Steroid Production Research Articles

An adrenal incidentaloma that had appeared to produce dehydroepiandrosterone-sulfate in excess before immunohistochemical study of the tumor.

Adrenal incidentaloma is a clinically unapparent adrenal mass more than one cm in diameter detected during imaging performed not for adrenal disease. A 34-year-old man was evaluated for AI with a diameter of 3.5 cm in the left adrenal. He was obese with body mass index of 33,9. Blood pressure was 110-120/90 mmHg. The general laboratory tests were unremarkable. An adrenal hormone screening set revealed that ACTH was 6.9 pg/mL, cortisol 14.9 μg/dL, renin activity 0.9 ng/mL/h, aldosterone 79.4 pg/mL, dehydroepiandrosterone-sulfate (DHEA-S) measured on two occasions 5,217 ng/mL and 6,477 ng/mL (gender- and age-adjusted reference values, 1,060-4,640 ng/mL). The levels of metanephrine and normetanephrine were normal. The tumor was thought to produce solely DHEA-S. The excised left adrenal tissue contained a tumor with a diameter of 26 mm and neighboring adrenal tissue. The tumor consisted mostly of acidophil cells without necrosis, capsular or vascular invasion, and mitosis. Immunohistochemical study revealed followings: the cells of the tumors were stained positive for 3β-hydroxysteroid dehydrogenase, and 17α-hydroxylase, and 11β-hydroxylase, weakly positive for DHEA sulphotransferase, and negative for aldosterone synthetase. The atrophy of neighboring tissue was presumably caused by excess cortisol production. Four months after surgery, the cortisol level was 11.2 μg/dL and DHEA-S level 1,462 ng/mL. The tumor is considered to be a cortisol-producing adenoma with modestly excessive DHEA-S production rather than isolated DHEA-S-producing adenoma. Immunohistochemical study of steroidogenic enzymes is a valuable addition to blood hormone measurement to clarify steroid production profile.

The Association between Vitamin D and the Components of Male Fertility: A Systematic Review.

Previous systematic reviews of the effects of vitamin D on the components of male fertility have been inconclusive. This article systematically reviews the latest research to examine the relationship between vitamin D, semen quality parameters, and sex hormones production. MEDLINE, Cochrane, and Web of Science databases were searched using the appropriate keywords. Observational studies indicate significant correlation between vitamin D levels and sperm parameters, with a particular emphasis on sperm motility, and partially suggest a relationship between higher serum testosterone and vitamin D levels. Additionally, interventional studies confirmed that vitamin D supplementation has a positive effect on sperm motility, especially progressive. However, most randomized clinical trials indicate that vitamin D treatment does not have any significant effect on testosterone or other hormone levels. Although our findings add to the discussion regarding the effect of vitamin D on male fertility, there is still no solid evidence to support the use of vitamin D supplementation to improve the outcomes of patients with impaired sperm parameters and hormonal disorders. Additional dedicated clinical studies are needed to clarify the relationship between vitamin D and male fertility, along with its components.

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis

The human adrenal cortex is composed of distinct zones that are the main source of steroid hormone production. The mechanism of adrenocortical cell differentiation into several functionally organized populations with distinctive identities remains poorly understood. Human adrenal disease has been difficult to study, in part due to the absence of cultured cell lines that faithfully represent adrenal cell precursors in the early stages of transformation. Here, Human Adrenocortical Adenoma (HAA1) cell line derived from a patient's macronodular adrenocortical hyperplasia and was treated with histone deacetylase inhibitors (HDACis) and gene expression was examined. We describe a patient-derived HAA1 cell line derived from the zona reticularis, the innermost zone of the adrenal cortex. The HAA1 cell line is unique in its ability to exit a latent state and respond with steroidogenic gene expression upon treatment with histone deacetylase inhibitors. The gene expression pattern of differentiated HAA1 cells partially recreates the roster of genes in the adrenal layer that they have been derived from. Gene ontology analysis of whole genome RNA-seq corroborated increased expression of steroidogenic genes upon HDAC inhibition. Surprisingly, HDACi treatment induced broad activation of the Tumor Necrosis Factor (TNF) alpha pathway. This novel cell line we developed will hopefully be instrumental in understanding the molecular and biochemical mechanisms controlling adrenocortical differentiation and steroidogenesis.

