Regulation Of Steroidogenesis Research Articles

WOMEN IN REPRODUCTIVE SCIENCE: Discovering science and the ovary: a career of joy.

My career has been about discovering science and learning the joys of the discovery process itself. It has been a challenging but rewarding process filled with many exciting moments and wonderful colleagues and students. Although I went to college to become a French major, I ultimately stumbled into research while pursuing a Masters Degree in teaching. Thus, my research career began in graduate school where I was studying NAD kinase in the ovary as a possible regulator of steroidogenesis, a big issue in the late 1960s. After a short excursion of teaching in North Dakota, I became a postdoctoral fellow at the University of Michigan, where radio-immuno assays and radio receptor assays had just come on the scene and were transforming endocrinology from laborious bioassays to quantitative science and of course these assays related to the ovary. From there I went to Baylor College of Medicine, a mecca of molecular biology, cloning genes and generating mouse models. It has been a fascinating and joyous journey.

Development of a Versatile and Sensitive Direct Ligand Binding Assay for Human NR5A Nuclear Receptors.

As regulators of steroidogenesis, development, and metabolism, the nuclear receptor 5A (NR5A) subfamily members steroidogenic factor 1 (SF-1) and liver receptor homologue 1 (LRH-1) are important pharmacological targets for cancers and metabolic diseases. Evaluation of small molecule modulators and candidate endogenous ligands for these orphan receptors has been hindered by the lack of accessible, robust direct-binding assays. Here, we leverage the potency of our new NR5A agonist (6N) to create a high-affinity probe for fluorescence polarization competition assays by conjugating 6N to fluorescein (FAM). The 6N-FAM probe tightly binds the NR5A receptors and detects direct binding of synthetic and phospholipid ligands. For 25 LRH-1 agonists, affinity predicts potency in cellular activation assays, demonstrating the potential for this assay in drug discovery. Moreover, phospholipids dilauroylphosphatidylcholine and phosphatidylinositol(4,5)phosphate bind with high affinity, demonstrating this assay is robust for evaluation of candidate endogenous ligands for human NR5A receptors.

Activation of PPARG inhibits dominant follicle development in cattle

The peroxisome proliferator-activated receptor gamma (PPARG, also called NR1C3) is a nuclear receptor of the peroxisome proliferator-activated receptor family (PPAR). PPARs are involved in the regulation of apoptosis, cell cycle, estradiol and progesterone synthesis, and metabolism. However, the role of PPARs and their regulation during follicular development and ovulation in monovular species remain poorly understood. In this study, a well-established intrafollicular injection model was used to investigate if the PPARG participates in the regulation of dominant follicle development and ovulation in cattle. Findings from this study revealed that the relative mRNA abundance of PPARG was similar between dominant and subordinate follicles around follicle deviation, decreased after the LH surge, and increased before ovulation. In addition, a quadratic correlation was found between PPARG mRNA levels in granulosa cells and progesterone concentration in the follicular fluid. Intrafollicular injection of 50 μM Troglitazone (TGZ; a PPARG agonist) inhibited follicular growth and decreased CYP19A1 mRNA abundance in granulosa cells. These findings indicate that PPARG is involved in the regulation of steroidogenesis, follicle growth and ovulation in cattle.

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.

