Impact of pituitary FSH purification on in vitro early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol and Folltropin) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol (50 ng/mL) or Folltropin (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, o nly Stimufol maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin than Stimufol. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol was better to preserve follicular morphology while Folltropin was more efficient to promote follicular growth.

Expression of mRNAs encoding tumor necrosis factor-alpha and its receptor-I in buffalo ovary.

The tumor necrosis factor-alpha (TNF-alpha) plays an important role in ovarian follicular development and ovulation process and acts through its receptor (TNFRI). The present investigation describes the expression of mRNAs encoding TNF-alpha and TNFRI in relation to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) and beta-actin as control genes, using RT-PCR, in granulosa cells, intact follicles and luteal tissues from buffalo ovary. There was significant higher expression of mRNAs encoding TNF-alpha in granulosa cells from medium follicles and TNFRI expression increased with increase in size of follicles. Post-ovulatory structures (corpus luteum and corpus albicans) exhibited significantly higher expression of TNFRI mRNAs as compared to that obtained in intact follicles suggesting its immediate and critical role just after ovulation, for mediating TNF-alpha action on these tissues. Though the expression of TNF-alpha mRNA was stimulated by treatment of granulosa cells with FSH during culture, the expression of TNFRI mRNA did not change. The FSH alongwith IGF-I did not exert any effect. These results suggested an important role of TNF-alpha and its receptor in buffalo ovarian functions.

Sitios de acción inhibitoria de la prolactina sobre la síntesis de estradiol inducida por la hormona folículo estimulante en las células de la granulosa / Sites of prolactin inhibition on gonadotropin-induced estrogen production in cultured rat granulosa cells

En este estudio se investigaron los sitios probables de la acción inhibitoria de prolactina (Prl) sobre la esteroidogénesis ovárica inducida por la hormona folículo estimulante (FSH). Para esta finalidad se estudió la capacidad de cultivos primarios de células de la granulosa de la rata de sintetizar estradiol y AMPc bajo la estimulación con FSH o de activadores de la vía dependiente de AMPc en presencia de Prl humana. La participación de otros sistemas de transducción de señal como los dependientes de PKC y proteínas Gi en los mecanismos de acción inhibitoria de la Prl fue también investigada utilizando inhibidores de estos sistemas como la calfostina C y la toxina pertusis. Los resultados demostraron la habilidad de la Prl de alterar la esteroidogénesis previa y posterior a la generación de AMPc, muy probablemente por mecanismos que involucran la activación de la subunidad catalítica de la adenilato ciclasa, así como a través de interactuar con sistemas de transducción de señal dependientes de PKC y proteínas sensibles a la toxina pertusis. Nuestros resultados sugieren un mecanismo de interacción entre receptores acoplados a proteínas G con aquéllos acoplados a cinasas de tirosinas mediado muy probablemente por vías de señalización dependientes de proteínas Gi.

We studied the sites of prolactin inhibition upon FSH induced ovarian steroidogenesis and the ability of prolactin (Prl) to inhibit the synthesis of estradiol and cAMP accumulation under the stimulation of FSH or cAMP dependent activators. The participation of other signal pathways such as PKC and Gi proteins on the inhibitory actions of Prl was also investigated using calfostine C andpertusis toxin as inhibitors. Results showed a dose dependent prolactin decrease in FSH-induced estradiol and cAMP production prior and after the generation of the cyclic nucleotide by a mechanism involving the catalytic subunit of adenyl cyclase and/or through activation of PKC or by the interaction with pertusin toxin sensitive G proteins. Our results suggest a mechanism by which G protein coupled receptors are linked with those coupled with tyrosine kinase through the involvement of a Gi protein mediated mechanism.

