Absence Of Luteinizing Hormone Research Articles

The effects of luteinizing hormone as a suppression factor for apoptosis in bovine luteal cells in vitro.

The fate of the corpus luteum, a transient endocrine gland formed and degraded during an oestrous cycle, is decided by various physiological factors, such as luteinizing hormone (LH). As a stimulator of progesterone, LH is known to maintain corpus luteum functional and structural integrity by inhibiting apoptosis, a programmed cell death. Therefore, we aim to investigate its action during the mid-luteal phase hypothesized that LH suppresses the death mechanism of bovine luteal steroidogenic cells (LSC) by analysing the expression of proteins involved. Cultured bovine LSC obtained from corpus luteum were treated for 24hr with recombinant TNF and IFNG in the presence or absence of LH. The result showed that LH proved to have a protective effect by increased cell viability (p<.05) and prevented DNA fragmentation (p<.05), as demonstrated by the WST-1 colorimetric assay and TUNEL assay. Expression analysis of mRNA and protein levels showed that LH altered the expression ofBCL2 (p<.05), CASP3 (p<.05), FAS (p<.05),andBAX(p<.05) to support cell survival. In conclusion, our study suggests that LH prolongs the corpus luteum life span through the anti-apoptotic mechanism by increasing cell viability and suppressing apoptosis-related genes and protein expression.

Luteal Lipids Regulate Progesterone Production and May Modulate Immune Cell Function During the Estrous Cycle and Pregnancy.

Although the corpus luteum (CL) contains high concentrations of lipid in the form of steroid hormone precursors and prostaglandins, little is known about the abundance or function of other luteal lipid mediators. To address this, 79 lipid mediators were measured in bovine CL, using ultra performance liquid chromatography-tandem mass spectrometry. CL from estrous cycle days 4, 11, and 18 were compared and, separately, CL from days 18 of the estrous cycle and pregnancy were compared. Twenty-three lipids increased as the estrous cycle progressed (P < 0.05), with nine increasing between days 4 and 11 and fourteen increasing between days 4 and 18. Overall, this indicated a general upregulation of lipid mediator synthesis as the estrous cycle progressed, including increases in oxylipins and endocannabinoids. Only 15-KETE was less abundant in the CL of early pregnancy (P < 0.05), with a tendency (P < 0.10) for four others to be less abundant. Notably, 15-KETE also increased between estrous cycle days 4 and 18. Ingenuity Pathway Analysis (IPA, Qiagen) indicated that functions associated with differentially abundant lipids during the estrous cycle included leukocyte activation, cell migration, and cell proliferation. To investigate changes in CL during maternal recognition of pregnancy, this lipid dataset was integrated with a published dataset from mRNA profiling during maternal recognition of pregnancy. This analysis indicated that lipids and mRNA that changed during maternal recognition of pregnancy may regulate some of the same functions, including immune cell chemotaxis and cell-cell communication. To assess effects of these lipid mediators, luteal cells were cultured with 5-KETE or 15-KETE. One ng/mL 5-KETE reduced luteal progesterone on day 1 of culture, only in the absence of luteinizing hormone (LH), while 1 ng/mL 15-KETE induced progesterone only in the presence of LH (10 ng/mL). On day 7 of culture, 0.1 ng/mL 15-KETE reduced prostaglandin (PG)F2A-induced inhibition of LH-stimulated progesterone production, while 1 ng/mL 15-KETE did not have this effect. Overall, these data suggest a role for lipid mediators during luteal development and early pregnancy, as regulators of steroidogenesis, immune cell activation and function, intracellular signaling, and cell survival and death.

Influence of luteinizing hormone support on granulosa cells transcriptome in cattle.

In cows, the use of follicle-stimulating hormone (FSH) to stimulate follicular growth followed by a short period of FSH withdrawal has been shown to be beneficial for oocyte developmental competence. Although this treatment represents a useful optimization to generate highly competent oocytes, the underlying physiological process is not completely understood. The goal of this study was to investigate the role of luteinizing hormone (LH) action during FSH withdrawal before ovulation. To accomplish this, LH release was pharmacologically inhibited during the coasting period with gonadotropin-releasing hormone (GnRH) antagonists. Granulosa cells samples were obtained from cows stimulated with FSH during 3days followed by a coasting period of 68h and treated with a GnRH antagonist (cetrorelix group) or not (control). A significant reduction in the number of follicles at >10mm diameter was observed with the cetrorelix group and gene expression of granulosa cells reveals that 747 transcripts are potentially regulated by LH. Further analysis indicates how the absence of LH may trigger early atresia, the upregulation of atretic agent as tumor protein P53 and transforming growth factor β1 and the inhibition of growth support. This work allows identification of genes that are associated with maintained follicular growth and conversely the ones leading to atresia in dominant pre-ovulatory follicles.

