Growth and function of thirty-four human benign and malignant thyroid xenografts in untreated nude mice.

Tissue was taken from 16 patients with benign thyroid lesions (10 nontoxic nodular colloid goiter, two follicular adenoma, one autonomous adenoma, one iodine-induced thyrotoxicosis, 2 Graves' disease) and 18 patients with malignant thyroid tumors [seven papillary, five follicular, five undifferentiated (anaplastic), and one medullalry carcinoma] and was xenotransplanted into the flanks of 124 syngeneic female BALB/c-nu/nu mice 6 weeks of age. Subsequently, without any further treatment, serum levels of thyroglobulin (TG), T3, T4, and thyroid-stimulating hormone were determined by radioimmunoassay at 4 or 5 weeks posttransplantation and at the end of the experimental time period of 4 months. All animals were autopsied. The grafts were examined by light microscopy and TG immunohistochemistry. Morphologically, the grafts of benign and malignant thyroid tumors showed features overall identical to the original tissue. Conversely, nontoxic nodular colloid goiter and Graves' disease grafts revealed a transformation to normofollicular structures. All benign thyroid grafts showed a stationary growth, as did most differentiated thyroid carcinomas. Tumor take rates in differentiated and in medullary carcinoma were 15%, and in undifferentiated carcinomas, 100%. In the cancer grafts, a correlation between resting phase (period until progressive tumor growth) and survival time of the corresponding patients was disclosed. All patients whose tumors were not taken by nude mice are still alive and show no signs of progressive tumor growth at 9 to 34 months after surgery. All but one patient with tumors revealing positive tumor take died within 3 months (resting phase, 3 weeks) or one year (resting phase, 7 to 14 weeks) after surgery. Integrity of hormonal function in benign and malignant xenografts at 4 months posttransplantation could be shown by significantly higher T3 and T4 serum concentrations in animals with benign thyroid tissues (T3, 1.69 +/- 0.13 nmol/liter; T4, 45.69 +/- 2.09 nmol/liter; S.E.) as compared to controls without grafted tissue [T3, 1.29 +/- 0.10 nmol/liter (p less than 0.05); T4, 33.39 +/- 2.71 nmol/liter (p less than 0.05)] and by increased TG serum concentrations in animals receiving benign (TG, 2.70 +/- 1.39 ng/ml) or malignant (e.g., TG in follicular carcinoma, 34.44 +/- 13.83 ng/ml; controls, 0.30 +/- 0.02 ng/ml) thyroid tissue. Thus, we conclude that benign and malignant thyroid xenografts in the nude mouse maintain full morphological and, regarding T3, T4, and TG serum levels, functional integrity for at least 4 months after transplantation.

The pre- and postnatal influence of hormones and neurotransmitters on sexual differentiation of the mammalian hypothalamus.

A number of brain structures and a great number of brain functions have been shown to be sexually dimorphic. It has also been shown that development and differentiation of these structures and functions proceeds during a critical pre- and postnatal period of increased susceptibility, and is controlled by gonadal steroids and neurotransmitter substances. The brain of male and female mammals seems to be still undifferentiated before the period of increased susceptibility to gonadal steroids and neurotransmitters starts. Feminization of brain structure and functions, e.g., establishment of the cyclic LH-surge mechanism and the expression of lordosis behavior, seems to depend on the moderate interaction of estrogens with the developing nervous system. Defeminization and masculinization of brain functions seem to be established during interaction of the developing nervous system with androgens, which have to be converted, at least in part, into estrogens. Structural differentiation of the male brain, e.g., the sexually dimorphic nucleus of the preoptic area (SDN-POA), seems to be exclusively estrogen-dependent, during differentiation of male brain functions, however, estrogens may be supportive, rather than directive, to the primary action of androgens. The molecular mechanisms of sexual differentiation of the brain are not yet fully understood. It seems, however, that the priming action of gonadal steroids during the period of increased susceptibility is either mediated by neurotransmitters, or neurotransmitters modulate the priming action of gonadal steroids. In particular, the adrenergic, the serotoninergic, the cholinergic, and possibly the dopaminergic system were shown to have strong influences on sexual differentiation of brain structure and functions. In contrast to the great number of available studies on the influence of gonadal steroids on sexual differentiation of the brain, there are rather few studies available concerning the influence of neurotransmitter systems. The available results are partly contradictory, so that an interpretation must be done with caution and will leave plenty of room for speculation. Postnatal application of compounds which stimulate or inhibit adrenergic activity mainly affected the neural control of gonadotropin secretion, and had only minor influences on differentiation of behavior patterns. It seems, however, that adrenergic participation in the differentiation of the center for cyclic gonadotropin release is very complex and stimulatory and inhibitory components may operate simultaneously. Activation or inhibition of beta-adrenergic receptors during postnatal development was shown to impair the responsiveness of the center for cyclic gonadotropin release to gonadal steroids, and impairs the expression of ejaculatory behavior in male rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes with age in levels of serum gonadotropins, prolactin and gonadal steroids in prepubertal male and female rats.