219 Use of a genetically-engineered swine line to elucidate the role of GnRH-II and its receptor in gilts

Abstract The second form of GnRH (GnRH-II) and its receptor (GnRHR-II) are produced in only one livestock species, the pig. Paradoxically, their interaction does not stimulate gonadotropin secretion. Instead, both have been implicated in autocrine/paracrine regulation of steroidogenesis. To elucidate their role in ovarian function, our laboratory generated transgenic swine with ubiquitous knockdown (KD) of GnRHR-II. Blood samples were collected from GnRHR-II KD (n = 8) and littermate control (n = 7) gilts at the onset of estrus (follicular) and 10 d later (luteal). Serum samples were subjected to HPLC-MS/MS to quantify concentrations of 16 steroid hormones. At euthanasia, ovarian weight, ovulation rate and weight of each excised corpus luteum (CL) were recorded; HPLC-MS/MS was also performed on CL tissue. A line (GnRHR-II KD versus control) x phase (follicular versus luteal) interaction was detected for serum progesterone concentrations; levels were reduced in transgenic compared with control gilts during the luteal phase (P = 0.0329). A tendency for a line effect was observed for 11-deoxycorticosterone and 11-deoxycortisol; transgenic females tended to produce less of these corticosteroids (P &lt; 0.10). A phase effect was detected for cortisone, 11-deoxycortisol, cortisol, corticosterone, androstenedione, androsterone, testosterone, estrone and 17β-estradiol (P &lt; 0.05); concentrations were greater in follicular versus luteal samples (P &lt; 0.05). Conversely, 17α-hydroxyprogesterone concentrations were elevated in luteal samples (P &lt; 0.05). Ovarian weight did not differ between lines, although ovulation rate was reduced in GnRHR-II KD versus control gilts (P = 0.0123). However, average CL weight was greater in GnRHR-II KD compared with control females (P &lt; 0.0001); therefore, total CL weight tended to be reduced in transgenic gilts (P = 0.0958). In tissue samples, concentrations of progesterone and estrone tended to be reduced in transgenics females (P ≤ 0.10). Ultimately, these data suggest that GnRH-II and its receptor may help regulate ovulation rate, CL development and progesterone production in gilts. Supported by USDA/NIFA AFRI-ELI predoctoral fellowship (2017-67011-26036; ATD) and AFRI (2017-67015-26508; BRW) funds.

Development of a Robust Direct Binding Assay for Phospholipid‐Sensing NR5A Nuclear Receptors

The human NR5A nuclear receptors, steroidogenic factor‐1 (SF‐1, NR5A1) and liver receptor homolog‐1 (LRH‐1, NR5A2), are phospholipid‐sensing regulators of steroidogenesis, development, and metabolism. Their control of such diverse biological processes renders them attractive pharmacological targets for the treatment of several cancers and metabolic diseases such as obesity, type II diabetes, and non‐alcoholic fatty liver disease. However, the evaluation of candidate endogenous ligands and the development of small molecule modulators have been hindered by the lack of a robust direct binding assay. Using a structure‐guided approach, we recently developed a potent NR5A agonist (EC50 of 15 nM) targeting the ligand‐binding pocket. We have leveraged this molecule to create a high‐affinity fluorescent probe that binds SF‐1 and LRH‐1 with affinites of 12 nM and 1 nM, respectively. This probe has enabled the creation of a fluorescence polarization (FP)‐based competition assay suitable for both synthetic small molecules and mammalian phospholipids. FP is solution‐based, allowing proteins to retain their native conformation, and the competition format of the assay ensures that binding will only be detected in the ligand‐binding pocket. We have found that for a set of 25 LRH‐1 agonists derived from the previously‐reported RJW100 scaffold, affinities determined by FP correlate with potencies in a luciferase reporter assay, suggesting that in vitro binding affinity may be predictive of in‐cell activity for this class of molecule. Moreover, binding affinities reported for other NR5A agonists harboring different chemical scaffolds agree with our FP‐based measurements. Finally, we show that the previously‐reported dilauroylphosphatidylcholine agonist binds LRH‐1 with a Ki of 80 nM, suggesting that this assay may be used to evaluate candidate endogenous ligands for these receptors.Support or Funding InformationR01 DK115213 02EHD has additional support from the National Science Foundation Graduate Research Fellowship and the Emory University NIH‐5T32GM008367‐27. SGM has additional support from F31 DK111171 03.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular profiling demonstrates modulation of immune cell function and matrix remodeling during luteal rescue†.

The corpus luteum (CL) is essential for maintenance of pregnancy in all mammals and luteal rescue, which occurs around day 16-19 in the cow, is necessary to maintain luteal progesterone production. Transcriptomic and proteomic profiling were performed to compare the day 17 bovine CL of the estrous cycle and pregnancy. Among mRNA and proteins measured, 140 differentially abundant mRNA and 24 differentially abundant proteins were identified. Pathway analysis was performed using four programs. Modulated pathways included Tcell receptor signaling, vascular stability, cytokine signaling, and extracellular matrix remodeling. Two mRNA that were less in pregnancy were regulated by prostaglandin F2A in culture, while two mRNA that were greater in pregnancy were regulated by interferon tau. To identify mRNA that could be critical regulators of luteal fate, the mRNA that were differentially abundant during early pregnancy were compared to mRNA that were differentially abundant during luteal regression. Eight mRNA were common to both datasets, including mRNA related to regulation of steroidogenesis and gene transcription. A subset of differentially abundant mRNA and proteins, including those associated with extracellular matrix functions, were predicted targets of differentially abundant microRNA (miRNA). Integration of miRNA and protein data, using miRPath, revealed pathways such as extracellular matrix-receptor interactions, abundance of glutathione, and cellular metabolism and energy balance. Overall, this study has provided a comprehensive profile of molecular changes in the corpus luteum during maternal recognition of pregnancy and has indicated that some of these functions may be miRNA-regulated.

