Over the last few years, our laboratory has demonstrated that different physiological conditions or stressors affect the posttranslational processing of hypophysiotropic and nonhypophysiotropic proTRH and, consequently, the output of TRH and other proTRH-derived peptides. These alterations in proTRH processing are generally associated with parallel changes in the levels of two members of the family of prohormone convertases 1/3 and 2 (PC1/3 and PC2). An important regulator of proTRH is thyroid hormone, which is the peripheral end product of the hypothalamic (TRH)-pituitary (TSH)-thyroid (T3/4) (HPT) axis. In this study we investigated the effect of thyroid status on the processing of proTRH inside and outside the HPT axis. Our data showed that high levels of thyroid hormone down-regulated PC1/3 and PC2 and TRH synthesis, which led to an accumulation of intermediate forms of proTRH processing. Conversely, low levels of thyroid hormone up-regulated proTRH synthesis and PC1/3 and PC2 levels. Control of the activity of PCs and proTRH processing occurred specifically in the paraventricular nucleus, whereas no change due to thyroid status was found in the lateral hypothalamus or preoptic area. The posttranslational regulation of proTRH processing in the paraventricular nucleus by thyroid status is a novel aspect of the regulation of the HPT axis, which may have important implications for the pathophysiology of hypo- and hyperthyroidism.

No abstract

Objective: To compare the efficacy of piezo-assisted micromanipulation with conventional micromanipulation for intracytoplasmic sperm injection (ICSI) into oocytes in patients with impaired semen parameters and no success with in vitro fertilization (IVF). Study Design: A retrospective randomized study was conducted on 204 cycles for 104 couples with piezo-assisted ICSI and 122 cycles for 96 couples with conventional ICSI. Piezo-assisted ICSI consists of two steps, namely penetration of the zona pellucida alone with a piezo-pulse and then puncturing of the oolemma with a light negative pressure without piezo, as with conventional ICSI. The tips of injection pipettes were prepared after pulling by breakage with a scalpel under a microscope, so that the inner diameter at and near the tip was 5 µm, as for conventional ICSI. Results: Piezo-assisted ICSI demonstrated significantly more favorable results, with a fertilization rate of 90.3% (conventional ICSI: 83.1%, p < 0.01) and a cleavage rate of 88.1% (conventional ICSI: 84.6%, p < 0.01). Conclusion: Piezo-assisted ICSI is easy to incorporate a spermatozoa exactly into the ooplasm with little deformation of the oocyte during insertion. Piezo-assisted ICSI can be used effectively for human oocytes to improve the fertilization, cleavage rates.

. Regulation of regional expression in rat brain PC2 by thyroid hormone/characterization of novel negative thyroid hormone response elements in the PC2 promoter. Am J Physiol Endocrinol Metab 288: E236 -E245, 2005; doi:10.1152/ajpendo.00144. 2004.-The prohormone convertases (PCs) PC1 and PC2 are involved in the tissue-specific endoproteolytic processing of neuropeptide precursors within the secretory pathway. We previously showed that changes in thyroid status altered pituitary PC2 mRNA and that this regulation was due to triiodothyronine-dependent interaction of the thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large proximal region of the human PC2 promoter. In the current study, we examined the in vivo regulation of brain PC2 mRNA by thyroid status and found that 6-n-propyl-2-thiouracil-induced hypothyroidism stimulated, whereas thyroxine-induced hyperthyroidism suppressed, PC2 mRNA levels in the rat hypothalamus and cerebral cortex. To address the mechanism of T3 regulation of the PC2 gene, we used human PC2 (hPC2) promoter constructs transiently transfected into GH3 cells and found that triiodothyronine negatively and 9-cis-retinoic acid positively regulated hPC2 promoter activity. EMSAs, using purified TR␣1 and retinoid X receptor-␤ (RXR␤) proteins demonstrated that TR␣ bound the distal putative nTRE-containing oligonucleotide in the PC2 promoter, and RXR bound to both nTRE-containing oligonucleotides. EMSAs with oligonucleotides containing deletion mutations of the nTREs demonstrated that the binding to TR and RXR separately is reduced, but specific binding to TR and RXR together persists even with deletion of each putative nTRE. We conclude that there are two novel TRE-like sequences in the hPC2 promoter and that these regions act in concert in a unique manner to facilitate the effects of thyroid hormone and 9-cis-retinoic acid on PC2. posttranslational processing; hypothyroidism; prohormone convertase; pituitary; regulation A VARIETY OF REGULATORY PEPTIDES AND PROTEINS are generated from inactive precursors by endoproteolytic processing. The majority of prohormones are cleaved at paired basic residues to generate bioactive hormones in a cell-specific manner by prohormone convertases (PCs), members of the mammalian family of the subtilisin-like endoproteases (47, 54). Seven members of the PC family have been cloned: both PC1 and PC2 are found exclusively in neural and endocrine cells equipped with a regulatory secretory pathway (10,18,49,50), PC4 is found in the testis (37), and the other PCs are ubiquitously distributed (46, 48). PC1 and PC2 process a variety of prohormones and proneuropeptides, including proopiomelanocortin (POMC), prosomatostatin, provasopressin, proneurotension, pro-thyrotropin-releasing hormone (pro-TRH), and proislet amyloid polypeptide (4,9,14,15,17,57).The important role of PC1 and PC2 in hormonal biosynthesis has been elucidated by the studies of PC1-and PC2-null mice and in a patient with defective PC1. PC1-null mice are s...

Most pro-neuropeptides are processed by the prohormone convertases, PC1 and PC2. We previously reported that changes in thyroid status altered anterior pituitary PC1 mRNA and this regulation was due to triiodothyronine (T 3 )-dependent interaction of thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large region of the human PC1 promoter. In this study, we demonstrated that hypothyroidism stimulated, while hyperthyroidism suppressed, PC1 mRNA levels in rat hypothalamus and cerebral cortex, but not in hippocampus. In situ hybridization was used to confirm real-time PCR changes and localize the regulation within the hypothalamus and cortex. Using a human PC1 (hPC1) promoter construct (with and without deletions in two regions that each contain a negative TRE) transiently transfected into GH3 cells, we found that T 3 negatively regulated hPC1 promoter activity, and this regulation required both of these two regions. Electrophoretic mobility shift assays (EMSAs) using purified thyroid hormone receptor 1 (TR 1) and retinoid X receptor (RXR ) proteins demonstrated that RXR and TR both bound the PC1 promoter. Addition of TR 1/RXR to the wild-type PC1 probe demonstrated binding as both homodimers and a heterodimer. EMSAs with oligonucleotides containing deletion mutations of the putative nTREs demonstrated that the proximal nTRE binds more strongly to TR and RXR than the distal nTRE, but that both regions exhibit specific binding. We conclude that there are multiple novel TRE-like sequences in the hPC1 promoter and that these regions act in a unique manner to facilitate the negative effect of thyroid hormone on PC1.