De novo cholesterol biosynthesis: an additional therapeutic target for the treatment of postmenopausal breast cancer with excessive adipose tissue.

The onset and development of breast cancer in postmenopausal women are associated with closely related individual-dependent factors, including weight gain and high levels of circulating androgens. Adipose tissue is the most peripheral site of aromatase enzyme synthesis; therefore, the excessive accumulation of visceral fat results in increased androgens aromatization and estradiol production that provides the microenvironment favorable to tumorigenesis in mammary epithelial cells expressing estrogen receptors (ERs). Moreover, to meet the increased requirement of cholesterol for cell membrane assembly and the production of steroid hormones to sustain their proliferation, ER-positive cells activate de novo cholesterol biosynthesis and subsequent steroidogenesis. Several approaches have been followed to neutralize the de novo cholesterol synthesis, including specific enzyme inhibitors, statins, and, more recently, metformin. Cumulating evidence indicated that inhibiting cholesterol biosynthesis by statins and metformin may be a promising therapeutic strategy to block breast cancer progression. Unlike antiestrogens and aromatase inhibitors (AIs) which compete for binding to ER and inhibit androgens aromatization, respectively, statins block the production of mevalonic acid by inhibiting the activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, and metformin hampers the activation of the sterol regulatory element-binding protein 2 (SREBP2) transcription factor, thus inhibiting the synthesis of several enzymes involved in cholesterol biosynthesis. Noteworthy, statins and metformin not only improve the prognosis of overweight patients with ER-positive cancer but also improve the prognosis of patients with triple-negative breast cancer, the aggressive tumor subtype that lacks, at present, specific therapy.

Restored Thymic Output after Androgen Blockade Participates in Antitumor Immunity.

The thymus is a hormone-sensitive organ, which involutes with age in response to production of sex steroids. Thymic involution leads to a decrease in the generation of recent thymic emigrants (RTEs), resulting in a reduced response to immune challenges such as cancer. Interestingly, the standard of care for prostate cancer patients is androgen deprivation therapy (ADT), which leads to thymic regeneration and an increase in thymic output. It remains unknown whether these newly produced T cells can contribute to the antitumor immune response. This study defines the kinetics of thymic regeneration in response to ADT in mice, determining that thymic epithelial cell proliferation is critical for the increase in RTE output. Using a mouse model to track RTE in vivo, we demonstrate that these newly generated RTEs can traffic to tumors, where they become activated and produce effector cytokines at levels similar to more mature T cells. Collectively, these data suggest that RTEs produced from ADT-induced thymic regeneration could be harnessed for the antitumor immune response.

Serum GDF9 and BMP15 as potential markers of ovarian function in women with and without polycystic ovary syndrome.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are critical paracrine regulators of female fertility and are predominantly expressed by oocytes. However, it is unknown if serum concentrations reflect changes in ovarian function and/or reproductive endocrine disorders. This study aimed to determine if serum GDF9/BMP15 are associated with ovarian, pituitary, oestrogenic, androgenic and metabolic characteristics and the ovarian pathologies, polycystic ovarian morphology (PCOM) and polycystic ovary syndrome (PCOS). Women aged 21-45 years (n = 381) were included from a cross-sectional study at the National University Hospital, Singapore. Participants were volunteers and patients with possible PCOS. Anthropometric measurements, transvaginal ultrasound scans and serum sampling were performed and a questionnairecompleted. Serum GDF9 and BMP15 concentrations were matched with menstrual cycle length, ovarian protein and steroid hormone production, pituitary hormone production and metabolic assessments in women with PCOM or PCOS and those with neither (control). Serum GDF9 and BMP15 were detectable in 40% and 41% of women, respectively and were positively correlated with each other (r = 0.08, p = 0.003). GDF9, but not BMP15, was positively correlated with ovarian volume (p = 0.02) and antral follicle count(AFC) (p = 0.004), but not with anti-Müllerian hormone(p = 0.05). However, serum GDF9 and BMP15 concentrations were not significantly different between control, PCOM and PCOS women, nor associated with androgenic or metabolic PCOS features. However, the relationship between GDF9 and AFC differed between control, PCOM and PCOS women (p = 0.02). Serum GDF9 and BMP15 concentrations somewhat reflect ovarian but not androgenic or metabolic characteristics of PCOS, with increased GDF9 reflecting high AFC as seen in PCOM/PCOS.

