher than normal levels of plasma prolactin.     

Changes in hypothalamic serotonin concentration following manipulations on the gonadotrophic gonadal axis of the constant estrous anovulatory (CEA) rat.

The effect of pinealectomy or of injection of luteinizing hormone (LH) containing pituitary extract on the serotonin concentration of the hypothalamus was comparatively investigated in female rats with three different types of constant estrous anovulatory (CEA) syndrome. CEA syndrome was provoked by frontal hypothalamic deafferentation (FHD), by neonatal androgen treatment (NA) and by exposure of continuous illumination (light induced constant estrous; LCE syndrome). Pinealectomy caused an increase in hypothalamic serotonin concentration in the FHD rat, but failed to increase it in the NA and LCE group. The injection of crude anterior pituitary extract, however, provoked significant elevation of the hypothalamic serotonin concentration equally in all three types of CEA syndrome. Ovariectomy in itself failed to cause any significant change in the serotonin level of the brain. However, pinealectomy or the injection of LH containing pituitary extract proved to be effective also in the ovariectomized CEA animals. It is concluded that the increase in the brain-serotonin concentration of CEA rats, observed after pinealectomy or after injection of LH containing crude pituitary estract, is running through a hypothalamo-pituitary mechanism rather than through the hormone secreting activity of the ovaries.     

Immunocytochemical and ultrastructural correlates of the pineal inhibition of prolactin cell activity in blind-anosmic female rats

Summary The effects of the pineal gland on the light microscopic-immunocytochemical and ultrastructural appearance of pituitary mammotrophs were studied in female rats eight weeks after prepubertal blinding and olfactory bulbectomy.Blinding and anosmia resulted in a marked decrease in the size of the pars distalis concomitant with a reduction in the apparent number and size of PRL cells as compared with intact animals. Ultrastructurally, these cells appeared much less active than those of intact rats. The small and angular-shaped mammotrophs of blind-anosmic rats characteristically exhibited scant arrays of rough endoplasmic reticulum, small Golgi complexes with few immature secretory granules, few mature secretory granules and rare exocytosis patterns.Pinealectomy tended to reverse the effects of blinding and anosmia on pars distalis size and PRL cell size, apparent number and ultrastructure. In fact, the mammotrophs of blind-anosmic-pinealectomized rats were quite similar in ultrastructural appearance to those of intact rats.From these data we conclude that the pineal causes mammotroph hypotrophy and hypoplasia in blind-anosmic female rats.Supported by USPHS Biomedical Research Support Grant # RR 05675. The authors thank Dr. Bruce A. Richardson for his kind help with the immunocytochemistry     

Partial maintenance of testes and accessory organs in blinded hamsters by homoplastic anterior pituitary grafts or exogenous prolactin

When blinded, golden Syrian hamsters undergo marked gonadal atrophy. This phenomenon is prevented by pinealectomy. The mechanisms involved in this pineal-mediated response were investigated through either the transplantation of pituitary homografts or treatment of blinded, male hamsters with exogenous prolactin. It was found that anterior pituitary homografts placed beneath the kidney capsule on the day of bilateral optic enucleation partially maintained testicular and accessory organ weights. Serum prolactin levels were reduced in blinded animals below that of intact controls. On the other hand, blinded hamsters bearing anterior pituitary homografts showed serum prolactin levels comparable to those of intact controls. In other experiments, the injection of either 3.2 or 6.4 I.U. of ovine prolactin/hamster/ day for a period up to seven weeks partially inhibited the atrophy of testes and accessory organ weights in blinded hamsters. These data suggest a possible role for prolactin in the pineal-mediated atrophic response to light deprivation.     

An immunocytochemical study of effects of light deprivation on prolactin cells in the adenohypophysis of the golden hamster.

