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.     

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     

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.     

Growth related changes in the content of heparin and 5-hydroxytryptamine of mast cells

Summary Fine structural changes of testicular interstitial cells of Leydig and secretory cells of seminal vesicles were studied in golden hamsters under different functional states of the pineal gland. Experiments were performed in the reproductive season (summer months). In the hamsters blinded for 8 weeks the testes and the seminal vesicles were markedly atrophic, and the Leydig cells and the secretory cells of seminal vesicles were extremely involuted. By contrast, both types of cells in the pinealectomized or superior cervical ganglionectomized hamsters exhibited cytological features suggestive of an enhanced secretory activity. This study shows that functional activity of Leydig cells as well as secretory cells of seminal vesicles in the hamster may be depressed or augmented by stimulating or inhibiting the pineal antigonadal function, respectively, without performing hypophysectomy or hormonal administration.Dedicated to Professor Shu Yeh on the occasion of his 70th birthday. This study was supported in part by a grant from the National Science Council, the Republic of China     

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.     

Pineal-independent regulation of photo-nonresponsiveness in the Siberian hamster (Phodopus sungorus)

The pineal hormone melatonin influences circadian rhythms and also mediates reproductive responses to photoperiod. The authors tested whether pinealectomy influences circadian oscillators responsible for induction of nonresponsiveness to short day lengths by preventing normal short-day patterns of circadian entrainment. Adult male Siberian hamsters were pinealectomized or sham operated, maintained in either 18 h light per day (18L) or 15L for 10 weeks, and then tested for responsiveness to 10L. Because pinealectomized hamsters do not show gonadal regression in short day lengths, responsiveness was assessed by measuring phase angle of entrainment and the length of the nightly activity period following transfer to 10L. The incidence of nonresponsiveness was significantly higher in 18L hamsters than in 15L hamsters but was unaffected by pineal status. Fully 88% of 18L hamsters failed to entrain to 10L in the normal short-day manner; the duration of nightly activity remained compressed, and the phase angle of entrainment was large and negative relative to lights off. The 15L hamsters entrained normally to 10L. Exposure to constant light after 10L treatment was equally effective in inducing arrhythmicity in pinealectomized and intact hamsters. Changes in the period of morning and evening circadian oscillators subsequent to 18L treatment did not predict circadian responsiveness to short photoperiod. Long-day induction of photo-nonresponsiveness, which prevents winter responses to short day lengths, occurs independently of pineal melatonin feedback on the circadian system.     

Depressed prolactin cell activity in long-term blind-pinealectomized female hamsters is due to loss of estrous cyclicity

Pinealectomy in the female golden Syrian hamster is not always completely effective in preventing the suppressive effects of long-term light deprivation due to blinding on pituitary prolactin (PRL) cell activity. We examined this curious phenomenon by measuring pituitary PRL mRNA levels, PRL synthesis, and radioimmunoassayable PRL, and correlating these changes with the status of estrous cyclicity. As expected, 12 weeks of light deprivation resulted in loss of estrous cyclicity and a greater than 90% decline in all indices of pituitary PRL cell activity, compared with intact cycling controls. Pinealectomy prevented only 40-50% of this decline. However, if noncycling light-deprived pinealectomized animals were excluded, pinealectomy was completely effective, i.e., cycling intact control animals were no different than cycling blind-pinealectomized. We conclude that the inability of pinealectomy to completely prevent the decline in prolactin cell activity seen after blinding is due to the loss of estrous cyclicity in some blind-pinealectomized females, with the attendant loss of the prolactin-stimulating hormone estrogen.     

Relationship between norepinephrine release in the hypothalamic paraventricular nucleus and circulating prolactin levels in the Siberian hamster: role of photoperiod and the pineal gland

The impact of norepinephrine (NE) and its metabolite, 3-methoxy4-hydroxyphenylglycol (MHPG), on circulating prolactin (PRL) was evaluated in the paraventricular region of the hypothalamus as a function of photoperiod and integrity of the pineal gland. In Experiment 1, whole tissue content of NE and MHPG was assessed in male and female hamsters that had been pinealectomized or sham-pinealectomized and exposed to long or short photoperiods for 5 weeks. The results revealed a marginal effect of photoperiod in males, but no overall effects of surgery. Because analysis of whole tissue content can be complicated by concurrent changes in synthesis and storage rates, Experiment 2 was conducted using microdialysis to assess extracellular levels of NE and MHPG in female hamsters. Pinealectomy completely prevented the short-day-induced suppression of luteinizing hormone, but it only partially prevented the effects of short days on PRL. Furthermore, both NE and MHPG levels were significantly elevated in short-day-exposed pinealectomized and sham-operated animals. These results suggest that NE release within the paraventricular nucleus inhibits the circulating PRL levels and is one mechanism by which direct photic information can influence the neuroendocrine axis independently of the pineal melatonin signal.     