Wild fish responses to wastewater treatment plant upgrades in the Grand River, Ontario

Municipal wastewater treatment plant (WWTP) effluent is one of several point sources of contaminants (nutrients, pharmaceuticals, estrogens, etc.) which can lead to adverse responses in aquatic life. Studies of WWTP effluent impacts on rainbow darter (Etheostoma caeruleum) collected downstream of WWTPs in the Grand River, Ontario have reported disruption at multiple levels of biological organization, including altered vitellogenin gene expression, lower levels of in vitro steroid production, and high frequency of intersex. However, major upgrades have occurred at treatment plants in the central Grand River over the last decade. Treatment upgrades to the Waterloo WWTP were initiated in 2009 but due to construction delays, the upgrades came fully on-line in 2017/2018. Responses in rainbow darter have been followed at sites associated with the outfall consistently over this entire time period. The treatment plant upgrade resulted in nitrification of effluent, and once complete there was a major reduction in effluent ammonia, selected pharmaceuticals, and estrogenicity. This study compared several key responses in rainbow darter associated with the Waterloo WWTP outfall prior to and post upgrades. Stable isotopes signatures in fish were used to track exposure to effluent and changed dramatically over time, corresponding to the effluent quality. Disruptions in in vitro steroid production and intersex in the darters that had been identified prior to the upgrades were no longer statistically different from the upstream reference sites after the upgrades. Although annual variations in water temperature and flow can potentially mask or exacerbate the effects of the WWTP effluent, major capital investments in wastewater treatment targeted at improving effluent quality have corresponded with the reduction of adverse responses in fish in the receiving environment.

MORPHOLOGICAL FEATURES OF STRESS MARKERS IN PLACENTA

Introduction. The multinucleated syncytiotrophoblast of human placental villi is responsible for transport functions between thematernal and fetal circulation and is the main site of protein synthesis and steroid production. It is formed by the fusion of underlyingcytotrophoblast cells. The nuclei of the multinucleated syncytiotrophoblast are nonmitotic, but the mechanism of cell cycle arrest in thesyncytiotrophoblast is not known. Single publications have described that cell fusion induces a cellular senescence, best known as acomponent of the stress response and an important factor in preventing tumor cell growth. For syncytiotrophoblast, one of the criteriaof its aging is the formation of so-called syncytial nodes - local accumulations of nuclear material with protrusion of these cells into theinterstitial space.The purpose of the study. To determine the morphological features in women's placentas under the influence of chronic stresscaused by coronavirus disease and internal irradiation, with the subsequent formation of criteria for diagnosing diseases and assessingthe severity of lesions in the mother and fetus.Material and methods. A morphological study of syncytial nodules in the villi of the placenta of groups of women with chronic stressdue to internal irradiation and COVID-19 (classified by severity of the disease) was performed in comparison with the physiologicalcourse of pregnancy and childbirth. Histopathological and ultrastructural examination of placentas was performed with the detection ofsyncytial nodes with morphometric analysis of their ultrastructural features of the nuclear component of syncytiotrophoblast nodules andtheir comparison in clinical groups. The number of syncytial nodes was estimated on histological specimens stained with hematoxylinand eosin. The effect of chronic stress in women was significantly (p<0.05) confirmed by increased cortisol levels (saliva test). Statisticalprocessing of the results was carried out using licensed programs «Microsoft Excel» and «Statistica». The design of the study and allmethods used in this study were reviewed and approved by the Bioethics Committee of the State Institution «Institute of Pediatrics,Obstetrics and Gynecology named after Academician O.M. Lukyanova of the National Academy of Medical Sciences of Ukraine», Kyiv,Ukraine.Results of the study. Morphological examination of the placental tissue of parturient women with COVID-19 showed an increasein the number of syncytial nodes in the villi, which we associated with the presence of stress. Ultrastructural studies revealed changes inthe nuclei of syncytial nodules depending on the pathology.Conclusion. An increase in the number of syncytial nodes in the chorionic structures in internal radiation and COVID-19 in pregnantwomen has been established, which is important as predictors of placental factors for the health of the individual in the future.

