Glycoprotein Hormone Alpha-subunit Research Articles

Exploring the molecular underpinnings of macrosomia in gestational diabetes mellitus: The role of EGFR signaling and placental syncytiotrophoblast

Macrosomia, which is frequently associated with gestational diabetes mellitus (GDM), is linked to maternal glycemic control during gestation. When GDM is complicated by macrosomia (GDMM), the placenta exhibits increased mass, underscoring its role as a critical nexus for maternal-fetal nutrient exchange. Despite this recognized correlation, the underlying mechanisms propelling placental hypertrophy have remained elusive. Our study leveraged single-cell RNA transcriptome sequencing of GDMM placental tissues to pinpoint the specific syncytiotrophoblast (SCT) subsets that regulate placental dimensions. Notably, we observed pronounced upregulation of the epidermal growth factor receptor (EGFR) and its corresponding ligands, with a particular emphasis on the autoregulatory cascade involving the glycoprotein hormone alpha subunit (CGA), EGFR, and the transcription factor GATA binding protein 2 (GATA2), as well as perturbations in hormonal homeostasis within the SCT. Furthermore, our cell interaction analysis revealed an enhanced interplay between myeloid cells and SCT3, augmenting the EGFR signaling pathway. These molecular exchanges underscore the pivotal role of the placental immune microenvironment in the etiology of macrosomia, shedding light on the pathophysiology of GDMM and paving the way for novel therapeutic approaches.

A challenging case of a pituitary macroadenoma and toxic thyroid adenoma with inappropriate TSH secretion.

Thyroid-stimulating hormone-secreting pituitary adenomas (TSHomas) are rare, accounting for less than 1% of all pituitary adenomas. We present a case of hyperthyroidism secondary to a likely TSHoma and coexisting functional thyroid adenoma. Laboratory errors and familial abnormalities in thyroid function tests were ruled out, and a diagnosis of the toxic thyroid adenoma was confirmed on a thyroid uptake scan. However, the triiodothyronine suppression test was contraindicated due to the patient's cardiovascular disease, and the thyrotropin-releasing hormone stimulation test, measurement of glycoprotein hormone alpha-subunit, and genetic testing were unavailable. Magnetic resonance imaging of the brain revealed a suprasellar pituitary macroadenoma measuring 40 mm × 20.3 mm × 17 mm. The patient was initiated on carbimazole; however, thyroid stimulating hormone and thyroxine levels remained elevated. The patient declined trans-sphenoidal surgery and was treated with radioactive iodine to manage the toxic thyroid adenoma, leading to reduced thyroxine levels and symptom improvement. Unfortunately, the patient passed away before long-acting somatostatin analogs became available. This case highlights the diagnostic and therapeutic challenges involved in managing thyrotoxicosis with dual etiology. Hyperthyroidism can have multiple etiologies, which can coexist in the same patient. Persistent discordant thyroid function tests warrant further investigation. The gold standard for diagnosis of TSHomas remains immunohistochemical analysis of the tumor tissue.

Thyroid Dysfunction in Pregnancy: A Literature Review

Due to structural similarities between HCG and thyroid-stimulating hormone, thyroid stimulation during pregnancy starts in the first trimester (TSH). The volume of thyroid hormone distribution increases along with placental metabolism, maternal thyroxine transport, and estrogen-mediated changes in thyroxine-binding globulin (TBG), all of which cause a 20–40% rise in early pregnancy thyroid hormone demand. Due to the human chorionic gonadotropin alpha subunit's cross-reactivity with the TSH receptor, the reference range of laboratory values for TSH is lower. High serum protein levels may alter estimates rather than direct measurements of free thyroxine (FT4), leading to reported results that are inaccurate. The clinical range of thyroid dysfunction during pregnancy can include both hyperthyroidism (Graves' disease and transitory gestational thyrotoxicosis) and hypothyroidism (overt hypothyroidism, subclinical hypothyroidism, and autoimmune thyroid disease). The risk of preterm and infant respiratory distress syndrome is increased by this malfunction.

