Investigation of the uterine structural changes in the experimental model with polycystic ovary syndrome and effects of vitamin D treatment: An ultrastructural and immunohistochemical study

In this study, we aimed to investigate the structural changes seen in the endometrium in experimental PCOS rat model and the effects of vitamin D treatment on these changes at immunohistochemical and electron microscopic levels. 24 prepubertal female rats were divided into three groups. Two groups were injected with dehydroepiandrosterone and one of them was treated with 1,25(OH)2 D3 at the same time. The control group was injected with sesame oil. At the end of the 28th day, the blood samples were collected. Uterus tissues were prepared for light and electron microscopic examinations. Epithelial, stromal and endometrial thickness measurements were investigated. Immunohistochemical staining was applied against caspase-3 and Ki-67. Serum AMH and estradiol levels were higher in PCOS group compared to the control group. Serum progesterone levels were similar in all groups. Endometrial, epithelial and stromal thickness measurements were increased in PCOS group compared to the control group, and decreased in the vitamin D treatment group compared to the PCOS group. Light and electron microscopic results of PCOS group showed an increase in apoptosis and proliferation. In the PCOS group, immunohistochemical staining of caspase-3 and Ki-67 were found to be higher than in the control group, but stainings were decreased with vitamin D treatment compared to PCOS group. Structural changes observed in endometrium may be related to implantation problems seen in patients with PCOS. Our studies suggest that vitamin D therapy may be beneficial in these patients.     

补充维生素D联合益生菌对多囊卵巢综合征患者肠道菌群多样性、代谢和激素水平的影响

目的探究补充维生素D联合益生菌治疗多囊卵巢综合征(PCOS)对患者菌群多样性、代谢和激素水平的影响。方法选取2019年1月至2019年3月在东南大学附属中大医院诊断并治疗的PCOS患者60例,采用随机数表法分为联合补充组(30例)和维生素D组(30例),联合补充组同时使用维生素D和益生菌,维生素D组只补充维生素D。对治疗前后患者肠道菌群进行检测,并比较代谢因子和激素水平,使用Pearson相关性分析探究指标间的相关性。结果治疗后两组菌群丰富度、Shannon-Wiener指数及最大Shannon-Wiener指数均较治疗前显著升高(均P<0.05),且联合补充组丰富度、Shannon-Wiener指数显著高于维生素D组(t=4.496,2.896,均P<0.05);治疗后两组GSH、TAC水平均显著升高,其中联合补充组TAC显著高于维生素D组(t=2.505,P=0.002);治疗后两组MDA、TT显著降低,其中联合补充组MDA、TT显著低于维生素D组(t=2.371,2.177,P=0.021,0.034)。副拟杆菌属与MDA呈显著正相关(r=0.339,P=0.040),肠杆菌科与TT呈显著正相关(r=0.330,P=0.046),粪杆菌属与TAC呈显著正相关(r=0.437,P=0.007),乳杆菌属与TAC呈显著正相关(r=0.350,P=0.033),与TT呈显著负相关(r=-0.371,P=0.024)。结论补充维生素D联合益生菌可有效改善PCOS患者代谢因子及激素水平,且其肠道微生物与PCOS代谢及激素指标存在一定相关性。     

Effects of scrotal hyperthermia on Leydig cells in long-term: a histological,immunohistochemical and ultrastructural study in rats

We have previously demonstrated that scrotal hyperthermia induce Leydig cell (LC) damage in short-term. The objectives of this pilot study were to investigate morphological changes and regulation of steroidogenesis on LC in long-term and the time of observation were extended to investigate whether the LC would eventually make a recovery after scrotal hyperthermia. The rats were randomly allotted into one of four groups: A (control), B (70 days after scrotal hyperthermia), C (105 days after scrotal hyperthermia), D (140 days after scrotal hyperthermia); each group contain seven animals. Scrotal hyperthermia was carried out in a thermostatically controlled water bath at 43°C for 30 min once daily for six consecutive days. Control rats were treated in the same way, except the testes were immersed in a water bath maintained at 22°C. Hyperthermia applied rats were sacrificed under 50 mg/kg ketamine anaesthesia after 70, 105 and 140 days, and biopsy materials of testes were obtained for light and electron microscopic examinations. Morphologically normal and the number of testosterone positive LC was significantly higher in 140 days after last heat than all other heat treatment groups. In heat treated groups, a dilated smooth endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. In the 140 days after scrotal hyperthermia, the severities of degenerative changes of LC were less than that observed in the other heat treated groups. We conclude that, scrotal hyperthermia cause morphological damaging and impaired steroidogenesis in LC and recovery of these findings were noted first time in 140 days after the last heat treatment.     

