Granulosa Cells Alone, Without Theca Cells, Can Mediate LH-induced Oocyte Meiotic Resumption.

Signaling in the granulosa cells of mammalian ovarian follicles is necessary for maintaining prophase arrest in the oocyte and for mediating the resumption of meiosis in response to luteinizing hormone (LH). However, the follicle also includes an outer layer of theca cells, some of which express receptors for LH. To investigate whether theca cells are required for maintaining meiotic arrest and reinitiating meiosis in response to LH, we mechanically separated the granulosa cells and oocyte from the theca and basal lamina. This was accomplished by cutting a slit in the outer surface of isolated follicles such that the mural granulosa cells and cumulus-oocyte complex were extruded from the theca shell, forming a lawn of cells on an organotypic membrane. The remnant of theca cells and basal lamina was then removed. The separation of the granulosa cells from the theca cells and basal lamina was demonstrated by immunofluorescence localization of endomucin (blood vessels of the theca) and laminin gamma (basal lamina). Cells comprising these granulosa cell-oocyte complexes expressed LH receptors and were connected by gap junctions. Oocytes within these granulosa cell complexes maintained meiotic arrest and resumed meiosis in response to LH, showing that the granulosa cells alone, without theca cells, transduce these signals. This semi-intact and mostly 2-dimensional preparation could facilitate imaging studies of follicle physiology.

The beneficial effect of Allium Cepa bulb extract on reproduction of rats; A two-generation study on fecundity and sex hormones.

Allium Cepa Linn. (Onions) has extensively been used in traditional medicine, is one of the important Allium species regularly used in our daily diet, and has been the source of robust phenolic compounds. The current study is intended to evaluate the fecundity-enhancing effect of A. Cepa on the reproductive performance of two successive generations of rats; F0 and F1. A. Cepa extract was initially tested for in vitro antioxidant assay via DPPH and ROS, followed by in vivo toxicity testing. In the fecundity assessment, eighteen pairs of male and female rats (n = 36, 1:1, F0 generation) were divided into three groups and dosed with 75mg/kg and 150 mg/kg daily of A. Cepa extract and saline respectively, up to pre-cohabitation, cohabitation, gestation and lactation period. The reproductive performance, including body weight, live birth index, fertility index, and litter size, was assessed. Various parameters like Hematological, Hormonal (FSH, LH, Testosterone, estradiol), antioxidant markers (SOD, Glutathione peroxidase) and lipid profile of F0 and F1 generations were assessed with evaluation of histopathology of male and female organs. Ethanolic extract of A. Cepa showed the greatest antioxidant potential in DPPH and ROS methods. The continued exposure of the F0 and F1 generations to A. Cepa extract did not affect body weight, fertility index, litter size, and survival index. However, semen pH, sperm motility, sperm count, sperm viability, and semen volume were significantly improved in both generations. We have found pronounced fecundity outcomes in both genders of F0 and F1 generations with A. Cepa 150mg/kg/day extract as compared to control. Results showed that A. Cepa significantly increased (P < 0.05) hemoglobin, follicular stimulating hormone (FSH), luteinizing hormone (LH), plasma testosterone and glutathione peroxidase activities, while total lipid, LDL, and cholesterol were significantly decreased (P < 0.05) in both generations. Histology of both generations of animals reveals enhanced spermatogenesis and enhanced folliculogenesis with improved architecture. Altogether, the present results suggest that A. Cepa extract improved fecundity in both male and female rats by improving hormonal activities and oxidative stress.

Effect of follicle-stimulating hormone and luteinizing hormone on apoptosis, autophagy, and the release and reception of some steroid hormones in yak granulosa cells through miR-23a/ASK1 axis.