PSUN346 TSHoma: A Rare Cause of Pituitary Adenoma

Abstract Introduction The TSH-producing adenoma (TSHoma) prevalence in the general population is 1 to 2 cases per million, accounting for less than 2% of all pituitary adenomas. Plurihormonal tumors account for about 30% of these cases, with the most common co-secreted hormone being GH. We present the case of a patient diagnosed with TSH adenoma many years after presentation. Case Presentation A 58 year old male with a history of incidentally diagnosed pituitary microadenoma on MRI in 2007, presented in 2014 following abnormal thyroid function testing (TFT), only reported symptoms being occasional headaches. TFT was significant for elevated TSH and FT4 (TSH 4.87—Ref range: 0.27-4.2. Free T4: 1.98–Ref range 0.7-1.7). Pituitary hormone profile was remarkable for a low IGF-1 and a borderline high Alpha subunit of glycoprotein hormone. A sellar MRI confirmed a 12mm pituitary macroadenoma. He was managed conservatively on beta blockers and low dose Methimazole, owing to a stable adenoma size and poor surgical candidacy with multiple comorbidities (e. g. Atrial fibrillation). Repeat labs in 2018 showed increasing TSH (12.85), with persistent elevations of FT3, FT4 and Thyroglobulin. He had follow up TFT monitoring and surveillance MRIs until 2021 when his lesion progressed to 14 mm with mass effect on right pre chiasmatic optic nerve and adjacent internal carotid artery. The patient underwent trans sphenoidal biopsy with debulking in 2021. The biopsy showed a plurihormonal adenoma (prolactin, GH and TSH secreting). Interestingly, prolactin, GH or IGF-1 were never elevated. Following surgery, MRI showed residual 13.2×9.7 mm tumor with no mass effect. His most recent labs showed a TSH: 2.5 and FT4: 1.7. Discussion The diagnosis of a TSHoma should be considered in all centrally hyperthyroid patients, to avoid complications and undue thyroid ablation. A TSHoma must be differentiated from Resistance to Thyroid Hormone (RTH)-beta, which occurs due to mutations in the Thyroid Hormone Receptor Beta gene. Serum a-subunit concentration and Sex Hormone Binding Globulin (SHBG) concentrations are normal in RTH-beta with no TSH suppression following somatostatin analog administration. The presence of a high TSH, FT3 and FT4, in the setting of an elevated a-subunit and pituitary macroadenoma on MRI is strongly suggestive of a TSHoma. Beta blockers are recommended for symptomatic individuals however anti thyroid drug therapy is not advisable for there is risk of thyroid hormone suppression with concomitant tumor overgrowth. Definitive therapy includes trans sphenoidal tumor resection with somatostatin analog therapy to achieve euthyroidism prior to surgery. Patients should be followed up with frequent monitoring of TFT's, along with MRIs every 2-3 years. For patients with residual disease following surgery, treatment with somatostatin analogs such as Octreotide or Lanreotide is favorable as opposed to pituitary irradiation. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

ODP297 Analysis of the responsible region of TRH in the hypothalamus-pituitary-thyroid axis using the Paraventricular nucleus-specific TRH deficient mice.