Population ratio and morphology of prolactin cells were studied by employing immunohistochemical methods in the adenohypophysis of normal and experimental golden hamsters of both sexes at 16 weeks of age. Prolactin cells occupied 29% of the total adenohypophyseal cells in the intact males exposed to 14/10 h light/dark schedule. After stimulation of the pineal activity by binding or exposure of males to continuous darkness for eight weeks, prolactin cells became atrophic and were reduced in population to 17% and 13%, respectively. Pinealectomy prevented to some extent the effects of the above treatments; thus, prolactin cells constituted 27% in the pinealectomized and blinded hamsters, and 19% in the pinealectomized and darkness-treated group; and their morphology was comparable with that of the intact controls. Prolactin cells in the normal females were apparently larger in size and more numerous as compared with those of the normal males, comprising 47% of cell population in the anterior pituitary. In response to light deprivation, prolactin cells were atrophic with a diminished cytoplasm and decreased in cell number as reflected in the population ratio of 27% in the blinded and 21% in the darkness-treated groups. In pinealectomized females combined with binding or darkness-treatment, prolactin cells contained an abundance of secretory granules in the cytoplasm and maintained the population ratio comparable to that in the intact females. The present study revealed that light deprivation exerts an inhibitory effect on the secretory activity of prolactin cells and also causes hypoplasia of prolactin cells in the hamster adenohypophysis, the effects being mediated by the pineal gland.     

Does pinealectomy,anopsia or exposure to constant darkness affect pituitary prolactin levels in the goldfish,Carassius auratus L.?

Synopsis The ef fects of exposure to constant darkness, anopsia and pinealectomy on pituitary prolactin levels in the goldfish were examined. Determination of prolactin was made in a polyacrylamide microelectrophoretic system coupled with densitometry. Neither pinealectomy nor anopsia had an effect on pituitary prolactin content in the fall. Exposure to constant darkness in the fall was without obvious effect, whereas such treatment in the spring resulted in significantly elevated pituitary prolactin content. Pinealectomy in the spring did not alter pituitary content, suggesting that the response to constant darkness is independent of the pineal organ, perhaps involving retinal or extra-retinal photoreception.     

Effects of pinealectomy on pituitary gonadotrophs, pituitary gonadotropin potency and hypothalamic gonadotropin releasing activity in Notemigonus crysoleucas

The effects of pinealectomy on pituitary gonadotrophs, pituitary gonadotropin potency and hypothalamic gonadotropin releasing activity were examined in the cyprinid teleost, Notemigonus crysoleucas, exposed to various photoperiod-temperature regimes. In fish exposed to a long photoperiod-warm temperature regime, pinealectomy resulted in a decrease in gonadal activity, in hypothalamic gonadotropin releasing activity and an increase in pituitary gonadotropin potency. Fewer gonadotrophs were present in the pituitary of sham operated fish than in the pituitary of pinealectomized fish. Ovarian development was more rapid in sham operated than in pinealectomized fish exposed to a long photoperiod–low temperature regime. Pituitary gonadotropin activity was also greater in shams than in pinealectomized fish. A short photoperiod-warm temperature regime retarded ovarian development in N. crysoleucas. Pinealectomy reversed this trend. Gonadotrophs made up a greater area of the pituitary in pinealectomized fish than in shams under these conditions. Gonadotropin potency of the pituitary and hypothalamic gonadotropin releasing activity were also greater in pinealectomized fish than in shams. The area of the pituitary occupied by gonadotrophs was greater in pinealectomized than in sham operated animals maintained on a short photoperiod-low temperature regime. Pituitary gonadotropin activity was also greater in pinealectomized fish as compared to shams. Pituitary gonadotropin potency varies diurnally in animals maintained on both short and long photoperiods; the rhythm of variation differs depending on photoperiod. Pinealectomy alters the diurnal rhythm of pituitary gonadotropin potency in animals exposed to both long and short photoperiods. It is concluded that pinealectomy has a pronounced effect on reproductive activity in N. crysoleucas. The effects of pinealectomy on reproduction vary with photoperiod, but are mediated via the hypothalamus and pituitary. In fish exposed to long daylengths the pineal favours reproductive activity, but the epiphysis retards reproductive processes in animals maintained on short photoperiods.     

The influence of blinding, olfactory bulbectomy and pinealectomy on plasma and anterior pituitary prolactin levels and on uterine and anterior pituitary weights in normal and neonatally androgenized rats