Felis catus ovary as a model to study follicle biology in vitro.

The current study was designed to evaluate the response of individual intact antral follicles from adult female domestic cats to a luteinizing hormone (LH) stimulus in vitro by assessing cumulus-oocyte expansion (C-OE) and steroid production. C-OE and steroid levels (estradiol [E2] and progesterone [P4]) obtained from individual antral feline follicles (n = 366 follicles; n = 56 cats) were analyzed after 12 or 24 h of culture in the presence or absence of LH (low [3.4 ng/ml] or high [100 ng/ml]). At the end of the culture, the highest percentage of expanded cumulus-oocyte complexes (COCs) was observed in the LH groups at 12 or 24 h in comparison to their controls (p < 0.001). There was a significant increase in expanded COCs when comparing LH concentrations (high vs. low) at 12 or 24 h. Higher levels of both E2 and P4 were observed in the media from antral follicles after 12 and 24 h of culture in the presence of LH (both concentration, p < 0.05). There was no association between hormone levels and follicle diameter; high variability was observed in the steroid levels produced by antral follicles within all treatment groups. These data indicate, for the first time, that feline antral follicles (0.5-2 mm) from different stages of the natural estrous cycle can be cultured and will respond to an LH stimulus, based on an increase in steroid levels as well as C-OE after 12 or 24 h in culture.

Effect of Vitamin D on basal and Luteinizing Hormone (LH) induced testosterone production and mitochondrial dehydrogenase activity in cultured Leydig cells from immature and mature rams

The objectives of this study were to investigate the potential effects of 1α,25-(OH)2VD3 (biologically active form of Vitamin D) on basal and LH-induced testosterone production and mitochondrial dehydrogenase activity in Leydig cells from immature and mature rams cultured in vitro. Leydig cells were isolated from testes of immature and mature rams, treated without (control) or with increasing concentrations of LH (1, 10, 100ng/ml) and/or 1α,25-(OH)2VD3 (1, 10, 100nM). After 24h, concentrations of testosterone in culture media were measured. After 96h, mitochondrial dehydrogenase activity in Leydig cells were measured. In immature and mature ram Leydig cells, treatment with 10 and 100ng/ml LH increased testosterone production and mitochondrial dehydrogenase activity. Treatment with 1α,25-(OH)2VD3 in the absence of LH did not increase testosterone production, but 10 and 100nM 1α,25-(OH)2VD3 increased LH induced testosterone production for both immature and mature ram Leydig cells. Treatment with all doses of 1α,25-(OH)2VD3 in the absence of LH and 10 and 100ng/ml LH in the absence of 1α,25-(OH)2VD3 increased mitochondrial dehydrogenase activity for cultured Leydig cells from immature and mature rams and 1 and 10nM 1α,25-(OH)2VD3 treatment enhanced the LH induced increase in mitochondrial dehydrogenase activity. Result demonstrate Vitamin D3 induced regulation of function of Leydig cells from immature and mature rams cultured in the presence or absence of LH and support a potential role for Vitamin D3 in regulation of gonadal function in rams.

The crucial role of mechanical heterogeneity in regulating follicle development and ovulation with engineered ovarian microtissue

Contemporary systems for in vitro culture of ovarian follicles do not recapitulate the mechanical heterogeneity in mammalian ovary. Here we report microfluidic generation of biomimetic ovarian microtissue for miniaturized three-dimensional (3D) culture of early secondary preantral follicles by using alginate (harder) and collagen (softer) to fabricate the ovarian cortical and medullary tissues, respectively. This biomimetic configuration greatly facilitates follicle development to antral stage. Moreover, it enables in vitro ovulation of cumulus–oocyte complex (COC) from the antral follicles in the absence of luteinizing hormone (LH) and epidermal growth factor (EGF) that are well accepted to be responsible for ovulation in contemporary literature. These data reveal the crucial role of mechanical heterogeneity in the mammalian ovary in regulating follicle development and ovulation. The biomimetic ovarian microtissue and the microfluidic technology developed in this study are valuable for improving in vitro culture of follicles to preserve fertility and for understanding the mechanism of follicle development and ovulation to facilitate the search of cures to infertility due to ovarian disorders.