Abstract Objectives Thyrotropin-releasing hormone (TRH) was the first hypothalamic hormone isolated that stimulates pituitary thyroid-stimulating hormone (TSH) secretion. TRH was later found to be distributed throughout the brain, gastrointestinal tract and pancreaticβ-cells. We previously reported the TRH null mice (conventional TRHKO) which exhibit characteristic tertiary hypothyroidism, however the responsible regionfor the hypothalamic-pituitary-thyroid (HPT) axis remained obscure because of the broad expression of TRH in hypothalamus. Previous studies suggested that TRH in the hypothalamic paraventricular nucleus (PVN) was secreted through the median eminence (ME), however it was not directly demonstrated . To determine the region functionally responsible for the HPT axis, we established and analyzed PVN-specific TRH knock-out (PVN-TRHKO) mice. Methods For the targeting vector, a Neo-loxP cassette was inserted into the 3′-UTR of the Trh gene in ES cells. Another loxP site was placed at intron 1. After we obtained the Trh +/lox [neo+] founder mice, we crossed them with the CAG-FLPe transgenic mice to remove the Neo cassette. The offspring were backcrossed to wild-type mice, producing conditional knock-out mice without the Neo cassette, which were labeled as Trh +/lox . We crossed Trh +/lox with transgenic mice expressing Single-minded homolog (Sim)1-Cre recombinase and created PVN-TRHKO mice (Sim1-Cre; Trh lox/lox mice). We confirmed that most Sim1 was expressed in the PVN using Sim1-Cre/tdTomato mice. We performed the following experiments using PVN-TRHKO andTrh lox/lox mice as control (Ct). 1) immunostaining of TRH using antibody forproTRH in cerebrum, 2)qPCR analysis detecting the expression of preproTrh mRNA in hypothalamic PVN, 3) measurement of the level of serum free-T4 and TSH, 4)qPCR analysis detecting theexpression levels of Tshβ, Prl, Cga, Gh, Pomc and Lhβ gene mRNAin the anterior pituitary and 5)immunohistochemical analysis with antibodies for TSHβ and Prolactin in anterior pituitary. Results In the PVN-TRHKO mice, 1) ProTRH-immunopositive cells were lost in the area of PVN and ME, whereas proTRH detected in the medial preoptic area(MPA) and medial preoptic nucleus (MPO) in the hypothalamus.2)The expression level of proTRHmRNA was decreased to7.7% compared with Ct.3) Free-T4 levels decreased to 50% and the TSH levels increased to 170% of those in Ct. 4) TshβmRNA levels were decreased by 65% and thePrlmRNA levels were reduced to 55% of those in Ct. 5) The number of TSH-positive andProlactin-positive cells in the pituitary significantly decreased to 50% and 60% of those in Ct. Conclusions PVN-TRHKO mice exhibited tertiary hypothyroidism similar to conventional TRHKO. The pathway of TRH neuron from PVN to ME was lost in the PVN-TRHKO mice. These findings are conclusive evidence that the TRH neuron in the PVN is the center of the HPT axis. Presentation: No date and time listed

L3MBTL2-mediated CGA transcriptional suppression promotes pancreatic cancer progression through modulating autophagy

SummaryL3MBTL2 is a crucial component of ncPRC1.6 and has been implicated in transcriptional repression and chromatin compaction. However, the repression mechanism of L3MBTL2 and its biological functions are largely undefined. Here, we found that L3MBTL2 plays a distinct oncogenic role in tumor development. We demonstrated that L3MBTL2 repressed downstream CGA through an H2AK119ub1-dependent mechanism. Importantly, the binding of the MGA/MAX heterodimer to the E-box on the CGA promoter enhanced the specific selective repression of CGA by L3MBTL2. CGA encodes the alpha subunit of glycoprotein hormones; however, we showed that CGA plays an individual tumor suppressor role in PDAC. Moreover, CGA-transcript1 (T1) was identified as the major transcript, and the tumor suppression function of CGA-T1 depends on its own glycosylation. Furthermore, glycosylated CGA-T1 inhibited PDAC, partly by repression of autophagy through multiple pathways, including PI3K/Akt/mTOR and TP53INP2 pathways. These findings reveal the important roles of L3MBTL2 and CGA in tumor development.

