Differential response to sustained stimulation by hCG & LH on goat ovarian granulosa cells.

Background & objectives: Chapekar established a model of ovarian tumourigenesis in mice by splenic transplantation of ovaries, resulting in sustained luteinizing hormone (LH) levels because of absence of feedback inhibition. There is increasing evidence of the differential response to LH or hCG under various experimental conditions. The effect of sustained hormonal stimulation in long term cultures is sparsely investigated. The study is aimed to determine the role of hCG and LH stress on caprine ovarian granulosa cells and their downstream signaling in short and long term cultures. Methods: To study the response of hCG and LH stress and downstream signaling, short term cultures were set up by exposing goat ovarian granulosa cells in primary cultures to hCG and LH stress (levels beyond their physiological doses) for 5 days (P0). Cells were sub-cultured at sixth day and subjected to prolonged LH/ hCG stress for two weeks in passage 1(P1) (long term cultures). Downstream cell signaling molecules were assessed. Intracellular cAMP was estimated by ELISA. For PKA and PKC, activity assays were performed. pERK protein expressions in short term cultures were assessed by Western blot and flowcytometry; in long term cultures, pERK expression was analyzed by flowcytometry. Results: Differential effects on cell proliferation were observed in long term cultures, where the untreated and hCG exposed cells showed markedly reduced cell proliferation after second week of exposure while LH treated cells continued to proliferate. Different levels of cAMP, PKA, PKC and phosphorylated ERK1/2 were observed on short term and long term LH stimulation. On sustained hormonal stimulation, cAMP levels were significantly (P<0.05) higher in hCG treated cultures as compared to controls and LH treated cultures. LH led to maximal elevation of ERK in long term cultures. Interpretation & Conclusions: As pERK1/2 promotes cellular proliferation, activation of ERK1/2 in LH treated cultures may be responsible for sustained growth. Prolonged LH treatment promoted growth and proliferation in caprine ovarian granulosa cells whereas prolonged exposure to hCG led to elevated levels of cAMP and decreased the rate of proliferation. Defining the signals and second messengers that act as survival or apoptotic mediators may help in elucidation of the mechanisms controlling proliferation or programmed cell death in granulosa cells.

effect of LH and GnRH analogues on induction ovulation in Bactrian camel [Camelus bactrianus]

Ovarian follicle response and corpus luteum formation following induction of ovulation using gonadotropin-releasing hormone [GnRH] analogues and luteinizing hormone [LH] in Bactrian camel were characterized. Bactrian camels with a mature follicle [13-19.6 mm] received: 1] natural porcine LH [25 mg, IV, n = 4], 2] Buserelin [20 microg, IV, n = 4] and 3] Alarelin [25 microg, IM, n = 4]. Daily ultrasonography and blood samplings were conducted between day -3 and +15 of the experiment [day 0 = Induction of ovulation]. Data were analyzed by univariat analysis with repeated measures analysis included in the model. Following treatment, mature follicle ovulated within 2 days and a new follicle wave emerged after 2-3 days. New mature follicle reached a size of 13.5 +/- 0.14 mm by day 12. Corpus luteum was detected on day 6 and reached the maximum size of 19.73 +/- 0.81 mm on day 9. Progesterone concentration initiated to increase on day 5, reached maximum concentration on day 9 and decreased significantly on day 11. In conclusion, due to the lack of significant difference among treatment groups [P>0.05], Alarelin may be considered as a drug of choice for inducing ovulation in Bactrian camel because of its effectiveness, simple route of administration [IM vs. IV], lower price, and local availability

Serum insulin, glucose and non esterified fatty acids after administration of follicle-stimulating and luteinizing hormones in bitches

This paper reports the effect of the simultaneous administration of follicle-stimulating (FSH) and luteinizing hormones (LH) on serum glucose, insulin and nonesterified fatty acid responses after glucose or insulin challenge. The animals were originally at anestrous. FSH (dose 2.5 U/kg body wt.) and LH (0.27 U/kg body wt.) were s.c. injected on days 1, 4, 8 and 11. Vaginal smears were obtained daily. Six untreated controls at anestrous and six treated bitches reaching proestrous were used. Glucose tolerance tests were done with a dose of 1 g of glucose per kg of body weight. Bovine insulin was administered at the dose of 0.25 U/kg body wt. During these tests, neither serum glucose and nonesterified fatty acids nor glucose distribution space and glucose clearance were affected by the treatment. The serum insulin response to hyperglycemia was greatly increased. The distribution space and clearance rate of this hormone were not affected by FSH + LH treatment. We conclude that, in the bitch, FSH + LH treatment, at doses that trigger "sex seasons", increases the serum insulin response to glucose load and produces a moderate resistance to the hypoglycemic, lipogenic and antilipolytic insulin actions. These phenomena are evident during hyperglycemia

Effect of FSH and LH on testis during nonbreeding season in Calotes versicolor (Daud.).