Radioimmunological determination of serum LH, FSH, and estradiol concentrations in prepubertal female rats demonstrates the temporal coincidence of increased serum levels of these hormones between days 9 and 21. Serum FSH and estradiol levels are continuously high during that time, whereas interindividual fluctuations in LH levels were enormous. No high LH, FSH, and estradiol levels were observed between day 21 and puberty, during which time serum prolactin and progesterone gradually increased. Serum testosterone in the female immature rats stayed uniformly low. It is suggested that increased serum estradiol levels in the presence of low prolactin levels (between day 10 and 20) act in a positive feedback fashion on the CNS-pituitary axis. The resulting increased gonadotropin levels are later (between day 20 and puberty) decreased by an inhibitory action of prolactin and/or progesterone on pituitary gonadotropin release. In male rats serum FSH and prolactin, which were low during the first 3 weeks, increased later to reach high levels during puberty. Serum LH was slightly elevated during the 2nd and 3rd week of life at which time serum progesterone also increased to reach the highest levels in the prepubertal period. Serum testosterone was higher in male than in female rats for the first 3 weeks of life; the difference between both sexes was significant but not striking. Between day 21 and the prepubertal period the testosterone levels were relatively low, but they increased again during puberty. Sex differences in androgen levels (measured with a less specific antibody) were more pronounced whereas estradiol levels in males showed the same pattern between birth and puberty as in the female littermates. These results suggest that not only testosterone but also other, not yet identified, androgens may be involved in the masculinzation of the brain.

Corelease of dynorphin-like immunoreactivity, luteinizing hormone, and follicle-stimulating hormone from rat adenohypophysis in vitro.

Rat anterior pituitary quarters or acutely dispersed rat anterior pituitary cells were incubated in vitro, and the release of dynorphin A1-13-like immunoreactivity (Dyn A1-13-IR) into the incubation medium was studied. Addition of LHRH led to a concentration-dependent enhancement of the release of Dyn A1-13-IR with a maximum secretory rate which was about 4-fold higher than basal secretion. Dyn A1-13-IR was released by LHRH concomitantly with LH and FSH, and the concentration-response relationships as well as the time course were virtually identical. Gel filtration and HPLC revealed a single peak of Dyn A1-13-IR, with an apparent mol wt of about 6000. In addition to Dyn A1-13-IR, alpha-neo-endorphin-like immunoreactivity was released by LHRH. The LHRH-stimulated release of Dyn A1-13-IR was mimicked by the LHRH analog D-Ala6,des-Gly10-LHRH ethylamide and blocked in a competitive manner by the LHRH antagonist D-pGlu1,D-Phe2,D-Trp3,6-LHRH. Addition of TRH (5 microM), rat corticotropin-releasing factor (100 nM), arginine vasopressin (1 microM), or synthetic human pancreatic GH-releasing hormone (10 nM) produced no effect on Dyn A1-13-IR release. An extract of the rat medial basal hypothalamus stimulated the release of Dyn A1-13-IR and beta-endorphin-like immunoreactivity, and the former, but not the latter, effect was blocked by the LHRH antagonist D-pGlu1,D-Phe2,D-Trp3,6-LHRH. These results demonstrate that dynorphin-like material and other proenkephalin B-derived peptides are released concomitantly with LH and FSH from rat adenohypophysis in vitro upon activation of LHRH receptors. This may indicate that proenkephalin B-derived peptides coexist with LH and/or FSH in at least some gonadotrophs of the normal rat anterior pituitary gland.