A Rare Case of a Plurihormonal Pituitary Adenoma

Background: Pituitary adenomas are usually benign tumors that arise from adenophypophyseal cells and produce one or two types of hormones. Plurihormonal adenomas are a rare subtype that produce two or more hormones and represent less than 1% of all pituitary adenomas. Clinical Presentation: A 76-year-old female presented for evaluation of abnormal thyroid function test results. She was found to have an elevated free T4 of 1.92 ng/dL and total T4 of 14.4 ug/dL with an inappropriately normal TSH of 2.11 uIU/mL. Physical examination was significant for tachycardia, tremors, diaphoresis, coarse facial features, and enlarged hands. Further biochemical evaluation of her pituitary hormone levels demonstrated an elevated prolactin (PRL) of 237.2 ng/mL, elevated insulin-like growth factor 1 (IGF-1) of 787 ng/mL, normal morning ACTH of 47 pg/mL, normal morning cortisol of 17.0 ug/dL, an inappropriately suppressed FSH of <5.0 mIU/mL, an elevated alpha subunit of pituitary glycoprotein hormones (PGH) of 6.9 ng/mL, and an elevated free T4 of 3.5 ng/dL by equilibrium dialysis. She underwent an MRI of the pituitary and brain which demonstrated a pituitary adenoma measuring 1.2 x 1.3 x 1.8 cm with a portion herniating into the sella turcica with no mass effect of the optic chiasm. A formal visual field examination was normal. The patient underwent workup for Cushing’s Disease with a low dose overnight dexamethasone suppression test, resulting in an appropriate response with an 8 AM cortisol of <1.0 ug/dL. Glucose suppression test confirmed the diagnosis of acromegaly with growth hormone at 120 minutes of 19.90 ng/mL. Neurosurgery performed a trans-sphenoidal pituitary adenoma resection. Thyroid function tests on post-operative day 4 demonstrated a suppressed TSH of 0.01 uIU/mL and an elevated free T4 of 2.30 ng/dL. Histological evaluation revealed dual expression of transcription factors pituitary-specific positive transcription factor 1 (PIT1) and steroidogenic factor 1 (SF1) as well as PRL, GH, TSH and FSH expression. Immunostaining for LH and ACTH were negative. Post-operative IGF-1 and GH levels were 106 ng/mL and 0.51 ng/mL at 17 weeks, respectively. Post-operative TSH normalized to 0.82 uIU/mL, free T4 normalized to 1.04 ng/dL, and PRL normalized to 8.1 ng/mL at 12 weeks. The patient remained symptom free after successful surgical resection. Discussion: Our case demonstrates the clinical course of a unique patient with clinical and biochemical manifestations of thyroid dysfunction and acromegaly with a pituitary adenoma immunoreactive for GH, TSH, FSH, and PRL. The co-secretion of GH, TSH, PRL, and FSH as well as positivity for the alpha-subunit is extremely unusual. This case emphasizes the importance of considering pituitary abnormalities as a cause for abnormal thyroid function tests.

Identification and semi-relative quantification of intact glycoforms of human chorionic gonadotropin alpha and beta subunits by nano liquid chromatography-Orbitrap mass spectrometry

The human chorionic gonadotropin (hCG) protein belongs to a family of glycoprotein hormones called gonadotropins. It is a heterodimer made of two non-covalently linked subunits. The α-subunit structure, hCGα, has 2 N-glycosylation sites, while the beta subunit, hCGβ, has 2 N- and 4 O-glycosylation sites. This leads to numerous glycoforms. A method based on the analysis of hCG glycoforms at the intact level by nano-reversed phase liquid chromatography coupled to high resolution mass spectrometry (nanoLC-HRMS) with an Orbitrap analyzer was previously developed using a recombinant hCG-based drug, Ovitrelle®, as standard. It allowed the detection of about 30 hCGα glycoforms, but didn't allow the detection of hCGβ glycoforms. This method was thus here significantly modified (addition of a pre-concentration step of the sample to increase the sample volume from 70 nl to 1 µl, optimization of the gradient slope and the nature and content of the acidic additive in the mobile phase). It led to an improvement of the separation of hCGα and hCGβ glycoforms, which allowed for the first time the detection of 33 hCGβ glycoforms at intact level. In addition, a higher number of hCGα glycoforms (42 in total, i.e. a 40% increase) was detected. The figures of merit of this new method were next assessed. The relative standard deviations (RSDs) of the retention time ranged between 0.02 and 0.95% (n = 3), with an average value of 0.36% for the alpha glycoforms and between 0.01 and 1.08% (n = 3) with an average value of 0.23% for the beta glycoforms. The RSDs of the relative peak area measured on the extracted ion chromatogram of each glycoform were below 20% (n = 3), with an average value of 9.8%, thus allowing semi-relative quantification. Therefore, this method has a high potential for rapid quality control aiming for the detection and comparison of glycoforms present in glycoprotein-based pharmaceutical preparations.

Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production.