Preparation and characterization of crosslinked starch-albumin films for coating of prednisolone tablets for use in Covid-19 related respiratory disease

Steroidal products have been found useful in inflammations associated with Covid 19. Prednisolone is one readily available steroid, which is often found as uncoated normal release tablets. Modified release prednisolone may be desirable in Covid 19 for sustained actions. This is expected to reduce dosing frequency and enhance compliance. This study is concerned with development of controlled release prednisolone using coating technology with bio-compatible, cross-linked starch-albumin films. Starches and proteins are excellent film formers with good flexibility, transparency, and bio-compatibility. The cross-linked starch-albumin films were prepared using glycerol as the plasticizer: starch (A), starch-albumin (B), starch-albumin cross-linked with formaldehyde at 1 % (C), 5 % (D) and 10 % (E). Equilibrium moisture sorption (EMS) at 100 % relative humidity, equilibrium swelling (ESC) in buffer solutions of pHs 2, 7 and 9, and DSC thermal properties were evaluated. In-vitro drug release from the film coated prednisolone tablets were evaluated in 0.1N HCl, water and phosphate buffer 8.0 as dissolution media. Films showed ESC in the order A>D>E>B>C; D>C>A>E>B and A>D>C>B>E in acidic, neutral and alkali media respectively. EMS was in the order B>E>A>D>C; with slight shift in the melting temperatures. In-vitro release at 240 min varied from 78 to 117 % (E>D>C>A>B); 19 to 60 % (D>B>C>E>A) and 49 to 60 % (B>A>C>D>E) in 0.1N HCl, water and PBS respectively. Cross-linking improved the stability and swelling of films. The in-vitro release in alkaline medium suggests their usefulness for controlled drug delivery. New pH-responsive polymers, with improved physicochemical properties for coating prednisolone tablets were developed.

Autophagy: A Double-Edged Sword in Male Reproduction

Autophagy, an evolutionarily conserved cell reprogramming mechanism, exists in all eukaryotic organisms. It is a fundamental and vital degradation/recycling pathway that removes undesirable components, such as cytoplasmic organelles, misfolded proteins, viruses, and intracellular bacteria, to provide energy and essential materials for organisms. The success of male reproduction depends on healthy testes, which are mainly composed of seminiferous tubules and mesenchyme. Seminiferous tubules are composed of Sertoli cells (SCs) and various germ cells, and the main functional part of mesenchyme are Leydig cells (LCs). In recent years, a large amount of evidence has confirmed that autophagy is active in many cellular events associated with the testes. Autophagy is not only important for testicular spermatogenesis, but is also an essential regulatory mechanism for the ectoplasmic specialization (ES) integrity of SCs, as well as for the normal function of the blood-testes barrier (BTB). At the same time, it is active in LCs and is crucial for steroid production and for maintaining testosterone levels. In this review, we expanded upon the narration regarding the composition of the testes; summarized the regulation and molecular mechanism of autophagy in SCs, germ cells, and LCs; and concluded the roles of autophagy in the process of spermatogenesis and testicular endocrinology. Through integrating the latest summaries and advances, we discuss how the role of autophagy is a double-edged sword in the testes and may provide insight for future studies and explorations on autophagy in male reproduction.

Gender Differences in the Indirect Effect of Psychosocial Work Environment in the Association of Precarious Employment and Chronic Stress: A Cross-Sectional Mediation Analysis.