Release of dynorphin-like immunoreactivity from rat adenohypophysis in vitro during inhibition of anterior pituitary hormone secretion from individual cell types.

LHRH has previously been found to be the only known hypothalamic releasing factor which can specifically stimulate the release of the opioid dynorphin and other proenkephalin B-derived peptides from the rat adenohypophysis in vitro. In the present study the mechanisms that regulate dynorphin release were further characterized. It was examined whether or not dynorphin release from the adenohypophysis in vitro is altered during inhibition of the secretion of various anterior pituitary hormones. Rat anterior pituitary quarters were incubated in vitro and hormone release into the incubation medium was measured by RIAs. Somatostatin, dopamine, T3, dexamethasone, and 5 alpha-dihydrotestosterone were used to inhibit the secretion of GH, PRL, TSH, ACTH/beta-endorphin, or LH/FSH, respectively. GH, PRL, or beta-endorphin release was inhibited without affecting the simultaneous release of dynorphin A-(1-13)-like immunoreactivity (Dyn A1-13-IR). Concentrations of T3, somatostatin, or dopamine which were effective in suppressing the evoked and/or basal release of TSH, GH, or PRL, respectively, produced no effect on Dyn A1-13-IR release caused by high potassium concentration (40 mM) or LHRH (500 pM). The LHRH-induced release of LH and FSH was inhibited by the glucocorticoid dexamethasone or the androgen 5 alpha-dihydrotestosterone. Under these conditions, Dyn A1-13-IR release was also reduced. However, whereas LH release was completely blocked by 5 alpha-dihydrotestosterone, FSH and Dyn A1-13-IR release was reduced only by 50%. The release of FSH and Dyn A1-13-IR in vitro from anterior pituitary glands taken from rats, castrated 3 weeks before, was enhanced to a similar extent (about 2.5-fold); the simultaneous enhancement of LH release was significantly (P less than 0.005) greater (about 5-fold). We conclude that the mechanisms which regulate the release and/or biosynthesis of dynorphin and other proenkephalin B-derived peptides of the adenohypophysis are similar to those of the gonadotropins but different from those of any other anterior pituitary hormone, and may be more closely related with FSH release than LH release. These data support the view that dynorphin of the normal rat adenohypophysis may be localized in at least a subpopulation of gonadotrophs.

Effect of human growth hormone-releasing hormone on the release of dynorphin-like immunoreactivity, luteinizing hormone, and follicle-stimulating hormone from rat adenohypophysis in vitro.

The effect of GH-releasing hormone (GHRH) on the release of the endogenous opioid dynorphin from rat adenohypophysis was investigated in vitro. Rat anterior pituitary quarters were incubated in vitro, and hormone release into the incubation medium was measured by RIAs. Human pancreatic GHRH [hpGHRH-(1-44)] as well as human Leu27,Gly45-GHRH [GHRH-(1-45)] enhanced the secretion of dynorphin A1-13-like immunoreactivity (Dyn A1-13-IR) in a concentration-dependent manner. The concentrations of hpGHRH-(1-44) that stimulated the release of Dyn A1-13-IR were about 100-fold higher than those that enhanced GH secretion. GH release induced by hpGHRH-(1-44) was blocked by somatostatin (IC50, approximately 10 nM) without affecting hpGHRH-(1-44)-induced release of Dyn A1-13-IR. GH release was elicited by prostaglandin E2, while Dyn A1-13-IR secretion remained unchanged. At concentrations that enhanced Dyn A1-13-IR release, hpGHRH-(1-44) also elicited LH and FSH secretion. The LHRH antagonist D-pGlu1, D-Phe2,D-Trp3,6-LHRH blocked the secretion of Dyn A1-13-IR, LH, and FSH induced by hpGHRH-(1-44), whereas the LHRH antagonist did not influence the simultaneous GH release elicited by hpGHRH-(1-44). A possible direct effect of GHRH on the LHRH receptor was examined in radioligand binding studies using iodinated D-Ala6, des-Gly10-LHRH ethylamide (LHRH-A). The binding of [125I]iodo-LHRH-A to rat anterior pituitary membranes was completely displaced by hpGHRH-(1-44) and GHRH-(1-45). The deduced apparent dissociation constants were about 3 orders of magnitude higher than that of LHRH-A, but were close to those concentrations that enhanced Dyn A1-13-IR release. We conclude that GHRH-induced release of Dyn A1-13-IR is unrelated to GH release. High concentrations of GHRH may interact directly with LHRH receptors on gonadotrophs and thereby enhance the release of LH, FSH, and Dyn A1-13-IR.