The serum concentration of thyrotropin (thyroid stimulating hormone, TSH) is drastically reduced by small increase in the levels of thyroid hormones (T3 and its prohormone, T4); however, the mechanism underlying this relationship is unknown. TSH consists of the chorionic gonadotropin α (CGA) and the β chain (TSHβ). The expression of both peptides is induced by the transcription factor GATA2, a determinant of the thyrotroph and gonadotroph differentiation in the pituitary. We previously reported that the liganded T3 receptor (TR) inhibits transactivation activity of GATA2 via a tethering mechanism and proposed that this mechanism, but not binding of TR with a negative T3-responsive element, is the basis for the T3-dependent inhibition of the TSHβ and CGA genes. Multiple GATA-responsive elements (GATA-REs) also exist within the GATA2 gene itself and mediate the positive feedback autoregulation of this gene. To elucidate the effect of T3 on this non-linear regulation, we fused the GATA-REs at -3.9 kb or +9.5 kb of the GATA2 gene with the chloramphenicol acetyltransferase reporter gene harbored in its 1S-promoter. These constructs were co-transfected with the expression plasmids for GATA2 and the pituitary specific TR, TRβ2, into kidney-derived CV1 cells. We found that liganded TRβ2 represses the GATA2-induced transactivation of these reporter genes. Multi-dimensional input function theory revealed that liganded TRβ2 functions as a classical transcriptional repressor. Then, we investigated the effect of T3 on the endogenous expression of GATA2 protein and mRNA in the gonadotroph-derived LβT2 cells. In this cell line, T3 reduced GATA2 protein independently of the ubiquitin proteasome system. GATA2 mRNA was drastically suppressed by T3, the concentration of which corresponds to moderate hypothyroidism and euthyroidism. These results suggest that liganded TRβ2 inhibits the positive feedback autoregulation of the GATA2 gene; moreover this mechanism plays an important role in the potent reduction of TSH production by T3.

ASSESSMENT AND COMPARISON OF HORMONAL IMMUNOEXPRESSION AND THE CLINICAL PICTURE IN PATIENTS WITH PITUITARY ADENOMAS.

Symptoms related to hypersecretion of hormones in patients with pituitary adenomas do not always correlate with immunohistochemical staining results. To evaluate the relationship between the pituitary adenomas hormone immunoexpressions and endocrine presentations. The clinical status and immunoexpression of 72 patients who underwent transsphenoidal surgery for pituitary adenomas were analyzed. Macroadenomas were diagnosed in 51 cases (70.84%), while microadenomas were found in 21 cases (29.16%). The 72 adenoma specimens were divided into 22 monohormonal, 21 plurihormonal, 21 immunonegative and 8 unreliable specimens. The positive immunohistochemical staining results occurred as follows: prolactin and growth hormone 25% each, adrenocorticotropic hormone 13.89%, thyroid-stimulating hormone 5.56%, leuteinizing hormone and follicle-stimulating hormone 12.5%, glycoprotein hormone alpha-subunit 22.22%. Statistically significant relationships between the immunohistochemical presentation and the preoperative diagnosis were found for prolactin and hyperprolactinemia, growth hormone and acromegaly and adrenocorticotropic hormone and Cushing's syndrome. The lack of full concordance between the clinical presentations and immunohistochemical staining was mainly a result of the presence of nonfunctioning adenomas, plurihormonal adenomas and unreliable specimens. The morphometric method introduced in this study, utilizing the immunoexpression index, provided a very precise evaluation of pituitary adenomas pathology.

Multigormonal secretory activity of cells of clinically non-functioning pituitary tumors in vitro and the effect of tyroliberin on it

Experiments on cell cultures demonstrated that isolated cells of clinically inert pituitary tumors release several hormones in small amounts into the medium: luteinizing and follicle-stimulating hormones, alpha-subunit of glycoprotein hormones, prolactin, and growth hormone. Multihormonal secretion of these cells indicates their poor morphofunctional differentiation. In contrast to normal pituitary cells, cells of clinically inert pituitary tumors respond nonspeciflcally to hypothalamic thyrotropin releasing hormone: by increased secretion of prolactin, gonadotropins, glycoprotein hormone alpha-subunit, and growth hormone. This capacity of tumor cells detected in vitro agrees with the probability of increased levels of gonadotropins and glycoprotein hormone alpha-subunit in the serum of patients with clinically inert pituitary tumors during pharmacodynamic thyrotropin releasing hormone test.

Clinical challenges of a co-secreting TSH/GH pituitary adenoma.