Design and Development of a Field Applicable Gold Nanosensor for the Detection of Luteinizing Hormone

In this paper, we describe a novel strategy for the fabrication of a nanosensor for detecting luteinizing hormone (LH) of sheep using a gold nanoparticle-peptide conjugate. A new peptide sequence "CDHPPLPDILFL" (leutinizing hormone peptide, LHP) has been identified, using BLAST and Clustal W analysis, to detect antibody of LH (sheep). LHP has been synthesized and characterized, and their affinity toward anti-LH was established using enzyme linked immunosorbant assay (ELISA) technique. The thiol group in LHP directly binds with gold nanoparticles (AuNPs) to yield AuNP-LHP construct. Detailed physicochemical analysis of AuNP-LHP construct was determined using various analytical techniques. Nanosensor using gold nanoparticle peptide conjugate was developed on the basis of competitive binding of AuNP-LHP and LH toward anti-LH. Nitrocellulose membrane, precoated with anti-LH, was soaked in the mixture of AuNP-LHP and sample of analysis (LH). In the absence of LH (sheep), anti-LH coated on the membrane binds with AuNP-LHP, leading to a distinctive red color, while in the presence of LH, no color appeared in the membrane due to the interaction of anti-LH with LH thereby preventing the binding of AuNP-LHP with membrane bound anti-LH. The sensor assay developed in this study can detect LH (sheep) up to a minimal concentration of ∼50 ppm with a high degree of reproducibility and selectivity. The gold-nanoparticle-peptide based nanosensor would be a simple, portable, effective, and low cost technique for infield applications.

Regulation of Steroid Production in Bovine Fetal Ovaries.

The formation of the primordial follicle pool, a process essential to female reproductive capacity, begins around day 90 of bovine gestation (gestational length= 281 days). Previous results indicate that bovine fetal ovaries produce steroids and that ovarian estradiol (E2) and progesterone (P4) decline around the time of follicle formation. Furthermore, we showed that in vitro E2 and P4 can inhibit follicle formation and prevent primordial follicles from acquiring the capacity to activate (initiate growth). Although it is known that the fetal ovary is capable of producing steroids and that E2 and P4 can affect the formation and development of follicles, little is known about the mechanisms that regulate fetal steroid production in vivo. To begin to investigate the regulation of fetal steroid production, pairs of fetal ovaries (n=4, 81-97 days) were collected at an abattoir and cut into pieces (1 mm3). Ovarian pieces were cultured in 24-well culture plates (2 pieces/well) and treatments were applied to duplicate wells within each experiment (fetus). Medium was collected and replaced every 2 days for 10 days and measured for E2 and P4 by radioimmunoassay. To test the hypothesis that gonadotropins can stimulate steroid production, ovarian pieces were cultured in the presence or absence of LH (luteinizing hormone) and/or FSH (follicle-stimulating hormone) at 100 ng/ml. Average cumulative levels of E2 and P4 in control cultures over 10 days of culture were 0.40 and 0.72 ng/well, respectively. LH, FSH, and LH+FSH stimulated E2 production, whereas only LH+FSH stimulated P4 accumulation above control levels (P < 0.05). The combination of LH+FSH enhanced E2 levels more than FSH alone (P < 0.05). To test the hypothesis that availability of androgen substrate limits fetal ovarian E2 production, ovarian pieces were cultured with testosterone (T, 0.5 μM) in the presence or absence of LH and/or FSH (n=4 fetus). Estradiol secretion was stimulated by T (18-fold), LH+T (24-fold) and FSH+T (35-fold), compared with control medium and FSH+T increased E2 values more than T alone (P < 0.05). The high levels of E2 observed in the presence of exogenous T support our hypothesis that androgen is limiting to E2 synthesis in vitro. Unexpectedly, T decreased P4 production by 98% compared to control medium (P < 0.05). Testosterone was also inhibitory to P4 production in the presence of gonadotropins (P < 0.05). To determine if T inhibits P4 directly or indirectly through aromatization to E2, ovarian pieces (n=3 fetuses) were cultured in control medium or with T or E2 (0.5 μM). The average cumulative level of P4 in control medium was 1.18 ng/well. Treatment with T or E2 decreased P4 accumulation by 92 and 81%, respectively, relative to control medium (P < 0.05), suggesting that the effects of T on progesterone production are exerted, at least in part, by its aromatization to E2. Further studies are needed to elucidate the mechanism(s) of steroidal inhibition of P4 production and the physiological role of this inhibition. Together these results show that fetal bovine ovaries are responsive to both LH and FSH, that ovarian production of androgens may be limiting to E2 synthesis and that steroid hormones may play a role in regulating fetal steroid production. (This project was supported by National Research Initiative Competitive Grant no. 2008-35203-05989 from the USDA National Institute of Food and Agriculture).

