Effect of hypothalamic extracts and synthetic LH-RH on proteic synthesis by rat hypothalamus in vitro (author's transl)]

The incorporation of 14C-Leucine in pituitary proteins in rats, in vitro, has been studied. In absence of stimulation, the pituitaries of adult female rats have shown approximately twice the capacity of protein biosynthesis in vitro than the pituitaries of prepuberal female rats (21 days old). For the stimulation in vitro of the pituitaries, synthetic LH-RH or hypothalamic extracts from adult or prepuberal female rats were used. The pituitaries of adult female rats did not respond to any of the stimulation tests employed. The pituitaries of prepuberal female rats increased their biosynthetic activity significantly, when synthetic LH-RH or adult female rat hypothalamic extract was added to the culture medium. The addition of prepuberal female rat hypothalamic extract did not alter the basic response. The female prepuberal rats injected during 5 consecutive days with FSH and LH, have shown a greater sensibility to LH-RH in vitro than the ones injected with estradiol and progesterone, or with synthetic LH-RH.     

Effect of hydrocortisone on immunohistochemically demonstrable phenylethanolamine-N-methyltransferase in cultures of embryonic and postnatal superior cervical ganglia

Pre- and postnatal superior cervical ganglia of the rat were cultured in Rose chambers for 1-7 days with or without hydrocortisone. Phenylethanolamine-N-methyltransferase (PNMT) was demonstrated by indirect immunofluorescence technique. In cultures without added hydrocortisone, no cells or fibres showed PNMT-immunoreactivity, without regard to the time in culture or the developmental stage at the time of explantation. The first PNMT-immunoreactive cells in hydrocortisone-containing cultures appeared 3 days after the explantation of E14 ganglia, or 1 day after the explantation of E15 ganglia, i.e. at the developmental stage E16-E17. The cultures of neither E14 nor E15 ganglia showed marked fibre growth from the PNMT-immunoreactive cell bodies. On the other hand, in the hydrocortisone-containing cultures of newborn or postnatal rats, there was extensive nerve fibre formation from the PNMT-immunoreactive cells in the course of the culture. PNMT-immunoreactive cells did not appear in hydrocortisone-containing cultures of ganglia taken from rats older than 17 postnatal days.     

Effect of hydrocortisone on immunohistochemically demonstrable phenylethanolamine-N-methyltransferase in cultures of embryonic and postnatal superior cervical ganglia

Summary Pre- and postnatal superior cervical ganglia of the rat were cultured in Rose chambers for 1–7 days with or without hydrocortisone. Phenylethanolamine-N-methyltransferase (PNMT) was demonstrated by indirect immunofluorescence technique. In cultures without added hydrocortisone, no cells or fibres showed PNMT-immunoreactivity, without regard to the time in culture or the developmental stage at the time of explantation. The first PNMT-immunoreactive cells in hydrocortisone-containing cultures appeared 3 days after the explantation of E14 ganglia, or 1 day after the explantation of E15 ganglia, i.e. at the developmental stage E16–E17. The cultures of neither E14 nor E15 ganglia showed marked fibre growth from the PNMT-immunoreactive cell bodies. On the other hand, in the hydrocortisone-containing cultures of newborn or postnatal rats, there was extensive nerve fibre formation from the PNMT-immunoreactive cells in the course of the culture. PNMT-immunoreactive cells did not appear in hydrocortisone-containing cultures of ganglia taken from rats older than 17 postnatal days.     

Suspension culture of myocardial cells from newborn rats.

Myocardial cells from newborn rats were held in a spinner type culture for 2 days and then explanted into culture flasks. Three main cell types were observed: single multipolar cells of embryonic type, cell aggregates containing 10 to 50 connected cells, and bipolar cells retaining some adult characteristics. Except for the latter, up to 95% of intact cells settled and were beating 6 hours after explantation. The percentage of fibroblast-like cells was drastically reduced when compared with conventional cultures. Cell debris could be removed 2 hours after explantation by changing the culture medium, or more effectively by a density step centrifugation using Lymphoprep or Lymphoprep-Ficoll mixtures.     

Steady-state amounts of alpha- and luteinizing hormone (LH) beta-subunit messenger ribonucleic acids are uncoupled from pulsatility of LH secretion during sexual maturation of the heifer.