Co-secreting thyrotropin/growth hormone (GH) pituitary adenomas are rare; their clinical presentation and long-term management are challenging. There is also a paucity of long-term data. Due to the cell of origin, these can behave as aggressive tumours. We report a case of a pituitary plurihormonal pit-1-derived macroadenoma, with overt clinical hyperthyroidism and minimal GH excess symptoms. The diagnosis was confirmed by pathology showing elevated thyroid and GH axes with failure of physiological GH suppression, elevated pituitary glycoprotein hormone alpha subunit (αGSU) and macroadenoma on imaging. Pre-operatively the patient was rendered euthyroid with carbimazole and underwent successful transphenoidal adenomectomy (TSA) with surgical cure. Histopathology displayed an elevated Ki-67 of 5.2%, necessitating long-term follow-up. Thyrotropinomas are rare and likely under-diagnosed due to under-recognition of secondary hyperthyroidism. Thyrotropinomas and other plurihormonal pit-1-derived adenomas are more aggressive adenomas according to WHO guidelines. Co-secretion occurs in 30% of thyrotropinomas, requiring diligent investigation and long-term follow-up of complications.

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

The prevalence of microRNA binding sites around the CGA, FSHB, LHB and TSHB genes in mammals with a different number of dominant follicles

The prevalence of microRNA binding sites around the CGA, FSHB, LHB and TSHB genes in mammals with a different number of dominant follicles

Posttranscriptional actions of triiodothyronine on Tshb expression in TαT1 cells: New insights into molecular mechanisms of negative feedback.

Rapid actions of triiodothyronine (T3) on thyrotropin (TSH) synthesis and secretion have been described in hypothyroid male rats. However, the molecular mechanisms remain unknown. TαT1 cells, a thyrotroph cell line, was used herein to characterize the possible non-genomic actions of T3 on the expression of alpha (Cga) and Tshb genes, and the posttranscriptional processing and translation of both transcripts. The involvement of αVβ3 integrin was also assessed. T3 quickly reduced Tshb mRNA content, poly(A) tail length and its association with ribosomes. The effect of T3 on Tshb gene expression was detected even in the presence of a transcription inhibitor. The decrease in Tshb mRNA content and polyadenylation depend on T3 interaction with αVβ3 integrin, while T3 reduced Cga mRNA content by transcriptional action. The translational rate of both transcripts was reduced by a mechanism, which does not depend on T3-αVβ3 integrin interaction. Results indicate that, in parallel with the inhibitory transcriptional action in Cga and Tshb gene expression, T3 rapidly triggers additional posttranscriptional mechanisms, reducing the TSH synthesis. These non-genomic actions partially depend on T3-αVβ3 integrin interaction at the plasma membrane of thyrotrophs and add new insights to the molecular mechanisms involved in T3 negative feedback loop.

Study of association the single nucleotide polymorphisms (rs6631) in 3′-UTR CGA gene with male infertility in Iranian population

ObjectiveStudying the possible association between SNP (rs6631) residing in the miRNA-binding-site of spermatogenesis-related gene (CGA) with azoospermia male infertility in Iranian population was our purpose of this research. CGA has essential role in gonads function. SNP is located in the miRNA-binding site in 3′ untranslated region (3′-UTR) gene may lead to disruption of CGA and elevate the risk of male infertility. MethodsBlood samples were collected from different ethnicities and their DNA was extracted by using salting out method. Sixty-eight infertile men suffer from azoospermia and sixty-three fertile men were genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique and some of them were sequenced randomly. Statistical analyses were performed to investigate the association between rs6631 and azoospermia. ResultsCGA-215AA genotype was significantly associated with azoospermia infertility. Logistic regression analysis of the genotype frequency between fertile and infertile men revealed significant difference (p < 0.05) in all models of inheritance except recessive model. The most genotype frequencies for T215A in our studied population was heterozygote genotype (T/A), it was same as the results in NCBI. hsa-miR-1302 and hsa-miR-557 bind to rs6631-A (mutant allele) more stably than rs6631-T, so CGA expression may be dysregulated. As a result, this SNP may elevate risk of male infertility. ConclusionOur results indicated that the mentioned polymorphism in CGA gene could be associated with azoospermia male infertility in Iran. Further studies are required to support the obtained result.

Association of T/A polymorphism in miR-1302 binding site in CGA gene with male infertility in Isfahan population.

