The role of nitric oxide in reproduction

Nitric oxide (NO) plays a crucial role in reproduction at every level in the organism. In the brain, it activates the release of luteinizing hormone-releasing hormone (LHRH). The axons of the LHRH neurons project to the mating centers in the brain stem and by afferent pathways evoke the lordosis reflex in female rats. In males, there is activation of NOergic terminals that release NO in the corpora cavernosa penis to induce erection by generation of cyclic guanosine monophosphate (cGMP). NO also activates the release of LHRH which reaches the pituitary and activates the release of gonadotropins by activating neural NO synthase (nNOS) in the pituitary gland. In the gonad, NO plays an important role in inducing ovulation and in causing luteolysis, whereas in the reproductive tract, it relaxes uterine muscle via cGMP and constricts it via prostaglandins (PG).

Oxytocin is a cardiovascular hormone

Oxytocin (OT), a nonapeptide, was the first hormone to have its biological activities established and chemical structure determined. It was believed that OT is released from hypothalamic nerve terminals of the posterior hypophysis into the circulation where it stimulates uterine contractions during parturition, and milk ejection during lactation. However, equivalent concentrations of OT were found in the male hypophysis, and similar stimuli of OT release were determined for both sexes, suggesting other physiological functions. Indeed, recent studies indicate that OT is involved in cognition, tolerance, adaptation and complex sexual and maternal behaviour, as well as in the regulation of cardiovascular functions. It has long been known that OT induces natriuresis and causes a fall in mean arterial pressure, both after acute and chronic treatment, but the mechanism was not clear. The discovery of the natriuretic family shed new light on this matter. Atrial natriuretic peptide (ANP), a potent natriuretic and vasorelaxant hormone, originally isolated from rat atria, has been found at other sites, including the brain. Blood volume expansion causes ANP release that is believed to be important in the induction of natriuresis and diuresis, which in turn act to reduce the increase in blood volume. Neurohypophysectomy totally abolishes the ANP response to volume expansion. This indicates that one of the major hypophyseal peptides is responsible for ANP release. The role of ANP in OT-induced natriuresis was evaluated, and we hypothesized that the cardio-renal effects of OT are mediated by the release of ANP from the heart. To support this hypothesis, we have demonstrated the presence and synthesis of OT receptors in all heart compartments and the vasculature. The functionality of these receptors has been established by the ability of OT to induce ANP release from perfused heart or atrial slices. Furthermore, we have shown that the heart and large vessels like the aorta and vena cava are sites of OT synthesis. Therefore, locally produced OT may have important regulatory functions within the heart and vascular beds. Such functions may include slowing down of the heart or the regulation of local vascular tone.

Biosynthesis and metabolism of steroid hormones by human adrenal carcinomas

Over a 15-year period, our university-based laboratory obtained 125 adrenal tumors, of which 15 (12%) were adrenal cortical carcinomas. Of these, 6 (40% of the carcinomas) occurred in patients with clear clinical manifestations of steroid hormone excess. Adrenal cortical carcinoma cells derived from the surgically resected tumors in 4 of these patients were isolated and established in primary culture. Radiotracer steroid interconversion studies were carried out with these cultures and also on mitochondria isolated from homogenized tissues. Large tumors had the lowest steroidogenic activities per weight, whereas small tumors had more moderately depressed enzyme activities relative to cells from normal glands. In incubations with pregnenolone as substrate, 1 mM metyrapone blocked the synthesis of corticosterone and cortisol and also the formation of aldosterone. Metyrapone inhibition was associated with a concomitant increase in the formation of androgens (androstenedione and testosterone) from pregnenolone. Administration of metyrapone in vivo before surgery in one patient resulted in a similar increase in plasma androstenedione, though plasma testosterone levels were not significantly affected. In cultures of two of four tumors examined, dibutyryl cAMP stimulated 11ß-hydroxylase activity modestly; ACTH also had a significant stimulatory effect in one of these tumors. Unlike results obtained with normal or adenomatous adrenal cortical tissues, mitochondria from carcinomatous cells showed a lack of support of either cholesterol side-chain cleavage enzyme complex or steroid 11ß-hydroxylase activity by Krebs cycle intermediates (10 mM isocitrate, succinate or malate). This finding is consistent with the concept that these carcinomas may tend to function predominantly in an anaerobic manner, rather than through the oxidation of Krebs cycle intermediates.