Vitamin D3 mediated regulation of steroidogenesis mitigates testicular activity in an aged rat model

Testicular aging leads to a decrease in spermatogenesis and steroidogenesis. Vitamin D plays an important role in reproduction by modulating testicular steroidogenesis. As the role of vitamin D3 in testicular steroidogenesis during aging has not been explored, the aim of this study was to evaluate the effects of vitamin D3 on testicular functions in d-gal-induced aged rats. Vitamin D3 treatment on d-gal-induced aged rats resulted in significant improvement in sperm parameters, histoarchitecture, serum testosterone, and rostenedione and estrogen levels. The results of both in vivo and in vitro studies showed that vitamin D3 directly regulates testicular steroidogenic markers. Vitamin D3 treatment also increased CYP19A1 and decreased AR expression in the testes of d-gal-induced aged and normal rats. These results suggest that estrogen-mediated action may be responsible for an improvement in spermatogenesis in aged testis. Furthermore, it may be suggested vitamin D3 has a protective role in the aged testis and unaffected spermatogenesis in normal rats treated with vitamin D3 could be due to a balance between estrogen and androgen action

Correlation of follicle-stimulating hormone receptor gene Asn 680 Ser (rs6166) polymorphism with female infertility.

Background and Aims:Female infertility is a complex multifactorial, and polygenic disease associated with genetic factors plays an essential role in its formation and follicle development, oocyte maturation, and steroidogenesis regulation in the ovary. The aim here is too study the genetic association between follicle-stimulating hormone receptor (FSHR) Asn680Ser; (rs6166) gene polymorphism with female Infertility in our population.Methods:In this prospective case-control study, we enrolled 106 infertile and 164 unrelated healthy control individuals. Genomic DNA was extracted from the 5 ml of venous blood using the modified salting-out method. A polymerase chain reaction-amplified exon 10 of FSHR and purified PCR products were sequenced on an ABI 3730XL DNA sequencer. The data were analyzed statistically.Results:We found that the presence of rare allele ”G” and heterozygous and common homozygous genotypes significantly increased the risk of female infertility. No significant change in the FSHR 191756 G >A genotype frequency was observed, regardless of chromosomal integrity. The genotype frequency distribution of locus 680 was consistent with the Hardy–Weinberg Equilibrium (HWE) in both groups (P > 0.05).Conclusion:No significant differences were found in allelic variants frequency and genotype distribution between each category of subjects when analyzing the FSHR SNPs in the exonic region (P value >0.05). FSHR Asn680Ser polymorphisms and female infertility (P > 0.05). Variations in FSHR gene have an essential influence on ovarian function and can account for several defects of female fertility. FSHR Asn680Ser (rs6166) gene polymorphism is associated with female infertility and can be used as a relevant molecular biomarker to identify the risk of infertility in our population. This finding can be important for disease pathogenesis.

Level of the nuclear factor с-Fos in human aldosteroma cells after potassium treatment

Potassium ions play an important role in the regulation of steroidogenesis, water-salt balance, apoptosis, proliferation. However, the nuclear phase of the agonist signaling in adrenocorticocytes remains poorly understood. The aim of the work was to elucidate the effect of potassium on the level of nuclear transcription factor c-Fos in human tumor adrenocortical tissue (aldosteroma). Methods. Expression of c-Fos transcription factor in aldosteromas was detected using Western-blot analysis. Results. The increase of c-Fos level in human aldosteroma cells at potassium physiological concentration of 3.5 mmol/l compared to medium without potassium was observed. The level of cFos did not change after 8.5 mmol/l K+ administration. The possible participation of transcription factor c-Fos in steroidogenic effects of K+ is being discussed. Conclusions. The results of study suggest that K+ can have a direct effect on the expression level of nuclear transcription factor c-Fos in tissues from human aldosteromas.

Gestational age-dependent gene expression profiling of ATP-binding cassette transporters in the healthy human placenta.