The Regulators of VEGF Expression in Mouse Ovaries

The objectives of this study were to explore whether ovarian vascular endothelial growth factor (VEGF) expression in mice can be regulated by IL-6 (interleukin-6), angiotensin II, FSH, and hCG; and to test whether the mouse ovarian VEGF expression can result in angiogenesis. The ICR mice were sacrificed, and their ovaries were recovered. Recovered ovaries were treated with IL-6, angiotensin II, FSH, and hCG separately and incubated for 24 hours in alpha-MEM. Expression of mRNA and protein of VEGF were assessed by RT-PCR and immunohistochemistry. The resulting angiogenesis was evaluated through immunohistochemical analysis for CD34. Treatment of mice ovaries with IL-6, FSH, and hCG resulted in a significant increase of VEGF mRNA, and IL-6 was the most potent inducer of VEGF. IL-6 and FSH resulted in increased neovascularization in the follicular phase of mouse ovaries. In contrast, angiotensin II could not increase VEGF expression or neovascularization. We documented an in vitro increase in VEGF expression by IL-6, FSH, and hCG; and reaffirmed that the proliferative response of murine ovarian endothelial cells paralleled an increase of VEGF expression.

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.

Regulation of gonadotropin releasing hormone receptor mRNA expression in cultured rat granulosa cells

The homologous regulation of pituitary Gonadotropin Releasing Hormone Receptor (GnRH-R) mRNA expression by GnRH has been well demonstrated. However, the regulation of the ovarian GnRH-R is poorly understood. The present study was performed to demonstrate the presence of GnRH transcripts in addition to GnRH-R mRNA and the regulation of GnRH-R mRNA expression in the granulosa cells isolated from small antral follicles. The GnRH and GnRH-R mRNA levels were determined by a competitive reverse transcription-polymerase chain reaction (RT-PCR). The granulosa cells were obtained from immature rats implanted with diethylstilbestrol for 3 days. When GnRH transcript expression was examined in isolated granulosa cells by RT-PCR, the PCR products showed two bands. The larger band contained intronic sequences and the smaller band was a fully processed GnRH gene transcript identical to hypothalamic GnRH. This suggests that authentic GnRH gene transcripts are expressed in ovarian granulosa cells and may act on the granulosa cells in a paracrine or autocrine manner. Since GnRH action in the granulosa cells is mediated by specific GnRH-R, it is of interest to examine whether GnRH-R is synthesized in the granulosa cells. When the granulosa cells were cultured in media only, GnRH-R mRNA levels increased abruptly within 3 h and gradually decreased thereafter during the 24 h culture period. However, GnRH itself did not alter the GnRH-R mRNA expression levels in cultured granulosa cells. Interestingly, treatment with FSH decreased the GnRH-R mRNA levels in a dose-dependent manner. A time-course analysis revealed that the GnRH-R mRNA levels were significantly lower up to 9 h after FSH treatment, and returned to the basal level between 12 h-24 h. Activation of adenylate cyclase with forskolin also decreased the GnRH-R mRNA levels. It is therefore concluded that in the granulosa cells of the small antral follicles GnRH-R mRNA expression was not homologously regulated by GnRH, while FSH may negatively regulate GnRH-R mRNA expression in the granulosa cells possibly through a cAMP-protein kinase A pathway.

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.

Produçäo de embriöes a partir do desenvolvimento de oócitos imaturos in vitro: valor de estimulaçäo curta com FSH / In vitro development of immature oocytes to viable embryos: brief ovarian stimulation with FSH

OBJETIVO: Avaliar os recentes avanços nas técnicas de maturaçäo oocitária que têm possibilitado o desenvolvimento de embriöes a partir de oócitos imaturos. Desta forma objetivamos avaliar a maturaçäo ovular, taxas de fertilizaçäo e desenvolvimento embrionário em oócitos imaturos aspirados sob pequenas doses de estímulo gonadal. MATERIAL E MÉTODOS: 8 mulheres entre 28 e 39 anos foram submetidas a estimulaçäo com urofolitrofina purificada (Metrodin) e seus oócitos coletados quando pelos menos 2 deles atingiram diâmetro médio de 10mm. Os oócitos coletados foram cultivados em meio TCM-199 suplementado com soro sintético. Metrodin, Profasi (hCG) e Piruvato, ambiente controlado a 37 graus Celsius e concentraçäo de CO2 a 5 por cento por 24 até 72h. Após avaliaçäo da maturidade ovular, procedeu-se a ICSI e transferência de embriöes morfologicamente normais por via transcervical para cavidade uterina. RESULTADOS: 43 oócitos coletados de 7 pacientes foram submetidos a maturaçäo in vitro. Em uma das pacientes näo evidenciou presença de oócitos. Observou-se desenvolvimento até primeiro corpúsculo polar em 27 (63 por cento) ocócitos, sendo que destes, 17 (63 por cento) fertilizaram e 15 (88 por cento) apresentaram clivagem em seu seguimento. Como resultado, obtivemos 1 gravidez após 6 transferências (1/6 - 17 por cento por transferência) que resultaram em gestaçäo viável, com evoluçäo normal, atualmente no segundo trimestre. CONCLUSÖES: Estes resultados demonstram a viabilidade de cultura e conseqüente maturaçäo in vitro de oócitos imaturos, observando-se resultados em termos de taxas de fertilizaçäo, clivagem e gravidez, próximas as encontradas em ciclos induzidos até o completo desenvolvimento ovular.