4H syndrome: a rare cause of leukodystrophy

A 24-year-old woman suffered from gait unsteadiness and tetraparesis since childhood. Her medical history was characterized by a normal delivery of non-consanguineous parents. Walking with support was acquired at 10 months. Childhood development was characterized by occurrence of a progressive cerebellar ataxia, short stature, mental development retardation (IQ = 46), and hypodontia (i.e., absence of deciduous teeth eruption and short tooth roots). Walking without support was never acquired. At the age of 14, a partial growth hormone (GH) deficit (insufficient peak of GH in standard condition and with stimulation tests) and an hypogonadotropic hypogonadism [absence of luteinizing hormone (LH) and follicle stimulating hormone (FSH), lack of response to LH-releasing hormone (LH-RH) injection with LH peak at 0.6 UI/l and FSH peak at 1.4 UI/l] were found. Dental panoramic radiographs showed short tooth roots and absence of dental pulp chamber (Fig. 1). At the age of 24, neurological examination observed a severe static and kinetic cerebellar syndrome, spastic tetraparesis, multidirectional nystagmus, vertical down and up-gaze palsy, and mental retardation. Walking perimeter was 10 m with human assistance. Neuro-ophthalmologic examination confirmed vertical gaze palsy, and revealed amblyopia (with visual acuity of 4/10 on both sides), severe myopia, and bilateral temporal papillary atrophy. Nerve conduction studies and electromyography were normal. Neuroimaging performed at 3 Tesla (Philips, Best, The Netherlands) showed marked atrophy of the corpus callosum (reflecting the global white matter (WM) volume) and the cerebellum. In addition, spinal cord atrophy was moderate (data not shown). Myelinated areas including the pyramidal tracts, the internal capsule, the deep cerebellum, and the brainstem were hyperintense relative to cortical grey matter (CGM) on T1-WI and hypomyelinated areas (=rest of the white matter) were diffusely hypointense relative to CGM on T1-W1 (Fig. 2a–c). On T2-FLAIR images, the myelinated area appeared iso/hypointense relative to CGM and the hypomyelinated area appeared diffusively hyperintense relative to CGM (Fig. 2d–f). The pons appeared small with widening of the prepontine cystern, which was probably due to hypomyelinated corticopontine tracts as well as atrophy of crossing cerebellar tracts (Fig. 2a, c, d, f). Single voxel proton-magnetic resonance spectroscopy (HMRS) spectra showed low choline/creatine and N-acetylaspartate/creatine ratios within the left semi-ovale WM, left basal ganglia and pons. A prominent myo-inositol peak was also found within the pons (Fig. 2g). Tractography obtained from a Diffusion O. Outteryck (&) D. Devos L. Hopes P. Vermersch Department of Neurology, Salengro Hospital, Universite Lille Nord de France, CHRU Lille, Rue Emile Laine, 59037 Lille Cedex, France e-mail: Olivier_outteryck@hotmail.com

Triptorelin 6-Month Formulation in the Management of Patients with Locally Advanced and Metastatic Prostate Cancer

Triptorelin 6-month formulation was developed to offer greater convenience to both patients and physicians by reducing the injection frequency. The efficacy, pharmacokinetics and safety of a new 6-month formulation of triptorelin were investigated over 12 months (48 weeks). The primary objective was to evaluate the formulation in achieving castrate serum testosterone levels (< or = 1.735 nmol/L or < or = 50 ng/dL) on day 29 and in maintaining castration at months 2-12. Absence of luteinizing hormone (LH) stimulation and change in prostate-specific antigen (PSA) level were also assessed. An open-label, non-comparative, phase III study in 120 patients with advanced prostate cancer was conducted from July 2006 to August 2007 in private and public institutions in South Africa. Each patient received two consecutive intramuscular injections of triptorelin embonate (pamoate) 22.5 mg at an interval of 24 weeks. In all patients, testosterone (primary outcome measurement) was measured at baseline and then every 4 weeks; LH was measured before and 2 hours after the two injections. PSA was measured on day 1 and at weeks 12, 24, 36 and 48. Adverse events were recorded at each visit. In the intent-to-treat population, 97.5% (95% CI 92.9, 99.5) of patients achieved castrate serum testosterone levels by day 29, and 93.0% (95% CI 86.8, 97.0) maintained castration at months 2-12. After the second injection, 98.3% of patients showed absence of LH stimulation. The most frequent drug-related adverse events were hot flushes (71.7% of patients). No patient withdrew from the study as a result of an adverse event. The triptorelin 6-month formulation was well tolerated and was able to achieve and maintain castration for the treatment of locally advanced and metastatic prostate cancer. By reducing the frequency of required injections, this new formulation offers a more convenient treatment regimen. (Clinical Trial Registration,NCT00751790 at www.clinicaltrials.gov).