Histological and biochemical studies carried out on the male reproductive organs of the Indian garden lizard, Calotes versicolor after the treatment with pituitary gonadotrophins (FSH and LH), showed a significant increase in the weight, protein content and diameter of testis, but decrease in its cholesterol. The spermatogonia, spermatocytes and spermatids increased significantly in the germinal epithelium of seminiferous tubule, and spermatozoa appeared in its lumen. The Leydig and Sertoli cells were hypertrophied with increase in their nuclear diameter. The epidymal weight, diameter and protein content also increased after gonadotrophins treatment. There was a significant decrease in the testicular cholesterol indicating the utilization of cholesterol for steroid hormone synthesis. The combined gonadotrophin (FSH + LH) treatment was more effective than the individual gonadotrophin treatment.

Aspectos subcelulares de la inervacción de las células intersticiales del ovario de embrion de pollo cultivado con LH o h CG / Subcellular aspects of interstitial cells innervation in chick embryo ovari cultured with LH or hCG

Diversos autores han demostrado el efecto de LH y hCG sobre la secreción esteroidea del ovario del embrión de pollo. Sin embargo, no se ha investigado el efecto de las hormonas luteinizante y gonadotrofina coriónica humana sobre su inervación. El propósito del presente trabajo fue estudiar los aspectos subcelulares de la inervación de las células intersticiales de ovarios de embrión de pollo cultivados con LH o hCG. Explantos de ovarios izquierdo (funcionante) y derecho (regresivo) de 14 embriones de pollo de 7, 11, 15 u 19 días de desarrollo in ovo de raza Cobb's White Rock fueron cultivados por separado durante 4 días en : 1 - Medio básico (contro); minimun essential medium (MEM-GIBCO)CON 10 de suero fetal bovino, 2-Medio básico con el agregado de LH o hCG (problemas). Los ovarios fueron seccionados en trozos de 2 a 3 mm de diámetro, lo que permitió realizar aproximadamente 30 cultivos de cada ovario. Controles: las características de las células intersticiales y la aparición de las fibras y terminaciones nerviosas observadas en los explantos de ambos ovarios 11 días cultivados durante cuartro días eran similares a los 15 días "in ovo". Problemas: con LH o hCG las fibras y terminaciones nerviosas fueron observadas en el ovario derecho y la médula del ovario izquierdo de 7 días cultivados durante cuatro días, en íntimo contacto con las células intersticiales productoras de esteroides. El presente estadio se correspondía con los 11 días de desarrollo "in ovo"Estos hallazagos sugieren que la inervación de los ovarios estaría controlada por un mecanismo indirecto vía hipotálamo-hipoficiario y por otro local con la producción de factores neurotróficos modulada por LH

Effects of ovine LH, GH and prolactin, and testosterone on serum testosterone and estradiol-17 beta levels, and seminal vesicle and testicular activity in the catfish Clarias batrachus (L.).

Intraperitoneal administrations of testosterone (0.5 microgram/g body wt), and ovine LH (1.0 microgram/g body wt), GH (5 micrograms/g body wt) and prolactin (10 micrograms/g body wt) daily for 7 days during early prespawning phase (May) in C. batrachus produced varied effects on seminal vesicle (SVSI) and testicular (GSI) weights and biochemical correlates. Testosterone and LH treatments significantly increased serum testosterone level and concentrations of total proteins, fructose, hexosamines and sialic acid in both seminal vesicles and testis. Serum E2 levels increased significantly only after testosterone treatment. GH treatment increased significantly serum testosterone level and only the concentrations of SV hexosamines and testicular protein. Prolactin, however, significantly lowered serum testosterone level and concentrations of total protein, hexosamines in both SV and testis, and testicular fructose and sialic acid levels. The results show that the stimulating effect of LH and GH on SV and testicular activity is mediated through the increased secretion of testosterone and the inhibitory effect of prolactin by decreased testosterone secretion.

