Presence Of Luteinizing Hormone Research Articles

Maternal MicroRNA Profile Changes When LH Is Added to the Ovarian Stimulation Protocol: A Pilot Study.

While the use of follicle-stimulating hormone (FSH) in ovarian stimulation for in vitro fertilization (IVF) is an established practice, the use of luteinizing hormone (LH) remains debatable. MicroRNAs (miRNAs) are short, endogenous, non-coding transcripts that control a variety of cellular functions, such as gonadotrophin production and follicular development. The goal of this pilot study was to investigate whether the employment of recombinant LH (rLH) in ovarian stimulation protocols results in changes in the miRNA profiles in human oocytes. Patients were divided into two groups: seven received recombinant FSH (rFSH, 225 IU), and six received rFSH (150 IU) plus rLH (75 IU). MiRNA predesigned panels and real-time PCR technology were used to analyze the oocytes retrieved from the follicular ovarian retrieval. Among the miRNAs evaluated, a series of them evidenced upregulation or downregulation in their expression in the FSH plus LH group compared to the FSH group. Considering the results obtained from the functional and network analysis, the different maternal miRNA profiles in the two groups revealed a differential modulation of pathways involved in numerous biological functions. Overall, based on the pathways associated with most of these maternal miRNAs, the presence of LH may result in a different modulation of pathways regulating survival under the control of a Tp53-related mechanism. Interestingly, among the miRNAs differentially expressed in oocytes of the two groups, we have found miRNAs already investigated at ovarian, follicular, oocyte, and embryonic levels: hsa-miR-484, hsa-miR-222, hsa-miR-520d-5p, hsa-miRNA-17, hsa-miR-548, and hsa-miR-140. Thus, investigation into the role of these miRNAs in oocyte molecular pathways may help determine how LH affects oocyte competence and eventually leads to the clinical improvement of IVF.

FRI376 Regulation Of Theca Cell Function By Salt-Inducible Kinases

Abstract Disclosure: M. Rodriguez Esquivel: None. E. Hayes: None. O. Lakomy: None. M. Hassan: None. R. Filzen: None. C.O. Stocco: Grant Recipient; Self; NIH R01HD097202. The most common identifiable factors of female infertility are ovulatory disorders. Ovulation is the pinnacle of folliculogenesis, a process that requires an interplay between the oocyte, the granulosa cells, and the theca cells (TCs). TCs are the only source of ovarian androgens. Androgens play vital roles in female fertility acting as substrates for estrogen production in granulosa cells and directly regulating ovarian function via the activation of the androgen receptor. However, abnormally elevated androgen levels reduce fertility in humans and animals, suggesting that a delicate equilibrium exists between the beneficial and harmful effects of androgens in the ovary. Therefore, uncovering novel mechanisms regulating androgen synthesis in TCs is of great significance. The capacity of TCs to synthesize androgens relies on the expression of the rate-limiting enzyme CYP17A1, which converts progestogens into androgens, and its stimulation by the luteinizing hormone (LH). Previously, our group showed that salt-inducible kinases (SIKs) regulate granulosa cell steroidogenesis. Here, we investigated whether SIKs play any role in the regulation of androgen production in TCs. SIK2 and SIK3 were detected by IHC in the TCs of rat ovaries. Western blot confirmed this finding using isolated rat TCs. Next, rat TCs in culture were treated with LH in the presence or absence of HG, a highly specific SIK inhibitor. SIK inhibition enhanced the stimulatory effect of LH on Cyp17a1 mRNA expression. HG alone also increased Cyp17a1 expression to levels higher than those found in the presence of LH. Similarly, HG alone increased testosterone and potentiated LH stimulation of testosterone production. SIK inhibition also enhanced the stimulatory effect of LH on the expression of StAR and Cyp11a1. Concentration-response experiments for the SIK inhibitor in the presence or absence of LH show that SIK inhibition stimulates Cyp17a1 in a concentration-dependent manner from 0.1 to 1 µM. Of note, at 3 µM, HG was less effective in inducing Cyp17a1 than at 1 µM, suggesting that higher concentrations of HG may have alternative unknown effects. Activation of adenyl cyclase with forskolin or emulation of increased intracellular cAMP with dbcAMP stimulated Cyp17a1 an effect that was also enhanced by the inhibition of SIK. Since the main target of cAMP is PKA, we used lentivirus to express constitutively active PKA (caPKA). Overexpression of caPKA was sufficient to stimulate testosterone production, an effect that was significantly augmented by SIK inhibition, suggesting that SIKs regulate factors downstream of PKA. This evidence shows that TCs express SIKs and reveal novel roles for SIKs in the regulation of TC function and androgen production. This information could contribute to uncovering therapeutic targets to treat hyperandrogenic diseases such as PCOS. Presentation: Friday, June 16, 2023

Isolation of Leydig cells from adult rat testes by magnetic-activated cell sorting protocol based on prolactin receptor expression.