Our primary objective for this study was to determine whether steady-state amounts of alpha- and LH beta-subunit mRNAs in the anterior pituitary are altered during sexual maturation in the bovine female. A secondary objective was to determine whether 17 beta-estradiol (E2) alters amounts of LH subunit mRNAs before onset of puberty. Heifers (7 mo old) were assigned to one of three treatments: 1) ovariectomized (OVX, n = 16); 2) OVX and administered E2 (OVXE, n = 16); or 3) ovary-intact (INTACT, n = 20). Pituitaries were collected at an estimated 120 days before onset of puberty (prepuberty) or 25 days before onset of puberty (peripuberty). Six INTACT heifers were used to determine time of puberty during the experimental period, and their pituitaries were collected 40 h after administration of prostaglandin F2 alpha (postpubertal INTACT group). Relative amounts of mRNAs for LH subunits in each pituitary were determined by Northern analysis and scanning densitometry. Amounts of alpha- and LH beta-subunit mRNAs were lower in pituitaries of INTACT heifers and OVXE heifers, regardless of stage of sexual maturation, than in those of OVX heifers. Amounts of alpha-subunit mRNA were similar in OVXE and INTACT heifers regardless of stage of sexual maturation. Amounts of LH beta-subunit mRNA did not change during sexual maturation in heifers in the INTACT group. Concentrations of E2 were higher and LH beta-subunit mRNA were lower in heifers from the prepubertal OVXE group than in heifers in all other treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histological and ultrastructural studies of the rabbit pars intermedia in organ culture

Pars intermedia (PI) tissue from fetal, perinatal, neonatal and juvenile rabbits has been maintained in organ culture for up to nine weeks after explantation. Autoradiography showed that DNA synthesis took place for at least 22 days of culturing. PI-glandular cells and interstitial cells remain identifiable throughout this period but ACT-type cells were recognised only up to six weeks. Material from fetal and perinatal animals had a higher proportion of surviving cells than that from adult animals. The degree of differentiation achieved by PI-glandular cells in vitro appears to depend on three factors: i) the stage of development reached before explantation; ii) the original topographic position in the PI tissue before explantation; and iii) the position in the explant in relation to the gas-liquid interphase.     

Sensitivity of the fetal rat pituitary-adrenal system to corticotropin-releasing factor in organ culture.

Six groups of adrenal glands from 17-day fetal rats were explanted to organ culture for 2 days. In one group, adrenal gland was cultured alone, and in the remaining five groups adrenal gland was cultured with pituitaries from fetuses ranging in age from 14 to 18 days. In each of the groups, half of the cultures had corticotropin-releasing factor (CRF) added to the medium. A histometric parameter utilized the size of adrenocortical cells as an indicator of sensitivity of the pituitary-adrenal system. When 17-day adrenal gland was cultured alone, addition of CRF did not cause any enlargement of cortical cells. When the adrenal gland was cultured with two 14-day pituitaries, cortical cells were enlarged. Addition of CRF to this culture induced no further change. With two 15-day pituitaries in the presence of CRF, cortical cells were slightly larger than those in the absence of CRF. With 16- to 18-day pituitaries, a marked hypertrophy of cortical cells was induced, and the addition of CRF caused further acceleration in their enlargement. These results suggest that, in organ culture, 14-day pituitary can release some adrenocorticotropic hormone (ACTH) with or without additional CRF. Older pituitaries (16- to 18-day) can apparently release an amount of ACTH in the presence of CRF that is greater than their own spontaneous ACTH secretion.     

A similar distribution of gonadotropin isohormones is maintained in the pituitary throughout sexual maturation in the heifer.

Our working hypotheses for this study were that 1) the profile of intrapituitary LH and FSH isoforms would be shifted toward acidic forms as sexual maturation progresses in the bovine female; and 2) concentration of 17 beta-estradiol (E2) in circulation during sexual maturation would be a major factor modulating the percentage of the more acidic isoforms. In addition, the biological-immunoreactive (B:I) ratios of each isoform of LH were evaluated at selected stages of sexual maturation. Heifers (7 mo of age) were assigned to one of three treatment groups: 1) ovariectomized (OVX; n = 16); 2) OVX and administered E2 (OVXE; n = 16); or 3) ovary-intact (INTACT; n = 14). Pituitaries were collected from heifers in each group at an estimated 120 days (prepubertal) of 25 days before puberty (peripubertal). A fourth group of 6 heifers remained intact (postpubertal INTACT) to determine time of puberty during the experimental period. Pituitaries of heifers assigned to the postpubertal INTACT group were collected during the follicular phase of the first or second estrous cycle postpuberty. Pituitaries were used for determination of relative amounts of gonadotropin isohormones. Tissue extracts of the pituitaries were chromatofocused on pH 10.5-4.0 gradients. The LH of all pituitaries resolved into thirteen isoforms that were designated isoforms A-L, and S, with isoform A the most basic form. Isoforms F and G (basic pH range) were the predominant isoforms of each chromatofocusing profile and comprised 50-60% of the immunoreactive LH. Isoforms J and K were the major isoforms eluting in the acidic pH range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of treatment with cortisol in vivo on secretion of gonadotropins in vitro