Follicle-stimulating hormone increases primordial follicle reserve in mature female hypogonadal mice

Ovarian primordial follicle reserve is considered hormonally independent or subject to depletion by FSH-driven follicle recruitment. To explore specific in vivo effects of FSH on early follicle populations in the absence of luteinizing hormone (LH) activity, we examined mature hypogonadal (hpg), gonadotrophin-deficient mice expressing transgenic (tg) human FSH. Sustained expression of tg-FSH (5.3 +/- 0.3 IU/l) increased ovary weights fourfold and significantly elevated total primordial follicle numbers twofold in tg-FSH hpg (4209 +/- 457) relative to non-tg hpg (2079 +/- 391) and wild-type (2043 +/- 195) age-matched ovaries. Absolute primary follicle numbers in tg-FSH hpg ovaries were similar to non-tg hpg and wild-type ovaries. Furthermore, tg-FSH quantitatively increased secondary and antral follicles in hpg ovaries to numbers equivalent to wild-type, but did not induce ovulation, indicating a selective FSH response without LH. Circulating inhibin B and inhibin A levels were significantly increased in tg-FSH hpg females compared with hpg controls, and inhibin B correlated with antral number, consistent with FSH-driven antral follicle formation. These findings revealed that sustained pituitary-independent FSH activity, in the absence of endogenous gonadotrophins, promotes an increase in primordial follicle reserve despite also stimulating follicular growth in mature females. Therefore, the tg-FSH hpg ovary presents a novel paradigm to evaluate specific gonadotrophin effects on follicle reserve and recruitment.

Insulin-like growth factor-II stimulates steroidogenesis in cultured bovine thecal cells

The objective of the present study was to determine the effects of insulin-like growth factor-II (IGF-II) on luteinizing hormone (LH)-induced progesterone and androstenedione production by bovine thecal cells and compare it to that of insulin and IGF-I. Cells from large (>7.9 mm) bovine follicles were collected and cultured for 2 days in the presence of 10% fetal calf serum. Then cells were cultured for an additional 1 or 2 days in serum-free medium with various doses of recombinant human IGF-II, bovine LH (30 ng/ml), IGF-I, and(or) insulin. Cell numbers were determined at the end of treatments via Coulter counting and used to correct steroid production data. In the presence of LH, 1-day treatment with 3–300 ng/ml of IGF-II had no significant effect on progesterone or androstenedione production, whereas 2-day treatment with 30, 100 and 300 ng/ml of IGF-II increased ( P < 0.05) both progesterone and androstenedione production by 2–3-fold. The estimated effective dose of IGF-II stimulating 50% of the maximal steroidogenic response was calculated to be 25 ng/ml. In the absence of LH, 2-day treatment of IGF-I or -II had no effect on thecal androstenedione production but increased ( P < 0.05) thecal progesterone production. In the presence of LH, 100 ng/ml of IGF-I increased progesterone and androstenedione production to a greater degree than did 100 ng/ml of IGF-II. Maximal effects of IGF-I and insulin on thecal steroidogenesis were similar and were not additive. Anti-IGF type I receptor antibodies attenuated ( P < 0.05) the stimulatory effect of both IGF-I and IGF-II on thecal cell steroidogenesis. Use of radioligand assays demonstrated that specific receptors for 125I-IGF-II existed in thecal cells with a 25 ng/well of IGF-II causing 50% inhibition of binding. IGF-I cross-reactivity with 125I-IGF-II receptors averaged 3% whereas cross-reactivity of IGF-II with 125I-IGF-I receptors averaged 114%. These results indicate that the stimulatory effects of IGF-II on thecal cell steroidogenesis is mediated by IGF type I receptors and thus IGF-II, like IGF-I, may play a significant role in thecal cell steroidogenesis during follicular development.

Insulin and Insulin-Like Growth Factors (IGFs) promote Vascular Endothelial Growth Factor (VEGF-A) secretion by human luteinizing granulosa cells following Controlled Ovarian Stimulation (COS)

