Sex steroid hormone determination of the maternal brain: effects beyond reproduction.

Herein we discuss the effects of hormones on reproduction, but with a focus on the ripples that emanate from the main effects. That is, the role of hormones in reproductive events is both well-known and well accepted; less studied and understood are effects that appear to be ancillary to the primary objectives of the hormonal effects, which support, complement and extend their primary effects. We present evidence for how the hormonal stimulation of pregnancy constructs the maternal brain; makes it more efficient; enhances cognition; regulates stress responsiveness; modifies sensory systems (we discuss mainly olfaction); neurogenesis; and learning. Thus, steroid and other hormones and neuropeptides restructure the nervous system, particularly of females, to produce and regulate maternal behavior as well as behaviors and physiological systems that contribute to and support what is arguably the primary function of the hormones: survival and effective nurturance of the female's metabolic and genetic investment.

Dietary calcium and vitamin D intake in elderly women: effect on serum parathyroid hormone and vitamin D metabolites.

In this study, the effect of dietary calcium and vitamin D on serum parathyroid hormone and vitamin D metabolites was measured in 376 free-living women aged 65-77 y. Mean calcium intake in both groups was close to the recommended dietary allowance of 800 mg/d. Mean vitamin D intake in the 245 women not taking vitamin D supplements was 3.53 microg/d (141 IU/d), which is below the recommended dietary allowance of 5 microg/d (200 IU/d). To test the hypothesis that vitamin D is more important than calcium in reducing serum parathyroid hormone, the source of dietary calcium intake was subdivided into milk, which is fortified with vitamin D, and nonmilk sources. The serum parathyroid hormone concentration was inversely correlated with calcium intake derived from milk (r = -0.20, P < 0.01) but not from nonmilk sources (r = -0.06). Furthermore, serum calcidiol correlated with milk calcium intake (r = 0.35, P < 0.001) but not with nonmilk calcium intake (r = 0.10). Multivariate analysis showed a significant effect of season on serum calcidiol but not on serum parathyroid hormone. Serum parathyroid hormone was inversely correlated with serum calcidiol (r = -0.33, P < 0.001) and the regression predicted that mean serum parathyroid hormone would be reduced in the elderly to concentrations considered normal in the young when serum calcidiol is 122 nmol/L (49 ng/mL); this would require a much higher recommended dietary allowance for vitamin D than 5 microg/d (200 IU/d).

Comparative response to UV irradiation of cytoskeletal elements in rabbit and skate lens epithelial cells.

PURPOSE: This work reports a differential effect of ultraviolet A (UVA) irradiation on the three major cytoskeletal structures, actin and vimentin filaments and microtubules of lens cells in primary culture. The effect on cells from lens of the skate (a bottom-dwelling marine elasmobranch) was compared with that on rabbit lens, in order to assess UVA sensitivity as a function of exposure to these wavelengths in the native habitat. METHODS: Exposure intervals of irradiation time up to 6 hours were selected, at fluences from 13.5 to 54.4 J/cm2 and at 365 +/- 45 nm wavelength, to represent mild to moderate physiological levels. Cultures were fixed and processed with anti-alpha-tubulin-FITC and rhodamine phalloidin, or with anti-vimentin FITC and rhodamine phalloidin conjugates. RESULTS: With epifluorescence microscopy, it was found that microtubules were most sensitive to UVA irradiation (in depolymerizing), followed by actin, with vimentin hardly at all affected. Irradiation for 6 hours followed by incubation for 3 days in fresh medium showed no recovery of actin but good recovery of microtubule organizing centers, followed by mitosis in many (rabbit) cells. Skate cells were more sensitive and showed no recovery. CONCLUSIONS: In view of the role of cytoskeletal elements in intracellular structure, cell division and transport, their disruption supports the hypothesis that UVA may damage lens epithelial cells in vivo so as to contribute to cataract formation. In addition, the data suggest that the lenses of animals exposed to sunlight require effective cytoskeletal repair mechanisms to avoid loss of function.

Serum vitamin D metabolites and calcium absorption in normal young and elderly free-living women and in women living in nursing homes.

