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Preview: Molecular Human Reproduction - current issue

MHR: Basic science of reproductive medicine Current Issue





Published: Wed, 11 Oct 2017 00:00:00 GMT

Last Build Date: Mon, 06 Nov 2017 08:49:06 GMT

 



Evaluation of apoptotic- and autophagic-related protein expressions before and after IVM of fresh, slow-frozen and vitrified pre-pubertal mouse testicular tissue

2017-10-11

AbstractSTUDY QUESTIONDo freezing and in vitro culture procedures enhance the expression of proteins involved in apoptotic or autophagic pathways in murine pre-pubertal testicular tissue?SUMMARY ANSWERIVM strongly modified apoptosis- and autophagy-related relative protein levels in mice testicular tissue whereas the impact of cryopreservation procedures was minimal at the end of the culture.WHAT IS KNOWN ALREADYIn vitro spermatogenesis remains a challenging technical issue as it imposes to find a very close balance between survival and death of germ cell natural precursors (i.e. gonocytes and spermatogonia), which will eventually undergo a complete spermatogenesis close to in vivo conditions. The establishment of efficient culture conditions coupled with suitable cryopreservation procedures (e.g. controlled slow freezing [CSF] and solid surface vitrification [SSV]) of pre-pubertal testicular tissue is a crucial step in the fields of fertility preservation and restoration to improve the spermatic yield obtained in vitro.STUDY DESIGN, SIZE, DURATIONHere, we study cryopreservation procedures (i.e. CSF or SSV) and the impact of culture media compositions. A first set of 66 mouse pre-pubertal testes were directly cultured during 30, 36, 38 and 60 days (D) from 2.5 to 6.5-day-old CD-1 mice to evaluate the impact of time-aspect of culture and to endorse the reverse phase protein microarrays (RPPM) technique as an adapted experimental tool for the field of in vitro spermatogenesis. Ninety others fresh, slow-frozen and vitrified pre-pubertal testes were cultured during 30 days for the principal study to evaluate the impact of cryopreservation procedures before and after culture. Thirty-four testes dissected from 2.5, 6.5, 36.5, 40.5, 42.5 and 62.5 days postpartum (dpp) mice, corresponding to the time frames of spermatogenesis orchestrated in vitro, were used as in vivo controls.PARTICIPANTS/MATERIALS, SETTING, METHODSAfter in vitro culture, testicular tissue samples originated from 2.5 or 6.5-day-old CD-1 male mice were analyzed using RPPM. This targeted proteomic technique allowed us to assess the expression level of 29 apoptosis- and autophagy-related factors by normalizing blank-corrected signal values. In addition, morphological analyses (e.g. HES, PAS, TRA98 and CREM) and DNA fragmentation in intra-tubular cells (i.e. terminal deoxynucleotidyl transferase dUTP nick end labeling; TUNEL) were assessed for the distinct experimental conditions tested as well as for in vivo control mouse testes.MAIN RESULTS AND THE ROLE OF CHANCEA validation of the RPPM procedure in the field of in vitro spermatogenesis was completed with assay and array robustness before a principal study concerning the evaluation of the impact of in vitro culture and cryopreservation procedures. The proportion of elongated spermatids and the total cell number per seminiferous tubule tended to be very different between the in vivo and in vitro conditions (P < 0.05), suggesting the presence of a beneficial regulation on the first spermatogenesis wave by intrinsic apoptosis (Caspase_9) and autophagy (Atg5) factors (P < 0.0003 and r2 = 0.74). Concerning the impact of culture media compositions, a basic medium (BM) composed of αMEM plus 10% KnockOut™ serum replacement and gentamicin supplemented with retinol (Rol) and vitamin E (Vit. E) was selected as the best culture medium for fresh 6.5 dpp tissue cultured during 30D with 27.7 ± 8.10% of seminiferous tubules containing elongated spermatids. Concerning the impact of cryopreservation procedures, SSV did not have any impact on the morphological parameters evaluated after culture in comparison to fresh tissue (FT) controls. The proportion of tubules with elongated spermatids on testicular explants cultured with BMRol+Vit. E was not different between SSV (6.6 ± 1.6%) and CSF (5.3 ± 1.9%); however, round spermatids were observed more frequently for SSV (19 ± 6.2%) than CSF (3.3 ± 1.9%, P = 0.0317). Even if the proportion of TUNEL-positive cells for BMRol+Vit. E was higher at D30 after SSV (4.12 ± 0.26%) [...]