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 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.

Efectos de estrógenos y andrógenos sobre la esteroidogénesis de células de granulosa de ratas inmaduras en condiciones de cultivo / Effects of estrogens and andorgens on the steroidogenesis of granulosa cells of immature rats in culture conditions

Estudiamos el efecto de la androstenediona y del dietilestilbestrol sobre la secreción de esteroides de células de granulosa de ratas inmaduras, para ello empleamos cultivos primarios de estas células y métodos de análisis radioinmunológicos. Se encontró que el dietilestilbestrol no fue capaz de modificar la esteroidogénesis ni en condiciones basales ni en condiciones estimuladas por la hormona estimulante del folículo. La androstenediona produjo un incremento en la secresión de estradiol y la de progesterona dependiente de la concentración del andrógeno y del tiempo de incubación. El efecto de este andrógeno sobre la secreción de progesterona fue sinérgico en relación con las acciones de la hormona estimulante del folículo. Estos resultados sugieren que en este modelo experimental los estrógenos no parecen desempeñar el papel de reguladores

Contraceptive potential of an antiprogestin ZK 98.734: reversal of ZK 98.734--induced blockade of folliculogenesis with FSH and LH and their differential effects in bonnet monkeys.

Studies were undertaken in adult bonnet monkeys to investigate whether treatment with an antiprogestin ZK 98.734 at weekly intervals, starting from day one of menstrual cycle, could arrest ovulation and also to determine if ZK 98.734 induced blockade of ovulation could be reversed with gonadotropins. Adult animals have ovulatory menstrual cycles of normal duration were treated at weekly intervals with ZK 98.734 (25 mg/dose, sc, oil base) for 10 consecutive weeks and its effects on serum levels of estradiol, bioactive LH and progesterone, and endometrial histology were investigated. Following treatment with the antiprogestin they were treated with hMG or hFSH alone. Ovulation was blocked during treatment period in all the animals (n = 14). Typical follicular phase rise in estradiol levels was inhibited, mid cycle surge in the levels of bioactive LH was abolished and serum progesterone levels remained below 1 ng/ml throughout the treatment period. However, prolonged treatment had no significant effect on the basal levels of estradiol which were around 50 pg/ml. ZK 98.734 also had no significant effect on cortisol levels. In animals (n = 4) followed for recovery after the last dose, the treatment cycle length was increased to 117.8 + 6.8 days. In three animals the treatment cycles were anovulatory, whereas in one delayed ovulation with luteal insufficiency was observed. The endometrium had become atrophic. Treatment with hMG (Pergonal: 35 I.U. hLH and 35 I.U. hFSH) or hFSH (Metrodin, 35 I.U.) for 7 consecutive days initiated folliculogenesis and the animals ovulated either spontaneously or after a single im injection of hCG (100 I.U.) on day 8 in ZK 98.734 treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of fertility regulating agents on motility and zona-free hamster egg penetration by spermatozoa of bonnet monkey.