Objective: To investigate the role of insulin-related growth factors and luteinizing hormone (LH) in the physiological regulation of VEGF-A production by luteinized granulosa cells (LGC) from preovulatory follicles in humans. Design: Prospective, comparative, experimental study. Materials and Methods: LGC were obtained from women (n=6), with IRB approval, by follicular aspiration after undergoing COS for in-vitro fertilization. Subjects (33.5 ± 1.2 years, mean age ± SEM) had regular menstrual cycles with no evidence of PCOS or other endocrinopathies. LGC were isolated and plated at 50,000 cells/well on fibronectin in DMEM-Ham’s F-12 medium with 5 μg/ml transferrin, 5 ng/ml selenium, 10 μg/ml aprotinin and 25 μg/ml human LDL. Cells were incubated in the presence and absence of 100 ng/ml recombinant human (r-h) LH (Ares Serono) and 0, 1, 10, 100 ng/ml of r-h IGF-1, IGF-II or insulin (Sigma). Media were collected daily and assayed for free VEGF-A (Quantikine VEGF ELISA, R&D Systems). After 3 days (d), cells were fixed, Crystal Violet stained and analyzed for DNA content to estimate cell numbers. Data were analyzed by two-way repeated ANOVA, using a randomized block design. Results: In the absence of LH or insulin-related growth factors, LGCs secreted low but detectable levels of VEGF-A through d 3 of culture (179–304 pg/ml on d 3). However, exposure to IGF-I or -II resulted in a dose-related increase in VEGF-A levels by d 1 (e.g., 568 vs 183 pg/ml or 453 vs 278 pg/ml,100 ng/ml IGF-I or-II vs controls, respectively; p<0.05), which was sustained through d 3 (1001 vs 179 pg/ml or 701 vs 255 pg/ml, p<0.05). Conversely, the addition of insulin (100 ng/ml) did not increase VEGF-A levels until d 2 (737 vs 436 pg/ml, p<0.05). Although LH alone did not increase VEGF-A levels above controls on d 1–3, the combination of LH and insulin-related growth factors increased VEGF-A levels to a greater extent than IGFs alone at all doses (1181 vs 717 or 800 vs 567 pg/ml, LH + 10 ng/ml IGF-I or -II vs IGF-I or -II alone, respectively, on d 2; p<0.05). The synergistic effect of LH with IGF-I/-II was evident earlier on d 2 and at lower doses (10 ng/ml), compared to insulin and LH, which was not significant until d 3. There was no change in DNA content following incubation for 3 days in the presence of IGF-I, -II, insulin or LH when compared to controls. However, DNA content decreased (p<0.05) in the presence of LH + IGF-II at the maximum dose. Conclusion: Exposure to both insulin-related growth factors and LH is required for optimal VEGF-A secretion by human LGC. Thus, local (IGFs) as well as endocrine (LH, insulin) factors may combine to regulate VEGF-A production, and thus angiogenesis and vascular permeability, in the primate ovulatory follicle and corpus luteum. Evidence of insulin/IGFs, as well as LH, regulation of VEGF-A production supports further studies on their role in regulating angiogenic factors during COS cycles in women with endocrinopathies, such as PCOS. Supported by: NICHD U54 HD18185, RR 00163.

In Vitro Modulation of Steroidogenesis and Gene Expression by Melatonin: A Study with Porcine Antral Follicles

The effects of melatonin on steroidogenesis and gene expression of CYP 11A, CYP 17, and CYP 19 were investigated using a porcine antral follicle culture model. Follicles were cultured with melatonin at doses of 10, 50, and 100 ng/mL in the presence and absence of an optimum dose of luteinizing hormone (LH) (100 ng/mL) for a period of 30 hours. It was found that melatonin stimulated progesterone production both in the presence and absence of LH. Androstenedione production was stimulated by melatonin at the highest dose of 100 ng/mL but melatonin had an inhibitory effect in the presence of LH. Estradiol production was not affected by melatonin alone, while in the presence of LH it showed a bimodal effect. Expression of genes for steroidogenic enzymes specific for the production of progesterone, androstenedione and estradiol (CYP 11A, CYP 17, and CYP 19, respectively) were also analyzed in the theca of follicles cultured with and without LH. Results showed an inhibition of CYP 11A and CYP 17 expression both in the presence and absence of LH. However, the expression of CYP 19 was not affected. Our results indicate that melatonin modulates ovarian theca cell steroidogenesis at the molecular level and this modulation may be mediated by its effects on the transcriptional activity of the steroidogenic enzymes.

Evidence Suggesting that the Potentiating Action of Neuropeptide Y on Luteinizing Hormone (LH)-Releasing Hormone-Induced LH Release Remains Unaltered in Aged Female Rats