The ATP‐binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal‐fetal interface. We and others have demonstrated a gestational age‐dependent expression pattern of two ABC transporters, P‐glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational‐age dependent manner in normal human pregnancy. Using the TaqMan® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.

Seasonal regulation of steroidogenic enzyme expression within the green anole lizard (Anolis carolinensis) brain and gonad

Steroid hormones, such as testosterone and estradiol, are necessary for reproductive behavior. Seasonally breeding animals have increased sex steroid hormone levels during the breeding compared to non-breeding season, with increased reproductive behaviors and altered brain morphology in breeding individuals. Similar to other seasonally breeding animals, green anole lizards (Anolis carolinensis) have high sex steroid hormone levels and increased reproductive behaviors in the breeding season. Relatively less is known regarding the regulation of steroidogenesis in reptiles and this experiment examined whether enzymes involved in sex steroid hormone synthesis vary seasonally within the brain and gonads in wild-caught anole lizards. Specifically, we examined mRNA expression of steroidogenic acute regulatory protein (StAR), P450 17α-hydroxylase/C17-20lyase (Cyp17α1), 17 beta-hydroxysteroid dehydrogenase type 3 (17βHSD 3), and aromatase (Cyp19α1). We found that the mRNA for each of these genes was expressed in the lizard brain. Interestingly, Cyp19α1 mRNA expression in the brain was increased during the non-breeding season, potentially revealing a role for aromatase expression in the non-breeding brain. In the anole gonads, StAR mRNA expression levels were increased in both males and females during the breeding season, while the mRNA expression levels of CYP17α1 and 17βHSD 3 are increased when StAR mRNA expression was decreased, suggesting that the enzymes in the steroidogenic pathway are potentially regulated independently of StAR. This work reveals the seasonal regulation of steroidogenesis in the reptilian brain and gonad, although more work is necessary to determine the regulatory mechanisms that control these expression patterns.

Apelin in Reproductive Physiology and Pathology of Different Species: A Critical Review.

Apelin has been isolated from the bovine stomach extracts as an endogenous ligand of the previously orphan receptor APJ. Expression of the apelinergic system (apelin and APJ) was described in many organs where pleiotropic effects like regulation of food intake, body weight, or cardiovascular and immune function were described. Recent studies have shown that apelin also plays an important role in the regulation of female and male reproduction. Some data showed that the gene and protein of apelin/APJ are expressed in the hypothalamic-pituitary-gonad (HPG) axis tissue. Thus, apelin is synthesized locally in the hypothalamus, pituitary, ovaries, and testis of many species and has autocrine and/or paracrine effects. Most research indicates that apelin has an inhibitory effect on gonadotropin secretion and participates in the direct regulation of steroidogenesis, cell proliferation, and apoptosis in gonads. The article summarizes also results of a series of recent studies on the effect of apelin on reproduction pathology, like polycystic ovarian syndrome, endometriosis, and ovarian cancer. Many of these pathologies are still in critical need of therapeutic intervention, and recent studies have found that apelin can be targets in reproductive pathological states.

Calretinin Participates in Regulating Steroidogenesis by PLC-Ca2+-PKC Pathway in Leydig Cells

Calretinin, a Ca2+-binding protein, participates in many cellular events. Our previous studies found the high expression of calretinin in testicular Leydig cells. In this study, (MLTC-1 cells were infected with LV-calb2, R2C cells with LV-siRNA-calb2. The primary mouse Leydig cells were also used to confirm those data from cell lines. Testosterone level was significantly higher in the MLTC-1 cells with over-expressed calretinin than in the control, while progesterone was lower in the R2C cells in which down-regulated calretinin. The expressions of StAR changed in synchrony with hormones. Cytoplasmic Ca2+ level was significantly increased when calretinin was over-expressed. When MLTC-1 cells were infected with LV-calb2 and then stimulated using Clopiazonic, a Ca2+-releasing agent, testosterone was significantly increased. Interestingly, the expression levels of PLC, p-PKCµ (PKD), p-MARCKS and CREB, were significantly increased in the MLTC-1 cells with over-expressed calretinin, while PLC, p-PKD, p-MARCKS, MARCKS and CREB were decreased in the R2C cells with down-regulated calretinin. We also observed the increased expression of calretinin up-regulated testosterone production and the expressions of StAR and PLC in primary mouse Leydig cells. So, calretinin as a Ca2+-binding protein participates in the regulation of steroidogenesis via the PLC-Ca2+-PKC pathway in Leydig cells.