Ultrastructural features of the pineal gland in normal and light deprived golden hamsters

Summary Although current physiological findings imply that the mammalian pineal organ liberates an antigonadal agent, microscopic examinations of this organ have afforded little information regarding the possible storage and release of such a substance. Since it is known that light deprivation for six weeks results in pineal-induced atrophy of certain reproductive organs in adult golden hamsters, one might expect that any morphological manifestations of this activity in the pineal organ would be enhanced in hamsters which had heen deprived of light for that length of time. A comparison at the ultrastructural level of pineal glands from normal and experimentally blinded hamsters revealed that pineal cells from the blinded animals exhibited a greater number of vesicles and contained complex membranous whorls. The possible significance of the vesicles and lamellar whorls is discussed in terms of similar structures found in other tissues.A feature common to pineal tissue of both the normal and experimental hamsters was the apparent cellular segregation of two morphologically distinct types of mitochondria. Pinealocytes containing small, cristaform mitochondria were designated as P1 cells; those containing larger mitochondria characterized by a dense, plexiform array of cristae were designated as P2 cells.Supported by A. D. Williams 3558, Medical College of Virginia, and National Institutes of Health 5FI-GM-31, 981-02.The author is grateful to Dr. Hugo R. Seibel of the Department of Anatomy at the Medical College of Virginia for assistance with the surgical procedures employed in this study.     

Morphometric analysis of the pineal complex of the golden hamster over a 24-hour light:dark cycle: I. The superficial pineal in untreated and optically enucleated animals

Morphometric analysis of the superficial pineal gland of intact and blinded golden hamsters was conducted at both the light and electron microscopic level. The volume of the superficial gland was estimated to be 151 X 10(6) micron 3, comprising 90-94% of the total pineal parenchymal tissue. Analysis of structural rhythms in animals maintained under a 14:10 L:D cycle showed significant 24-hr variations in values for pinealocyte nuclei, nucleoli, rough and smooth endoplasmic reticulum, Golgi bodies, dense bodies, and dense-cored vesicles. Peak values for these structures generally occurred at the light:dark interface. These results provide morphological correlates for known rhythmic variations in the synthesis of pineal-gland products. Superficial pineals examined 8 weeks following optic enucleation exhibited a decrease in the volume of pinealocyte nuclei and cytoplasm, while nucleolar size and the amounts of smooth and rough endoplasmic reticulum, Golgi bodies, dense bodies and dense-cored vesicles were enhanced. The latter changes are interpreted as indications of increased synthetic activity by the superficial pineal gland in response to light deprivation.     

Origin of prolactin cells in the pituitary autografts in the renal capsule of young male and female rats.

Origin of the prolactin cells in the pituitary grafts autotransplanted into the renal capsule was electron microscopically investigated in young male and female rats. Prolactin cells may mainly originate from the completely degranulated acidophils after grafting in both sexes. The comprehensive degranulation of acidophils may be essential condition for the development of prolactin cells. Formation of initial prolactin granules is related to the Golgi apparatus in 3 and 6 days in the males. Even prolonged transplantation for the duration of 10 and 20 days failed to develop the typical prolactin cells storing large polymorphic granules in the grafts in the males. In the females, however, the population of prolactin cells became higher (20-30%) and their granulation was more rapidly advanced than in the male. Nine and 16 days after grafting quite numerous typical prolactin cells, characterized by the pronounced vesiculation of ER stored large polymorphic granules in the females. Progressive vesiculation of ER in degranulated acidophils may be the second necessary condition for establishing the prolactin cells. Thus, the entire course of cell conversion of acidophils into prolactin cells was observed in this study. All the graft cells including prolactin cells finally turned into the cells losing the respective individuality (10 days in the males; 32 days in the females). This fine structural similarity was striking in the males. We could not, however, deny the possibility of origination of prolactin cells from the follicular cells or their adjoining undifferentiated cells.     