To investigate the site of action of glucocorticoids in modulating secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from pituitaries of male rats, we implanted intact male rats with 250-mg pellets of cortisol (F) or cholesterol (C). Four days later, we collected and enzymatically dispersed the pituitaries. After the dispersed pituitaries had been in culture for 2 days, we treated the cells with gonadotropin-releasing hormone (GnRH) (0-150 nM) and determined the concentrations of LH and FSH in the medium after 6 h of incubation. Cells from donor animals pretreated with F secreted 30-60% more LH approximately 75% more FSH than cells from donor animals pretreated with C. This increase occurred regardless of the presence of F or C in the incubation medium in vitro. The slopes and ED50s of the GnRH dose-response curves were not altered. These data show that glucocorticoids have stimulatory effects on both LH and FSH. The inhibitory effects observed in vivo must be exerted by some mechanism that is not carried over to the in vitro model, and perhaps involve sites of action in addition to the pituitary.     

Sexual behaviour in castrated rabbits treated with varying doses of testosterone

Sexual behaviour was studied in castrated rabbits injected every third day with 1, 7·5, 15 or 30 mg TP, during a 120-day period. Castration led to a very pronounced reduction of sexual activity. This was restored to precastration levels by the three larger doses used about 60 days after the beginning of hormone treatment. The lowest dose did not cause any significant increase in sexual activity. About 70 days after the hormone treatment ceased, almost all sexual activity had disappeared. The correlation coefficients obtained between the various parameters of sexual activity ranged between 0·21 and 0·54. The present observations are discussed in relation to results obtained with other species.     

Effect of salmon melanin-concentrating hormone and mammalian gonadotrophin-releasing hormone on somatolactin release in pituitary culture of Cichlasoma dimerus

We detected a close morphological association between melanin-concentrating hormone (MCH)-immunoreactive (ir) fibers and somatolactin (SL)-ir cells in the pars intermedia of the cichlid fish Cichlasoma dimerus by double-label immunofluorescence. Male pituitaries obtained from adult C. dimerus were incubated with 0.1-10 muM salmon MCH, and the amount of SL released into the culture medium was semi-quantified by Western blot. This assay showed an increase of SL release in a dose-dependent manner (linear regression: P < 0.05). A close association of GnRH-ir fibers with SL-ir cells was also detected at the pars intermedia level. Male pituitaries were also incubated with 0.1-10 muM of mammalian GnRH, and SL release was semi-quantified by Western blot, showing an increase of released SL levels in a dose-dependent manner (linear regression: P < 0.05). In contrast, SL release was unaffected from female pituitaries incubated with salmon MCH; however, an increasing tendency was observed when mammalian GnRH was used. Hypothalamic close association of MCH-ir perikarya and GnRH-ir fibers was found by double-label immunofluorescence indicating a possible relationship between them. These results suggest that SL, like other pituitary hormones, is under hypothalamic control and is involved in diverse physiological processes including background adaptation and reproduction. This study has also shown that the in vitro culture of a single C. dimerus pituitary is a feasible method for studying the control of SL release and other pituitary hormones.     

Testosterone synthesis in vitro by the fetal testis of the guinea pig

Synthesis and secretion of testosterone by testes of guinea pig fetuses were studied in organ culture. The amount of testosterone secreted into the culture medium was estimated by radio-immunoassay. It was demonstrated that testosterone was already secreted by testes explanted at 25 days. The amount of testosterone secreted during the first day in culture by testes from fetuses of different ages (25–30 days) increased with fetal age. The amount of testosterone extracted from fetal guinea pig testes at ages used for explantation (25–30 days) were much lower than the amount secreted during the first 24 hours in culture. During subsequent days in culture, an increase in the amount of testosterone secreted was observed only for testes explanted at 25 days. The amount secreted by testes from older fetuses (26–30 days) stayed constant or decreased. Aminogluthetimide phosphate (100 μM) decreased significantly testosterone secretion by testes explanted at 25 days.These findings indicate that the capacity for biosynthesis of testosterone is present in fetal guinea pig testes at 25 days and increases during the subsequent days.     

