Effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism & sex ratio in mouse preimplantation embryos.

Background & objectives: There are potential risks of major birth defect in IVF (in vitro fertilization) pregnancy as well as IVF-ICSI (intra cytoplasmic sperm injection) pregnancies in comparison with naturally conceived human pregnancies. This increase risk could be due to either gonadotropins used for ovarian stimulation or in vitro culture conditions or multiple pregnancy or combinations of all the factors. The effects of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio on mouse preimplantation embryos were evaluated through the use of fl uorescence in situ hybridization (FISH). Methods: The study material consisted of 111 preimplantation mouse embryos (2-16 cell stage) in control group and 405 preimplantation mouse embryos in gonadotropin stimulated group from genetically identical Swiss Albino young (6-8 wk) mouse kept in a similar environmental conditions. The study was designed to investigate effect of gonadotropins on chromosome aneuploidy, chromosome mosaicism and sex ratio through the use of FISH technique using chromosome X, Y and 19 probes. All blastomeres of embryos in both groups were assessed. Results: Interpretable FISH results were obtained in 66 embryos in control group and 128 embryos in gonadotropin stimulated group. There was no excess of chromosome aneuploidy (only one case of sex chromosome trisomy in study group; 19, 19, X, Y, Y) or chromosome mosaicism or deviations in sex ratio between the two groups. However, deviation (1.36 M: 1 F in control group & 1.25 M : 1 F in study group) was seen from expected sex ratio (1 M : 1 F) i.e., skewed sex ratio in both the groups. Interpretation & conclusions: Our results showed that gonadotropins used for ovarian stimulation had no effects in causing increase in chromosome X, Y, 19 aneuploidy and mosaicism and skewing of sex ratio in mouse model. A large scale study with more FISH probes on a larger sample size need to be done to confi rm the findings.

Avaliação de protocolos para indução de inatividade ovariana em gatas domésticas / Evaluation of protocols for induction of inactivity ovarian in domestic cats

Os índices de prenhez após inseminação artificial em felídeos selvagens não são satisfatórios devido ao variável ambiente endócrino após a estimulação com gonadotropinas. O objetivo deste estudo consistiuem aumentar a taxa de sucesso em programas de inseminação artificial em gatas domésticas (animal modelo). As fêmeas (n=9) foram divididas em três grupos, cada um com três animais, sendo: 1) controle(C), somente 200 UI eCG/ 100 UI hCG ; 2) levonorgestrel oral (L)(0,075 mg) durante 37 dias + eCG/hCG; 3) etonogestrel (E), implante subdérmico durante 37 dias + eCG/hCG. Foram submetidas ao exame laparoscópico 29-39 horas após a administração de hCG para verificação da resposta ovariana e realização de esfregaço vaginal para monitoração da fase do ciclo estral. Foram coletadas amostras de fezes 60 dias antes e 60 dias após o tratamento com gonadotropinas para dosagem hormonal de estrógenos. Os resultados foram avaliados através do Teste ANOVA. Os níveis de significância mostraram que o Grupo E, em contraste com o Grupo C e o Grupo L, apresentou inibição satisfatória das concentrações de estrógenos durante a sua utilização. O grupo L não apresentou inibição ovariana durante o tratamento e diferença significativa em relação ao Grupo C. No exame laparoscópico todas as fêmeas dos grupos C, L e E apresentaram folículos e 77% das fêmeas apresentaram corpo lúteo. Também apresentaram células epiteliais superficiais anucleadas e nucleadas características de estro. Concluiu-se que a utilização de implantes de etonogestrel em gatas domésticas mostrou-se eficaz, possibilitando asua utilização prévia aos programas de inseminação artificial, aspiração folicular e também para a contracepção.