Extracellular calcium-sensing receptor: structural and functional features and association with diseases

The recently cloned extracellular calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays an essential role in the regulation of extracellular calcium homeostasis. This receptor is expressed in all tissues related to this control (parathyroid glands, thyroid C-cells, kidneys, intestine and bones) and also in tissues with apparently no role in the maintenance of extracellular calcium levels, such as brain, skin and pancreas. The CaR amino acid sequence is compatible with three major domains: a long and hydrophilic aminoterminal extracellular domain, where most of the activating and inactivating mutations described to date are located and where the dimerization process occurs, and the agonist-binding site is located, a hydrophobic transmembrane domain involved in the signal transduction mechanism from the extracellular domain to its respective G protein, and a carboxyterminal intracellular tail, with a well-established role for cell surface CaR expression and for signal transduction. CaR cloning was immediately followed by the association of genetic human diseases with inactivating and activating CaR mutations: familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism are caused by CaR-inactivating mutations, whereas autosomal dominant hypoparathyroidism is secondary to CaR-activating mutations. Finally, we will comment on the development of drugs that modulate CaR function by either activating (calcimimetic drugs) or antagonizing it (calcilytic drugs), and on their potential therapeutic implications, such as medical control of specific cases of primary and uremic hyperparathyroidism with calcimimetic drugs and a potential treatment for osteoporosis with a calcilytic drug.

Galactosaminoglycans from normal myometrium and leiomyoma

In many tumors, the amount of chondroitin sulfate in the extracellular matrix has been shown to be elevated when compared to the corresponding normal tissue. Nevertheless, the degree of chondroitin sulfate increase varies widely. In order to investigate a possible correlation between the amount of chondroitin sulfate and tumor size, several individual specimens of human leiomyoma, a benign uterine tumor, were analyzed. The glycosaminoglycans from eight tumors were extracted and compared with those from the respective adjacent normal myometrium. The main glycosaminoglycan found in normal myometrium was dermatan sulfate, with small amounts of chondroitin sulfate and heparan sulfate. In leiomyoma, both dermatan sulfate and chondroitin sulfate were detected and the total amounts of the two galactosaminoglycans was increased in all tumors when compared to normal tissue. In contrast, the heparan sulfate concentration decreased in the tumor. To assess the disaccharide composition of galactosaminoglycans, these compounds were incubated with bacterial chondroitinases AC and ABC. The amounts of L-iduronic acid-containing disaccharides remained constant, whereas the concentration of D-glucuronic acid-containing disaccharides increased from 2 to 10 times in the tumor, indicating that D-glucuronic acid-containing disaccharides are responsible for the elevation in galactosaminoglycan concentration. This increase is positively correlated with tumor size.

Dopaminergic modulation of grooming behavior in virgin and pregnant rats

Dopamine receptors are involved in the expression of grooming behavior. The pregnancy-induced increase in self-licking observed in rats is important for mammary gland development and lactation. This study focuses on the role of dopamine receptor subtypes in grooming behavior of virgin and pregnant female rats. General and mammary gland grooming were measured in virgin rats treated with 0.25 mg/kg of the D1-like agonist SKF-81297 and antagonist SKF-83566 and the D2-like agonist lisuride and antagonist sulpiride. The effects of 0.01 and 0.25 mg/kg doses of the same agonists and antagonists were evaluated in pregnant rats as well. In virgin animals both SKF-83566 and sulpiride treatments significantly reduced the time spent in general grooming, while none of the dopamine agonists was able to significantly change any parameter of general grooming. Time spent in grooming directed at the mammary glands was not affected significantly by any of the drug treatments in virgin rats. All drugs tested significantly decreased the frequency of and the time spent with general grooming, while SKF-81297 treatment alone did not significantly reduce the duration of mammary gland grooming in pregnant rats. These data show that in female rats the behavioral effects of D1-like and D2-like dopamine receptor stimulation and blockade differ according to physiological state. The results suggest that dopamine receptors may play specific roles modulating grooming behavior in pregnant rats. Since grooming of the mammary gland during pregnancy may influence lactation, this aspect is relevant for studies regarding the perinatal use of dopamine-related drugs.

Reproductive experience influences grooming behavior during pregnancy in rats

The pregnancy-induced increase in self-licking observed in rats is important for mammary gland development and lactation. Reproductive experience has epidemiologial implications such as a decrease in the incidence of mammary gland cancer in women and it also influences various behavioral, neurochemical and endocrine parameters. The aim of the present study was to investigate the influence of reproductive experience on grooming behavior patterns during pregnancy in rats. Self-grooming behavior was measured in age-matched virgin, primi- and multigravid (days 7, 8, 9, 19, and 20 of pregnancy) rats. General grooming (head, forelimbs and shoulders) was not significantly different among virgin, primi- and multigravid rats during pregnancy. Confirming previous work, pregnant rats spent significantly more time in specific grooming (mammary glands, nipple lines, genital and pelvic regions) than did virgin animals. In addition, self- licking of mammary glands was significantly increased in multi- as compared to primigravid rats on days 8, 9, 19 and 20 of pregnancy. The increase in mammary gland grooming observed in multigravid rats appears to be a consequence of previous reproductive experience. These data show that reproductive experience modulates mammary gland grooming during pregnancy, possibly contributing to successful reproduction.