Daily rhythm in serum melatonin and leptin levels in the Syrian hamster (Mesocricetus auratus)

Melatonin is produced and secreted by the pineal gland in a rhythmic manner; circulating levels are high at night and low in the day. Leptin is a hormone secreted by adipocytes as a product of the obese gene and plays an important role in regulating body energy homeostasis and reproductive function in rodents and humans. The present study was conducted to examine daily fluctuations in serum levels of melatonin and leptin in Syrian hamster. We measured serum leptin and melatonin levels by ELISA in (a) intact and pinealectomized (pinx) male hamsters kept under long daylight conditions [14 h of light (14L)]; (b) intact and pinx hamsters under short daylight (10L); and (c) intact hamsters in constant light (24L). Blood samples were obtained every 2 h throughout a 24-h period. Statistically significant circadian variations were found in both melatonin and leptin profiles. Their relationship was inverse, i.e. when melatonin was high in the serum, leptin was comparably low. These results suggest that there is a rhythm in leptin levels in the adult male Syrian hamster and this rhythm is pineal gland (melatonin) and/or photoperiod dependent.     

Immunocytochemical analysis of prolactin cells in the adult rat adenohypophysis: distribution and quantitation relative to sex and strain

Prolactin immunocytochemistry was conducted 1) in serial sections of whole fixed pituitaries, for cell distribution analysis; and 2) on preparations of enzymatically dispersed anterior pituitary cells maintained for 1-4 days in vitro, for quantitation of the relative population of prolactin (PRL) cells. Both types of analysis were conducted on glands from young adult rats of both sexes in Long-Evans, Sprague-Dawley, and Fischer 344 strains. Cell quantitation results were compared with serum levels, intracellular content, and in vitro release of prolactin, for individual cell preparations. The results show that both PRL cell distribution and relative population numbers are similar between sexes in all three rat strains. Average percentages ranged from 25.9% to 32.1% in all young males and diestrous females. Prolactin-positive numbers of cells correlated positively with intracellular hormone content but not with serum or medium PRL levels. The prolactin cell population was significantly larger in aged Fischer 344 males but was not correlated with intracellular PRL levels. The prolactin cell population was heterogeneous in staining intensity and distribution in both sexes of all three strains.     

Reception of photoperiodic information by fetal Siberian hamsters: role of the mother's pineal gland

The rate of reproductive development in juvenile male Siberian hamsters is strongly influenced by daylength (photoperiod). Recent studies indicate that reception of photoperiodic cues begins during fetal life. The present experiments yielded a further demonstration that developing male Siberian hamsters receive information about the photoperiod to which their mother is exposed during pregnancy. The possibility that photoperiodic information is transmitted from mother to young after birth was investigated by cross-fostering young gestated on 12L and 16L to mothers from the other photoperiod. Litters were cross-fostered on the day of birth and then were transferred, along with their foster mothers, to 14L. We found no influence of the mother after birth, indicating that transmission of photoperiodic information from mother to young must occur during gestation. To determine if the pineal gland of the mother is required for this response, adult females were pinealectomized or sham-operated and paired with intact males in 12L, 14L, or 16L. After parturition parents and offspring were exposed to 14L. The influence of prenatal photoperiod on postnatal testicular development in 14L was blocked by pinealectomy of the mother. Postnatal testicular development was retarded in offspring that experienced a photoperiod transfer from either 15L to 14L or 8L to 12L at birth. In contrast, the inhibitory effect of a transfer from 16L to 14L at birth was abolished when juvenile males were exposed to a single long photoperiod (16.3 h light) at age 17-21 days and then were returned to 14L.     

The differentiation of prolactin cells in an organ culture of chick embryo adenohypophysis

We have studied differentiation of prolactin cells in explants of cephalic and caudal parts of Rathke's pouch of 4.5 day and 5.5 day old chick embryos after their incubation in vitro lasting for 7-8 days. Indirect immunofluorescence using an antiserum against bovine prolactin was used to detect prolactin cells in the cultures. Differentiation of prolactin cells was detected regularly in explants of the cephalic lobe of the adenohypophysis anlage in 5.5 day old embryos; under certain growth conditions prolactin cells were found in explants of the same lobe in 4.5 day old embryos. Prolactin cells were either absent or found in small numbers in cultures of the caudal part of adenohypophysis of 5.5 day old embryos. Our results provide evidence for the appearance of the committed precursors of prolactin cells in the Rathke's pouch at late stages of its formation and for their regional localization in the cephalic part of the anlage. This localization is in correspondence with the distribution of differentiated cells of this type in definitive adenohypophysis.     