Post-Transcription Regulation of mRNA and Hormone Synthesis and Release at the Anterior Pituitary Gland: Information derived from the recovery of pituitary gonadotrope cell function after with a GnRH agonist

This review presents a summary of post-transcription regulation of mRNAs with a focus on the anterior pituitary gland. The control of gene transcription and production of mRNAs is the predominant form of regulation of hormone synthesis. However, post-transcription regulation of mRNAs provides another level of control of hormone synthesis. Examples of how hormone synthesis can be controlled at the level of mRNA include mRNA nuclear export and subcellular localization, mRNA stability and turnover, and regulation of mRNA translation. The gonadotrope cells of the anterior pituitary have multiple internal effector systems and provide an ideal model cell to study post-transcription regulation of mRNAs. Gonadotrope cells are stimulated to release LH and FSH by hypothalamic GnRH that binds to GnRH receptors. GnRH receptors are coupled to G-proteins and second messenger signaling pathways that involve cAMP and IP3. These signaling pathways are associated with the release of LH and FSH and transcription of mRNAs for LH and FSH. The stability of these mRNAs can be influenced by androgens, estrogens and progestagens. Therapy with a GnRH agonist leads to desensitization of gonadotrope cells to GnRH and a depletion of cellular stores of LH and FSH mRNAs, and content of LH and FSH. After discontinuation of therapy with GnRH agonist, levels of LH and FSH mRNAs return to normal some time before LH and FSH content and secretion are restored. This is indicative of post-transcription regulation of LH and FSH mRNAs. Future studies on post-transcription regulation of mRHAs will provide new molecular insights into how gonadotrope cells balance and integrate stimulation by GnRH with feedback modulation by the gonads.

Influence of gonadotropins on adrenalectomy induced changes in the testis of albino mouse.

Effects of adrenalectomy and administration of gonadotropins on cell counts of different cell types of spermatogenesis and morphology of the Leydig cells were studied in 30 day old mice. Adrenalectomy (duration, 12 days; age at autopsy 42 days) caused a significant decrease in the diameters of seminiferous tubules and Leydig cell nucleus and, cell counts of intermediate spermatogonia, round and elongated spermatids. Administration of FSH (75 micrograms/0.1 ml saline) + LH (25 micrograms/0.1 ml saline) everyday for 12 days to adrenalectomized mice restored testicular activity as revealed by significant increases in mean diameter of the Leydig cell nuclei and cell counts of intermediate spermatogonia and elongated spermatids over those of adrenalectomized mice. The results indicate that (i) testis of adrenalectomized mouse responds to gonadotropin treatment and (ii) impairment in gonadotropin secretion is possibly a major factor in inducing testicular regression following adrenalectomy.

Effects of gonadotropins and prolactin on ovarian activity of a wild avian species, the tree pie Dendrocitta vagabunda.

Ovine FSH (40: g per bird daily for 10 days) increased ovarian weight, follicular size, phosphatase activities, and RNA and protein levels in tree pie (Dendrocitta vagabunda), but exogenous ovine LH (40 micrograms per bird daily for 10 days) with the same dose and duration caused depletion of ovarian cholesterol and ascorbic acid concentrations with a rise in sialic acid and glycogen levels of the ovary. In contrast, prolactin (LTH: 5 I.U. per bird daily for 10 days) administration showed reverse biochemical changes to those of FSH. The findings suggest that FSH induces mainly ovarian follicular growth and LH stimulates ovarian steroidogenesis, but LTH is antigonadal in this wild avian species.

Hormonal modulation of secretion of immunoreactive riboflavin carrier protein by adult rat Leydig cells in vitro.

Adult rat Leydig cells in culture synthesize and secrete riboflavin carrier protein (RCP) as demonstrated by [35S]-methionine incorporation into newly synthesized proteins followed by immunoprecipitation as well as specific radioimmunoassay. LH stimulates the secretion of RCP 4-fold which could be inhibited upto 75% by an aromatase inhibitor. 8-bromo-cyclic AMP and cholera toxin could mimic the LH stimulated secretion of the carrier protein. The extent of stimulation of RCP secretion brought about by exogenous estradiol-17 beta is comparable to that of LH. The antiestrogen tamoxifen, when added along with either LH or estrogen, inhibited the stimulated levels significantly. These results show that the estrogen-inducible riboflavin carrier is secreted by Leydig cells under positive regulation of LH.