Oestrogens and prolactin as possible regulators of puberty.

At day 15 after birth, high serum oestradiol levels, high FSH levels and occasionally high LH levels were observed in control female rats. Injections of potent and specific antisera for oestrogen between days 8-15 prevented LH peaks and decreased basal LH levels. Serum FSH levels were increased after treatment. High oestradiol levels at this time appear to exert a positive feedback action on phasic as well as tonic LH release. Injections of ovine prolactin (0.5 mug/g body wt twice daily) between days 8-25 of life significantly advanced the day of vaginal opening in immature rats and initiated regular oestrous cycles. Such treatment completely prevented high LH levels at day 15 but serum FSH levels remained high. Prolactin and progesterone levels were higher at day 25 than at day 15 in control rats and at that time LH and FSH levels were low. Prolactin treatment had a depressant effect on endogenous prolactin and progesterone levels. It is proposed that after day 20 the positive feedback threshold of oestradiol on LH release is at a mature, highly sensitive level. The steady increment in serum prolactin and/or progesterone levels between day 20 and puberty, however, inhibited phasic LH release thus preventing early puberty. The possible mechanisms of action of prolactin are discussed.

Activity of the pituitary-adrenocortical system and thyroid gland during the oestrous cycle of the rat.

The concentrations of LH, progesterone, ACTH, corticosterone and thyroxine in the plasma were estimated at various times during the oestrous cycle of the rat. The well-established patterns of LH and progesterone secretion were confirmed. On each day of the cycle the plasma concentrations of ACTH and corticosterone were lowest in the morning and rose in the afternoon. Conversely, during oestrus and dioestrus, the plasma concentrations of thyroxine were higher in the morning than in the evening. However, during the afternoon of pro-oestrus the concentrations of ACTH, corticosterone and thyroxine in the plasma rose and, like the concentrations of LH and progesterone, all reached levels far higher than those attained at any other time of the cycle.

Effects of tri-iodothyronine, thyroxine and isopropyl-di-iodothyronine on thyroid-stimulating hormone in serum and pituitary gland and on pituitary concentrations of prolactin, growth hormone, luteinizing hormone and follicle-stimulating hormone in hypothyroid rats.

Replacement of the 3'-halogen of the tri-iodothyronine (T3) molecule by a propyl-group produces a thyromimetic analogue, 3'-isopropyl-3,5-di-iodo-L-thyronine (T2iPr), with high biological potency. A serum thyroid-stimulating hormone (TSH) suppression test with one single intraperitoneal injection of 3 or 30 nM-T3 or T2iPr or with 30 or 300 nM-thyroxine (T4) per kg body weight was performed on 56 adult male Lewis rats which were maintained for 3 weeks on an iodine-deficient diet containing 0.2% 6n-propyl-2-thiouracil (PTU). Blood was withdrawn from each rat by cardiac puncture 24 h before and 3, 7, 24 and 48 h after application of the iodothyronines. Raised serum levels of TSH, due to the treatment with PTU, were significantly reduced within 3 h of treatment with 30 nM-T3, 300 nM-T4, 3 or 30 nM-T2iPr and they remained low throughout the observation period. Treatment with 3 nM-T3, or 30 nM-T4 per kg body weight was less effective. Pituitary concentrations of growth hormone, TSH, prolactin and FSH were significantly reduced by the treatment with PTU. There was also a slight, but insignificant reduction of pituitary concentrations of LH. Treatment with T3, T4 or T2iPr stimulated the reaccumulation of growth hormone, TSH, prolactin, LH and FSH in the pituitary gland.

Circannual variations in serum concentrations of pituitary, thyroid, parathyroid, gonadal and adrenal hormones in male laboratory rats.