Follicle-stimulating hormone (FSH), luteinizing hormone (LH), miR-23a, apoptosis signal-regulating kinase 1(ASK1)/c-Jun N-terminal kinase (JNK), autophagy and apoptosis play crucial roles in follicular development. However, their role in yak granulosa cells (GCs) remains unknown. Therefore, we examined the effect of miR-23a, ASK1, FSH, and LH on apoptosis, autophagy, and the release and reception of some steroid hormones in these cells. Our results showed that miR-23a overexpression significantly increased the abundance of Beclin1, the LC3II/I ratio, and the number of Ad-mRFP-GFP-LC3-labeled autophagosomes, and decreased p62 abundance. Additionally, Bax abundance and the number of terminal deoxynucleotidyl transferase deoxynucleotide triphosphate nick end labeling-positive cells were reduced, while Bcl2 expression was increased. Overexpression of miR-23a also significantly increased the abundance of estradiol receptor α (ER-α) and ß (ER-ß) and the concentrations of estradiol (E2), progesterone (P4) in yak GCs. Here, treating yak GCs with miR-23a decreased ASK1 expression, which regulates ASK1/JNK-mediated apoptosis, autophagy, E2 and P4 levels, and ER-α/ß abundance. In contrast, treatment of yak GCs with FSH (10 µg/mL) and LH (100 µg/mL) increased miR-23a abundance, regulating the subsequent effect on ASK1/JNK-mediated apoptosis, autophagy, ER-α/ß abundance, and E2 and P4 concentrations. In conclusion, miR-23a enhances autophagy in yak GCs, attenuates apoptosis, and increases ER-α/ß abundance and E2 and P4 concentrations by downregulating ASK1. Additionally, FSH and LH can regulate these effects of miR-23a by altering its expression. These results provide important insights that can inform the development of strategies to reduce abnormal follicular atresia and improve the reproductive rate of yaks.

Exogenous FSH/LH modulates TGF beta signaling genes in granulosa cells of Simmental heifers without affecting IVP results.

Follicular wave synchronization and follicular superstimulation with FSH are commonly used in OPU-IVP programs to increase oocyte developmental competence. Factors like Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), from the TGF beta superfamily, are produced by the oocyte and modulate follicular function. The aim of this study was to analyze the FSH-induced effects on (1) embryo production in dual-purpose Simmental cattle, and (2) TGF beta-mediated effects on oocyte-granulosa cell communication. Simmental heifers (n = 12, age 484 ± 62 days) underwent two OPU-IVP cycles in a cross-over design. Follicular waves were synchronized using 0.5 mg cloprostenol on Day 0, followed by 10 µg buserelin on Day 2. Subsequently, half of the heifers were randomly assigned to receive FSH/LH (four injections of 75 IU FSHp and 75IU LHp, 12 h apart on Days 4 and 5) before the first OPU, while the remaining heifers received FSH/LH before the second OPU. At the time of OPU, i.e. 7 days after the start of synchronization, granulosa cells were collected for RT-qPCR analysis. FSH treatment did not affect the number of oocytes collected (17.3 vs. 13.3, P > 0.05), but increased the percentage of quality 1 oocytes compared to controls (45.7 % vs. 22.0 %, P < 0.001). Neither cleavage (86.4 % vs. 85.7 %), nor blastocyst (42.1 % vs. 39.3 %) rate, or the number of transferable embryos produced by IVP (4.1 vs 4.8) was influenced by FSH treatment (P > 0.05 in all cases). FSH treatment increased HIF1A and FSHR levels in granulosa cells, while STAR was decreased (P = 0.008 in all cases). FSH treatment did not affect BMP15 or GDF9 mRNA expression (P > 0.05) but appeared to modulate the expression of genes involved in the BMP signaling pathway. Transcriptional levels of BMP15 receptor (BMPR1A, P = 0.016), and its downstream signaling factor SMAD1 (P = 0.008) were affected by FSH treatment. Our results demonstrated no benefit of this FSH stimulation protocol on IVP results in Simmental heifers. Further, our results suggest that the effects of FSH on bovine oocytes during acquisition of developmental competence may be mediated through BMP, but do not involve the regulation of transcriptional availability of GDF9, providing new insights into possible paracrine effects of the oocyte on granulosa cells.

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

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.

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

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.

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