Reproductive success in endangered captive small felids species is veryl ow. Due to great variability in endocrine environment post gonadotropin treatment, after artificial insemination pregnancy rates are very low. Nowadays, ovarian activity controll improves the AI success in many species. In this study, new protocols were compared to improve the fertilization rates in artificial insemination programs in domestic cat. Female domestic cats were divided in three treatments: 1) control (eCG/hCG); 2) levonorgestrel (0.075 mg) orally during 37days + eCG/hCG; 3) etonogestrel subdermal implant during 37days + eCG/hCG: Laparoscopies were done 29-39 hours post hCG treatment to verify ovarian activity. Vaginal swabs were collected at laparoscopic procedures. Fecal samples were colected 60 days before, during and 60 days after the gonadotropin treatment for estradiol assay. Means comparisons were done by ANOVA test. Results demonstrated that etonogestrel (implant) and not oral levonorgestrel successfully suppressed ovarian activity. The levonorgestrel group didnot show ovarian inactivity during the administration, presenting oestradiol peaks and without significative diference comparing to control group. All females presented anuclear and nuclear superficial vaginal epithelial cells at laparoscopies. In conclusion, the etonogestrel implant used in the domestic cat was efficient and can be used previous to gonadotropin protocol in artificial insemination programs, follicular aspiration and contraception

Insulin alone can lead to a withdrawal of meiotic arrest in the carp oocyte.

Meiotic arrest of oocyte in an Indian carp, Labeo rohita Ham. has been found for the first time to be withdrawn by insulin only. Addition of insulin to oocytes in vitro caused germinal vesicle breakdown (GVBD), one of the first visual markers to determine initiation of the final maturational process. Under the influence of insulin the germinal vesicle (GV) of the oocyte migrated towards the animal pole, reached the micropyle and then dissolved (GVBD). By using different concentrations of insulin i.e., 0.063, 0.63, 6.3 and 12.6 mM, optimum amount required was found to be 6.3 mM. Induction of GVBD by insulin could be blocked by cycloheximide (Chx), a translation inhibitor, while actinomycin D (AcD) had no effect suggesting non-involvement of transcriptional activity in this process. Addition of the maturation-inducing steroid 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) stimulated (P<0.01) GVBD of carp oocytes and its combination with insulin showed an additive effect. Gonadotropin (GtH) caused GVBD but its effect was greatly augmented by insulin. Our results demonstrate that not only can insulin alone induce GVBD in carp oocytes, but it also augments the stimulatory effect of DHP or IGF-I or GtH on GVBD. This information will be important in hormonal manipulation during induced breeding of carp.

Efficacy of exogenous gonadotropins on the maintenance of spermatogenesis in pethidine treated albino rats.

An attempt is made to induce the pethidine suppressed gonadal activities by the administration of exogenous gonadotropins (hCG, PMSG, hCG + PMSG). Administration of 5 IU gonadotropins either separately or in combination to the rats treated with pethidine for 30 days resulted in the significant increase in the weight of testis, diameter of testis and seminiferous tubules. Gonadotropin(s) treatment stimulated the spermatogenic activity which was inhibited by pethidine. Therefore the number of spermatogonia, spermatocytes, spermatids in the seminiferous tubules and spermatozoa in cauda epididymis is increased significantly. Decreased testicular cholesterol, increased protein content and weight of accessory sex organs indicate the rejuvenation of steroidogenesis. Combination of both the gonadotropins is more effective in bringing all these activities.

Gonadotropin triggers the generation of proteinaceous factor in vitellogenic perch (Anabas testudineus) oocyte which stimulates ovarian aromatase activity.

Oocytes of vitellogenic stage were collected from A. testudineus and incubated in vitro for 4 hr in the absence (control) or presence of 500 ng of piscine gonadotropic hormone (GtH). After the termination of incubation, oocytes were repeatedly washed and then homogenized and ultracentrifuged at 100,000 g to obtain the supernatant fraction (100K sup). Addition of 100K sup from GtH treated oocytes to the oocyte incubation caused a 3-fold increase in ovarian aromatase activity as compared to the control, whereas 100K sup from control oocytes had no such stimulatory activity. Addition of cycloheximide (50 micrograms/ml) along with GtH blocked the stimulatory effect of 100K sup. Treatment of 100K sup from GtH incubate with pepsin or heat also destroyed its stimulatory effect. All these indicate proteinaceous nature of the factor. This factor was purified to 161-fold by utilizing Sephadex G-75 and DEAE Sephacel chromatography. Addition of increasing concentrations of partially purified GtH induced protein (GIP) to oocyte incubation caused a dose dependent increase in ovarian aromatase activity. Both dbcAMP and forskolin mimicked GIP activity. Results indicate that GtH induces the synthesis of a protein factor in perch oocytes which stimulates aromatase activity via the mediation of cAMP.