Seasonal effects were studied on basal levels of hormones in the serum of adult male Sprague-Dawley rats, which were born and raised under rigorously controlled laboratory conditions. Groups of 90-day-old rats were killed at monthly intervals by rapid decapitation. Significant fluctuations were observed throughout the observation period of 19 months in serum levels of TSH, prolactin, androgens, tri-iodothyronine and LH. Minor fluctuations were observed in serum levels of FSH, corticosterone, parathyroid hormone and thyroxine. The results indicate that male laboratory rats exhibit circannual and semi-annual fluctuations in serum levels of several hormones even though the animals were born, raised and maintained in constant laboratory conditions.

Pre- and postnatal influence of testosterone propionate and diethylstilbestrol on differentiation of the sexually dimorphic nucleus of the preoptic area in male and female rats.

The volume of the sexually dimorphic nucleus in the preoptic area (SDN-POA) of the rat brain is several fold larger in males than in females. When female rats were treated pre- and postnatally with testosterone propionate (TP) or with diethylstilbestrol (DES) they became anovulatory and their SDN-POA developed equivalent in size to that of normal males. Identical treatment of male rats resulted in deficient testicular development, but had no influence on SDN-POA volume. The results indicate that the gross morphological sex difference in SDN-POA volume can exclusively be controlled by the hormonal environment during the critical period of sexual brain differentiation, and that non-steroidal estrogens are just as effective as convertible androgens in stimulating SDN-POA differentiation.

Postnatal treatment of rats with the beta 2-adrenergic agonist salbutamol influences the volume of the sexually dimorphic nucleus in the preoptic area.

Sexual differentiation of the brain seems to be influenced by postnatal interaction of gonadal steroids with neurotransmitter systems, in particular the adrenergic system. Stimulation or inhibition of adrenergic receptors during early postnatal development had previously been shown to influence steroid-induced sexual differentiation of rat brain function. In the present study newborn male and female rats were treated daily for 5 days with salbutamol, a specific beta 2-receptor agonist, or with alprenolol, a beta-receptor antagonist and the volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was examined in adulthood. This nucleus, one of the most striking sex differences in brain anatomy, is several-fold larger in male than in female rats. Postnatal treatment with salbutamol increased SDN-POA volume in female and in male rats. The effect was particularly striking in males, because any previous pre- and/or postnatal treatment of male rats with large amounts of gonadal steroids had been unable to increase the volume of the SDN-POA above normal. The beta-receptor antagonist alprenolol had no effect on SDN-POA differentiation. The results indicate that beta 2-adrenergic stimulation influences development and differentiation of the SDN-POA.

Some blood vessels in the rat median eminence are surrounded by a dense plexus of vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI) immunoreactive nerves.

Using immunohistochemistry at the light and electron microscopic level some blood vessels along the median eminence were shown to be surrounded by dense networks of vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI)-positive fibers. VIP and PHI released from these fibers may contribute to the elevated levels of these two peptides measured in portal blood as compared to peripheral blood by radioimmunoassay. VIP and PHI may also be important in the control of blood flow through the median eminence.

Differentiation of the sexually dimorphic nucleus in the preoptic area of the rat brain is determined by the perinatal hormone environment.

The volume of a sexually dimorphic nucleus in the preoptic area (SDN-POA) of the rat brain is several fold larger in males than in females. When female rats were treated pre- and postnatally with testosterone propionate (TP) their SDN-POA developed equivalent in size to that of normal males. The results indicate that the gross morphological sex difference in SDN-POA volume is exclusively controlled by the hormonal environment during the critical period of sexual brain differentiation.

Effects of adult or perinatal hormonal environment on ultradian rhythms in locomotor activity of laboratory LEW/Ztm rats.