Identification of a highly specific and versatile vitamin D receptor antibody

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is critical for regulation of serum calcium and phosphorus levels and for proper maintenance of bone mineralization and neuromuscular function. Biological effects of 1,25(OH)₂D₃ are mediated through a nuclear steroid hormone receptor, known as the vitamin D receptor (VDR). The discovery of VDR in a number of different cell and tissue types, suggests that the physiological role of vitamin D may extend beyond the regulation of calcium homeostasis and bone function. Unfortunately, identification of tissues expressing VDR has been controversial due to low abundance of the receptor and quality of the antibodies used. Therefore, we elected to characterize a panel of commercially available VDR antibodies in order to identify antibodies with high specificity and sensitivity. To address these objectives, we have used multiple immunoassays to determine VDR expression in tissues from several organs from multiple species employing tissues from VDR knockout mice as critical negative controls. Many of the antibodies tested showed nonspecific binding that can account for divergent reports. However, one antibody, identified as D-6, is highly specific and extremely sensitive. The specificity, sensitivity, and versatility of this antibody make it the preferred antibody for identifying VDR expression in target tissues using immunological methods.     

Myo-inositol and D-chiro-inositol (40:1) reverse histological and functional features of polycystic ovary syndrome in a mouse model

Mice exposed to continuous light undergo functional and histological changes that mimic those of human Polycystic Ovary Syndrome (PCOS). We herein induced the syndrome by exposing 30-day-old females to 10 weeks of permanent light. Ovarian morphology and histology, as well as reproductive parameters (time of observed pregnancy/delivery) were investigated. Ovaries of PCOS-modeled mice showed lack of tertiary follicles and corpora lutea, altered ovarian architecture, and increased thickness of the theca layer. When mice were returned to a normal light-dark regimen for 10 days, a slight, spontaneous improvement occurred, whereas a quick and almost complete recovery from PCOS signs and symptoms was obtained by treating animals with a daily supplementation of 420 mg/kg myo-inositol and D-chiro-inositol (MyoIns/DCIns) in a 40:1 molar ratio. Namely, ovaries from mice treated by this protocol recovered normal histological features and a proper ratio of theca/granulosa cell layer thickness (TGR), suggesting that the androgenic phenotype was efficiently reversed. Indeed, we identified TGR as a useful index of PCOS, as its increase in PCOS-modeled mice correlated linearly with reduced reproductive capability ( r = 0.75, p < 0.0001). Mice treated with a 40:1 formula regained low TGR values and faster recovery of their fertility, with a physiological delivery time after mating. On the other hand, a higher D-chiro-inositol treatment formula, such as MyoIns versus DCIns 5:1, was ineffective or even had a negative effect on clinical-pathological outcomes.     

Estimation of the cutoff value of vitamin D: the Dong-gu study

Background

Vitamin D plays an essential role in bone health and growth, but the optimal serum 25-hydroxyvitamin D (25(OH)D) concentration is not known. This study was performed to investigate the optimal 25(OH)D concentration in regard to parathyroid hormone (PTH) concentration in the Korean general population aged 50 years or older.

Findings

The study population consisted of 8,857 subjects (3,545 men and 5,312 women) who participated in the baseline survey of the Dong-gu study, conducted in Korea between 2007 and 2010. Serum 25(OH)D and PTH concentrations were measured by chemiluminescent microparticle immunoassay. The optimal 25(OH)D concentration was estimated by using nonlinear regression model. Our data show that PTH concentration reached a theoretical plateau at 38.2 pg/ml and corresponding 25(OH)D concentration was 21.1 ng/ml in men and PTH concentration at 42.9 pg/ml and 25(OH)D concentration at 13.8 ng/ml in women.

Conclusions

These results indicate that, for Korean general population aged 50 years or older, the optimal 25(OH)D concentration is 21.1 ng/ml in men and 13.8 ng/ml in women.     