Current knowledge on the acute regulation of steroidogenesis.

How rapid induction of steroid hormone biosynthesis occurs in response to trophic hormone stimulation of steroidogenic cells has been a subject of intensive investigation for approximately six decades. A key observation made very early was that acute regulation of steroid biosynthesis required swift and timely synthesis of a new protein whose role appeared to be involved in the delivery of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane where the process of steroidogenesis begins. It was quickly learned that this transfer of cholesterol to the inner mitochondrial membrane was the regulated and rate-limiting step in steroidogenesis. Following this observation, the quest for this putative regulator protein(s) began in earnest in the late 1950s. This review provides a history of this quest, the candidate proteins that arose over the years and facts surrounding their rise or decline. Only two have persisted-translocator protein (TSPO) and the steroidogenic acute regulatory protein (StAR). We present a detailed summary of the work that has been published for each of these two proteins, the specific data that has appeared in support of their role in cholesterol transport and steroidogenesis, and the ensuing observations that have arisen in recent years that have refuted the role of TSPO in this process. We believe that the only viable candidate that has been shown to be indispensable is the StAR protein. Lastly, we provide our view on what may be the most important questions concerning the acute regulation of steroidogenesis that need to be asked in future.

Leydig cells: formation, function, and regulation.

Herein we summarize important discoveries made over many years about Leydig cell function and regulation. Fetal Leydig cells produce the high levels of androgen (testosterone or androstenedione, depending upon the species) required for differentiation of male genitalia and brain masculinization. Androgen production declines with loss of these cells, reaching a nadir at postpartum. Testosterone then gradually increases to high levels with adult Leydig cell development from stem cells. In the adult, luteinizing hormone (LH) binding to Leydig cell LH receptors stimulates cAMP production, increasing the rate of cholesterol translocation into the mitochondria. Cholesterol is metabolized to pregnenolone by the CYP11A1 enzyme at the inner mitochondrial membrane, and pregnenolone to testosterone by mitochondria and smooth endoplasmic reticulum enzymes. Cholesterol translocation to the inner mitochondrial membrane is mediated by a protein complex formed at mitochondrial contact sites that consists of the cholesterol binding translocator protein, voltage dependent anion channel, and other mitochondrial and cytosolic proteins. Steroidogenic acute regulatory protein acts at this complex to enhance cholesterol movement across the membranes and thus increase testosterone formation. The 14-3-3γ and ε adaptor proteins serve as negative regulators of steroidogenesis, controlling the maximal amount of steroid formed. Decline in testosterone production occurs in many aging and young men, resulting in metabolic and quality-of-life changes. Testosterone replacement therapy is widely used to elevate serum testosterone levels in hypogonadal men. With knowledge gained of the mechanisms involved in testosterone formation, it is also conceivable to use pharmacological means to increase serum testosterone by Leydig cell stimulation.

Seasonal variations in cellular expression of neuropeptide Y (NPY) in testis of the catfish, Clarias batrachus and its potential role in regulation of steroidogenesis

The present study demonstrates seasonal variation in the cellular expression of neuropeptide Y (NPY), a known orexigenic neuropeptide, in the testis of the catfish, Clarias batrachus and its relation with testicular steroids. In vitro effects of NPY on androgen production and activities of steroidogenic enzymes were also analyzed to reaffirm the relation between NPY and steroids. NPY-immunoprecipitation was observed in Sertoli cells, interstitial cells and germ cells in recrudescing testis. Intensity of NPY-immunoreaction in the interstitial cells increased steadily with initiation of spermatogenesis and reached maximal in fully grown testes, and then decreased suddenly in the spermiating/spent testis. NPY was also expressed considerably in Sertoli cells in recrudescing testis, but disappeared in the fully grown testis. A moderate NPY-immunoreactivity was also seen in spermatogonial cells in recrudescing testis, but intense NPY-immunoprecipitation was detected in advanced germ cells (spermatids/spermatozoa) in fully mature testis. NPY-immunoreation intensity in interstitial cells showed positive correlation with increasing levels of testicular testosterone and 11-ketotestosterone, and with activities of 3β-HSD & 17β-HSD coinciding with advancing testicular activities. NPY treatment of testicular fragments in vitro stimulated the activities of 3β-HSD & 17β-HSD and increased testosterone & 11-ketotestosterone levels. This study for the first time demonstrates the existence of NPY peptide at cellular levels in fish testis, which stimulates androgen production by acting directly at testicular level.