Evaluación morfologia de las uniones intercellulares en el ovario del embrión de pollo: acción de hormonas esteroideas y gonadotroficas in vitro / Morphological evaluation of intercellular junctions in the ovary of chick embryos: action of steroid and gonadotropic hormones in vitro

Se determinaron las variaciones de las uniones intercelulares de las células germinales y epiteliales en el epitelio ovárico producidas por hormonas gonadotróficas y esteroideas sobrelos ovarios del embrión de pollo a los 7 días de desarollo. Se cultivaron explantos de ovarios derecho e izquierdo Sin (controles) y con adición de hormonas (experimental durante 4 días. Los cultivos fueron procesados para su estudio ultraestructural (MET). En ambos ovarios controles los complejos de unión eran similares a los identificados in ovo. En el ovario izquierdo se observó aumento y mayor desarollo de las uniones adherens y desmosomas; en el ovario derecho los mismos disminuyeron por acción de 17 Beta-estradiol. La respuesta del ovario izquierdo a la progesterona y testoterona fue similar a la obtida con estrógeno. En la gónada derecha no se observaron cambios. En ambos ovarios se produjo una dismunucíon de las uniones intercelulares por accíon de FSH. Los cambios producidos por LH y hCG fueron semejantes a los encontrados en el ovario izquierdo por efecto del estrógeno, consistentes en un incremento de los complejos de uníon, principalmente los de tipo adherens. Estos análisis indican que las hormonas esteroideas y gonadotróficas actúan modificando las uniones intercelulares y participarían en los procesos de crecimiento y atrofia que ocurren en los ovarios del embríon de pollo.

Effect of difluoromethyl ornithine [DFMO] on ovulation in rats

This study was conducted to investigate the effects of difluoromethyl ornithino [DFMO] on ovulation and proceeding in adult female Albino rats. These were divided into four groups. The control groups [I [n= 6] and III [n= 4]] were injected vehicle [0.9% normal saline] and in the experimental groups [II [n= 6] and IV [n= 4]] each rat was injected 50 mg DFMO subcutaneously at 0900 hrs on pro-oestrous day. Rats of group I [control] and II [experimental] were sacrificed at 1400 hrs the same day and their plasma were collected for LH radioimmunoassay. There was highly significant reduction in the level of LH [p < 0.001] in experimental [group III] as compared to controls [group I]. Rats of groups III [control] and IV [experimental] were sacrificed next day at 0800 hours on oestrous. It was observed that there was highly significant [p <0.001] reduction in number of ova shed in DFMO treated rats as compared to controls. These results suggested that DFMO blocks the enzyme ornithine decarboxylase [ODC] required for polyamines synthesis

Effect of exogenous gonadotrophins on restoration of ovarian activities in nicotine treated unilaterally ovariectomized albino rats.

Adult cycling albino rats were hemispayed and administered nicotine for 15 days. FSH/LH or FSH+LH was then administered to these rats. Nicotine inhibited ovarian compensatory hypertrophy significantly and increased cholesterol and lipid levels in the ovary. Administration of FSH alone or in combination with LH restored the ovarian compensatory hypertrophy and decreased the cholesterol and lipid levels significantly, but LH alone was not effective. The results suggest that the inhibition of ovarian growth in nicotine treated rats may be due to lack of availability of pituitary gonadotrophins and these effects can be rectified by the administration of gonadotrophins.

Hormonal modulation of biosynthesis of prostatic specific antigen, prostate specific acid phosphatase and prostatic inhibin peptide.

Hormonal modulation of in vitro biosynthesis of three prostatic secretory proteins, prostate specific acid phosphatase (PSAP), prostate specific antigen (PSA) and prostatic inhibin peptide (PIP) by human benign hyperplasia (BPH) tissue was studied. LH and inhibins caused increase in the synthesis of all three proteins whereas FSH enhanced the synthesis of PIP and PSA only but decreased PSAP synthesis. Prolactin and thyroid releasing hormone decreased synthesis of PIP and PSAP. However, PSA synthesis was enhanced by TRH and was decreased by prolactin. Estradiol caused significant increase in PSA and PSAP but no discernible changes in PIP synthesis were noticed. Testosterone caused an increase in PIP, PSA and PSAP. These data indicate that biosynthesis of PIP, PSA and PSAP by BPH tissue is under multihormonal regulation.

Hormonotoxins: synthesis, characterization and bioefficacy of some defined disulfide linked conjugates of ovine LH with a ribosome inactivating protein, gelonin.