Role of endogenous glucocorticoids in hyperalgesia and edema in old arthritic rats

We compared the intensity and frequency of arthritis in old (8-12 months, N = 12) and juvenile (2 months, N = 10) rats and determined the role played by adrenal glands in this disorder. Arthritis was induced by subcutaneous injection of Mycobacterium butyricum at the base of the tail of female Holtzman rats at day zero. Paw edema and hyperalgesia were monitored from day zero to day 21 after induction as signs of arthritis development. Some (N = 11) old animals were adrenalectomized bilaterally and treated with dexamethasone or celecoxib immediately following surgery. All bilaterally adrenalectomized old animals became susceptible to arthritis and the onset of disease was shortened from the 10th to the 5th day. Hyperalgesia and paw edema responses were less frequent in older animals (50 and 25% compared to control juvenile rats, respectively), although old responder animals showed responses of similar intensity to those of their juvenile counterparts: by the 14th day the data for hyperalgesia were juvenile = 0.8 ± 0.07/old = 0.8 ± 0.09, and for paw edema juvenile = 56.6 ± 6.04/old = 32.24 ± 12.7, reported as delta% increase in paw edema. Chronic treatment of adrenalectomized old animals with dexamethasone (0.01 or 0.1 mg/kg) but not celecoxib (3 mg/kg), once daily for 21 days by gavage, abolished the effects of adrenalectomy, in particular those related to the hyperalgesia response (old = 0.95 ± 0.03/dexamethasone = 0 ± 0; 14th day), thus suggesting a specific participation of circulating corticosteroids in the modulation of pain in old arthritic rats.

Distribution of versican and hyaluronan in the mouse uterus during decidualization

Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-µm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation.

Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Câncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8%) of the tumors were HPV DNA positive while 29.1% of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P = 0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P = 0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

Relationships between fetal body weight of Wistar rats at term and the extent of skeletal ossification

We investigated the relationship between fetal body weight at term (pregnancy day 21) and the extent of ossification of sternum, metacarpus, metatarsus, phalanges (proximal, medial and distal) of fore- and hindlimbs and cervical and coccygeal vertebrae in Wistar rats. The relationships between fetal body weight and sex, intrauterine position, uterine horn, horn size, and litter size were determined using historical control data (7594 fetuses; 769 litters) of untreated rats. Relationships between body weight and degree of ossification were examined in a subset of 1484 historical control fetuses (154 litters) which were subsequently cleared and stained with alizarin red S. Fetal weight was independent of horn size, uterine horn side (left or right) or intrauterine position. Males were heavier than females and fetal weight decreased with increasing litter size. Evaluation of the skeleton showed that ossification of sternum, metacarpus and metatarsus was extensively complete and independent of fetal weight on pregnancy day 21. In contrast, the extent of ossification of fore- and hindlimb phalanges and of cervical and sacrococcygeal vertebrae was dependent on fetal body weight. The strongest correlation between body weight and degree of ossification was found for hindlimb, medial and proximal phalanges. Our data therefore suggest that, in full-term rat fetuses (day 21), reduced ossification of sternum, metacarpus and metatarsus results from a localized impairment of bone calcification (i.e., a malformation or variation) rather than from general growth retardation and that ossification of hindlimb (medial and proximal) phalanges is a good indicator of treatment-induced fetal growth retardation.

A connection between extracellular matrix and hormonal signals during the development of the human fetal adrenal gland

The human adrenal cortex, involved in adaptive responses to stress, body homeostasis and secondary sexual characters, emerges from a tightly regulated development of a zone-specific secretion pattern during fetal life. Its development during fetal life is critical for the well being of pregnancy, the initiation of delivery, and even for an adequate adaptation to extra-uterine life. As early as from the sixth week of pregnancy, the fetal adrenal gland is characterized by a highly proliferative zone at the periphery, a concentric migration accompanied by cell differentiation (cortisol secretion) and apoptosis in the central androgen-secreting fetal zone. After birth, a strong reorganization occurs in the adrenal gland so that it better fulfills the newborn's needs, with aldosterone production in the external zona glomerulosa, cortisol secretion in the zona fasciculata and androgens in the central zona reticularis. In addition to the major hormonal stimuli provided by angiotensin II and adrenocorticotropin, we have tested for some years the hypotheses that such plasticity may be under the control of the extracellular matrix. A growing number of data have been harvested during the last years, in particular about extracellular matrix expression and its putative role in the development of the human adrenal cortex. Laminin, collagen and fibronectin have been shown to play important roles not only in the plasticity of the adrenal cortex, but also in cell responsiveness to hormones, thus clarifying some of the unexplained observations that used to feed controversies.