Administration of STS-557 (17 alpha-cyanomethyl-17 beta-hydroxyestra 4,9(10)-dien-3-one; 12 mg/monkey daily) for 4 weeks either alone or in combination with 20 Aet-1 (testosterone-trans-4-n-butyl cyclohexyl carboxylate; code CDB 1781; 40 mg/monkey single administration) had no significant effect on motility and zona free hamster egg penetration by spermatozoa of bonnet monkey, but continuation of the treatment for 12 weeks reduced (in one monkey treated with STS-557) or abolished (one treated with STS-557 and two with STS-557 + 20 Aet-1) the motility as well as zona-free hamster egg penetration (by spermatozoa of all treated monkeys). Motility and the ability to penetrate zona-free hamster egg returned to normalcy after 10 weeks of withdrawal of treatments. Active immunization of monkeys with ovine FSH (4 weeks after booster) had no adverse effect on motility of spermatozoa but none of the zona-free hamster eggs was fertilized. The correlation between motility and the capacity to penetrate the zona-free hamster eggs by monkey spermatozoa varies with the treatment. Such correlation was apparent in monkeys treated with STS-557 but not in monkeys immunized with ovine FSH.

Sperm production and fertility of bonnet monkeys (Macaca radiata) following immunization with ovine follicle stimulating hormone.

Adult male bonnet monkeys were rendered oligospermic but not azoospermic following active immunization with ovine follicle stimulating hormone. The percentage of sperms in the semen having good motility was reduced with a concomitant increase in the sperm ATPase activity. Eight out of 10 immunized monkeys failed to impregnate females of proven fertility after mating for consecutive three cycles while the remaining two impregnated the cohabitated females during the third cycle at a time when the antibody titer was reduced. Active immunization with ovine follicle stimulating hormone may not produce complete azoospermia but renders adult male monkeys infertile provided sufficient antibody titer is maintained.

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.

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.

Ovulatory response to the sequential administtration of follicle stimulating hormone and human chorionic gonadotropin by autografted ovary in unilaterally ovariectomized adult rat with peripheral devervation induced by guanethidine treatment

Ratas hembras adultas con cilos estrales regulares de 4 días de duración fueron unilateralmente ovariectomizadas (se extirpó el ovario derecho) en el día del diestro 1; en el mismo acto, el ovario fue injertado en el tejido celular subcutáneo. Um grupo de estos animales fue inyectado con guanetidina (20 mg/Kg) cada 72 horas, durante 30 días. Todos los animales (tratados o no con guanetidina) fueron autopsiados, en el primer día de estro vaginal luego del período de 30 días. En otro experimento, ratas hemicastradas con auntoinjerto de ovario, tratadas o no con guanetidina como en el experimento anterior, a los 30 días luego de la hemiovariectomia y el autoinjerto, estando en diestro, fueron tratadas con 45 i.u de hormona folículo estimulante ovina (FSH); 56 horas después recibieron 25 u. i. de gonadotropina coriónica humana (hCG) y fueron autopsiadas 20 horas después. Otro grupo de animales con hemicastración y autoinjerto, con o sin tratamiento con guanetidina, en el día treinta del postoperatório, también en diestro, fueron inyectados con 10 ng de benzoato de benzoato de estradiol (BE) y se les autopsió en el primer día de cornificación vaginal que siguió al tratamiento. La tasa de animales ovulantes en el ovario in situ no fue modificada por el tratamiento con guanetidina, FSH y hCG o BE. La administración de guanetidina provocó disminución del número de ovocitos liberados por animal ovulante (8.3 ñ 0.8 vs 10.4 ñ 0.3, - < 0.05) y menor peso final del ovario remanente que en el grupo no tratado (18.8 ñ 0.7 vs 23.7 ñ 0.7, - < 0.01). Aunque el grado de hipertrofia compensadora fue similar en ambos grupos (45.5% y 48.8%). La respuesta ovulatoria (tasa de animales ovulantes y número de ovocitos liberados por animal ovulante) y el aumento del peso del ovario remanente inducidos por la administración de FSH y hCG o de BE, no fueron modificados por el tratamiento previo con guanetidina. El estudio histológico del injerto de ovario realizado en los animales sin tratamiento, o que fueron inyectados con guanetidina, FSH y hCG o benzoato de estradiol, no mostró signos de ovulación...