The neuropeptide orexin A - search for its possible role in regulation of steroidogenesis in adult mice testes.

Orexin A, a hypothalamic neuropeptide, regulates food intake and sleep-wake cycle by binding to orexin receptor 1. Besides brain, orexin A and orexin receptor 1 are also present in peripheral organs. In our earlier studies, localization and expression of orexin A and orexin receptor 1 have been shown in adult mouse testis, and further their role in testicular development in neonatal mouse was also demonstrated. In this study, role of orexin A and orexin receptor 1 in the testis of adult mouse by blocking binding of orexin A to orexin receptor 1 using an orexin receptor 1 antagonist, SB-334867, was investigated under invivo and exvivo conditions. Mice were given a single bilateral intratesticular injection of the antagonist at doses of 4 and 12μg/mouse and were sacrificed 24h post-injection. The antagonist treatment caused degenerative changes in the seminiferous tubules in the testis and also caused alterations in steroidogenesis, with a concomitant decrease in the level of testosterone (T) and an increase in the level of 17β-estradiol (E2 ) in serum and in testis. Further, expressions of SF1, StAR, P450scc and 17β-HSD were downregulated, while the expressions of 3β-HSD and P450arom were upregulated in antagonist-treated mice compared with controls. Also, the oxidative stress in the testis was increased in treated mice. In exvivo study, antagonist treatment to the testis caused a marked decrease in the level of T and an increase in the level of E2 in the media, accompanied by downregulated expression of SF1, StAR, P450scc and 17β-HSD and an upregulation in the expression of 3β-HSD and P450arom, indicating a direct role of orexin A in regulation of testicular steroidogenesis. The results of exvivo study supported the findings of invivo study. In conclusion, the results suggest potential involvement of orexin A and orexin receptor 1 in regulation of steroidogenesis and spermatogenesis in the testis of adult Parkes mice.

Localization of EFA6 (exchange factor for ARF6) isoform D in steroidogenic testicular Leydig cells of adult mice

EFA6 (exchange factor for ARF6) activates Arf6 (ADP ribosylation factor 6) by exchanging ADP to ATP and the resulting activated form of Arf6 is involved in the membrane trafficking and actin remodeling of cells. Our previous study has shown the selective expression/localization of EFA6D in steroidogenic adrenocortical cells in situ of adult mice. In view of the previous finding, the present study was undertaken to examine its localization in mouse Leydig cells representing another steroidogenic cell species in order to further support the possible involvement of the EFA6/Arf6 cascade via membrane trafficking in the regulation of steroidogenesis and/or secretion. A distinct band for EFA6D with the same size as that of the brain was detected in the testis of adult mice. In immuno-light microscopy, immunoreactivity for EFA6D was seen throughout the cytoplasm in most Leydig cells without any distinct accumulation along the plasmalemma. Lack of immunoreactivity for EFA6D was seen in the seminiferous tubular epithelium. In immuno-electron microscopy, the immune-labeling was seen in sporadic/focal patterns on plasma membranes and some vesicles and vacuoles subjacent to the plasma membranes. More constant and rather predominant is the labeling on numerous mitochondria. No immuno-labeling was seen in lipid droplets. The present study suggests that EFA6D is somehow involved in regulation of the synthesis and/or secretion of testosterone through the membrane-traffic by activation of Arf6. In addition, EFA6D is suggested to play in mitochondria some yet unidentified roles rather independent of Arf6-activation, which remains to be elucidated.

Melatonin Regulates the Synthesis of Steroid Hormones on Male Reproduction: A Review.

Melatonin is a ubiquitous molecule and exhibits different effects in long-day and short-day breeding animals. Testosterone, the main resource of androgens in the testis, is produced by Leydig cells but regulated mainly by cytokine secreted by Sertoli cells. Melatonin acts as a local modulator of the endocrine activity in Leydig cells. In Sertoli cells, melatonin influences cellular proliferation and energy metabolism and, consequently, can regulate steroidogenesis. These suggest melatonin as a key player in the regulation of steroidogenesis. However, the melatonin-induced regulation of steroid hormones may differ among species, and the literature data indicate that melatonin has important effects on steroidogenesis and male reproduction.