Infertility occurs in 10-15% of couples worldwide and close to half of it is caused by male factors. One of the genes that can affect male infertility is CGA. Polymorphisms in CGA gene may affect gene expression, therefore affecting male infertility by disrupting the regulation of this gene. One of the polymorphisms is the substitution of T with A in the miR-1302 binding site in the 3' untranslated region of the CGA gene. In this study, we explored this polymorphism in Isfahan population. In this case-control study, by the use of Tetra primer-ARMS-PCR technique, rs6631 has been investigated in 224 infertile men and 196 controls. Infertile men were recruited from Isfahan Fertility and Infertility Center. Analysis of genotype and allele frequencies indicated that the differences between case and control populations were significant for rs6631 because P = 0.00 which is above the threshold. We found a significant relationship between this polymorphism and male infertility. This study which performed for the first time in Iran suggests that polymorphism in CGA gene can affect male infertility. Also, this polymorphism has high heterozygosity, so it can be used for further studies in different populations.

Mitogen- and stress-activated protein kinase 1 is required for gonadotropin-releasing hormone–mediated activation of gonadotropin α-subunit expression

Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific β-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.

Molecular characterization, modeling, in silico analysis of equine pituitary gonadotropin alpha subunit and docking interaction studies with ganirelix

Equine pituitary gonadotropins (eLH, eFSH, eCG) are heterodimeric glycoprotein hormones with alpha (α) and beta (β) subunits. It is responsible for maintenance of pregnancy in mares during early gestation and fairly valuable for inducing superovulation in animals other than equines. The alpha subunit is common, while beta subunit is species-specific in all glycoprotein hormones. In the present investigation, molecular cloning and in silico characterization including homology modeling and molecular docking analysis of the equine chorionic gonadotropin (eCG) alpha subunit was carried out for gaining structural and functional insights into the eCG alpha subunit and its possible interaction with ganirelix, a gonadotropin-releasing hormone (GnRH) antagonist. The equine chorionic gonadotropin (eCG) alpha subunit expressed in pituitary gland was selected, amplified from total RNA, cloned and sequenced. The in silico analyses were made for homology modelling, structural details, epitope identification and chromosomal localization. Molecular docking studies of eCG alpha were undertaken with a drug ganirelix which is used to control ovulation and has antagonistic activity against GnRH. The protein sequence corresponding to selected open reading frame (ORF) was 99–100% similar with domesticated horse, Przewalski’s horse, and 92–93% with Burchell’s zebra and donkey. Molecular docking studies revealed the possible interaction of eCG alpha with ganirelix. The possible drug-macromolecule interactions were visualized between eCG alpha and ganirelix. The study will provide structural insight into unique sites and an alternate route of gonadotropin suppression applicable to assisted reproductive technologies.

Seasonal alterations in the daily rhythms in hypothalamic expression of genes involved in the photoperiodic transduction and neurosteroid-dependent processes in migratory blackheaded buntings.

The present study investigated seasonal alterations in the daily rhythms of hypothalamic expression of genes involved in the photoperiodic regulation of annual cycles in birds. We measured the 4-hourly mRNA expression of genes involved in the photoperiodic transduction (OPN5, EYA3, CGA, TSHβ, DIO2, DIO3) and neurosteroid-dependent processes (AR, CYP19, ERα, ERβ) in the hypothalamus of migratory blackheaded buntings photoinduced with photosensitive, photostimulated (early and late stimulated) and photorefractory seasonal states. There were significant differences in daily mRNA profiles between the photoperiodic states. Particularly, increased CGA, TSHβ and DIO2 and decreased DIO3 mRNA levels in the early photostimulated state, compared to the photosensitive state, suggest that thyroid hormones have a role in photostimulation in buntings. Similar differences in the expression of genes coding for the aromatase enzyme (CYP19) and receptors for oestrogen (ERα, ERβ) (but not androgen; AR) indicate that there is seasonal alteration in the neuro-oestrogen-mediated functions. Furthermore, peak expression times of CGA, TSHβ and DIO2 genes at hours 14-15 of the day in the early stimulated state indicated molecular regulation of the daily rhythm of photoinducibility in buntings. Most significantly, however, we found an attenuated daily rhythm in thyroid hormone modulatory genes and a switch of peak expression time from day to night in CYP19 mRNA rhythm in the subsequent late photostimulated state, although testicular maturation still persisted. These alterations in daily rhythms may have signalled the initiation of processes underlying other seasonal phenologies in parallel with the gonadal response, such as a manifestation of the night-time flight in buntings. These results show alterations in daily rhythms underlying the transcriptional regulation of the photoperiod-induced seasonal states in migratory blackheaded buntings.