Talla baja por resistencia a la hormona de crecimiento / Low height due to growth hormone resistance

El retraso de crecimiento de un niño por alteraciones en la hormona de crecimiento puede producirse por déficit en su producción o por falta de respuesta a su efecto. Ambos grupos de niños tienen características clínicas bastante similares. La hormona de crecimiento ejerce su efecto biológico a través de receptores tisulares, y si éstos están alterados se producirá un cuadro de resistencia periférica a dicha hormona. Presentamos un niño que consultó a los 14 años y 7 meses de edad por retraso severo del crecimiento, con talla de 95 cm, obesidad centrípeta, manos y pies pequeños, micropene y facies infantil, características sugerentes de un déficit de hormona de crecimiento. El estudio de laboratorio mostró concentraciones plasmáticas elevadas de hormona de crecimiento elevados y disminución de las de somatomedina-C. El paciente fue tratado con hormona de crecimiento biosintética, sin respuesta bioquímica ni clínica, lo que sugiere resistencia periférica a la acción de la somatotropina

Oocyte maturation in Clarias batrachus: in vitro effect of various gonadotropins and homologous pituitary homogenate.

The effects of five different gonadotropins and homologous pituitary homogenate (HP) on germinal vesicle breakdown (GVBD) were investigated in vitro using folliculated oocytes of Clarias batrachus. Among all the gonadotropins, salmon gonadotropin (SG-G100) was the most potent in vitro inducer of oocyte maturation. At concentrations of 1, 0.1, 0.01 and 0.001 microgram/ml it induced 86.98 +/- 2.71, 68.74 +/- 2.85, 44.56 +/- 1.75 and 25.90 +/- 2.36% GVBD. Next to SG-G100 in inducing GVBD was luteinizing hormone (LH) which was consistently found to be effective at all the concentrations used. Human chorionic gonadotropin was also found to be effective at all the concentrations but when compared to SG-G100 and LH, it was less effective. Follicle stimulating hormone and pregnant mare serum gonadotropin were found to be effective at higher concentrations but were ineffective at the lowest concentration. HP treatment resulted in a significant number of GVBD at all the three concentrations used.

Interstitial cells of the ovaries of the chick embryo: ultraestructural aspects of their innervation

En trabajos previos demostramos que en el ovario derecho atrófico y en la médula del ovario izquierdo funcionante del pollo experimental regresión las células germinales y epiteliales y se preservan las células intersticiales (Avila y col. 1987). El objetivo del presente trabajo fue analizar la relación de las fibras y terminaciones nerviosas con las células intersticiales en ambos ovarios durante la embriogénesis en el pollo. Para ello se utilizaron ovarios izquierdos y derechos de embriones de 7 a 19 días de desarrollo, los que fueron procesados para su estudio ultraestructrual y determinación ultracitoquímica de fosfatasa ácida. A los 7 días se encontraron células intersticiales aisladas en la región central de ambos ovarios, las cuales contenían REL, Golgi, mitocondrias con crestas tubulares y abundantes vacuolas lipídicas. Las fibras y teminaciones nerviosas eran escasas. A partir de los 11 días las células intersticiales se encontraron agrupadas en el ovario derecho y en la médula del ovario izquierdo y se relacionaban con fascículos nerviosos rodeados de células de Schwann. Los terminaciones nerviosas, en íntimo contacto con la membrana de las células intersticiales, contenían mitocondrias, microtúbulos y vesículas grandes y pequeñas, algunas con contenido electrodenso. Esta relación entre las células intersticiales y las fibras y terminaciones nerviosas fue más frecuente desde los 15 días. La actividad de fosfatasa ácida localizada en las células germinales y epiteliales, presentó una reacción más intensa a los 15 días en el ovario derecho y en la médula del ovario izquierdo. No se detectó actividad enzimática en las células intersticiales ni en las fibras y terminaciones nerviosas en las distintas edades estudiadas. Estos hallazgos sugieren que las fibras y terminaciones nerviosas serían necesarias para la diferenciación de las células intersticiales en ambos ovarios del embrión de pollo