Lactating Sprague-Dawley rats had their litters adjusted to 8-10 pups on day 3 of lactation favoring females and some litters were injected with 1.25 mg of testosterone propionate to neonatally androgenize (NA) them. At 22-25 days of age both normal and NA animals were ovariectomized (OVX) and subjected to either sham olfactory bulbectomy (ANOS) + sham pinealectomy (PX), blinding (BLD) + ANOS or BLD + ANOS + pinealectomy (PX). At 13 weeks of age all animals were injected with 0.5 mg of polyestradiol phosphate. At 15 weeks of age the animals were fitted with atrial catheters and at 16 weeks of age blood samples (0.3 ml) were obtained every 3 hours over a 24 hour period. Uterine and anterior pituitary (AP) weights were recorded at sacrifice. Plasma and AP were assayed for prolactin (PRL) by RIA and AP were also assayed for PRL using the Nb2 lymphoma cell PRL bioassay. In both normal and NA animals, BLD + ANOS suppressed plasma PRL levels and PX partially prevented this response. Uterine weights were similar among groups while AP weights were significantly lower for sensory deprived animals. Anterior pituitaries extracted at pH 7.6 had a PRL concentration that was higher for the BLD + ANOS groups when estimated by either RIA or BA, a result that was not observed when the AP were extracted at pH 10.6. The amount of PRL extracted at pH 10.6 was twice that obtained at pH 7.6. Sensory deprived animals that were OVX prepubertally and administered estrogen as adults had a small but significant increase in mean plasma prolactin at 1700 hr. Both normal and NA animals responded in a similar manner to experimental manipulation.     

Studies on the minimal dosage of melatonin required to inhibit pineal antigonadotrophic activity in male golden hamsters.

Blinding adult male golden hamsters was followed by atrophy, within 12 weeks, of the testes and accessory sex organs (seminal vesicles and coagulating glands) and by a significant reduction in pituitary prolactin levels. In experiment 1 blind hamsters received subcutaneously implanted melatonin-beeswax (1:24 mg) pellets at the following intervals: once per week, per 2, 3, 4, 6 weeks, or only one pellet during the 12-week experimental period. The melatonin-beeswax pellets, regardless of the frequency of implantation, overcame completely the inhibitory effects of blinding on reproduction and nearly completely the depressant action of light deprivation on pituitary prolactin levels. In the second study the melatonin-beeswax pellets were implanted subcutaneously into blind hamsters every 2 weeks. The pellets contained either 1 mg, 500, 100, 50, or 1 mug melatonin. With the exception of the 1-mug dosage, melatonin again negated almost totally the inhibitory action of darkness on the gonads and accessory organs and also, for the most part, prevented the drop in pituitary prolactin levels. Based on these studies, when melatonin is chronically administered subcutaneously in a beeswax pellet the minimal dosage of melatonin required to counteract the inhibitory effect of darkness on reproduction seems to be less than 3.6 mug/day. The effects of chronic melatonin treatment are similar to those of pinealectomy.     

Prolactin Induces Apoptosis of Lactotropes in Female Rodents

Anterior pituitary cell turnover occurring during female sexual cycle is a poorly understood process that involves complex regulation of cell proliferation and apoptosis by multiple hormones. In rats, the prolactin (PRL) surge that occurs at proestrus coincides with the highest apoptotic rate. Since anterior pituitary cells express the prolactin receptor (PRLR), we aimed to address the actual role of PRL in the regulation of pituitary cell turnover in cycling females. We showed that acute hyperprolactinemia induced in ovariectomized rats using PRL injection or dopamine antagonist treatment rapidly increased apoptosis and decreased proliferation specifically of PRL producing cells (lactotropes), suggesting a direct regulation of these cell responses by PRL. To demonstrate that apoptosis naturally occurring at proestrus was regulated by transient elevation of endogenous PRL levels, we used PRLR-deficient female mice (PRLRKO) in which PRL signaling is totally abolished. According to our hypothesis, no increase in lactotrope apoptotic rate was observed at proestrus, which likely contributes to pituitary tumorigenesis observed in these animals. To decipher the molecular mechanisms underlying PRL effects, we explored the isoform-specific pattern of PRLR expression in cycling wild type females. This analysis revealed dramatic changes of long versus short PRLR ratio during the estrous cycle, which is particularly relevant since these isoforms exhibit distinct signaling properties. This pattern was markedly altered in a model of chronic PRLR signaling blockade involving transgenic mice expressing a pure PRLR antagonist (TG^{Δ1–9-G129R-hPRL}), providing evidence that PRL regulates the expression of its own receptor in an isoform-specific manner. Taken together, these results demonstrate that i) the PRL surge occurring during proestrus is a major proapoptotic signal for lactotropes, and ii) partial or total deficiencies in PRLR signaling in the anterior pituitary may result in pituitary hyperplasia and eventual prolactinoma development, as observed in TG^{Δ1–9-G129R-hPRL} and PRLRKO mice, respectively.     