Several lines of evidence suggest that one of the mechanisms by which the hypothalamic neuropeptide Y plays an obligatory role in the preovulatory luteinizing hormone (LH) discharge in young rats is to potentiate the action of LH-releasing hormone (LHRH) on LH release at the level of the pituitary. This study examined whether an alteration in the potentiating action of neuropeptide Y on LHRH-induced LH release may contribute to the attenuation or absence of LH surges during female reproductive ageing. Young regularly cycling (2-3-month-old) and old constant oestrous (19-20-month-old) rats ovariectomized for 7 days were primed with oestradiol-17beta-filled Silastic capsules. Two days later, rats received s.c. progesterone at 09.00 h and then were injected i.p. with either saline or pentobarbital at 13.30 hours. Pentobarbital-treated rats received i.v. pulses of neuropeptide Y, LHRH, a combination of neuropeptide Y and LHRH, or saline, every 30 min from 14.00 to 18.00 h via a jugular cannula. Hourly blood samples were collected between 11.00 and 21.00 h. In old rats, the progesterone-induced LH surge was significantly attenuated and delayed as compared to that of young rats. Pentobarbital injection completely blocked the LH surge. Neuropeptide Y pulses alone had no significant effect on LH release. In contrast, LHRH pulses increased LH release in both age groups, although the response was significantly reduced in older rats. While combined pulses of neuropeptide Y and LHRH significantly increased LH release in both young and old rats as compared to that of LHRH alone, the potentiating action of neuropeptide Y on LHRH-induced LH release remained unchanged between the two age groups. These results, together with our recent demonstration of altered hypothalamic neuropeptide Y neuronal activity in middle-aged pro-oestrous rats, suggest that a deficit in neuropeptide Y secretion and action in the hypothalamus, rather than a decrease in pituitary responsiveness to neuropeptide Y, may partially be responsible for the absence of LH surges in old rats.

Phenanthrenequinone disrupts progesterone production in rat luteal cells

The ability of the environmental contaminant phenanthrene (PH) and its photooxidized product phenanthrenequinone (PHQ) to disrupt progesterone secretion was examined in a model system of in vitro suspensions of luteal cells from the rat. Treatment with PHQ dramatically inhibited luteinizing hormone (LH) stimulated progesterone secretion. PHQ also generated a significant increase in reactive oxygen species (ROS). In the absence of LH, however, PHQ stimulated a small increase in basal progesterone secretion. The parent compound, PH, did not alter progesterone or ROS release. Since there is evidence that PHQ lowers the activity of nitric oxide synthase (NOS) and that nitric oxide (NO) affects progesterone production, we examined the response to the NOS inhibitors N-monomethyl-L-arginine, Zn protoporphyrin-9, and aminoguanidine in luteal cells. However, there was no effect of these agents on LH stimulated progesterone secretion. These results indicated that PHQ is a potent disrupter of progesterone secretion and should perhaps be considered in assessing the risk of PH to humans.

Oestradiol feedback stimulation of androgen biosynthesis by human theca cells.

This study analysed the effect of oestradiol on basal and LH-stimulated production of androstenedione and progesterone by human theca cells in monolayer culture. Incubations were carried out for either 2 days (seven experiments) or 4 days (four experiments), in the presence or absence of luteinizing hormone (LH), oestradiol (10(-9)-10(-6) M) or inhibin. Medium collected at 48 and 96 h was stored until radioimmunoassay for steroid content. Theca pooled from small follicles (<10 mm) was used in all but two experiments; in these, ovaries were obtained from ovulatory women in the mid-follicular phase of their cycle and theca from small and large follicles was pooled. Oestradiol inhibited progesterone production in a dose-dependent manner in all experiments, irrespective of follicle size, ovulatory status and ovarian morphology, with maximum effect at 10(-6) M. At this dose, oestradiol had no effect on androstenedione production by theca from four anovulatory women with polycystic ovaries but produced a significant augmentation of both basal and LH-stimulated androstenedione production in theca from five of the seven ovulatory women, with maximal response in theca from the two pre-ovulatory subjects. During the 48-96 h period of incubation, oestradiol augmented androstenedione production in all four experiments and had a greater stimulatory effect than the physiological dose of inhibin (10 ng/ml). This is the first report of oestradiol regulating human theca cell steroidogenesis in a dose-dependent manner.

Requirement for heparan sulphate proteoglycans to mediate basic fibroblast growth factor (FGF-2)-induced stimulation of Leydig cell steroidogenesis.

This study reports that, in contrast to previous findings, basic fibroblast growth factor (FGF-2) stimulates immature Leydig cell steroidogenesis in the absence of luteinizing hormone (LH). Heparan sulphate proteoglycans (HSPGs) are essential for this action of FGF-2 and the data suggest that HSPG/FGF-2 interactions have a significant role in the maintenance of immature Leydig cell steroidogenesis. Culture conditions were established for the maintenance of immature rat Leydig cells steroidogenesis in vitro for at least 2 days. Under these conditions the effect of exposure to FGF-2 at doses ranging from 0.1-10 ng/ml was shown to cause a significant stimulation of basal, but not LH-stimulated, 5 alpha-androstane 3 alpha,17 beta-diol production over 24h in culture. This stimulatory action on basal steroidogenesis is mediated through HSPG, as it was blocked by the addition of heparin (100 micrograms/ml), sodium chlorate (25mM) and protamine sulphate (5 micrograms/ml). These data demonstrate the involvement of HSPG in regulating FGF-2 action on Leydig cells and a potential role for Leydig cell HSPG in mediating paracrine regulatory actions of other heparin binding growth factors.