Coumestrol induces mitochondrial dysfunction by stimulating ROS production and calcium ion influx into mitochondria in human placental choriocarcinoma cells

2017-10-11

Abstract
STUDY QUESTION
Does coumestrol inhibit proliferation of human placental choriocarcinoma cells?
SUMMARY ANSWER
Coumestrol promotes cell death in the choriocarcinoma cells by regulating ERK1/2 MAPK and JNK MAPK signaling pathways and through disruption of Ca2+ and ROS homeostasis.
WHAT IS KNOWN ALREADY
A number of patients who suffer from choriocarcinomas fail to survive due to delayed diagnosis or a recurrent tumor and resistance to traditional chemotherapy using platinum-based agents and methotrexate. To overcome these limitations, it is important to discover novel compounds which have no adverse effects yet can inhibit the expression of a target molecule to develop, as a novel therapeutic for prevention and/or treatment of choriocarcinomas.
STUDY DESIGN, SIZE, DURATION
Effects of coumestrol on human placental choriocarcinoma cell lines, JAR and JEG3, were assessed in diverse assays in a dose- and time-dependent manner.
PARTICIPCANTS/MATERIALS, SETTING, METHODS
Effects of coumestrol on cell proliferation, apoptosis (annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of reactive oxygen species (ROS), lipid peroxidation, glutathione levels and endoplasmic reticulum (ER) stress proteins in JAR and JEG3 cells were determined. Signal transduction pathways in JAR and JEG3 cells in response to coumestrol were determined by western blot analyses.
MAIN RESULTS AND THE ROLE OF CHANCE
Results of the present study indicated that coumestrol suppressed proliferation and increased apoptosis in JAR and JEG3 cells by inducing pro-apoptotic proteins, Bax and Bak. In addition, coumestrol increased ROS production, as well as lipid peroxidation and glutathione levels in JAR and JEG3 cells. Moreover, coumestrol-induced depolarization of mitochondrial membrane potential (MMP) and increased cytosolic and mitochondrial Ca2+ levels in JAR and JEG3 cells. Consistent with those results, treatment of JAR and JEG3 cells with a Ca2+ chelator and an inhibitor of IP3 receptor decreased coumestrol-induced depolarization of MMP and increased proliferation in JAR and JEG3 cells.
LARGE SCALE DATA
N/A.
LIMITATIONS, REASONS FOR CAUTION
A lack of in vivo animal studies is a major limitation of this research. The effectiveness of coumestrol to induce apoptosis of human placental choriocarcinoma cells requires further investigation.
WIDER IMPLICATIONS OF THE FINDINGS
Our results indicate that coumestrol induces apoptotic effects on placental choriocarcinoma cells by regulating cell signaling and mitochondrial-mediated functions, with a potential to impair progression of the cancer.
STUDY FUNDING/COMPETING INTEREST(S)
This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.).



Oleic acid stimulation of motility of human extravillous trophoblast cells is mediated by stearoyl-CoA desaturase-1 activity