Synthesis of VS-105: A novel and potent vitamin D receptor agonist with reduced hypercalcemic effects

We have synthesized a novel vitamin D receptor agonist VS-105 ((1R,3R)-5-((E)-2-((3αS,7αS)-1-((R)-1-((S)-3-hydroxy-2,3-dimethylbutoxy)ethyl)-7α-methyldihydro-1H-inden-4(2H,5H,6H,7H,7αH)-ylidene)ethylidene)-2-methylenecyclohexane-1,3-diol). Preparation of a-ring phenylphosphine oxide 11, followed by Wittig–Horner coupling of 11 with the protected 25-hydroxy Grundmann’s ketone 22 generated the precursor 12. Deprotection of the TBDMS groups of 12 produced the target compound VS-105. The biological profiles of VS-105 were evaluated using in vitro assays (VDR receptor binding, VDR reporter gene and HL-60 differentiation) in comparison to calcitriol (the endogenous hormone) or paricalcitol. Furthermore, the PTH suppressing potency and hypercalcemic side effects of VS-105 were evaluated in the 5/6 nephrectomized uremic rats in comparison to paricalcitol. Combining various changes at 20-epi, 22-oxa, 24-methyl, and 2-methylene yielded VS-105 that not only is highly potent in inducing functional responses in vitro, but also effectively suppresses PTH in a dose range that does not affect serum calcium in the 5/6 nephrectomized uremic rats.     

LHCGR and ALMS1 defects likely cooperate in the development of polycystic ovary syndrome indicated by double-mutant mice

Polycystic ovary syndrome(PCOS) is a heterogeneous disorder with evidence of polygenetic components,and obesity may be a risk factor for hyperandrogen ism.Previous studies have shown that LHCGR is en riched in the ovary and LHCGR deficiency causes infertility without typical PCOS phenotypes.ALMS1 is implicated in obesity and hyperandrogenism,the common phenotypes among PCOS patients.Through whole-exome sequencing of 22 PCOS families and targeted candidate gene sequencing of additional 65 sporadic PCOS patients,we identified potential causative mutations in LHCGR and ALMS1 in a sibling-pair PCOS family and three sporadic PCOS patients.The expression of LHCGRL638 P in granulosa-like tumor cell line(KGN) cells promoted cyclic adenosine monophosphate production and granulosa cell proliferation,indicating that LHCGRL638 P is an activating mutation.Lhcgr~(L642 P/L642 P) mice showed an irregular estrous cycle,reduced follicles with dynamic folliculogenesis,and increased testosterone(T),estradiol(E2),and dehydroepiandrosterone.Lhcgr~(+/L642 P) AIms1~(+/PB) mice displayed increased T and E2 but decreased late secondary and preovulatory follicles.We showed that activating mutation of LHCGR likely plays important roles in the pathophysiology of PCOS involving abnormal reproductive physiology,whereas ALMS1 deficiency may promote anovulatory infertility via elevated androgens,suggesting that the disturbed LHCGR and ALMS1 cooperatively induce PCOS phenotypes,characterized as anovulation and hyperandrogenemia frequently observed in PCOS patients with obesity.     

Real-life use of vitamin D3-fortified bread and milk during a winter season: the effects of CYP2R1 and GC genes on 25-hydroxyvitamin D concentrations in Danish families,the VitmaD study

Common genetic variants rs10741657 and rs10766197 in CYP2R1 and rs4588 and rs842999 in GC and a combined genetic risk score (GRS) of these four variants influence late summer 25-hydroxyvitamin D (25(OH)D) concentrations. The objectives were to identify those who are most at risk of developing low vitamin D status during winter and to assess whether vitamin D₃-fortified bread and milk will increase 25(OH)D concentrations in those with genetically determined low 25(OH)D concentrations at late summer. We used data from the VitmaD study. Participants were allocated to either vitamin D₃-fortified bread and milk or non-fortified bread and milk during winter. In the fortification group, CYP2R1 (rs10741657) and GC (rs4588 and rs842999) were statistically significantly associated with winter 25(OH)D concentrations and CYP2R1 (rs10766197) was borderline significant. There was a negative linear trend between 25(OH)D concentrations and carriage of 0–8 risk alleles (p < 0.0001). No association was found for the control group (p = 0.1428). There was a significant positive linear relationship between different quintiles of total vitamin D intake and the increase in 25(OH)D concentrations among carriers of 0–2 (p = 0.0012), 3 (p = 0.0001), 4 (p = 0.0118) or 5 (p = 0.0029) risk alleles, but not among carriers of 6–8 risk alleles (p = 0.1051). Carriers of a high GRS were more prone to be vitamin D deficient compared to carriers of a low GRS. Furthermore, rs4588-AA carriers have a low but very stable 25(OH)D concentration, and interestingly, also low PTH level.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0413-7) contains supplementary material, which is available to authorized users.     