Bursal antisteroidogenic peptide alters the activity of steroidogenic enzymes in chicken granulosa cells

We have previously reported that a peptide from chicken bursa of Fabricius, bursal antisteroidogenic peptide (BASP), inhibits luteinizing hormone-stimulated progesterone biosynthesis by chicken ovarian granulosa cells. The objective of this study was to determine the site(s) of BASP inhibition within the steroidogenic pathway of chicken granulosa cells. The effects of BASP on key steroidogenic enzymes, including adenylyl cyclase (AC), phosphodiesterase, the cholesterol side-chain cleavage enzyme complex and 3ß-hydroxysteroid dehydrogenase were determined. Luteinizing hormone (10 ng/tube) stimulated a fivefold increase in granulosa cell progesterone production that was inhibited by BASP (0.06, 0.12 or 0.25 bursal equivalents) in a dose-dependent manner. Luteinizing hormone stimulated a sixfold increase in cyclic 3′,5′-adenosine monophosphate (cAMP) formation, and this increase was potentiated by BASP in a dose-dependent manner. In addition, BASP stimulated cAMP formation in the absence of luteinizing hormone without affecting progesterone production. The AC activator forskolin (0.1 mM) stimulated a 4.5-fold increase in progesterone synthesis, which was inhibited by BASP. In the presence of forskolin. BASP increased cAMP formation in a dose-dependent manner. A fivefold increase in progesterone synthesis induced by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (1.0 mM) was inhibited by BASP. In the presence of 3-isobutyl-l-methylxanthine, BASP increased cAMP formation in a dose-dependent manner. Finally, 22(R)-hydroxycholesterol (250, 500, 1,000, or 2,500 ng/tube) or pregnenolone (50, 100, 200, or 500 ng/tube) resulted in up to 15- or 10-fold increases in progesterone production, respectively. Increasing concentrations of BASP caused a dose-dependent suppression of the conversion of 22(R)-hydroxycholesterol, but not pregnenolone, to progesterone. The inhibition of steroidogenesis by BASP is not associated with reduced cAMP levels, and BASP appears to strongly stimulate AC activity. In addition, these findings suggest that BASP may limit the availability of progesterone precursors by inhibiting the activity of the cholesterol side-chain cleavage enzyme complex.

HCG-induced changes in LH/CG receptor mRNA transcript levels in the testis of adult hypophysectomized, ethane dimethyl sulphonate-treated rats

To elucidate the molecular mechanisms involved in the development of precursor cells into functional Leydig cells, effects of hCG on the expression of the luteinizing hormone (LH)/chorionic gonadotrophin (CG) receptor gene were investigated in the testis of adult hypophysectomized, ethane dimethyl sulphonate (EDS)-treated rats. The alkylating drug EDS specifically destroys Leydig cells in the adult rat testis. In hypophysectomized rats, in the absence of LH, this is not followed by the formation of new Leydig cells, leaving a testis in which Leydig cell precursors are presumed to be the only cell type that possess LH receptors. The absence of Leydig cells was confirmed by determining the gene expression of two key steroidogenic enzymes, 3β-hydroxysteroid dehydrogenase/isomerase (3βHSD) and P-450 17a-hydroxylase/C17–20 lyase (P-45017α) in these animals. Northern blot analysis re followed by EDS treatment decreased the total amount of LH/CG receptor mRNA and drastically changed the pattern of LH/CG receptor mRNA transcripts. In the testis of intact adult rats, the expression of the LH/CG receptor gene results in multiple mRNA transcripts with molecular sizes of approximately 7.0,4.2 2.5 1.8 and 1.2kb. The most abundant species observed in the testis of adult hypophysectomized, EDS-treated rats was the 1.8kb mRNA transcript, whereas the levels of the other mRNA transcripts were decreased. To stimulate the development of precursor cells into functional Leydig cells, hypophysectomized, EDS-treated rats were treated with high doses of hCG (100 IU) for different periods of time. Continued treatment with hCG for 7 days increased the level of total LH/CG receptor mRNA and the levels of the different transcripts such as the 7.0 kb mRNA which presumably codes for the full length LH receptor. Concomitantly with the appearance of newly formed Leydig cells, a strong stimulatory effect on the synthesis of 3βHSD and P-45017α mRNAs was found. After 7 days of hCG treatment, although the number of newly formed Leydig cells was only 4.5% of the control values, the pattern of LH/CG receptor mRNA transcripts had become similar to that found in the testis of intact animals containing functional Leydig cells. In contrast to control animals the 1.8 kb mRNA transcript remained the most predominant species in the testis of the hypophysectomized, EDS-treated rats.