The primary function of testicular Leydig cells (LCs) is to produce testosterone (T). In vitro culture of the cells represents a very important approach to study androgen production and its regulations. Various methods have been developed for the enrichment of the cells from the testes. However, getting cells in large numbers with high purity and viability is still challenging. Here, we describe a new way to isolate LCs from rat testes in large quantity with high purity and viability. Enzymatic digested testicular cells from adult rats were labelled with prolactin receptor (PRLR) antibody. The positive cells were isolated by magnetic-activated cell sorting (MACS) protocol. Purified LCs were tested in vitro for their steroidogenic (T production) and non-steroidogenic (25-OH-vitamin D production and Insl3 and Cyp2r1expressions) functions in the presence of Luteinizing Hormone (LH) for up to 24h. Reanalysis of scRNA-seq data indicates that Prlr expression is highly specific in LCs of adult rat testis. MACS procedure based on PRLR expression was able to isolate LCs with very high yield (about 106 cells/testis), high purity (about 95%) and viability (>93%). Purified LCs retained high steroidogenic and non-steroidogenic functions in responding to maximal LH stimulations, with more than 10-fold increases in T production in 3h and 42% and 103% increases in Insl3 and Cyp2r1 expressions in 24h. We have established an excellent way to purify high quality LCs from adult rat testis that can serve as a useful tool to study the physiology, pharmacology and toxicology of the cells in vitro.

Clauberg C. (Zentrbl. F. Gyn., 1933, No. 1, p. 47)

Clauberg S. (Zentrbl. F. Gyn., 1933, No. 1, p. 47), based on the presence of luteinizing hormone in the blood of pregnant women of the anterior lobe of the pituitary gland, recommends in cases of uterine bleeding, depending on the persistence of follicles in the ovary with insufficient development corpus luteum, pour over bleeding women about 300-400 cc, sant.

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.

Gossypol inhibits LH-induced steroidogenesis in bovine theca cells.

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0-25 μg/mL) for 24 h. Gossypol inhibited LH-stimulated theca cell A4 and P4 production in a dose-dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down-regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down-regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.

Gossypol inhibits LH-induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0-25 μg/mL) for 24 h. Gossypol inhibited LH-stimulated theca cell A4 and P4 production in a dose-dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down-regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down-regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.

Time course of the meiotic arrest in sheep cumulus-oocyte complexes treated with roscovitine.

Temporary meiosis arrest with cyclin-dependent kinases inhibitors has been proposed in order to improve the quality of in vitro matured oocytes. In sheep, however, this phenomenon has been rarely investigated. Therefore, the present study aimed to evaluate the effect of different incubation times with roscovitine on nuclear maturation and cumulus cell expansion of sheep cumulus-oocyte complexes (COCs). For this, COCs were cultured for 0, 6, 12 or 20 h in basic maturation medium (Control) containing 75 μM roscovitine (Rosco). After, they were in vitro matured (IVM) for 18 h in the presence of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). At the end of each treatment, cumulus cell expansion and nuclear maturation were assessed under a stereomicroscope and by Hoechst 33342 staining, respectively. In the Control and Rosco groups, the absence of cumulus cell expansion prevailed at 0, 6, 12 and 20 h. After IVM for 18 h, total cumulus cell expansion in the Rosco treatments was dependent on the exposure time to roscovitine. A significantly high percentage of oocytes treated with roscovitine for 6 h (87%), 12 h or 20 h (65%) were arrested at the germinal vesicle (GV) stage. In contrast, 23% GVBD, 54% metaphase I (MI) and 61% MII oocytes were observed in the Control groups at 6, 12 and 20 h, respectively. In all treatments, a significant percentage of oocytes reached MII after IVM for 18 h. Therefore, roscovitine reversibly arrested the meiosis of sheep oocytes during different culture times with the maximal efficiency of meiotic inhibition reached at 6 h. In addition, reversibility of its inhibitory action on cumulus cells was exposure-time dependent.

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.