Gender differences in the association between precarious employment and chronic stress have been found but the mechanisms underlying this relationship have not been explored. The main objective of this study was to evaluate the mediating effects of psychosocial risk factors at work (i.e., demands, control, and support) and work-life conflicts in the relationship between precarious employment and chronic stress as measured through the production of steroid hormones (both adrenal and gonadal) for men and women separately. Cross-sectional data were derived from a sample of workers from Barcelona (n = 125-255 men; 130 women). A set of 23 markers were determined from hair samples to evaluate the production of both adrenal and gonadal steroids. Decomposition analyses were applied to estimate the indirect effects of psychosocial risk factors and work-life conflict using linear regression models. Gender differences in the association between precarious employment and steroids production were confirmed. Psychosocial risk factors and work-life conflicts had indirect effects only among women (βCortisol = 0.18; 95% CI: 0.04-0.32; βCortisol/Cortisone 0.19; 95% CI: 0.08-0.31; β%Cortisol 0.12; 95% CI: 0.05-0.20). Gender differences suggest that the physiological response to precarious employment could be determined by the social construction of gender identities, as well as by positions and roles in the labour market and family. Future studies should delve further into these differences to improve employment and working policies, thus mitigating gender inequalities in the labour market to prevent work-related stress.

Protective effect of biogenic selenium nanoparticles against diquat-induced acute toxicity via regulation of gut microbiota and its metabolites.

Selenium nanoparticles (SeNPs) with unique biological properties have been suggested as a safer and more effective platform for delivering of Selenium for biological needs. In this study, we investigated the association between gut microbiota altered by SeNPs supplementation and its metabolites under oxidative stress conditions through 16S rDNA gene sequencing analysis and untargeted metabolomics. The results showed that dietary supplementation with SeNPs attenuated diquat-induced acute toxicity in mice, as demonstrated by lower levels of inflammatory effector cells, and biochemical markers in serum such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH). SeNPs also reversed the perturbed gut microbiota composition induced by diquat, decreased the Firmicutes/Bacteroidetes ratio, and increased the abundance of beneficial bacteria such as Akkermansia, Muribaculaceae, Bacteroides and Parabacteroides. Untargeted fecal metabolomics showed that SeNPs can regulate the production of steroids and steroid derivatives, organonitrogen compounds, pyridines and derivatives and other metabolites. Microbiome-metabolome correlation analysis suggested that Parabacteroides was the key bacteria for the SeNPs intervention, which might upregulate the levels of metabolites such as trimethaphan, emedastine, berberine, desoxycortone, tetrahydrocortisone. This study demonstrated that dietary SeNPs supplementation can extensively modulate the gut microbiota and its metabolism, thereby alleviating diquat-induced acute toxicity.

Human ovarian follicular granulosa cells isolated during ART procedure reflect substantial changes in activation of hormonal signaling pathways, during long-term in vitro conditions

Abstract The ovary is commonly known as an endocrine gland responsible for sex steroid production. One of the outstanding cells in ovarian microenvironment - granulosa cells (GCs) are responsible for converting the androgens to estrogens during follicular growth and secreting progesterone after ovulation. These secretory processes within the ovary are directly involved in hormonal signaling pathways, and they depend on different stages of cholesterol and lipid biosynthesis during the ovarian cycle. The understating of the regulation and further investigation into the processes taking part in ovary will expose new clinical advantages in detection and treatment of female reproductive system diseases associated with sex hormone abnormalities. The expression of genes belonging to ontology groups associated with steroid biosynthesis and metabolism, such as “cholesterol biosynthetic process” (GO:0006695, “regulation of lipid biosynthetic process” (GO:0046890), “regulation of lipid metabolic process” (GO:0019216), “response to insulin” (GO:0032868) and “response to lipopolysaccharide” (GO:0032496) were analyzed by using the microarray approach. The patterns of gene expression in human GCs at days 1-day, 7-day, 15-day, and 30-day of primary in vitro culture have been analyzed. Based on the microarray results, a group of upregulated genes have been selected: CCL20, CXCL5, STAR, MSMO1, and AADAC. The genes STAT5B, OPA3, PPARG, PROX1, and SEC14L2 were decreased across all the experimental groups during the 30-day cell cultivation period. These results suggest that, the GCs in cell culture under in vitro express steroidogenic markers and it is important to understand associations with lipid and liposaccharide synthesis relative to reproductive medicine.

Cyp17a2 is involved in testicular development and fertility in male Nile tilapia, Oreochromis niloticus.