Effect of 17α-methyltestosterone,estradiol-17β and synthetic LHRH on production of gonadotropic hormone in pituitaries of rainbow trout (organ culture)

Pituitary glands from 6-month-old sexually immature female rainbow trout, Salmo gairdneri, were kept in organ culture for 48 or 72 h. Certain groups of pituitaries were cultivated for 48 h on either control medium or medium with 17 alpha-methyltestosterone (MT), or with estradiol-17 beta (E2) in concentrations of 8.5 X 10(-7) M. Other groups of pituitaries were cultivated for 72 h on control medium, or for 48 h on either control medium or MT-medium or E2-medium, and subsequently for 24 h on medium with synthetic LHRH in concentrations of 8.5 X 10(-7) M and 8.5 X 10(-10) M. Gonadotropic (GTH) cells are identified by Alcian Blue-Periodic Acid Schiff-Orange G staining and the double-antibody immunoenzyme-cytochemical technique using anti-carp beta GTH as the first antibody. A quantitative histological procedure was used to study the nuclear size of the GTH cells in response to the different hormones. Secretory activity was estimated by measuring the gonadotropin (GTH) content in extracts of pituitaries, plasma, and the culture media every 24 h by radioimmunoassay. Cultivation on MT- or E2-enriched medium results in an increase of the total amount of GTH in the pituitary and medium, an accumulation of GTH in GTH-cells (approximately 20 percentage points) and an increase in their nuclear size, indicating a stimulation of GTH synthesis. However, autonomous GTH-release is not affected by these steroids. Subsequent cultivation of the pituitaries for 24 h with LHRH causes stimulation of GTH synthesis (approximately 20 percentage points). Preincubation with steroids increases the GTH synthesis capacity of LHRH only when used in a concentration of 8.5 X 10(-10) M. Moreover, 8.5 X 10(-7) M LHRH causes a stimulation of GTH-release. Preincubation of the pituitaries with steroids increases the responsiveness of GTH-cells to LHRH. It is concluded that GTH-production in pituitaries of immature female rainbow trout can be directly influenced by gonadal steroids and by a hypophysiotropic substance.     

Mouse prolactin obtained by pituitary organ culture in a serum-free medium.

Mouse prolactin was purified by organ culture of pituitaries and electrophoresis of the medium. Mouse pituitaries were organ-cultured in 9-cm Petri dishes containing Waymouth's medium (MB 752/1) supplemented with penicillin (50 units/ml), streptomycin (100mug/ml), and bovine insulin (0.12 I.U./ml) for 8 days. Prolactin-rich culture medium was half-saturated with ammonium sulfate and centrifuged. The pellet was subjected to analytical disc electrophoresis (10% acrylamide). Gels were sectioned into 2-mm segments. Prolactin was eluted in 0.04M phosphate buffer (pH 7.2), dialyzed and lyophylized. Two hundred and forty ml medium in which 360 pituitaries were cultured yielded 29.3 mg lyophylized mouse prolactin. Although the preparation contained 2 other bands on acrylamide gel disc electrophoresis, a single precipitin line was seen in immunodiffusion and immunoelectrophoresis, showing the identity of their antigenicity. From these results, two other proteins in the preparation were suggested to be deamidated prolactin.     

Effect of synthetic LH-RH and hypothalamic extracts on RNA synthesis by rat pituitaries in vitro (author's transl)]

Incorporation of 14C-Uridine in pituitary RNA from adults or prepuberal female rats in vitro, incubated with synthetic LH-RH or hypothalamic extracts, was studied in this work. RNA synthesis was not increased by any of the assayed stimuli in adult female rat pituitaries. The 14C-Uridine incorporation in pituitary prepuberal female rats increased in presence of synthetic LH-RH or adult female hypothalamic extracts, but not in presence of prepuberal female hypothalamic extracts. The differences found in the in vitro behaviour between pituitaries from prepuberal and adult female rats, and between their respective hypothalamic extracts, are attributables to the evolution of these organs along the sexual maturing process.     

Hippocampus morphology in tissue culture]

Explants of the hippocampus of newborn rats were studied neurohistologically and with electron microscope within 5--35 days of explantation. Two zones are found in the culture of the hippocampus: a zone of explant, and a zone of outgrowth. Neurons, glial cells and a network of their fibres are compactly arranged in the center of the former, whereas, the latter involves a layer of migrated glial cells. The explant is surrounded by glia limiting cells. Three types of neurons are identified in the long living culture of the hippocampus: pyramidal, polymorphic and granule cells. Numerous nerve endings observed in the hippocampic explant can be recognized as axodendritic, axosomatic and glomerular synapses. The availability of several types of neurons, a variety of synapses and their complication during outgrowth of the culture are suggestive of a formation in the hippocampic explant of a functional reflex activity.     