Vitamin D deficiency, which causes osteomalacia, may also be important in the pathogenesis of age-related osteoporosis. We studied serum vitamin D metabolites in 52 young women (mean age: 30 +/- 3 y; range: 25-35 y), 64 elderly free-living women (mean age: 71 +/- 4 y; range: 65-82 y), and 60 elderly women living in nursing homes (mean age: 84 +/- 9 y; range: 61-102 y). Mean serum 25-hydroxyvitamin D (calcidiol) was 10.8 +/- 4.4 nmol/L (27 +/- 11 ng/mL) in women living in nursing homes and was similar to that of free-living young (11.3 +/- 4.2 nmol/L, or 28 +/- 10 ng/mL) and elderly (11.5 +/- 3.2 nmol/L, or 29 +/- 8 ng/mL) women. Vitamin D deficiency (defined as serum calcidiol < 4.8 nmol/L, or 12 ng/mL) occurred in 8% of women living in nursing homes, in 6% of the young women, and in 1.6% of the free-living elderly women. Serum calcidiol was significantly correlated with vitamin D intake (r = 0.25, P < 0.05) and inversely correlated with serum intact parathyroid hormone (iPTH) (r = -0.16, P < 0.03). Serum iPTH increased with age and secondary hyperparathyroidism was observed in 17% of the women living in nursing homes. Calcium absorption declined with age, but calcium absorption and serum 1 alpha,25-dihydroxyvitamin D (calcitriol) were significantly lower in women living in nursing homes, which probably contributed to the secondary hyperparathyroidism. In conclusion, normal serum calcidiol may avoid the problem of osteomalacia, but it does not correct malabsorption of calcium. Although calcitriol corrects the malabsorption of calcium, it remains to be seen whether higher amounts of vitamin D can normalize the calcium malabsorption of aging.

Agonist-induced rise in intracellular calcium of lens epithelial cells: effects on the actin cytoskeleton.

Primary cultures of rabbit and skate lens epithelia were used to investigate the effect of calcium release from intracellular stores upon the actin cytoskeleton. Primary cultures were loaded with fura-2 AM and intracellular calcium, i.e. (Ca2+)i, quantitated using a Hamamatsu Photonics digital imaging system. Agonists used were bombesin, inositol-1,4,5-trisphosphate (IP3), thapsigargin (Tg), neuropeptide Y (NPY) and calcium chloride. Recordings were typically made on seven cells in each case. We found that IP3 caused a 6-8-fold immediate release of (Ca2+)i in rabbit cells, but skate cells showed no response unless permeabilized with saponin, whereupon an increase of about 50% occurred. Tg induced release from internal stores in rabbit cells, but had no effect on skate cells. Bombesin caused a large increase in (Ca2+)i release in both, while NPY had no effect in either. Skate cells incubated in calcium-free EGTA-Ringer's solution responded rapidly to addition of 5 mM CaCl2, whereas only three of 35 rabbit cells responded, and in gradual fashion. After calcium imaging, the cells were fixed and stained with rhodamine phalloidin or with an antibody against IP3 receptor (IP3R) conjugated to FITC. Fluorescence microscopy revealed that the actin cytoskeleton had reorganized from the normal stress fiber pattern into polygonal networks. Tg caused the same structures to form in rabbit cells, but bombesin had no effect. IP3 receptor was located intracellularly, presumably on endoplasmic reticulum, and was not associated with plasma membranes. The rapid response of rabbit cells may have been caused by the DMSO in which fura-2 was dissolved. We have found an interesting difference in agonist-induced calcium release between rabbit and skate cells. The latter may utilize either a Ca-Na exchanger or capacitative calcium entry, which could reflect a difference in lens accommodative mechanisms. This seems relevant in view of the fact that the rabbit lens accommodates through change in shape, whereas the skate lens does so through translation of position.

Lens cytoskeleton and after-cataract.

Lens epithelial and other ocular cells contain complex arrays of actin filaments which might be expected to allow them to migrate following injury: specifically into the capsular sac in cases of extracapsular cataract extraction or traumatic cataract. To test the possibility, a culture system was developed using a melanotic strain of mice, in which migrating cells are often 'marked' by melanosomes. Injured lenses were cultured on permeable membranes in contact with nutrient medium, and surrounded by iridial tract fragments. After study by light and electron microscopy, it was established that both pigmented and unpigmented cells migrated from the surrounding explants, apparently along the substrate meniscus, to the outside of the lens capsule and then through the wound to the capsule interior. This study suggests a source of cells in development of after-cataract syndrome.

Growth potential of the cells of permanent lines (HeLa, BHK/21, NRK).

A variety of culture parameters was tested to determine optimum growth conditions for the permanent cell lines HeLa, BHK/21 and NRK. Although the lines grew as well at clonal density as in mass culture, clonal analysis of cells transferred on coverslip fragments showed that only 71%-85% were able to produce permanent sublines. Kinetic analysis of the growth of individual clones showed that they varied widely in growth rate, despite continuous low density propagation designed to select out slowly growing cells. In addition, cytopathology was frequently evident in all lines studied, either when cultured at clonal density or under "ideal" conditions at moderate density. The results indicate that defective cells are continuously produced and that they exist in stable proportion in equilibrium cultures. These findings are at variance with claims that some permanent lines (i.e., HeLa) plate with 100% efficiency. Results are discussed in terms of the methodology used to determine plating efficiency, and also in terms of stochastic theories of cell kinetics, which predict the occurrence of cell death in permanent lines and explain the interconversion of permanent and limited cell lines observed in other systems.

