Cognitive impairment and morphological changes in the dorsal hippocampus of very old female rats.

The hippocampus, a medial temporal lobe structure necessary for the formation of spatial memory, is particularly affected by both normal and pathologic aging. In previous studies, we observed a significant age-related increase in dopaminergic neuron loss in the hypothalamus and the substantia nigra of female rats, which becomes more conspicuous at extreme ages. Here, we extend our studies by assessing spatial memory in 4-6 month-old (young), 26-month-old (old) and 29-32-month-old (senile) Sprague-Dawley female rats as well as the age-related histopathological changes in their dorsal hippocampus. Age changes in spatial memory performance were assessed with a modified version of the Barnes maze test. We employed two probe trials (PTs), one and five days after training, respectively, in order to evaluate learning ability as well as short-term and longer-term spatial memory retention. A set of relevant hippocampal cell markers was also quantitated in the animals by means of an unbiased stereological approach. The results revealed that old rats perform better than senile rats in acquisition trials and young rats perform better than both aging groups. However, during short-term PT both aging groups showed a preserved spatial memory while in longer-term PT, spatial memory showed deterioration in both aged groups. Morphological analysis showed a marked decrease (94-97%) in doublecortin neuron number in the dentate gyrus in both aged groups and a reduction in glial fibrillary acidic protein-positive cell number in the stratum radiatum of aging rats. Astroglial process length and branching complexity decreased in aged rats. We conclude that while target-seeking activity and learning ability decrease in aged females, spatial memory only declines in the longer-term tests. The reduction in neuroblast number and astroglial arborescence complexity in the dorsal hippocampus are likely to play a role in the cognitive deficits of aging rats.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats.

Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30-31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.

Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.

The ependymal route for insulin-like growth factor-1 gene therapy in the brain.

I.c.v. administration of the peptide insulin-like growth factor-1 (IGF-1) has been shown to be an effective neuroprotective strategy in the brain of different animal models, a major advantage being the achievement of high concentrations of IGF-1 in the brain without altering serum levels of the peptide. In order to exploit this therapeutic approach further, we used high performance recombinant adenoviral (RAd) vectors expressing their transgene under the control of the potent mouse cytomegalovirus immediate early (mCMV) promoter, to transduce brain ependymal cells with high efficiency and to achieve effective release of transgenic IGF-1 into the cerebrospinal fluid (CSF). We constructed RAd vectors expressing either a chimeric green fluorescent protein fused to HSV-1 thymidine kinase (TK/GFP)(fus), or the cDNA encoding rat IGF-1, both driven by the mCMV promoter. The vectors were injected into the lateral ventricles of young rats and chimeric GFP expression in brain sections was assessed by fluorescence microscopy. The ependymal cell marker vimentin was detected by immunofluorescence and nuclei were labeled with the DNA dye 4',6-diamidino-2-phenylindole. Blood and CSF samples were drawn at different times post-vector injection. In all cerebral ventricles, vimentin immunoreactive cells of the ependyma were predominantly transduced by RAd-(TK/GFP)(fus), showing nuclear and cytoplasmic expression of the transgene. For tanycytes (TK/GFP)(fus) expression was evident in their cytoplasmic processes as they penetrated deep into the hypothalamic parenchyma. I.c.v. injection of RAd-IGF-1 induced high levels of IGF-1 in the CSF but not in serum. We conclude that the ependymal route constitutes an effective approach for implementing experimental IGF-1 gene therapy in the brain.

Dopaminergic mesencephalic systems and behavioral performance in very old rats.

Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinson's disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4-6 months), old (22-24 months) and senile (30-32 months) Sprague-Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species.

Restorative effect of insulin-like growth factor-I gene therapy in the hypothalamus of senile rats with dopaminergic dysfunction.

Insulin-like growth factor-I (IGF-I) is emerging as a powerful neuroprotective molecule that is strongly induced in the central nervous system after different insults. We constructed a recombinant adenoviral vector (RAd-IGFI) harboring the gene for rat IGF-I and used it to implement IGF-I gene therapy in the hypothalamus of senile female rats, which display hypothalamic dopaminergic (DA) neurodegeneration and as a consequence, chronic hyperprolactinemia. Restorative IGF-I gene therapy was implemented in young (5 months) and senile (28 months) female rats, which received a single intrahypothalamic injection of 3 x 10(9) plaque-forming units of RAd-betagal (a control adenoviral vector expressing beta-galactosidase) or RAd-IGFI and were killed 17 days post-injection. In the young animals, neither vector modified serum prolactin levels, but in the RAd-IGFI-injected senile rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase-positive cells in the hypothalamus of experimental as compared with control senile animals (5874+/-486 and 3390+/-498, respectively). Our results indicate that IGF-I gene therapy in senile female rats is highly effective for restoring their hypothalamic DA dysfunction and thus reversing their chronic hyperprolactinemia.