Steroidogenic differential effects in neonatal porcine Leydig cells exposed to persistent organic pollutants derived from cod liver oil.

Seafood products, including fish and fish oils, are major sources of persistent organic pollutants (POPs) which may cause endocrine disruption related to reproductive dysfunction in males. Primary porcine neonatal Leydig cells were exposed to three extracts of POPs obtained from different stages in production of cod liver oil dietary supplement, in the absence and presence of luteinizing hormone (LH). No reduced viability was observed and all POP extracts showed increased testosterone and estradiol levels in unstimulated cells and decreased testosterone and estradiol secretion in LH-stimulated cells. A decrease in central steriodogenic genes including STAR, CYP11A1, HSD3B and CYP17A1 was obtained in both culture conditions with all POP extracts. We implicate both small differences in composition and concentration of compounds as well as "old" POPs to be important for the observed steroidogenic effects.

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.

Can cabergoline prevent ovarian hyperstimulation syndrome in polycystic ovarian patients undergoing gonadotropin stimulation?

Can cabergoline prevent ovarian hyperstimulation syndrome in polycystic ovarian patients undergoing gonadotropin stimulation?

Localization of adiponectin and its receptor and its possible roles in the ovary of a vespertilionid bat, Scotophilus heathi

The aim of this study was to evaluate the seasonal variation in serum adiponectin levels and ovarian expression of adiponectin and its receptor in the Scotophilus heathi bat and their relationship to the changes in the body fat mass, serum insulin and luteinizing hormone (LH) levels, and ovarian activity. A very high level of circulating adiponectin was found during recrudescence, whereas a very low level of circulating adiponectin was observed during pre-ovulatory period. The increased circulating adiponectin level coincided with fat deposition, insulin resistance and hyperinsulinemia (HI) in S. heathi. Immunocytochemical study in the ovary of bat showed localization of adiponectin mainly in thecal-interstitial cells (TICs), and adiponectin receptor 1 (AdipoR1) in the granulosa cells of growing follicles, but showed no staining in atretic follicle. Seasonal changes in ovarian adiponectin and AdipoR1 levels showed two peaks (during recrudescence and ovulatory phases) coinciding with two periods of follicular development. Adiponectin in the absence of LH, as occur during recrudescence, stimulates androstenedione (A4) synthesis in vitro by up-regulating the insulin receptor (IR). Adiponectin in presence of LH, as occur during pre-ovulatory phase, inhibits A4 synthesis in vitro by down-regulating androgen receptor (AR). Further, the in vitro study showed that adiponectin, in presence of LH, also promotes luteinizing hormone receptor (LH-R) and steroidogenic acute regulatory protein (StAR) level in the ovary, which might help in development of ovulatory follicles. In brief, adiponectin in the absence of LH induces increased A4 synthesis and consequently the condition of delayed ovulation, whereas in the presence LH suppresses both synthesis and action of A4 and thus induces preovulatory condition in the ovary of S. heathi.

Expression of luteinizing hormone in rat brain

A recent report demonstrated that in human aging brain after menopause/andropause luteinizing hormone (LH) is localized in the cytoplasm of pyramidal neurons and hippocampus and a significant increase of LH is also detected in the cytoplasm of pyramidal neurons and neurofibrillary tangles of Alzheimer's disease brain compared to age-matched control brain. It was suggested that the decreased steroid hormone production and the resulting LH expression in the neurons vulnerable to Alzheimer's disease pathology may have some relevance to the development of Alzheimer's disease. lt is, however, unclear whether the presence of LH in neurons of human aging and Alzheimer's disease brain is due to intracellular LH expression or to LH uptake from extracellular sources since gonadotropins are known to cross the blood brain barrier. Moreover, there is no report by using the brain of an experimental animal that LH is expressed in such neurons as found in the human brain. In the present study, we found that LH immunoreactivity is localized in the pyramidal neurons of cerebral cortex and hippocampus of 12 and 18 months old rats but can not detect any immunoreactivity for LH in the young adult (3-5 months old) rats. To confirm that these LH immunoreactivity results from de novo synthesis in the brain but not the uptake from extracellular space，we performed RT-PCR and found that mRNA for LH is detected in several regions of brain including cerebral cortex and hippocampus. These findings suggest us that LH expression in old rat brain may play an important role in aging process of rat brain.