Pineal and retinal melatonin is involved in the control of circadian locomotor activity and body temperature rhythms in the pigeon

Summary Although pinealectomy or blinding resulted in loss of the clarity of the free-running rhythm of locomotor activity and body temperature and reduced the peak level of circulating melatonin rhythms to approximately a half in intact pigeons, neither pinealectomy nor blinding abolished any of these rhythms. However, when pinealectomy and blinding were combined, the rhythms of locomotor activity and body temperature disappeared in prolonged constant dim light, and melatonin concentration was reduced to the minimum level of detection. In order to examine the role of melatonin in the pigeon's circadian system, it was administered either daily or continuously to PX + EX-pigeons in LLdim. Daily administration of melatonin restored circadian rhythms of locomotor activity which entrained to melatonin injections, but continuous administration did not induce any remarkable change of locomotor activity. These results suggest that melatonin synthesized in the pineal body and the eye contributes to circulating melatonin and its rhythmicity is important for the control of circadian rhythms of locomotor activity and body temperature in the pigeon.Abbreviations LD Light-dark - LLdim constant dim light - LLbright constant bright light - PX pinealectomy - EX blinding - SCN suprachiasmatic nucleus     

Prolactin secretion and its response to stress during the estrous cycle of the rats

Serum and pituitary prolactin (PRL) concentrations were measured during the estrous cycle of the rat with particular attention to the afternoons of the days of proestrus and estrus. Homogenizing machines, a Polytron and Sonifier, were used to extract PRL from the pituitary gland. The effects of ether anesthesia and restraint were also examined on the afternoons of both proestrus and estrus. The occurrence of a surge in PRL secretion during proestrus was confirmed with a peak at 1500 h, and this was accompanied by a decline in pituitary PRL content. A relatively high level of serum PRL was observed in the afternoon of estrus, during which time pituitary PRL content increased progressively. Ether anesthesia had no effect on the proestrus PRL surge, while restraint enhanced it. On the afternoon of estrus, restraint completely suppressed the rise in serum PRL, but ether anesthesia failed to suppress it completely. From these results, the following conclusions were drawn: 1) the PRL surge on the afternoon of proestrus occurs without synthesis of the hormone in the pituitary; 2) PRL secretion on the afternoon of estrus is accompanied by its synthesis in the gland; 3) the PRL response is distinct for each type of stress applied; and 4) PRL secretion is thus regulated by different mechanisms in proestrus and estrus.     

The effects of blinding,pinealectomy and exposure to constant dark conditions on gonadal activity in the female goldfish,Carassius auratus

Synopsis The effects of pinealectomy, blinding, and exposure to constant darkness were examined in female goldfish during different seasons. Neither blinding nor pinealectomy under short or decreasing photoperiod conditions, nor exposure to constant darkness had an effect on ovarian activity in goldfish during fall and winter. In spring, constant darkness has an inhibitory effect on ovarian activity. Pinealectomy under increasing photoperiod conditions partially inhibits reproductive activity, but not to the extent of constant darkness. Blinding, under the same regime also inhibited to some extent the ovarian response to increasing photoperiod. Our data suggest that retinal pathways and the pineal organ are involved in the photosexual response to increasing day lengths.Address correspondence to: Dr. V. L. de Vlaming, Marquette University     

Restoration of fertility in light-deprived female hamsters by chronic melatonin treatment

Summary Blinding young adult female hamsters was followed by functional involution of the ovaries and uteri and by the cessation of cyclic vaginal phenomena. Light deprivation was also accompanied by elevated plasma and pituitary levels of luteinizing hormone and depressed levels of prolactin in both the blood and the pituitary gland. Only one of 15 blinded hamsters became pregnant when they were exposed to fertile males for 30 days. Both pinealectomy or chronic melatonin treatment (1 mg melatonin implanted subcutaneously per week in beeswax) prevented the changes in the reproductive organs and in pituitary hormone levels attendant on light-deprivation. Both treatment also returned vaginal cycles to normal and restored plasma prolactin titers. Unlike hamsters that were blinded only, light deprived hamsters that were either pinealectomized or melatonin treated were capable of reproducing when they were caged with fertile males. The reproductive capability (i.e., percent of animals that become pregnant and the sizes of their litters) of these animals was equivalent to that of the untreated control hamsters. This is the first report that chronic melatonin treatment restores fertility in blinded female hamsters.Supported by Grant GB-43233X from the National Science Foundation     