Kinetics of chick embryo cell types in culture.

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.

Cell population kinetics of the mouse lens epithelium.

The dividing lens epithelium of 8-week-old CF1 mice consists of a monocellular layer of about 31,000 cells and does not include the postmitotic cells of the meridional rows and another postmitotic zone of seven cell positions' width immediately anterior to the rows. The latter two populations contain approximately 3,600 and 9,000 cells, respectively, for a total of 44,000 cells in the entire lens epithelium. Autoradiographic analysis based upon mitotic index and cell cycle times indicates that the epithelium produces 207 new lens fibers a day. Throughout the 20-day period of study, labeled cells appeared almost entirely as pairs following a single dose of 3H-thymidine and clusters of labeled nuclei were not seen. Moreover, the number of labeled cells dropped only slowly with time, as did the grain counts. These observations indicate that logarithmic division "cascade" does not occur in the lens. The dividing cell population consists largely of a slowly cycling stem cell group, dividing once about every 17-20 days, and consisting of some 5,000 cells. A subpopulation may exist which undergoes two rapid consecutive divisions before becoming postmitotic, but this is too small to make a significant contribution to lens fiber production. Four days are required to transit the postmitotic zone, and an additional 43 or so are needed to transit the meridional rows and differentiate into anucleate lens fibers. Data from other laboratories indicate that the entire process, from mitosis to final differentiation, requires about 4 months. Hence, most of this time is spent in migration of nondividing cells.

Colony formation by simian virus 40-transformed human parapharyngeal cells cultured in semisolid agar.

A simian virus 40 (SV40)-transformed line of human parapharyngeal cells (SV-TGo) was cultured in semisolid agar to determine its ability to grow in the absence of an anchoring substratum and to evaluate any phenotypic changes that might have resulted during the isolation of sublines specifically selected for anchorage independence. After 2--3 weeks and 14--15 population doublings in culture, SV-TGo plated with over 1,000% higher efficiency than negative controls (F2408 cells). Sublines, 0.3--2.0 mm in diameter, were isolated and transferred to Leighton tubes in which they underwent an additional 0--7 divisions before senescence after 39--44 total population doublings. Subline phenotype was identical to the original parental phenotype, including epithelioid morphology, organized pattern of growth, extreme sensitivity to density-dependent inhibition of growth, and continuous production of infectious SV40 as detected by the combined tests of cocultivation and direct isolation. Limited division potential was within the range observed for the parental line. The ability to grow in agar without identifiable phenotypic changes was therefore confirmed for this line of SV40-transformed human epithelioid cells.

A virus-producing cell line developed by transformation of human parapharyngeal cells with SV40.

Normal cultures of epithelial appearance were initiated by trypsinization of a surgically resected, histologically normal branchial cyst. Cellular morphology was consistent with derivation from the stratified squamous epithelium of the cyst or from vascular endothelium, although electron micrographs of the cultured cells failed to show any junctional complexes. Infection with SV40 produced transformants which were also epithelioid in appearance. These grew vigorously for 22 to 50 population doublings (about 23 to 32 subcultures, depending upon regimen) and then became quiescent. During this evolution, virus was detectable at all stages by both direct isolation (cell extracts) and cocultivation with permissive cells. In two sublines in which selection for rapidly growing cell types occureed, virus was detected only by cocultivation. The work confirms that of others in the finding that normal human epithelial cells are susceptible to transformation by oncogenic viruses, but are apparently less responsive than are fibroblasts to such transforming agents. It also suggests that subcultivation techniques that maintain the populations of transformed cells at low density tend to select against cell strains that are continous producers of infectious virus.

Herpes viruses and cancer.

PIP: Type 2 herpes simplex virus belongs to the herpes virus group, members of which have been shown to cause cancer in animals -- kidney cancer in frogs, lymphoid cancer in chickens and rabbits, and lung cancer in sheep. A herpes virus causes Burkett's lymphoma in humans; another causes nasopharyngeal cancer in humans. Herpes simplex viruses are common in humans in cervical and vaginal sores in women and in the genital tract in men (an estimated 15% of men older than 15). It is transmitted venereally. Type 2 herpes simplex virus has been epidemiologically associated with cervical cancer. It has been found in prostate cancer cells. In a hybridization experiment with DNA from cervical cancer cells, DNA from type 2 herpes simplex virus was found, but 60% of the viral DNA molecule was missing. In the chicken lymphoid cancer caused by a herpes virus, live virus vaccine eradicated the disease. This suggests that, if type 2 herpes simplex virus is found to cause cervical cancer, a vaccination cure can be developed.^ieng