Steroidal modulation of in vitro gonadotropin secretion in prepubertal and adult male Siberian hamsters

The effects of exogenous gonadal steroids, testosterone (T), and 17beta-estradiol (E(2)) upon the hypothalamo-pituitary-gonadal axis were reported to be different between prepubertal and adult Siberian hamsters. Utilizing an in vitro static culture system, we investigated if age-related differences in steroid responsiveness occurs at the pituitary. Prepubertal (20 days old) or adult (140 days old) male Siberian hamsters were implanted with 1 mm silastic capsules containing undiluted T, E(2) or cholesterol (Ch, control). After 15 days, pituitaries were removed, incubated in vitro, and subjected to the following treatments: two baseline measurements, one challenge with 10ng/ml of D-Lys(6)-gonadotropin-releasing hormone (GnRH), and three post-challenge washes. Fractions were collected every 30 minutes and measured for follicle-stimulating hormone (FSH) and luteinizing hormone (LH). T and E(2 )reduced basal secretion of LH and FSH in juveniles but not adults. In juveniles, E(2) increased GnRH-induced FSH and LH secretion, while T augmented GnRH-induced FSH secretion but attenuated GnRH-induced LH secretion. Steroid treatment had no effect on GnRH-stimulated LH or FSH release in adults. The only effect of steroid hormones upon adult pituitaries was the more rapid return of gonadotropin secretion to baseline levels following a GnRH challenge. These data suggest both basal and GnRH-induced gonadotropin secretion are more sensitive to steroid treatment in juvenile hamsters than adults. Further, differential steroidal regulation of FSH and LH at the level of the pituitary in juveniles might be a mechanism for the change in sensitivity to the negative effects of steroid hormones that occurs during the pubertal transition.     

Differentiation of embryonic mouse GH cells and ACTH cells in vitro

The ability of cells that produce growth hormone (GH) and cells that produce adrenocorticotropic hormone (ACTH) to differentiate in various culture media was analyzed by means of ultrastructural immunocytochemistry on 13-day embryonic mouse pituitaries that were maintained in organ culture for 3-11 days. At the time of culture, relatively undifferentiated nongranulated or poorly granulated cells that were unreactive with anti-growth-hormone serum (anti-GH) and anti-adrenocorticotropic-hormone serum (anti-ACTH) were present in the pituitary. After 10-11 days in culture, immunoreactive GH cells were obtained only in media that were supplemented with cortisol, whereas ACTH cells were obtained in all media tested, including Medium 199 alone. In cortisol-supplemented media, the GH cells showed ultrastructural features typical of those that occur in vivo, and anti-GH immunoreactivity was obtained after as little as 3 days in culture, i.e., at a stage comparable to that which occurs in vivo. The results indicate that mouse GH cells are capable of differentiating in Medium 199 supplemented only with cortisol, without the addition of fetal calf serum or insulin; cortisol therefore appears to be an essential component of the embryonic milieu for the production of GH-secretory granules.     

The relation between cell division rate and 3 HTdR incorporation in organ culture

Rates of incorporation of labelled thymidine (RIT), radioautographic labelling index for DNA synthesis (LI) and mitotic incidence following colcemid metaphase arrest (MI) were measured in organ cultures of newborn and adult rat lung. In adult cultures these three parameters correlated well, being low after explantation and reaching a maximum after two to three days. In newborn cultures RIT fell several fold over the first 24 hours after explantation and, in this respect, did not correlate with LI and MI. The changes in RIT over the first 24 hours appear to be due to changes in the degree of competition between endogenous TdR and exogenous labelled TdR, probably caused by leakage of the intracellular thymidine pool following explantation. The report emphasizes the need to check RIT data against radioautographic evidence before accepting it as an index of DNA synthesis.     

Effect of fibroblasts from monolayer cultures of hematopoietic and lymphoid tissue on the immune response in vitro]

Stromal fibroblasts from the monolayer cultures of human bone marrow, guinea pig bone marrow, spleen, thymus and peripheral blood suppressed the response of the plagueforming cells against sheep erythrocytes in the suspension cultures of mouse spleen cells. Combined cultivation of 20 X 10(6) fibroblasts from all the mentioned sources led to complete suppression of the immune response. This suppression was less in mice immunized three days before the spleen cell explantation into the suspension cultures and was absent entirely in case the pre-immunization of spleen cell donors was accomplished nine days before the explantation.