Steroid hormones play an essential role in many reproductive processes of vertebrates. Previous studies revealed that teleost-specific Cyp17a2 (cytochrome P450 family 17 subfamily a 2) might be required for the production of cortisol in the head-kidney and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) in ovary during oocyte maturation. However, the role of Cyp17a2 in male reproduction remains to be largely unknown. The aim of this study was to explore the essentiality of cyp17a2 gene in male steroidogenesis, spermatogenesis, and male fertility. A homozygous mutation line of cyp17a2 gene was constructed in tilapia by CRISPR/Cas9 gene editing technology. The expression level of germ cell and meiosis-related genes and steroidogenic enzymes were detected by qRT-PCR, IHC, and Western blotting. EIA and LC-MS/MS assays were used to measure the steroid production levels. And sperm quality was examined by Sperm Quality Analyzer software. In this study, cyp17a2 gene mutation resulted in the significant decline of serum DHP and cortisol levels. On the contrary, significant increases in intermediate products of cortisol and DHP were found in cyp17a2-/- male fish. The deficiency of cyp17a2 led to the arrest of meiotic initiation in male fish revealing as the reduction of the expression of germ cell-related genes (vasa, piwil, oct4) and meiosis-related genes (spo11 and sycp3) by 90 dah. Afterwards, spermatogenesis was gradually recovered with the development of testis in cyp17a2-/- males, but it showed a lower sperm motility and reduced fertility compared to cyp17a2+/+ XY fish. Deletion of cyp17a2 led to the abnormal upregulation of steroidogenic enzymes for cortisol production in the head-kidney. Moreover, unaltered serum androgens and estrogens, as well as unchanged related steroidogenic enzymes were found in the testis of cyp17a2-/- male fish. This study proved that, for the fist time, Cyp17a2 is indispensable for cortisol and DHP production, and cyp17a2 deficiency associated curtailed meiotic initiation and subfertility suggesting the essentiality of DHP and cortisol in the male fertility of fish.

Seasonal Change in Adiponectin Associated with Ovarian Morphology and Function in Wild Ground Squirrels (Citellus dauricus Brandt)

The goal of this study is to explore the relationship between altered circulating adiponectin concentration, ovarian tissue morphology, ovarian steroidogenesis, and sex hormone production in ovaries of wild ground squirrels. The ovarian mass differed significantly during the breeding and non-breeding seasons, and the circulating estradiol and progesterone concentrations were significantly higher in the breeding season, while the circulating adiponectin level was significantly lower. The expression levels of gonadotropin receptors (FSHR and LHR) and steroidogenic enzymes (StAR, P450scc, P450arom, and 3β-HSD) were significantly higher during the breeding season. Comparing the ovarian transcriptome data of wild ground squirrels between the two periods, we found that some differentially expressed genes were enriched for ovarian steroidogenesis and the adipocytokine signaling pathway, which correlated with our present results. Notably, the MAPK signaling pathway was also enriched and its related genes (Erk1, p38 Mapk, Jnk) were up-regulated by qPCR during the non-breeding season. These findings suggested that adiponectin may be involved in the regulation of seasonal changes in the ovarian function of wild ground squirrels, possibly by acting on the MAPK signaling pathway to regulate sex steroidogenesis in the ovaries.

To explore the regulatory role of Wnt/P53/Caspase3 signal in mouse ovarian development based on LFQ proteomics

Early ovarian follicular development is regulated by multiple proteins and signaling pathways, including the Wnt gene. To explore the regulatory mechanism of Wnt signaling on early ovarian follicular development, ovaries from 17.5 days post coitum (17.5 dpc) mice were collected and cultured in vitro for four days in the presence of IWP2 as a Wnt activity inhibitor and KN93 as a CaMKII inhibitor. LFQ proteomics technique was then used to analyze the significant differentially abundant (P-SDA) 93 and 262 proteins in the IWP2 and KN93 groups, respectively. Of these, 63 up-regulated proteins and 30 down-regulated proteins were identified for IWP2, along with 3 significant KEGG pathways (P < 0.05). For the KN93 group, 168 up-regulated proteins and 94 down-regulated ones were P-SDA, with 9 significant KEGG pathways also noted (P < 0.05). In both IWP2 and KN93 groups, key pathways (Wnt signaling pathway, Notch signaling pathway, P53 signaling pathway, TGF-β signaling pathway, ovarian steroid production) and metabolic regulation (energy metabolism, metal ion metabolism) were found to be related to early ovarian follicular development. Finally, western blotting demonstrated the regulatory role of Wnt/P53/Caspase3 signaling pathway in mouse ovarian development. These results contribute new knowledge to the understanding of regulatory factors of early ovarian follicular development. SignificanceIn this study, label-free quantification (LFQ) was used in combination with liquid chromatography-mass spectrometer (LC-MS/MS) to study potential changes in the proteomic profiles of embryonic mice subjected to Wnt inhibitor IWP2 and CaMKIIinhibitor KN93. In addition, bioinformatics and comparative analyses were performed using publicly available proteomics databases to further explore the underlying mechanisms associated with early mouse ovarian growth and development.