Four experiments were performed with male and female rats of the inbred strain LEW/Ztm maintained under a light-dark schedule of 12:12 hours. The animals were subject to castration (GOX) or ovariectomy (OVX), estradiol 17 beta-implantation (E2-capsules), and perinatal hormonal treatments with testosterone propionate (TP) and an androgen antagonist (cyproterone acetate, CA). Results indicated a difference in the locomotor activity pattern between the two sexes as a result of the endogenous estradiol levels of the adult animals. The activity pattern of male LEW rats was characterized by ultradian rhythms of 4 and 4.8 hr periods. The female LEW rats, on the other hand, generally exhibited a clear circadian activity pattern and no ultradian activity rhythms. Following ovariectomy, each of the females showed distinct ultradian rhythms. These disappeared after E2-implantation. Castration of adult males had no effect on the ultradian activity pattern. Implantation of E2-capsules resulted in a marked decrease of the ultradian activity components. Perinatal treatment of the males with an androgen antagonist (CA) did not appear to effect ultradian rhythms during adulthood. Females treated perinatally with testosterone showed a significant increase in the ultradian activity components. This effect is assumed to be due to low estrogen levels in these animals during adulthood. Our study supports the assumption that ultradian rhythms are a result of changes in the phase relationships between several circadian oscillators. The synchrony of these oscillations seems to be facilitated by estradiol.

Corticotropin releasing hormone (CRH) increases beta-endorphin (beta-end like) concentration in cerebrospinal fluid of rats with vasospasm following subarachnoid hemorrhage.

The chronic stage of vasospasm occurring several days after subarachnoid hemorrhage (SAH) is characterized by the development of histopathologic changes in cerebral arteries causing cerebral ischemia. Numerous experimental data indicate the involvement of immune mechanisms in the angiopathy caused by SAH. Endogenous opioids play also an important role in the ischemic lesions of the brain. Corticotropin releasing hormone (CRH) induces the release of beta-endorphin (beta-END) from hypothalamic neurons and also from mononuclear white blood cells. The function of CRH and beta-END in vasospasm following SAH and the interrelationship between neuroendocrine and immune changes requires further elucidation. In the present study we investigated the influence of CRH injected into cerebral cisterna magna (CM) of rats on beta-END-like level in cerebrospinal fluid (CSF) in acute and chronic phase of cerebral vasospasm following artificial SAH. Acutely CRH induced a significant rise of beta-END-like in CSF both in SAH and sham SAH rats. However, in rats subjected to SAH, a single injection of CRH caused a prolonged rise of 5-END in CSF, which was also seen 2 days after SAH, during the chronic phase of vasospasm. The obtained results indicate that CRH increases neuroendocrine changes induced by SAH, probably by an activation of immune cells involved in the patomechanism of chronic vasospasm.

Influence of the corticotropin releasing hormone (CRH) on the brain-blood barrier permeability in cerebral ischemia in rats.

The increase in the blood-brain barrier (BBB) permeability and a developing cerebral oedema due to the ischemic infarction appear a few hours, and intensify during a few days, after closing the carotid arteries. It fails to be clear, however, what causes the increase in the microvessels damage, and whether the damage is a secondary result of the vasoactive substances released by the neurones and glia cells damaged by the ischemia. CRH, which plays an essential role in integrative the nervous, endocrine, and immunological systems, has a positive effect on the decrease in the permeability of the BBB damaged by various physical and chemical factors. Therefore, the examination of the CRH role in the cerebral ischemia may prove useful for explaining the processes taking place in the foci of the cerebral infarction and their environment. The experiment was carried out on rats which, 20 minutes before closing of both internal carotid arteries, was administered 10 microg CRH to cerebrospinal fluid via cisterna magna of the brain. The BBB permeability was measured 30 minutes, 3 hours, 3 days, and 7 days after closing the arteries. The experiment has shown the CRH protective effect on the BBB and its consequent effect on the decrease in the BBB permeability which appears in the 3 hours after closing the arteries (p<0.05), and is high significant during the chronic phase of the cerebral ischemia (p<0.03). It can be thus concluded that CRH, by affecting directly the endothelium of the cerebral vessels, decreases the endothelial damage in the acute phase of the ischemia. The decrease is noted to be more significant in the chronic phase of the ischemia; such an effect can be attributed to CRH stimulating the hypothalamic-adrenal axis, and to the secondary activation of the mechanisms decreasing the BBB permeability.

Stimulation of rat pituitary tumoral cell proliferation in vitro by tetra- and pentagastrin.

The effects of the active gastrin fragments, penta- and tetragastrin on the incorporation of tritiated thymidine into the rat pituitary tumoral cells were investigated in vitro. The significant stimulation of 3H-thymidine incorporation was observed as an effect of the investigated substances. The putative role of gastrin as an autocrine growth factor active in pituitary tumorigenesis was discussed.