Validity of radioimmunoassay for blood and pituitary prolactin in the male rat. Effect of bromocriptine and thyrotropin-releasing hormone

Validity of a radioimmunoassay for rat prolactin (PRL) in serum and pituitary is analysed in adult male rats. Data are presented bearing on the accuracy, precision and sensitivity of the method. Serum levels and pituitary content are respectively ranged from 2.56 to 28.03 ng PRL RP2 ml-1 and from 7.36 to 21.44 microgram PRL RP2 per gland in intact animals. Treatment with bromocriptine (10 days) results in a decrease of serum PRL levels and pituitary PRL contents. In progesterone-estradiol benzoate pretreated rats, serum PRL levels are increased 20 min after the injection of TRH.     

Does prolactin influence the hypothalamo-pituitary GnRH-LH system in preovulatory-phase ewes?

The effects of prolonged infusions of prolactin (PRL) into the third ventricle of the brain of cycling ewes on the secretory activity of hypothalamic GnRH neurons and pituitary LH cells in the pars distalis during the proestrous day were studied. Mature Blackhead ewes were infused with vehicle (control, n=5) or with prolactin (200 mug/day, n=5) during 4 consecutive days prior to the next spontaneous ovulation. The dose of PRL was infused each day in 4 series of 50 mug/100 mul/h at 30-min. intervals, from 8.30 to 14.00 h. The animals were slaughtered on the 16th (proestrous) day of the estrous cycle immediately after the last infusion and their brains were fixed in situ. Plasma samples were collected for 6 h at 10 min. intervals, on days 12 (before the infusions) and 16 of the cycle. The distribution pattern, number and morphology of GnRH neurons in vehicle- and PRL-infused ewes were found to be similar and typical for the proestrous phase of the cycle. The immunoreactive (ir) GnRH stores in the median eminence were high and similar in both groups. There were no differences between control and PRL-treated ewes in the number or features of irLH cells. The area fraction and optical density for irLH cells and mRNA LHbeta-expressing cells did not differ between control and experimental groups. Irrespective of the kind of infusion, changes in LH secretion during the estrous cycle were similar in control and PRL-infused ewes. Mean plasma LH concentrations were higher (p<0.001) on day 16 compared to day 12 of the cycle. There were no differences in plasma LH concentrations or in the parameters of pulsatile LH secretion between groups. In conclusion, repeated, several-hour-long infusions of PRL into the CNS prior to the next spontaneous ovulation in ewes has no direct effect on the secretory activity of GnRH neurons, and/or the synthesis, accumulation, or tonic release of LH from the pituitary gonadotrophs.     

The proestrous prolactin surge is not the sole initiator of regressive changes in corpora lutea of normally cycling rats.

During the estrous cycle, secretion of prolactin is largely restricted to a surge on proestrus. We investigated whether this proestrous prolactin surge initiates regression of the corpora lutea of the preceding cycle. Adult rats were killed prior to the prolactin surge (Proestrus group), following the prolactin surge (Estrus group), after chemical blockade of the prolactin surge with bromocryptine (Estrus+BRC group), and after blockade of the prolactin surge and administration of prolactin (Estrus+BRC+PRL group). Corpora lutea of the current (proestrus) or preceding (estrus) cycle were dissected out, weighed, and sectioned for immunohistochemistry or cultured for examination of in vitro progestin production. Numbers of luteal monocytes/macrophages, differentiated macrophages, and apoptotic nuclei per high-power field were greater for Estrus and Estrus+BRC+PRL than for Estrus+BRC, which in turn had greater numbers than Proestrus (P< 0.05). In contrast, BRC completely reversed the decline in luteal weight observed between Proestrus and Estrus (P<0.05). Number of major histocompatibility complex II-positive cells was not different between groups (P>0.05). Finally, progestin production by corpora lutea in vitro was lower for Proestrus than for the other groups (P<0.05). The results indicate that the prolactin surge alone is not responsible for initiation of apoptosis or immune cell infiltration in regressing corpora lutea of the estrous cycle, although prolactin increases these markers of regression. Prolactin does cause a decline in luteal weight; however, the corpora lutea retain the capacity for steroidogenesis. We conclude that although prolactin has a role in luteal regression, it is not solely responsible for the initiation of this process.