Immunolocalization of androgen and vitamin D receptors in the epididymis of mature ram (Ovis aries)

This study illustrated the immunohistochemical distribution of androgen and vitamin D receptors of epididymis in 20 sexually mature ram (Rahmani breed) with average age ranged from (2^_4) years and average weight ranged from (50^_65kg). Androgen receptor was localized in the cytoplasm of both ciliated and non ciliated cells of efferent ductules, besides the principal cells via the entire epididymal duct. The principal cells of both corpus and proximal cauda epididymis showed the highest immunoreactivity to androgen receptors. Furthermore, vitamin D receptor was localized in the cytoplasm of all epithelium of the efferent ductules besides principal cells of all epididymal regions, however the immunoreaction was significantly higher in the efferent ductules, distal caput and distal cauda epididymis. In conclusion, these results suggest that the function of ram epididymis is regulated by both androgen and Vitamin D.     

Active vitamin D possesses beneficial effects on the interaction between muscle and bone

Vitamin D deficiency and advanced glycation end products (AGEs) are suggested to be involved in the pathogenesis of osteoporosis and sarcopenia. However, the effects of vitamin D and AGEs on myogenesis and the interaction between muscle and bone remains still unclear. We previously showed that osteoglycin (OGN) is secreted from myoblasts and stimulates osteoblastic differentiation, suggesting that it plays important roles in the interaction between muscle and bone. The aim of this study is thus to examine the effects of vitamin D and AGEs on myoblastic differentiation of C2C12 cells and osteoblastic differentiation of osteoblastic MC3T3-E1 cells through OGN expression. 1α,25-dihydroxyvitamin D₃ (1,25D) and eldecalcitol, an active vitamin D analog, induced the expression of MyoD, myogenin and OGN, and these effects were abolished by vitamin D receptor (VDR) suppression by siRNA in C2C12 cells. Moreover, conditioned medium from 1,25D-pretreated C2C12 cells stimulated the expression of type 1 collagen and alkaline phosphatase in MC3T3-E1 cells, compared to control medium from 1,25D-untreated C2C12 cells. In contrast, conditioned medium from VDR-suppressed and 1,25D-pretreated C2C12 cells showed no effects. AGE2 and AGE3 suppressed the expression of MyoD, myogenin and OGN in C2C12 cells. Moreover, 1,25D blunted the AGEs’ effects. In conclusion, these findings showed for the first time that active vitamin D plays important roles in myogenesis and muscle-induced osteoblastogenesis through OGN expression. Active vitamin D treatment may rescue the AGEs-induced sarcopenia as well as – suppressed osteoblastic differentiation via OGN expression in myoblasts.     

A study on the immunological vitality of an inflammatory biomarker explored with rs5743708 polymorphism in TLR2 gene among Saudi women confirmed with polycystic ovarian syndrome

IntroductionPolycystic ovary syndrome (PCOS) is an ovarian health condition as well as a long-term endocrine dysfunction that affects reproductive-aged women. Toll-like receptor 2 (TLR2) gene was linked to PCOS and chronic inflammation, and the prevalence of obesity was rising in Saudi women. Previous studies on rs5743708 polymorphism were documented in the obesity as well as in PCOS women.AimIn this study, we investigated the molecular role of rs5743708 polymorphism in TLR2 gene among Saudi women diagnosed with PCOS using the Rotterdam criteria.MethodsBlood samples were collected from 220 Saudi women in this hospital-based case-control study; 110 were PCOS women and remaining 110 were non-PCOS (control women). Biochemical analysis was performed on serum samples, and molecular analysis was performed on EDTA blood. Genotyping for rs5743708 polymorphism was performed with polymerase chain reaction-restriction fragment length polymorphism analysis.ResultsIn both groups, clinical data was calculated using t-test, which revealed both positive (p < 0.05) and negative (p > 0.05) associations. HWE analysis supported the rs5743708 polymorphism (p < 0.05). In the rs5743708 polymorphism, none of the genotypes, genetic models, or allele frequencies were found to be associated with PCOS and non-PCOS women. However, both ANOVA and regression analyses revealed a positive relationship in PCOS with weight and BMI (p < 0.0001).ConclusionThe rs5743708 polymorphism was not associated to PCOS in Saudi women. One of the predictions could be that 42.7% of PCOS and 73.6% of non-PCOS women were obese, and the rs5743708 polymorphism has been linked to both obesity and PCOS in the previous studies. This study suggests screening for additional polymorphisms with a large sample size.     