In order to synthesise a bioeffective hormonotoxin for selective targeting to specific cells in the gonads, gelonin, a single chain RIP obtained from an Indian plant, Gelonium multiflorum of Euphorbeaceae family was covalently linked to oLH with the use of N-succinimidyl-3-(2-pyridyldithio)propionate, generating a linkage containing a disulfide bond and a amide bond. The hormonotoxins were separated according to their molecular weight (indirectly according to oLH:gelonin molar ratio) and a complete biochemical analysis was performed. The linkage occurred through the epsilon-NH2 group of alpha oLH as judged from RP-HPLC analysis. The conjugates were devoid of ingredients as determined by SDS-PAGE and RP-HPLC analysis. The hormonotoxins retained substantial receptor binding, steroidogenic activity and immunoreactivity of oLH and gelonin to their antibodies. Hormonotoxins bind to the Leydig tumour cells via oLH part leaving gelonin free as judged by competitive displacement analysis. The hormonotoxin was internalized to the sufficient degree to effectively inhibit protein synthesis. The cytotoxicity of 1:1 molar ratio conjugate was relatively higher than that of others. The cytotoxicity of presently described more defined hormonotoxins exhibited higher receptor binding and cytotoxicity than the hormonotoxins reported earlier [Singh, et al., J Biol Chem, 264 (1989) 3089].

Influence of pituitary on histology of Bidder's organ in castrated toad Bufo melanostictus.

Weight, number of follicles and corpora lutea of Bidder's organ increased significantly after castration of adult toad in breeding season. Removal of pituitary along with testes decreased both weight and number of follicles and corpora lutea. Administration of luteinizing hormone (LH) in castrated and hypophysectomized toad showed more or less similar result as that of control indicating that LH has an effect on the histology of Bidder's organ in the toad.

Effect of LH, FSH, LH + FSH and homoplastic pituitary extract on developing testis and epididymis of pigeon Columba livia.

Ovine LH is needed for differentiation of juvenile Leydig cells and for their maintenance and steroidogenic potential, while FSH is necessary for Sertoli cell activity and spermatogonial multiplication suggesting that LH is steroidogenic hormone and FSH is gametogenic in the developing pigeon, C. livia. Homoplastic pituitary extract is more potent than ovine LH + FSH in stimulating gametogenic and endocrine components of the developing testis.

In vitro effects of gonadotrophic and steroid hormones on the interstitial cells of the chick embryo ovaries

Diversos estudios demostraron la importancia de la producción de esteroides por las células intesticiales del ovario del embrión de pollo tanto in ovo como in vitro. El presente trabajo se efectuó para analizar las modificaciones que LH, hCG, 17-ß-estradiol, propionato de testosterona y progesterona producen in vitro sobre las células intersticiales de gónadas femeninas del embrión de pollo. Explantos de ovarios derecho e izquierdo de 7 a 19 días de desarrollo in ovo fueron cultivados y procesados para su estudio estructural y ultraestructural. En los cultivos controles los nidos de células intersticiales aumentaron con la edad, en ambos ovarios, presentando aquellas abundante REL, mitocondrias con crestas tubulares e inclusiones lipídicas. Por acción de LH y hCG, las células intersticiales agrupadas incrementaron sus inclusiones lipídicas y organoides relacionados con la síntesis de esteroides en el ovario derecho y en la médula del ovario izquierdo enh todas las edades investigadas. En cambio, en presencia de progesterona, estrógeno o testosterona se observaron células intersticiales aisladas o en grupos, con escasos organoides e inclusiones lipídicas. Se concluye que las hormonas esteroideas deprimirían la actividad de las células intersticiales supliendo aquéllas la función de las mismas, mientras que la LH y hCC actuarían sobre dichas células estimulando la síntesis de esteroides los que serían el factor intrínseco responsable de la diferenciación sexual del ovario izquierdo funcionante y de la atrofia del ovario derecho

In vitro effect of mammalian pituitary hormones on germinal vesicle breakdown in oocytes of major carps, Labeo rohita, Cirrhinus mrigala, Catla catla and Cyprinus carpio.

Among all the mammalian pituitary hormones, luteinizing hormone (LH) was the most potent in vitro inducer of oocyte maturation in L. rohita, C. mrigala, C. catla and C. carpio. It induced significant germinal vesicle breakdown (GVBD) at concentrations of 10, 1, 0.1 and 0.01 micrograms/ml. At the highest concentration used, LH induced 77.9 +/- 5.9, 73.8 +/- 4.6, 50.3 +/- 2.8 and 40.8 +/- 1.4% GVBD in oocytes of L. rohita, C. mrigala, C. catla and C. carpio, respectively. Among other hormones, follicle stimulating hormone (FSH) induced only a marginally significant GVBD (13.2 +/- 0.8%) in the oocytes of C. carpio, but not in other three species. Thyroid stimulating hormone (TSH), growth hormone (GH) and prolactin (PRL) had no effect on GVBD.