Gene therapy in the neuroendocrine system.

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

Gene therapy for long-term restoration of circulating thymulin in thymectomized mice and rats.

Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.

Potential of gene therapy for the treatment of pituitary tumors.

Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy-the transfer of genetic material for therapeutic purposes-has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. We close the article by discussing the future of molecular therapies. We point out that although, gene therapy represents a key step in the development of molecular medicine, it has inherent limitations. As a consequence, it is our view that at some point, genetic therapies will have to move from exogenous gene transfer (i.e. gene therapy) to endogenous gene repair. This approach will call for radically new technologies, such as nanotechnology, whose present state of development is outlined.

Glucocorticoid-induced apoptosis in lymphoid organs is associated with a delayed increase in circulating deoxyribonucleic acid.

Pathological processes like cancer, chronic inflammation and autoimmune phenomena, all of which involve massive cell death, are associated with significant increases in circulating DNA. In order to clarify whether massive apoptosis occurring under physiological circumstances also causes DNA release into the circulation, we correlated the time-course of dexamethasone-induced intra thymic cell apoptosis with plasma DNA dynamics in rats. Animals were given 10 mg/l dexamethasone in their drinking water for up to 7 days. Sequential plasma samples were obtained during the treatment and DNA was quantitated by a micro fluorometric assay. Thymus and spleen weight as well as apoptotic cell levels were assessed at different times. Seven days of glucocorticoid treatment reduced thymic and spleen mass by 82 and 31%, respectively. Intra thymic apoptosis was maximal 24 h after the beginning of glucocorticoid treatment, declining markedly by 48 h. Very little apoptosis was observed in the spleen. Plasma DNA increased steadily during the first 4 days of glucocorticoid treatment (11.8 +/- 1.2 microg/ml on day 0; 24.2 +/- 1.6 microg/ml on day 4) beginning to decline afterward. Thymectomy but not splenectomy, drastically reduced the glucocorticoid-induced increase in plasma DNA. It is concluded that hormone-induced massive intra thymic cell death is followed by a delayed release of nucleosomal DNA into the circulation.

Concentraciones séricas de hormona de crecimiento en perras con tumores mamarios espontáneos antes y después de la mastectomía / Growth hormone serum concentrations in bitches with spontaneous mammary tumors before and after mastectomy

La producción no pituitaria y autónoma de hormona del crecimiento (GH) en la grándula mamaria ha sido reportada en perros. Se determinó el impacto de la mastectomía de glándulas mamarias tumorales sobre las concentraciones séricas de GH, en casos clínicos de tumores mamarios espontáneos de dieciocho perras enteras, a las que se les realizó mastectomía de todas las glándulas tumorales. Tres perras sanas fueron mantenidas solo bajo protocolo anestésico por un período similar al del procedimiento quirúrgico. Se tomaron muestras sanguíneas por venopunción periférica para determinar GH sérica, antes (día -1), 2 y 11 horas y 7 y 14 días después de la mastectomía o de la anestesia. Se realizó el estudio histopatológico de las glándulas mamarias tumorales extirpadas en la cirugía. . La GH sérica fue determinada por un radioinmunoensayo homólogo de fase líquida. La media ñ SEM (ng/ml) del grupo tratado antes y después de la cirugía fue de 17.3ñ 2.8; 8.5ñ1.5; 6.3ñ1.1 y 6.1ñ1.5 para el día -1, 2 horas, 7 y 14 días respectivamente. Los mismos valores para el grupo control fueron de 4.2 ñ1.0; 5.5 ñ 1.3 y 5.2 ñ 1.5 para el día -1, 2 y 11 horas luego de la anestesia. Después de la cirugía, la GH sérica descendió (media ñ SEM) -22.6 por ciento ñ 4.9 a las 2, -31,8 por ciento ñ 5,1 al día 7, y -29,7 por ciento ñ 9 al día 15, mientras que en el grupo control se incrementó en un 31.1 por ciento ñ 08 y 26.3 por ciento ñ 9 a las 2 y 11 horas, respectivamente luego de la anestesia. La histopatología de las mamas tumorales analizadas reveló carcinoma tubular y papilar, adenocarcinoma, carcinosarcoma y carcinoma complejo. Se concluyó que en perras la GH ectópica es también producida por la glándula mamaria con tumores espontáneos malignos (AU)

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: impact on hormone secretion.