The effects of obesity and polycystic ovary syndrome on serum lipocalin-2 levels: a cross-sectional study

Background  

Lipocalin-2 is a novel adipokine that appears to play a role in the development of insulin resistance. Serum lipocalin-2 levels are elevated in obese patients. Obesity and insulin resistance are cardinal characteristics of the polycystic ovary syndrome (PCOS). However, there are limited data on serum lipocalin-2 levels in patients with PCOS. The aim of the present study was to assess serum lipocalin-2 levels in PCOS.     

Polymorphism of the vitamin D binding protein (DBP) among primates: an evolutionary analysis

The distribution of the DBP (vitamin D binding protein) polymorphism is now well characterized among human populations but for primates only limited results are known. The aim of this paper is to describe the electrophoretic polymorphism of this protein among various species. Using three different electrophoretic methods, we are able to detect an unknown polymorphism and to classify the different alleles observed. These results may be used to set an international nomenclature for further comparisons. The different electrophoretic mobilities between Old and New World Monkeys show that: 1) the Cercopithecoïdea are presenting the largest genetic heterogeneity; 2) the DBP among the Galago corresponds to the lowest isoelectric points observed among Primates; 3) during the evolution from nonhuman Primates to Man, the DBP is able to keep its affinity for vitamin D derivatives despite the occurrence of significant molecular modifications; 4) among Anthropoïdea, the electrophoretic patterns of DBP are very close to the human Gc 1 proteins. These results show that evolution at the DBP level can be considered as a continous mechanism of structural modifications. A significant transition occurs during the differentiation between Cercopithecoïdea and Anthropoïdea. It is not too speculative to consider that some electrophoretic forms detected among Gorilla, Pongo, or Pan may be identical to rare variants observed among humans.     

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.     

Exons and functional regions of the human vitamin D receptor gene around and within the main 1a promoter are well conserved among mammals

The human vitamin D receptor (hVDR) gene encompasses eight exons (2–9) in the so-called coding region and six more exons (1a–1f) in the so-called regulatory region, which contains several reported promoters. Evolutionary comparison performed on the VDR promoter sequences of a dozen of mammalian species shows a very high conservation of numerous regions around and in the 1a promoter, including exons 1e, 1a and 1d, and the Sp1 site region. This suggests that the so-called 1a promoter is well conserved among mammals. Homology among mammals also concerns three functional SNP site regions of the hVDR 1a promoter, the 1e-G-1739A SNP region (a Cdx-2 binding site), and both 1a-G-1521C and 1a-A-1012G sites, the 1a-1012A being located within a GATA site. Interestingly, the 1521G and 1012A nucleotides are being evolutionary conserved, suggesting that the 1521C/1012G haplotype, which is found in human chromosomes (43% of Caucasians), is a human specificity.     

Intervention in autoimmunity: the potential of vitamin D receptor agonists

Vitamin D receptor (VDR) agonists are well known for their capacity to control calcium metabolism and to regulate growth and differentiation of many cell types. More recently, it has become clear that VDR agonists possess immunoregulatory properties and, in particular, pronounced pro-tolerogenic activities. VDR agonists can act directly on T cells, but DCs appear to be their primary targets. The capacity of VDR agonists to modulate DC and T cell functions is mediated by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor NF-kappaB that is downregulated by VDR agonists in APCs and in T cells. A potentially very important activity of VDR agonists is their capacity to induce in vitro and in vivo tolerogenic DCs able to enhance CD4+CD25+ suppressor T cells that, in turn, inhibit Th1 cell responses. These mechanisms of action can explain some of the immunoregulatory properties of VDR agonists in the treatment of Th1-mediated autoimmune diseases, but may also represent a physiologic element in the VDR-mediated regulation of innate and adaptive immune responses.