Growth hormone in fertility and infertility: Mechanisms of action and clinical applications

Secreted by the anterior pituitary gland, growth hormone (GH) is a peptide that plays a critical role in regulating cell growth, development, and metabolism in multiple targeted tissues. Studies have shown that GH and its functional receptor are also expressed in the female reproductive system, including the ovaries and uterus. The experimental data suggest putative roles for GH and insulin-like growth factor 1 (IGF-1, induced by GH activity) signaling in the direct control of multiple reproductive functions, including activation of primordial follicles, folliculogenesis, ovarian steroidogenesis, oocyte maturation, and embryo implantation. In addition, GH enhances granulosa cell responsiveness to gonadotropin by upregulating the expression of gonadotropin receptors (follicle-stimulating hormone receptor and luteinizing hormone receptor), indicating crosstalk between this ovarian regulator and the endocrine signaling system. Notably, natural gene mutation of GH and the age-related decline in GH levels may have a detrimental effect on female reproductive function, leading to several reproductive pathologies, such as diminished ovarian reserve, poor ovarian response during assisted reproductive technology (ART), and implantation failure. Association studies using clinical samples showed that mature GH peptide is present in human follicular fluid, and the concentration of GH in this fluid is positively correlated with oocyte quality and the subsequent embryo morphology and cleavage rate. Furthermore, the results obtained from animal experiments and human samples indicate that supplementation with GH in the in vitro culture system increases steroid hormone production, prevents cell apoptosis, and enhances oocyte maturation and embryo quality. The uterine endometrium is another GH target site, as GH promotes endometrial receptivity and pregnancy by facilitating the implantation process, and the targeted depletion of GH receptors in mice results in fewer uterine implantation sites. Although still controversial, the administration of GH during ovarian stimulation alleviates age-related decreases in ART efficiency, including the number of oocytes retrieved, fertilization rate, embryo quality, implantation rate, pregnancy rate, and live birth rate, especially in patients with poor ovarian response and recurrent implantation failure.

Steroidogenic and innate immune responses in Atlantic salmon are influenced by dietary total lipid, long chain polyunsaturated fatty acids and dissolved oxygen