2017-09-28

Abstract
STUDY QUESTION
Do fatty acids regulate development and motility of human extravillous trophoblast cells (EVTs)?
SUMMARY ANSWER
Oleic acid is a promising lipid molecule that has beneficial effects on motility and development of human EVTs.
WHAT IS KNOWN ALREADY
Fatty acid uptake into trophoblast cells is important for maintaining cellular events during pregnancy, but the molecular mechanisms of action of various fatty acids, including trans fatty acids, saturated fatty acids and monounsaturated fatty acids, in EVT cell lines are not clear.
STUDY DESIGN, SIZE, DURATION
Effects of oleic acid, elaidic acid, palmitic acid and stearic acid on HTR8/SVneo cells were assessed in diverse assays in a dose- and time-dependent manner.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Effects of fatty acids on cell proliferation, migration, invasion and apoptosis (Annexin V expression, propidium iodide staining, TUNEL and invasion assays) of HTR8/SVneo cells were determined. Signal transduction pathways in HTR8/SVneo cells in response to fatty acids were determined by Western blot analyses. Regulation of fatty acids on oxidative conditions in EVTs were determined and validated by measurement of production of cellular reactive oxygen species, intracellular concentrations of free Ca2+and lipid peroxidation assays.
MAIN RESULTS AND THE ROLE OF CHANCE
In present study, we confirmed different effects of oleic acid and elaidic acid on migration, invasion, proliferation and apoptosis of the EVT cell line, HTR8/SVneo. We also investigated stearoyl-CoA desaturase-1 (SCD1) to determine if its activity contributed to oleic acid-induced migration of HTR8/SVneo cells. Next, we analyzed cell signaling molecules mediated by oleic acid and elaidic acid treatment, including MAPK and PI3K/AKT pathways in HTR8/SVneo cells. We further established whether selective inhibition of signaling molecules altered the ability of fatty acids to cause changes in migration and proliferation of HTR8/SVneo cells. Last, we examined the regulatory effects of oleic acid and SCD1 on oxidative stress in HTR8/SVneo cells.
LARGE SCALE DATA
N/A.
LIMITATIONS, REASONS FOR CAUTION
The lack of in vivo animal studies is a major limitation of this research. Effectiveness of oleic acid to stimulate migration of human EVT cells requires further investigation.
WIDER IMPLICATIONS OF THE FINDINGS
Our results suggest that oleic acid can play an important role in promoting invasion of human EVT cell lines while both trans fatty acids and saturated fatty acids are not conducive to normal placentation. This may have implications for the prevention of pre-eclampsia and intrauterine growth restriction.
STUDY FUNDING AND COMPETING INTEREST(S)
This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810) awarded to G.S. and (No. HI17C0929) awarded to W.L. There are no conflicts of interest.



Treatment with AICAR inhibits blastocyst development, trophectoderm differentiation and tight junction formation and function in mice

2017-09-28

Abstract
STUDY QUESTION
What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function?
SUMMARY ANSWER
AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur.
WHAT IS KNOWN ALREADY
AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK.
STUDY DESIGN, SIZE, DURATION
Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0–2000 μM) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 μM AICAR.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR.
MAIN RESULTS AND THE ROLE OF CHANCE
Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible.
LARGE SCALE DATA
Not applicable.
LIMITATIONS, REASONS FOR CAUTION
Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin.
WIDER IMPLICATIONS OF THE FINDINGS
Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles.
STUDY FUNDING AND COMPETING INTEREST(S)
Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.



Glycerol and testicular activity: the good, the bad and the ugly

2017-09-28

ABSTRACT
Over the past decades, there have been several studies suggesting that semen quality is declining. Interestingly, these observations are paired with a significant increase in the number of individuals diagnosed with metabolic diseases, including obesity and diabetes mellitus. Hence, it is tempting to hypothesize that obesity and its associated comorbidities and risk factors (such as a hypercaloric diets) impair the homeostasis of the male reproductive health, with a possible direct effect on the testes. The blood and interstitial fluids of obese individuals usually have increased levels of glycerol, notably due to triglyceride and phospholipid catabolism and high fructose intake. Glycerol is metabolized via intermediary metabolism by a group of reactions centred at the glycerol-3-phosphate shuttle, which links the metabolic pathway of glucose, lipids and oxidative phosphorylation, illustrating its high relevance for biological systems. Glycerol enters and exits the cells by the action of specialized carriers, known as aquaglyceroporins, whose functional importance for male reproductive health has emerged in the last few years. Notably, glycerol has antispermatogenic properties. When present in high concentration in the testis, it causes blood–testis barrier disruption, impairing tubular fluid homeostasis. Nevertheless, glycerol metabolism in testicular cells remains a matter of debate. Herein we discuss previous and current research concerning the role of glycerol and its metabolism in testicular cells, and how it can influence testicular activity.