Effects of stress on catecholamine stores in central and peripheral tissues of long-term socially isolated rats

Both the peripheral sympatho-adrenomedullary and central catecholaminergic systems are activated by various psycho-social and physical stressors. Catecholamine stores in the hypothalamus, hippocampus, adrenal glands, and heart auricles of long-term socially isolated (21 days) and control 3-month-old male Wistar rats, as well as their response to immobilization of all 4 limbs and head fixed for 2 h and cold stress (4ºC, 2 h), were studied. A simultaneous single isotope radioenzymatic assay based on the conversion of catecholamines to the corresponding O-methylated derivatives by catechol-O-methyl-transferase in the presence of S-adenosyl-l-(³H-methyl)-methionine was used. The O-methylated derivatives were oxidized to ³H-vanilline and the radioactivity measured. Social isolation produced depletion of hypothalamic norepinephrine (about 18%) and hippocampal dopamine (about 20%) stores and no changes in peripheral tissues. Immobilization decreased catecholamine stores (approximately 39%) in central and peripheral tissues of control animals. However, in socially isolated rats, these reductions were observed only in the hippocampus and peripheral tissues. Cold did not affect hypothalamic catecholamine stores but reduced hippocampal dopamine (about 20%) as well as norepinephrine stores in peripheral tissues both in control and socially isolated rats, while epinephrine levels were unchanged. Thus, immobilization was more efficient in reducing catecholamine stores in control and chronically isolated rats compared to cold stress. The differences in rearing conditions appear to influence the response of adult animals to additional stress. In addition, the influence of previous exposure to a stressor on catecholaminergic activity in the brainstem depends on both the particular catecholaminergic area studied and the properties of additional acute stress. Therefore, the sensitivity of the catecholaminergic system to habituation appears to be tissue-specific.

131I-induced changes in rat thyroid gland function

Therapeutic doses of 131I administered to thyrotoxic patients may cause thyroid failure. The present study used a rat model to determine thyroid function after the administration of different doses of 131I (64-277 µCi). Thirty male Fisher rats in the experimental group and 30 in the control group (untreated) were followed for 6 months. The animals were 4 months old at the beginning of the experiment and were sacrificed at an age of 9 months. Hormone concentration was determined before 131I administration (4-month-old animals) and three times following 131I administration, when the animals were 7, 8, and 9 months old. The thyroid glands were removed and weighed, their volume was determined and histopathological examination was performed at the end of the experiment. Significant differences in serum triiodothyronine and thyroid-stimulating hormone concentration, measured at the age of 7, 8, and 9 months, were found in the experimental group. During aging of the animals, the concentration of thyroxin fell from 64.8 ± 8.16 to 55.0 ± 6.1 nM in the control group and from 69.4 ± 6.9 to 25.4 ± 3.2 nM in the experimental group. Thyroid gland volume and weight were significantly lower in the experimental than in the control group. Thyroid glands from the experimental group showed hyaline thickness of the blood vessel wall, necrotic follicles, a strong inflammatory reaction, and peeling of necrotic cells in the follicles. In conclusion, significant differences in hormone levels and histopathological findings indicated prolonged hypothyroidism after 131I administration to rats, which was not 131I dose dependent.

Effects of strain and age on ear wound healing and regeneration in mice

Round holes in the ears of MRL mice tend to close with characteristics of regeneration believed to be absent in other mouse strains (e.g., C57BL/6). We evaluated the kinetics and the histopathology of ear wound closure in young (8 weeks old) C57BL/6 and BALB/c mice. We also used middle-aged (40 weeks old) C57BL/6 mice to evaluate the influence of aging on this process. A circular through-and-through hole was made in the ear, photographs were taken at different times after injury and wound area was measured with digital analysis software. The percentages of closed area measured on day 100 were: 23.57 ± 8.66% for young BALB/c mice, 56.47 ± 7.39% for young C57BL/6 mice, and 75.31 ± 23.65% for middle-aged C57BL/6 mice. Mice were sacrificed on days 1, 3, 5, 25, 44, and 100 for histological evaluation with hematoxylin and eosin, Gomori’s trichrome, periodic acid-Schiff, or picrosirius red staining. In young mice of both strains, healing included re-epithelialization, chondrogenesis, myogenesis, and collagen deposition. Young C57BL/6 and BALB/c mice differed in the organization of collagen fibers visualized using picrosirius-polarization. Sebaceous glands and hair follicles regenerated and chondrogenesis was greater in young C57BL/6 mice. In middle-aged C57BL/6 mice all aspects of regeneration were depressed. The characteristics of regeneration were present during ear wound healing in both young BALB/c and young C57BL/6 mice although they differed in intensity and pattern. Greater ear wound closure in middle-aged C57BL/6 mice was not correlated with regeneration.