Recent studies have found that feeding high levels of long chain polyunsaturated fatty acids (LC-PUFA) influenced the steroid biosynthesis pathway of Atlantic salmon on a transcriptomic level, although research observed on a metabolic level is lacking. Dietary lipid and chronic stress have also been suggested to play a role in steroidogenesis. A study was performed on Atlantic salmon post-smolts (184 g) fed with diets of high and low levels of lipid with and without high levels of LC-PUFA under hypoxic (stressed) or normoxic (unstressed) conditions. Liver, head kidney and blood samples were collected after 35 and 116 days to determine short- and long-term effects. On day 35, dissolved oxygen affected the levels of the steroid hormones 11-deoxycortisol, cortisone and testosterone as well as the relative expression of cyp11a involved in steroidogenesis and ud2a2 involved in sex steroid metabolism. On day 116, an interaction between lipid x LC-PUFA x oxygen was found for cyp11a, hsd3b (steroidogenesis) and ud2a2 genes, whereas LC-PUFA and/or oxygen affected the steroid levels of 11-dehydrocorticosterone, corticosterone, cortisol, dehydroepiandrosterone, pregnenolone and testosterone. The observation that both LC-PUFA and oxygen affected cyp11a expression and the levels of pregnenolone, provides evidence for the influence of both parameters on the rate-limiting point of steroid production, which can take >35 days to establish. The 3-way interaction between lipid, LC-PUFA and dissolved oxygen on the expression of steroid genes suggests that the chronic stressor of hypoxia may be over-riding dietary influences of the lipid and LC-PUFA, though the relative proportion of LC-PUFA within the lipid may play a role as well. In terms of fatty acid composition, low dietary lipid and high LC-PUFA increased the levels of total n-6 and n-3 PUFA in the liver, particularly EPA and DHA, and the head kidney to a lesser extent. In terms of immune pathways, hypoxia influenced cytokine gene expression (tgfb, il10), cellular repair gene expression (gr, hsp70 and hsp90) and eicosanoid levels (PGE2 and LTB5) in the head kidney, while dietary lipid and LC-PUFA influenced cytokines (ifnγ and il1β) and eicosanoids (PGE2 and LTB5). These findings demonstrate that feeding different levels of LC-PUFA significantly alters steroidogenesis and innate immune response in Atlantic salmon post-smolts with additional interactions from dietary lipid and hypoxia stress. These results will significantly impact the aquaculture industry since it demonstrates salmon fed high levels of LC-PUFA are likely more resilient to cope with chronic stressors (e.g. prolonged hypoxia) due to lower corticosteroid levels and upregulated immune response.

Muscarinic receptors in the rat ovary are involved in follicular development but not in steroid secretion.

Acetylcholine (ACh) may be involved in the regulation of ovarian functions. A previous systemic study in rats showed that a 4-week, intrabursal local delivery of the ACh-esterase blocker Huperzine-A increased intraovarian ACh levels and changed ovarian follicular development, as evidenced by increased healthy antral follicle numbers and corpora lutea, as well as enhanced fertility. To further characterize the ovarian cholinergic system in the rat, we studied whether innervation may contribute to intraovarian ACh. We explored the cellular distribution of three muscarinic receptors (MRs; M1, M3, and M5), analyzed the involvement of MRs in ovarian steroidogenesis, and examined their roles in ovarian follicular development in normal conditions and in animals exposed to stressful conditions by employing the muscarinic antagonist, atropine. Denervation studies decreased ovarian norepinephrine, but ovarian ACh was not affected, evidencing a local, nonneuronal source of ACh. M1 was located on granulosa cells (GCs), especially in large antral follicles. M5 was associated with the ovarian vascular system and only traces of M3 were found. Ex vivo ovary organo-typic incubations showed that the MR agonist Carbachol did not modify steroid production or expression of steroid biosynthetic enzymes. Intrabursal, in vivo application of atropine (an MR antagonist) for 4 weeks, however, increased atresia of the secondary follicles. The results support the existence of an intraovarian cholinergic system in the rat ovary, located mainly in follicular GCs, which is not involved in steroid production but rather via MRs exerts trophic functions and regulates follicular atresia.

Mice deficient in ER protein seipin have reduced adrenal cholesteryl ester lipid droplet formation and utilization

Cholesteryl ester (CE)-rich lipid droplets (LDs) accumulate in steroidogenic tissues under physiological conditions and constitute an important source of cholesterol as the precursor for the synthesis of all steroid hormones. The mechanisms specifically involved in CE-rich LD formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich LDs. Seipin is an endoplasmic reticulum protein that forms oligomeric complexes at endoplasmic reticulum-LD contact sites, and seipin deficiency results in severe alterations in LD maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich LD formation, no studies have directly addressed whether seipin is important for CE-rich LD biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis, and ovary, steroidogenic tissues that accumulate CE-rich LDs under normal physiological conditions, were generated. We found that the steroidogenic-specific seipin-deficient mice displayed a marked reduction in LD and CE accumulation in the adrenals, demonstrating the pivotal role of seipin in CE-rich LD accumulation/formation. Moreover, the reduction in CE-rich LDs was associated with significant defects in adrenal and gonadal steroid hormone production that could not be completely reversed by addition of exogenous lipoprotein cholesterol. We conclude that seipin has a heretofore unappreciated role in intracellular cholesterol trafficking.