OBJECTIVE: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/beta-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli beta-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). DESIGN: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/beta-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. METHODS: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for beta-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. RESULTS: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/beta-gal showed widespread expression of the beta-galactosidase transgene around the injection areas. CONCLUSIONS: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.

Thyroid-stimulating hormone and growth hormone release alterations induced by mosquito larvae proteins on pituitary cells.

Mosquito larvae crude extract has shown to modulate cell proliferation of different mouse epithelial as well as human mononuclear cell populations in vivo and in vitro. A soluble fraction of the extract, with a molecular weight ranging from 12 to 80 kD, also showed an inhibitory effect on the proliferation of mouse hepatocytes. This effect disappeared after heating the extract at 90 degrees C for 60 min, suggesting that some proteinaceous molecule is involved. We report the effect of dialysed extract (MW >12 kD) on the concentration of both thyroid-stimulating hormone (TSH) and growth hormone (GH) in an incubation medium of pituitary cells from normal and oestrogenised rats. Time- and dose-dependent response of both hormones resulted in increasing TSH levels. Concentrations of GH were lower in the treated than in control pituitary cells. The time elapsed until the finding of differences suggests the presence in the mosquito extract of some protein binding the hormone. The differences were not due to lethal toxic effects since the Trypan blue viability test showed no differences between control and treated cells. Furthermore, the effect disappeared when the extract had previously been heated at 90 degrees C for 60 min. Finally, our results suggest the presence of some proteins in the mosquito Culex pipiens L. larvae, which would act as a pituitary hormone regulator.

Neuroendocrinology of aging: the potential of gene therapy as an interventive strategy.

OBJECTIVE: This paper reviews the current status of gene therapy in the neuroendocrine system and discusses the interventive potential of this methodology for neuroendocrine pathologies associated with aging. BACKGROUND AND RESULTS: A brief description is first presented of the viral-vector-based gene delivery systems being currently used in the neuroendocrine system, namely the adenoviral and herpetic (HSV1) vector systems. Next, an account of the neuroendocrine pathologies for which gene therapy approaches in animal models are being implemented is provided. This includes the treatment of experimental pituitary tumors by adenoviral-vector-mediated transfer of the suicide gene for the HSV-1 thymidine kinase. At the hypothalamic level, an adenovirus harboring the cDNA for arginine vasopressin has been used in Brattleboro rats to correct their diabetes insipidus. Next, the interventive potential of gene therapy for correcting age-associated neurodegenerative processes at neuroendocrine level is outlined. Finally, the role that emerging technologies may play in the development of future genetic therapies for aging is considered. CONCLUSION: Although effective implementation of gene therapy strategies still faces significant technical obstacles, these are likely to be progressively overcome as gene delivery systems are refined.

Study of the change of prolactin and progesterone during dopaminergic agonist treatments in pseudopregnant bitches.

In order to clarify the role of prolactin (PRL) and progesterone (P(4)) in the pathophysiology of canine pseudopregnancy (PSP) we designed an experiment, where we induced an abrupt pharmacological blockade of PRL secretion with dopaminergic agonists (DA) or placebo (PL). Thirty overtly pseudopregnant (PSPT) bitches were randomly allocated to three groups of 10 animals each: PL, bromocriptine (BR), and cabergoline (CA), which were treated with PL, 7.5microg/kg BR and 5microg/kg CA, respectively. On days 1, 7 and 14 (day 0: beginning of the treatment) all the animals were classified into grades of intensity of PSP clinical signs, considering serum or milk secretion and enlargement of the mammary glands. Presence or absence of treatment side effects were recorded and blood samples for PRL and P(4) determinations collected. Serum PRL and P(4) concentrations (ng/ml) of all the animals on day 1 were (least squares means [LSM]+/-S.E.M.) 17.70+/-2.05 and 1.13+/-0.13, respectively. During the experiment, serum PRL and P(4) concentrations decreased (day effect, P<0.05). During the experiment, serum PRL concentrations were lower in the DA treated group (BR and CA) compared with PL group (P<0.05). After a week of treatment, the percentage change of PRL was -62.52 versus 102.16+/-46.20 (P<0.01) for the treated (BR and CA) and PL groups, respectively. Conversely, no significant differences were found in the percent change in PRL between the BR and the CA groups nor in P(4) percentage change among all groups for the same week. Significant differences in the achievement of complete remission between treated and PL groups were found on days 7 (40 versus 0%, P<0.05) and 14 (90 versus 0%, P<0.01). No significant correlation between PRL and P(4) was found on day 1 in any of the animals. However, a significant correlation for the same hormones was found on days 7 and 14 for the DA treated groups (r=0.46, P<0.01). While in the PL group, PRL concentrations and intensity of clinical signs were not significantly correlated on days 1, 7 and 14; in the DA treated groups they were significantly correlated on days 7 and 14 (r=0.34, P<0.05). The presence of a positive correlation between PRL concentrations and the grades of intensity of clinical signs in the treated animals indicates the major role of PRL in PSP physiopathology. However, the lack of correlation during spontaneous involution of PSP in the PL group demonstrates that PRL concentrations do not completely explain the problem. In summary, abrupt changes in serum PRL seemed to be more important in ceasing PSP signs than total PRL concentrations in these groups of animals.