Cold exposure as a potentiating factor of pineal actions in nonhibernating mammals

Twenty-eight-day-old male rats were used in three experiments to study whether cold exposure potentiates pineal actions in nonhibernating mammals. The following questions were considered: (a) Can cold exposure increase the antigonadal effects of light deprivation? (b) Are the effects induced by blindness plus cold exposure pineal dependent? (c) Can cold exposure modify the response of the endocrine-reproductive axis to exogenously administered melatonin? Blind cold-exposed rats showed a significant loss in body weight as well as in weights of pituitary and reproductive tract organs compared with either intact or blind animals kept at 22 degrees C, or intact rats exposed to cold; serum testosterone levels were also lowest in blind cold-exposed rats. These effects were not present in blind cold-exposed animals that were pinealectomized at the beginning of the experiment. When intact animals placed at 22 or 10 degrees C were treated with daily injections of melatonin (50 micrograms) there was a reduction of body weight and weights of the hypophyso-gonadal axis organs. Those effects of melatonin were, however, significantly greater in cold-exposed rats than in rats placed at 22 degrees C. These results suggest that cold exposure should be considered as another state which potentiates the pineal-dependent actions of light deprivation. Cold exposure probably acts by increasing the sensitivity of sites at which pineal melatonin exerts its actions.     

The effect of pinealectomy, melatonin and leptin hormones on ovarian follicular development in female Syrian hamsters (Mesocricetus auratus)

We studied the effects of melatonin and leptin hormones on ovarian follicular development in intact and pinealectomized female Syrian hamsters. We first monitored the oestrous cycle of the hamsters by the vaginal smear samples throughout a ten day period to start the injections simultaneously in all groups and performed saline, melatonin and leptin hormone injection groups for both control and pinealectomized hamsters. Then the injections were applied for four days starting the oestrus phase of the cycle and the ovaries were removed for preparation of histological analysis. We measured the diameters and the numbers of the follicles and we classified the follicles according to the number of the granulosa cell layer. Leptin hormone injection increased melatonin hormone injection decreased the number and the diameter of the follicles. The stimulating effect of the leptin hormone was more pronounced in the pinealectomized group. The results of the present study indicate that the removal of the pineal gland and leptin hormone administration are playing a stimulatory while melatonin hormone administration is playing an inhibitory role on the follicular development in female Syrian hamsters.     

Maternal transfer of photoperiodic information in Siberian hamsters. IV. Peripubertal reproductive development in the absence of maternal photoperiodic signals during gestation

Peripubertal reproductive development of Siberian hamsters is influenced by photoperiodic information received during gestation; the maternal pineal is important for this process. We observed that in the absence of the maternal pineal, the fetus appears to receive no information about gestational photoperiods. This is not the equivalent of receipt of a long-day signal by the fetus. Pinealectomized and sham-operated pregnant females were exposed to photoperiods of 12L:12D, 14L:10D, 16L:8D, or constant light (LL); young were reared from birth to 28 days of age in LL or 14L:10D. Regardless of the gestational photoperiod, LL-reared male young born to pinealectomized dams had smaller testes than LL-reared young of pineal-intact dams exposed to 16L:8D while pregnant. Thus, pinealectomy did not result in transmission of a long-day signal, nor did young born of pinealectomized dams receive short- or intermediate-day signals. Unlike young of pineal-intact females exposed to 12L:12D or 14L:10D while pregnant, young born of pinealectomized dams had small testes when reared in 14L:10D, irrespective of gestational photoperiod. Uterine weights of female young presented similar patterns of responses. In a second experiment, adult females were entrained to 12L:12D, 14L:10D, or 16L:8D for 3 wk prior to pinealectomy to determine if the effect of maternal pinealectomy would be altered. Entrainment to the new photoperiod prior to surgery did not alter the effects of maternal pinealectomy.     

Immunocytochemical studies on prolactin cells in the adenohypophysis of the golden hamster

Mammotrophs or prolactin (PRL) cells were identified in the adenohypophysis of adult golden hamsters by immunocytochemical techniques with a polyclonal anti-PRL, that was proved to be specific to PRL by the dot immunoblotting test. Postembedding immunostaining was performed on Araldite thin sections by immunoperoxidase and immunogold methods. PRL cells were classified into three types according to the different size of the secretory granules. The Type A cells were usually small and angular or oval in shape, and had secretory granules ranging in diameter from 100-230 nm, and showed poorly developed organelles. The Type B and C cells were larger and round or ovoid in shape, contained larger granules, 230-280 nm and 280-570 nm, respectively, and displayed well developed organelles. Immunoreactive PRL cells in the male pituitaries were far less numerous than in the nonpregnant female glands, and were mostly of the Type A and B, whereas in the female the Type C and B cells predominated. In pregnant females, Type C cells became activated and increased in number, while the other two types decreased in proportion. In lactating females, Type A and B cells significantly increased in number at the expense of the Type C cells; meanwhile, the exocytosis of secretory granules was frequently found in all types of PRL cells. The present findings suggest that Type C and B PRL cells, especially the former, are potent in producing and releasing PRL and highly responsive to various physiological stimuli, while Type A cells are probably relatively inert in synthetic activity.