Gonadotropin regulation of genes differentially expressed in response to PKCζ inhibitor during ovulation in the rat

Aims The aim of the present study was to characterize genes regulated by protein kinase C (PKC)ζ inhibitor in the preovulatory granulosa cells following LH stimulation in the rat ovary. Main methods Annealing control primer (ACP)-based polymerase chain reaction (PCR) method was used to identify differentially expressed genes in granulosa cells of preovulatory follicles cultured in the presence of luteinizing hormone (LH) and myristoylated PKCζ pseudosubstrate peptide or a similarly sized control peptide. Key findings Among the 16 genes identified, five (testin, glypican-4, retrovirus SC1, aminolevulinic acid synthase 1 and serum-inducible kinase) experienced rapid and transient stimulation of gene expression upon exposure to human chorionic gonadotropin (hCG) in the ovary of immature rats primed with pregnant mare's serum gonadotropin (PMSG). In situ hybridization analysis revealed that hCG administration induced expression of these five genes in granulosa cells of preovulatory follicles. The Western analysis showed that the protein levels of testin and serum-inducible kinase were also increased by hCG. Expression of the eleven remaining genes in the ovary remained high at 24–72 h following hCG treatment. Significance The present data demonstrate the gonadotropin stimulation of genes differentially expressed by PKCζ inhibitor, implicating that PKCζ pathway possibly plays a role in controlling the ovulation process.

Oncostatin-M inhibits luteinizing hormone stimulated Leydig cell progenitor formation in vitro

BackgroundThe initial steps of stem Leydig cell differentiation into steroid producing progenitor cells are thought to take place independent of luteinizing hormone (LH), under the influence of locally produced factors such as leukaemia inhibitory factor (LIF), platelet derived growth factor A and stem cell factor. For the formation of a normal sized Leydig cell population in the adult testis, the presence of LH appears to be essential.Oncostatin M (OSM) is a multifunctional cytokine and member of the interleukin (IL)-6 family that also includes other cytokines such as LIF. In the rat OSM is highly expressed in the late fetal and neonatal testis, and may thus be a candidate factor involved in Leydig cell progenitor formation.MethodsInterstitial cells were isolated from 13-day-old rat testes and cultured for 1, 3 or 8 days in the presence of different doses of OSM (range: 0.01 to 10 ng/ml) alone or in combination with LH (1 ng/ml). The effects of OSM and LH on cell proliferation were determined by incubating the cultures with [3H]thymidine or bromodeoxyuridine (BrdU). Developing progenitor cells were identified histochemically by the presence of the marker enzyme 3beta-hydroxysteroid dehydrogenase (3beta-HSD).ResultsOSM, when added at a dose of 10 ng/ml, caused a nearly 2-fold increase in the percentage of Leydig cell progenitors after 8 days of culture. Immunohistochemical double labelling experiments with 3beta-HSD and BrdU antibodies showed that this increase was the result of differentiation of stem Leydig cells/precursor cells and not caused by proliferation of progenitor cells themselves. The addition of LH to the cultures consistently resulted in an increase in progenitor formation throughout the culture period. Surprisingly, when OSM and LH were added together, the LH induced rise in progenitor cells was significantly inhibited after 3 and 8 days of culture.ConclusionTaken together, the results of the present study suggest that locally produced OSM may not only play a role in the regulation of Sertoli cell proliferation and the initiation of spermatogenesis but may also play a role in the regulation of Leydig cell progenitor formation by keeping the augmenting effects of LH on this process in abeyance.

Gonadotropins mediate DNA synthesis and protection from spontaneous cell death in human ovarian surface epithelium

Gonadotropins have been implicated in the development of epithelial ovarian cancers. These tumors are derived from ovarian surface epithelium (OSE). The purpose of this study was to determine the effects of these hormones on DNA synthesis and spontaneous cell death in primary cultures of OSE and three immortalized OSE cultures. Primary cultures of OSE cells were generated from the ovaries of women with benign disease. The effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) on DNA synthesis and cell death were determined using [(3)H]thymidine incorporation and JAM assays. Significant inductions of DNA synthesis were demonstrated with LH in 4/12 (33%) primary cultures of OSE and 2/3 OSE cell lines and with FSH in 4/11 (36%) primary cultures of OSE and 2/3 OSE cell lines. A significant protection from cell death was also observed in the presence of FSH in 2/4 primary cultures of OSE and 1/3 OSE cell lines and in the presence of LH in 1/4 primary cultures of OSE and 2/3 OSE cell lines. The results indicate that while gonadotropins have the potential to induce cell proliferation and protect from cell death in OSE cells in vitro, their effects are variable in OSE cells from different women.

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.