Twenty-four-hour profiles of serum prolactin and luteinizing hormone in anoestrous crossbred bitches.

The dynamics of prolactin (PRL) and luteinizing hormone (LH) secretion in the anoestrous bitch is poorly known. Therefore, the objective of this study was to characterize the 24 h profiles of serum PRL and LH in crossbred anoestrous bitches and to assess whether a relationship exists between the secretory patterns of these two hormones. Serum PRL and LH concentrations were measured in 10 healthy anoestrous crossbred bitches at 145 min intervals for 24 h. During the experiment the animals received continuous artificial illumination and remained undisturbed except at the time of blood sampling. Serum PRL was measured by a homologous enzyme-linked immunosorbent assay, whereas LH and progesterone (P4) were determined by radioimmunoassay. The anoestrous state of the bitches was assessed by vaginal cytology, vaginoscopy and physical examination. Two groups of animals were identified according to their PRL levels: a high PRL group (n = 3, mean +/- SEM 12.3 +/- 2.7 ng/ml) and a low PRL group (n = 7, mean +/- SEM: 2.5 +/- 0.9 ng/ml). In the low PRL group, the PRL profiles were flat and did not show any significant circadian pattern. Nevertheless, occasional single-point peaks were detected in some of the bitches. In the high PRL group the individual PRL profiles were variable. To detect the presence of a circadian change of PRL concentrations, two different sets of time windows (TW) of sampling were studied. The first set was: day [TW1A, samples 1-5 (0900-1840 h)] and night [TW1B, samples 6-10 (2105-0645 h)]. The second set was chosen after visual inspection of the average PRL profiles for both (high and low) groups: [TW2A, samples 3-7 (1350-2330 h) and TW2B, samples 1-2 and 8-10 (0155-1125 h)]. PRL concentrations were not significantly different between day and night. In the high PRL group, but not in the low PRL group, average serum PRL was significantly (p < 0.01) higher in TW2A than TW2B. In both groups serum LH levels were more homogeneous than PRL levels. Neither TW showed circadian changes in LH patterns of secretion (TW1A versus TW1B, p < 0.69; TW2A versus TW2B, p < 0.88). On the other hand, bitches in the high PRL group showed significantly (p < 0.01) lower serum LH levels than those in the low PRL group of animals. Serum PRL concentrations presented a significant inverse correlation with LH concentrations (r=-0.21, p < 0.03) and a significant positive correlation with P4 concentrations across the study (0.92, p < 0.01). It is concluded that in anoestrous crossbred bitches serum PRL is highly variable and inversely related to LH. No circadian rhythm of PRL secretion appears to exist in most anoestrous bitches.

Altered functional responses with preserved morphology of gonadotrophic cells in congenitally athymic mice.

Neonatal thymectomy or congenital absence of the thymus induces severe reproductive deficiencies in female mice, which are associated with reduced levels of circulating and pituitary gonadotropins. In contrast, the reproductive function is well preserved in nude males. It was therefore of interest to assess gonadotrophic cell morphology and function in congenitally athymic male mice. Circulating gonadotropins were measured under basal and stressful conditions, taking as a reference their haired counterparts. Adult normal (+/+), heterozygous nude (nu/+), and homozygous (nu/nu) CD-1 mice were subjected to 1-h immobilization stress. Serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were assessed by RIA at 0, 30, and 60 min poststress. Athymic animals showed significantly lower basal levels of serum LH and FSH than their heterozygous littermates. Immunohistochemical assessment of LH and FSH cell populations revealed a normal morphology and cell number in the athymic animals compared to their normal littermates. Immobilization stress induced a significant reduction in gonadotrophin levels, particularly LH, in normal mice but had only a weak effect in athymic animals. It is concluded that congenital athymia in the adult male mouse is associated with decreased basal levels of serum LH and FSH, in the presence of a normal gonadotroph number and morphology. The anomalous responses of athymic mice to stress do not appear to be due to primary hypopituitarism but, rather, to an altered modulation of pituitary hormone secretion. .

Gene therapy in the neuroendocrine system: its implementation in experimental models using viral vectors.

Gene therapy, the transfer of genetic material for therapeutic purposes, has undergone an explosive development in the last few years. Within this context, development of gene therapy approaches for the neuroendocrine system, while incipient, has already generated a core of results which emerge as a promising area of research in neuroendocrinology. The present review presents a brief description of the viral vector-based gene delivery systems being currently used in neuroendocrinology, namely the adenoviral and herpes simplex type-1 (HSV-1)-derived vector systems, as well as an updated account of neuroendocrine pathologies for which gene therapy approaches in animal models are being implemented is provided. Current research efforts include treatment of experimental pituitary tumors by adenoviral vector-mediated transfer of the suicide gene for the HSV-1 thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. An adenoviral vector encoding the human retinoblastoma suppressor oncogene has also been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. At the hypothalamic level, an adenovirus harboring the cDNA for arginine vasopressin has been used in Brattleboro rats to correct diabetes insipidus for several weeks. The last part of the review outlines the potential of gene therapy to correct age-associated neurodegenerative processes at the neuroendocrine level. Although effective implementation of gene therapy strategies still faces significant technical obstacles, these are likely to be progressively overcome as gene delivery systems are being improved.

Mosquito larvae proteins modulate luteinizing hormone and prolactin release in pituitary cells of normal and estrogenized rats.

Whilst looking for vertebrate growth factor homologues in insects, we found that a soluble fraction of a 12-80 kDa molecular weight band peaking at 25 kDa, isolated from mosquito larvae extracts by gel permeation chromatography, had a modulatory effect on mouse hepatocytes and adult human mononuclear cell proliferation. The effect disappeared after heating the extract at 90 degrees C for 30 min, suggesting that the active factor may be a protein. In order to determine the activity of the extract on cell function, we assessed the effect of the extract on pituitary hormone secretion in vitro. We assayed a dialyzed fraction (MW greater than 12 kDa) of mosquito larvae for its effect on the release of luteinizing hormone (LH) and prolactin (PRL) from dispersed rat pituitary cells. In normal anterior pituitary (AP) cells we found that the extract had a stimulatory effect on LH release but an inhibitory action on prolactin secretion. In AP cells obtained from estrogen-induced hyperplasia, the extract had an inhibitory effect on prolactin secretion. In all cases the effects were time- and dose-dependent. Interference of the mosquito proteins with the radioimmunoassay was checked and found to be negligible. After a 60 min incubation, cell viability was comparable in control and treated cells. Furthermore, the biological effect of the extract was thermally unstable. Our results suggest that mosquito larvae may share common factors with mammals, probably peptidic in nature, which are able to modulate cell function.

Heterogeneity of circulating prolactin in the bitch.

Different molecular forms of circulating prolactin (PRL) are known to occur in several species. As no such information was available in dogs, we assessed the molecular profile of circulating PRL in bitches. Pooled sera from covertly (CTRL) and overtly pseudopregnant (PSPT) diestrous bitches with high or low (> 10 or < 10 ng x mL(-1), respectively) serum PRL (measured by ELISA) were analyzed by Sephadex G-100 and Concanavalin A-Sepharose column chromatography. Four serum PRL fractions were identified and termed big-big, big (> 67 kDa), native (23 kDa) and fragmented (< 20) kDa) PRL. The percentages of these fractions were roughly similar in CTRL and PSPT animals, irrespective of their serum PRL levels (higher in PSPT than in CTRL bitches). A large proportion of glycosylated PRL (between 69 and 100%) was also detected in these sera. We conclude that in dogs, circulating PRL occurs in multiple molecular forms, whose relative abundance is comparable in covertly and overtly pseudopregnant bitches.